The Antiarrhythmic Peptide Analog Rotigaptide (ZP123) Stimulates Gap Junction Intercellular Communication in Human Osteoblasts and Prevents
http://www.100md.com
《内分泌学杂志》
The Osteoporosis and Metabolic Bone Unit (N.R.J., S.C.T., Z.H., S.S.H., J.-E.B.J., O.H.S.), Department of Endocrinology and Clinical Biochemistry, Copenhagen University Hospital H:S, DK-2650 Hvidovre, Denmark
Zealand Pharma A/S (E.M., J.S.P.), DK-2600 Glostrup, Denmark
Abstract
Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body weight) or by continuous ip infusion (158 nmol per kilogram body weight per day). During metabolic stress, a high affinity-binding site (KD = 0.1 nM) with low density (15 fmol/mg protein) for [125I]di-I-AAP10 was demonstrated. During physiological conditions, specific binding sites for [125I]AAP10 could not be shown. Studies of the effects of rotigaptide on propagation of intercellular calcium waves and cell-to-cell coupling demonstrated that 10 nM rotigaptide produced a small increase in intercellular communication during physiological conditions (+4.5 ± 1.6% vs. vehicle; P < 0.05). During conditions with metabolic stress, 10 nM rotigaptide produced an increase in coupling measured by both methods. Four weeks after ovariectomy, bone strength of the femoral head was reduced by 20% in vehicle-treated ovariectomized rats, which was completely prevented in both rotigaptide-treated groups. Rotigaptide also prevented decreases in bone mineral. We conclude that the stable analog rotigaptide increases gap junctional communication in osteoblasts in vitro and preferably during conditions with metabolic stress. Rotigaptide further prevents ovariectomy-induced bone loss in vivo. Thus, gap junction modulation may be a promising new target for osteoporosis therapy.
Introduction
GAP JUNCTION CHANNELS are specialized protein pores through which cells communicate and coordinate intercellular signals and responses to hormones. Each gap junction channel is comprised of six connexin (Cx) proteins that are arranged as a hemichannel called the connexon. Placed in the plasma membrane, connexons from one cell form gap junction channels in conjunction with connexons located in membranes of apposing cells (1). Multiple gap junction channels are held together in a plaque of channels called the gap junction.
Twenty different Cxs have been identified with Cx43 being the most abundant Cx in cardiomyocytes and in bone (2, 3). Gap junctions are particularly well studied in the heart in which they are responsible for the electrical coupling of cardiomyocytes and synchronization of contraction of the myocardium. In bone, the functions of gap junctions are less well understood, but they seem to be important in embryonic bone development (4) and differentiation and maturation of the bone-forming cells, i.e. the osteoblasts (5). They also serve to link osteocytes together in a network throughout the bone, enabling them to propagate responses to mechanical forces to the osteoblasts (6, 7). Intracellular calcium is considered a major signaling molecule in bone, and in vitro studies have demonstrated that the intercellular calcium signal travels partly through gap junctions in cultures of chondrocytes (8, 9), osteoblastic cell lines (10), and human osteoblast-like cells (11). We previously reported that human osteoblast-like cells possess the ability to propagate calcium transients by two mechanisms, one involving purinergic receptors and the other involving passage of a messenger through gap junction channels (11). By desensitizing purinergic receptors with ATP, the gap junction wave is uncovered (11), and the number of cells in the gap junction-mediated wave can be determined. Intercellular calcium signaling might be a way for cells involved in regulation of bone turnover to communicate signals throughout the network of cells in bone. Both osteoblasts and osteocytes are coupled through gap junctional plaques between cells. Thus, improving intercellular signaling among these cells might affect the regulation of bone turnover.
The pathophysiological role of altered gap junction function has been hampered by the lack of selective pharmacological tools. Therefore, we found it interesting that the family of antiarrhythmic peptides (AAPs) has been reported to stimulate gap junction intercellular communication (GJIC) between cardiomyocytes, but effects in noncardiac and human tissue are unknown (12, 13, 14, 15). However, both the endogenous AAP (H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH) and the synthetic derivative AAP10 (H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH2) are enzymatically unstable, which render the use of these compounds as pharmacological tools difficult. Therefore, we developed a highly stable AAP analog called rotigaptide (Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2; formerly known as ZP123) that is closely related structurally to AAP10 but with a half-life in human plasma in vitro of 14 d relative to 3–4 min for AAP10 (16). Thus, in the present experiments, we used the stable AAP analog rotigaptide to examine the hypothesis that AAPs could affect calcium signaling and cell-to-cell coupling in primary human osteoblasts. In addition, we wanted to examine the in vivo effects of rotigaptide on bone strength and bone mass in both compact bone (femoral body) and trabecular bone (femoral head/neck). The effect was examined 4 wk after ovariectomy (OVX) in groups of rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body weight), or continuous ip infusion (158 nmol per kilogram body weight per day).
Materials and Methods
Cell culture
Human osteoblastic cells (hOBs) were isolated from human bone marrow obtained by puncture of the posterior iliac spine of healthy volunteers (aged 20–36 yr): 10–15 ml marrow material were collected in 15 ml PBS+Ca,Mg (Life Technologies, Grand Island, NY) with 100 U/ml Heparin (catalog no. H-3149; Sigma, St. Louis, MO). The mononuclear fraction of the marrow was isolated on a Lymphoprep gradient (Nycomed Pharma, Oslo, Norway) by centrifugation at 750 x g for 30 min. After harvesting, the mononuclear fraction was washed once with culture medium and centrifuged at 400 x g for 10 min. Subsequently cells were counted and plated in culture medium at 8 x 106 cells per 100-mm dish. hOB culture medium (all reagents obtained from Life Technologies) was composed of the following: MEM without Phenol Red with Glutamax supplemented with 10% heat-inactivated fetal calf serum and 0.1% penicillin/streptomycin. Culture medium was changed the following day, and the cells were cultured at 37 C in 5% CO2 with medium change every 7 d. After 3–4 wk of culture, the cells had reached 70% confluence. The medium was then supplemented with 100 nM dexamethasone (Sigma) for 7 d. Then cells were plated for video imaging experiments on a 25-mm no. 1 glass coverslip that was placed in a 35-mm dish (or in each well of a six-well multidish). Cells were plated at 2.5 x 105 cells/coverslip and cultured for 2–3 d before use.
The study complies with the Declaration of Helsinki, and was approved by the Danish Ethics Committee System.
Binding of [125I]di-I-AAP10 to hOB cells
hOB cells grown for 3–4 wk were replated in 24-multiwell dishes at a density of at 2.1 x 105 cells/cm2 (4 x 104 cells/well) and grown for 3 d in vitro in 0.50 ml/well of F-12K nutrient (Gibco BRL, Gaithersburg, MD), mixture supplemented with 10% fetal calf serum and 1000 U penicillin per 1000 μg streptomycin (Gibco BRL) in a normal atmosphere adjusted to 5% CO2 and 100% humidity at 37 C. [125I]di-I-AAP10 (2000 Ci/mmol) was custom synthesized by Amersham Pharmacia Biotech (Little Chalfont, Wales, UK).
In situ binding experiments with [125I]di-I-AAP10 were done in triplicate and performed essentially as described by Koenig (17). On the day of analysis, growth medium was removed and washed twice with 2 ml Dulbecco’s PBS (Gibco BRL). Increasing concentrations (0.05, 0.1, and 0.5 nM) of [125I]di-I-AAP10 solution (0.50 ml) with or without unlabeled 1 mM di-I-AAP10 (to estimate nonspecific binding) was added to the cell cultures and incubated for 2 h at 21 C. The binding reaction was stopped by rapidly rinsing each well, one at a time, with 2 x 1 ml ice-cold (0 C) Dulbecco’s PBS and left to dry. The cellular material with bound [125I]di-I-AAP10 was collected by adding 0.25 ml of 0.5% Triton X-100 (vol/vol) to each well and incubated for at least 1 h at room temperature to solubilize the cells. The extract was then transferred to counting vials, the wells rinsed with 0.25 ml water, and the rinse extract added to the corresponding vials. The vials were counted in a -counter. The protein contents of test samples were measured according to Lowry et al. (18). Data on specific binding were fitted to the equation:
where BMAX is maximum binding, s is the ligand concentration, and KD is the dissociation constant.
Intercellular calcium signaling
The cells cultured on coverslips were loaded with 5 μM fura 2-acetoxymethyl (AM) (Molecular Probes, Eugene, OR) for 30 min at 37 C and incubated in fresh medium for 20 min. Coverslips were then affixed to a PDMI-2 culture chamber (Medical Systems Corp., Greenvale, NY), maintained at 37 C with superfused CO2, on a Axiovert microscope (Zeiss, New York, NY). Intercellular calcium waves were induced by mechanical stimulation of a single cell using a borosilicate glass micropipette affixed to an Eppendorf 5171 micromanipulator (Eppendorf, Netheler, Germany). Imaging was performed using a MetaMorph imaging system (Universal Imaging, Downingtown, PA). The excitation light (340 and 380 nm) was provided by a monochromator (T.I.L.L. Photonics GmbH, Planegg, Germany). Images were acquired with an intensified CCD camera (Dage MTI, Michigan City, IN) and digitized with a Matrox MVP image processing board (Matrox, Dorval, Canada). The number of cells in the wave was counted, and triple determinations were performed for each experiment. The n denotes the number of experiments.
To suppress purinergic receptor-mediated calcium waves, cells were pretreated with 100 μM ATP immediately before the experiment. We have previously shown that this protocol effectively desensitizes purinergic receptors in hOB cells (10, 11).
Cell coupling
Microinjections were performed in monolayer of cells cultured on glass coverslips as described above using the Eppendorf 5171 micromanipulator and the Eppendorf Transjector 5346 system. A micropipette was loaded with a 10 mM Lucifer Yellow (LY) solution (Sigma), and one single cell was carefully injected with LY for 30 sec. The micropipette was removed from the cell and after 30 sec the number of cells that showed dye transfer were counted. The excitation light for LY was 430 nm, and images were acquired as described above.
The parachute assay is a noninvasive technique to monitor the diffusion of fluorescent dyes through gap junctions (19, 20). In this assay, cells are preloaded with an AM-ester of a dye that is gap junction permeant and the cells are parachuted down on an unloaded monolayer of cells. With this method, the cell membrane is not disrupted as it is in microinjection experiments. Within a few hours, gap junction channels are formed by docking of hemichannels from adjacent cells, which allows the cytoplasmatic fluorescent dye to pass through gap junction channels from the donor cell to a number of neighboring acceptor cells.
In these experiments, acceptor cells were prelabeled with the nontransferable dye PKH-26 (Sigma) 2 d before the parachute assay. Cells were labeled with PKH-26 according to the manufacturer’s instructions in a solution with a total PKH-26 concentration of 2 μM. To terminate the reaction, heat-inactivated fetal bovine serum was added. Cells were centrifuged, resuspended in medium, and replated in 35-mm plates with approximately 2.5 x 105 cells/plate. Donor cells were exposed to the membrane permeant dye calcein-AM (1 μM; Molecular Probes) for 45 min at 37 C. Cells were washed with PBS and harvested in a single cell suspension using trypsin digestion. Then 1000 donor cells for each plate were parachuted on the acceptor cells and incubated for 150 min at 37 C. After washing the cells with PBS, cells were harvested using trypsin digestion, and the cell suspension was diluted to a cell concentration of 1.5–2 x 105 cells/ml. The ratio of cells possessing both dyes relative to cells labeled with PKH-26 alone was used as a measure of GJIC. Fluorescent signals were measured by fluorescence activated cell sorting (FACS) using a model FACSCalibur machine (Becton Dickinson, Franklin Lakes, NJ).
In vivo study
Forty-six female nullipara Wistar rats, 8 months old (264 ± 5 g), were randomly allocated to four groups of treatment on d 0. Rats were anesthetized with a sc injection of 2 ml/kg of a 1:1 mixture of Hypnorm (fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml, Janssen, Beerse, Belgium) and midazolam (5 mg/ml, Hameln Pharmaceuticals, Hameln, Germany) diluted 1:1 in distilled water. When surgical anesthesia was established, a midline incision was made that allowed bilateral OVX or exposure of the ovaries without removal (sham-OVX). In addition, all rats had an Alzet osmotic minipump (flow rate 2.6 μl/h) inserted into the ip cavity for chronic administration of vehicle or rotigaptide. The pump was primed with vehicle or rotigaptide for 24 h before the operation. To relieve postoperative pain, the rats were treated with Temgesic (buprenorphine 20 μg/100 g sc twice daily, Schering-Plough, Brussels, Belgium) and Metacam (meloxicam 0.1 mg/100 g sc once daily, Boehringer Ingelheim, Mannheim, Germany) for 3 d after surgery.
Four groups of rats were studied: 1) sham + vehicle (n = 11); 2) OXV + vehicle (n = 11); 3) OVX + rotigaptide by continuous ip infusion (158 nmol per kilogram body weight per day) (n = 12); and 4) OVX + rotigaptide by sc injection (300 nmol per kilogram body weight twice daily) (n = 12).
On d 30, animals were killed, and femurs were collected for bone strength measurements and stored at –20 C. Furthermore, the opposite femur was collected for bone mineral density (BMD) measurements and stored in 70% ethanol at 5 C. For measurement of plasma concentrations of rotigaptide, blood samples were collected 2–4 h after the last dosing in rats treated with sc injection of rotigaptide. Plasma was handled and analyzed for rotigaptide as previously described in detail (16, 21). Bone strength measurements were performed on a compression device (Lloyd Instruments, Fareham, UK). BMD was performed as duplicate determinations on a PIXImus dual-energy x-ray densitometer (Lunar Corp., Madison, WI).
Drugs
Rotigaptide (proposed INN name for ZP123) and di-I-AAP10 were synthetized at Zealand Pharma A/S (Glostrup, Denmark). Tritiation of di-I-AAP10 was performed by Amersham (Aylesbury, UK).
Statistics
The nonparametric Wilcoxon test was used to examine statistical differences for data collected on a discontinuous scale. For data collected on a continuous scale, overall comparison among groups was performed using a one-way ANOVA followed by post hoc analysis using Fisher’s least significant difference test. Differences were considered significant at the 5% level.
Results
Binding of [125I]di-I-AAP10 to human osteoblastic cells during normoxia and hypoxia
When cells were deprived of oxygen and glucose and exposed to low Ca2+ (<5 μM), specific high-affinity binding sites for [125I]di-I-AAP10 could be demonstrated on intact hOB cells. The binding site had a KD = 0.4 nM but displayed a very low density with BMAX = 15 ± 3 fmol/mg protein. However, we were unable to demonstrate specific binding sites for [125I]di-I-AAP10 when hOB cells were exposed to physiological Krebs-buffer with normal concentrations of Ca2+ (1.8 mM) and glucose (6 mM) at atmospheric O2 tension (21%).
Effects of rotigaptide on intercellular calcium signaling in osteoblastic cells during normoxia
To assess the ability of rotigaptide to increase gap junction-mediated intercellular calcium signals, monolayers of human osteoblastic cells on glass coverslips were loaded with the calcium indicator dye fura 2. During real-time imaging, a mechanical stimulation with a glass micropipette was performed on hOB cells desensitized with ATP. After mechanical stimulation an increase in the intracellular calcium appeared with subsequent spreading of the signal to the surrounding cells (Fig. 1).
As illustrated in Fig. 2, vehicle and 0.1 nM rotigaptide had no effect on the spreading of intercellular calcium waves in hOB cells cultured during physiological conditions. However, 10 nM produced a consistent increase in the spreading of intercellular waves (P < 0.001), whereas the effect obtained after incubation with the highest concentration of rotigaptide (1 μM) was not statistically increased (P = 0.09). To compare responses during different conditions using different techniques, we used the 10 nM concentration of rotigaptide in the following studies.
Effects of rotigaptide on cell coupling in osteoblasts during normoxia
To assess whether the effect of rotigaptide on intercellular calcium signaling is a result of a direct effect on cellular coupling between osteoblastic cells, microinjection experiments were performed. The gap junction permeant dye LY was injected into a single human osteoblast in a monolayer. After 60 sec, the number of cells containing dye was assessed. During normoxia, the dye spread to a median of 10.4 cells (Fig. 3). Then rotigaptide was added to the bathing solution in a final concentration of 10 nM, and the microinjection with LY was repeated after 10 min. In this case the dye was distributed to a median of 9.2 cells, which was not significantly different from before adding the compound. Thus, during normoxic conditions rotigaptide did not affect the basic cell-to-cell gap junctional coupling in human osteoblastic cells as assessed by the microinjection technique (Fig. 3).
We also wanted to assess whether we could see an effect of rotigaptide on cell coupling in human osteoblasts using a noninvasive technique, the parachute assay. Parallel cultures were exposed to vehicle, rotigaptide (10 nM), or the gap junction inhibitor heptanol (3.5 mM). Two populations were identified: one positive for PKH-26 only and one positive for both calcein and PKH-26. The ratio between cells that were labeled with calcein (acceptor cells, positive for both calcein and PKH-26) and the total number of potential acceptor cells (all cells positive for PKH-26) was used as a measure for cell coupling. The parachute assay appeared to be more sensitive than LY dye transfer for detecting small increases in cell coupling because using this technique, an increase of 4.5 ± 1.6% (mean ± SEM) in cell-to-cell coupling was detected in cell populations treated with 10 nM rotigaptide for 48 h (Fig. 3). As expected, heptanol decreased coupling to 39.0 ± 2.7% of the coupling in control populations. Thus, during normoxia, a small rotigaptide-induced increase in cell-to-cell coupling in human osteoblasts could be detected but only with the noninvasive parachute assay.
Effects of rotigaptide on impaired cell coupling and signaling induced by different types of metabolic stress in bone cells
The degree of coupling between Cx43-expressing cells is regulated by intracellular pH (22, 23). Thus, acidification-induced closure of gap junctions during myocardial ischemia is considered an important substrate for lethal ventricular arrhythmias (24, 25). In this study, we examined the effect of rotigaptide on cellular uncoupling in bone cells exposed to metabolic stress. First, we investigated the effect of removal of glucose from medium on the propagation of intercellular calcium signaling in monolayers of human osteoblast-like cells. One single cell was stimulated mechanically. The stimulated cell showed an increase in intracellular calcium concentration, which subsequently spread to the neighboring cells, with a median wave propagation to 10.0 cells. ATP was added in a final concentration of 100 μM to desensitize purinergic receptors on the cell membrane. All cells in the field of view showed increases in intracellular calcium concentrations, which returned to resting calcium levels within 1–2 min. Another mechanical stimulus was applied, now with a median wave propagation to 3.0 cells. To induce metabolic stress, the medium was changed to a medium without glucose, and after 8 min of aglycemia, one single cell was poked. Again, an increase in intracellular calcium was observed in the stimulated cell, but in absence of glucose, the wave propagation decreased to a median of only 1.2 cells in the wave (P = 0.027). In vehicle-treated, time-control experiments, the uncoupling was sustained throughout the experiment (Fig. 4, top panel). In another group of cultures, rotigaptide was added in a concentration of 10 nM, and after 5 min of equilibration, the mechanical stimulation was repeated, still under glucose-free conditions. Now the wave was almost completely restored, with median wave propagation to 2.8 cells (P = 0.012) (Fig. 4). Thus, metabolic stress induced by removal of glucose decreases intercellular calcium signaling in human osteoblastic cells; however, calcium signaling is completely restored by adding 10 nM rotigaptide.
In addition, we investigated the effect of hypoxia on the propagation of intercellular calcium signaling in monolayers of fura 2-loaded hOB cells. Cells were incubated for approximately 48 h in a humidified atmosphere containing 3–6% O2, 5% CO2, and 89–92% N2. On the microscope, one single cell was stimulated mechanically, and the wave propagation was recorded. However, after 48 h of hypoxia, the intracellular calcium concentration was too high to determine the exact wave propagation properties.
To examine whether hypoxia affected the cellular coupling as assessed by LY dye transfer, we microinjected LY in single human osteoblastic cells grown in a monolayer. After 60 sec we counted the number of coupled cells. The osteoblasts were quite resistant to hypoxia in terms of cell coupling. Thus, after 24 h no difference was seen in coupling between cells cultured during hypoxia and normoxia. But after 48 h, cells cultured under hypoxic conditions showed a decreased dye transfer to a median of 8.2 cells (P = 0.01), compared with 11.7 in control cells cultured during normoxic conditions. After adding 10 nM rotigaptide, the cell-to-cell coupling was restored to a median of 10.5 cells, although this did not reach statistical significance (P = 0.09 vs. before rotigaptide) (Fig. 3).
Moreover, we tested the effect of rotigaptide on intercellular coupling using the parachute assay. Cells were kept under hypoxic conditions for 48 h and treated with either vehicle or 10 nM rotigaptide (Fig. 3). Rotigaptide-treated cells showed an increase of 34.4 ± 15.6% in coupling ratio, compared with vehicle (P = 0.06). Again cell coupling could be inhibited by heptanol, with a coupling ratio of only 38.7 ± 20.1% (P < 0.05 vs. vehicle).
Effects of rotigaptide on dichlorodiphenyl trichloroethane (DDT)-induced cellular uncoupling
The insecticide DDT is a well-known carcinogen and uncoupler of gap junctions, and it has been proposed that this effect may account for some of its oncogenic properties (26). To examine whether rotigaptide was able to counteract the DDT-induced effect on cell-to-cell coupling, microinjection experiments were performed on human osteoblastic cells grown in monolayers. The cells were incubated for 1 h with DDT at a concentration of 13 μM, which is considered a relevant concentration because it has been shown to affect depolarization and oxidative control of cells (24). Furthermore, concentrations of approximately one sixtieth of the ones used in this study have been detected in blood from healthy nonsymptomatic women (25), and because DDT is accumulated in human tissues, the concentrations used in the present study may be of relevance in human toxicology. Microinjection experiments were performed as described earlier, and the number of coupled cells was recorded. In hOB cells that were pretreated with DDT, 10 min of incubation with 10 nM rotigaptide increased the median number of coupled cells (Fig. 5), whereas cell coupling was unchanged throughout the experiment in pair-treated cells that received vehicle instead of rotigaptide. Thus, rotigaptide was able to reverse DDT-induced uncoupling in human bone cells.
Effect of rotigaptide on bone strength and BMD in OVX rats
The in vivo effect of rotigaptide on bone was examined 4 wk after OVX. BMD is usually used as a noninvasive indicator of bone status and bone strength, but the ultimate parameter for assessing bone quality is directly measured mechanical bone strength. Bone strength was determined at two different sites of the femur: the femoral bone shaft (i.e. femoral body), which is primarily comprised of compact bone, and the femoral head, which is mainly constructed by trabecular bone. Thus, the architecture of the femoral head is similar to the architecture of the femoral neck, in which hip fractures most often appear. Bone strength of the femoral body was performed using the three point bending test, and as shown in Fig. 6, no differences in bone strength of the femoral body were observed 4 wk following OVX. Bone strength of the femoral head was performed using a compression test, and as illustrated in Fig. 6, bone strength of the femoral head was significantly reduced in vehicle-treated OVX rats, compared with vehicle-treated, sham-operated control rats (167 ± 11 vs. 208 ± 13 N; P < 0.05). Interestingly, the OVX-induced decreased in bone strength of the femoral head was completely prevented in both rotigaptide-treated groups. To examine BMD in areas that were used for determination of femoral trabecular and cortical bone strength, respectively, BMD was measured at the femoral head and neck region and at the middle third of the femoral shaft. BMD measurements confirmed that the reduction in bone density 4 wk after OVX was greater in the femoral neck and head (–9.7 ± 2.2%; P < 0.01 vs. sham + vehicle) than in the femoral shaft (–1.1 ± 1.1%; n.s. vs. sham + vehicle). In agreement with bone strength data, pulse treatment with rotigaptide prevented the OVX-induced bone loss, whereas continuous ip infusion of rotigaptide had no significant effect on BMD in the femoral neck and head region (Fig. 7). At the time of killing, the plasma concentration of rotigaptide was 19 ± 2 nM in rats treated with continuous ip infusion and 635 ± 52 nM in rats treated with sc injections (2–4 h after the last sc injection).
Discussion
The purpose of this study was to examine the effects of AAPs on bone in vitro and in vivo. First, we wanted to establish whether we could show specific binding of the AAPs to primary hOB cells and investigate whether the stable AAP analog rotigaptide was able to affect gap junction intercellular coupling in these cells. Furthermore, we wanted to test the in vitro results in an in vivo setting because it has been shown that coupling among osteoblasts is related to production of bone-related proteins. Thus, a compound capable of increasing osteoblastic cell coupling in vitro might be able to affect bone turnover in vivo.
During metabolic stress (no oxygen, no glucose, low calcium), a high-affinity binding site for [125I]di-I-AAP10 was demonstrated (KD = 0.1 nM), which displayed a very low density (BMAX = 0.5 fmol/mg protein). However, during physiological conditions, specific binding sites for [125I]di-I-AAP10 could not be shown. Functional studies of the effects of rotigaptide on propagation of intercellular calcium waves and cell-to-cell calcein transfer (using the noninvasive parachute assay) demonstrated that 10 nM rotigaptide produced a small increase in cell-to-cell coupling during physiological conditions. Moreover, during physiological conditions, 10 nM rotigaptide did not affect gap junctional diffusion of microinjected LY. This is possibly related to the poor sensitivity (low number of observations) of this method relative to the noninvasive parachute assay (FACS recording of more than 20.000 cells/observation). However, during conditions with metabolic stress in hOB cells, 10 nM rotigaptide in most cases produced an increase in coupling measured by intercellular calcium waves (P < 0.05), LY dye coupling (P < 0.05), and in the noninvasive parachute assay (P = 0.06). Furthermore, 10 nM rotigaptide were able to revert cell-to-cell uncoupling induced by the insecticide DDT (P < 0.001). These findings suggest that hOB cells express binding sites for AAPs that are dormant during physiological conditions but appear during metabolic stress. Furthermore, the stable AAP analog rotigaptide increases gap junctional coupling in primary human osteoblasts.
However, no obvious differences between the parachute assay and the microinjection experiments were seen. In contrast, more convincing effects were seen when using the calcium wave assay. Using this technique, we found significant effects, even under normoxic conditions. Calcium wave propagation among osteoblasts is a far more complicated process than simple cell-cell coupling via gap junctions. First, it involves purinergic P2Y receptors, which we have desensitized in the current study, and second, it involves the passage of a soluble agent through the gap junctions, inducing opening of voltage-operated calcium channels in the neighboring cells, which subsequently leads to influx of calcium from the extracellular space. The nature of this soluble agent has not yet been clarified, but it could be an electrical current. Gating of gap junctions is also a complex process, and the opening of these intercellular junctions is highly regulated and specific. These basic differences between dye coupling/parachute assay and the calcium wave assay might be the explanation for the different responses we see of the compound. Furthermore, other explanations could also underlie the missing effect of the compound in some of the assays applied to the bone cells. The cells used are phenotypically mature osteoblastic cells at the end of the differentiation process. At this stage cells are highly coupled and might not be able to increase coupling more, at least at detectable levels. Thus, it might have been interesting to test the effect of the drug on cells earlier in the osteoblastic lineage, during which gap junctional communication/coupling is not so abundant as in later stages. This explanation is also supported by the fact that the most dramatic effects of the compound were seen under circumstances in which coupling was inhibited by DDT treatment or removal of glucose. These conditions reduced cell coupling and calcium wave propagation significantly, and to a higher degree than hypoxia alone, and the reduced coupling could be at least partly reversed by rotigaptide.
A large amount of work has demonstrated that in cardiomyocytes, hypoxia leads to uncoupling of GJIC, which is an important substrate for the development of cardiac arrhythmias (27). In a series of pioneering studies, Dhein and colleagues (13, 15) and Daleau (14) demonstrated that the synthetic AAP analog AAP10 prevented GJIC uncoupling in cardiomyocytes during hypoxia, but during normal physiological conditions AAP10 had no effect on GJIC or the electrical properties of the heart. Recent publications on rotigaptide (16, 21, 28) indicate that this novel stable compound has similar electrophysiological properties and that the effect of rotigaptide on cardiac GJIC is most pronounced during metabolic stress. In bone, the effect of oxygen deprivation on GJIC is less well studied, but it has been demonstrated that during hypoxia, osteoblast proliferation and differentiation are decreased (29). Thus, impaired osteoblast GJIC during hypoxia may be an important pathophysiological mechanism in metabolic bone diseases that involve poor vascularization. Therefore, we examined the effects of rotigaptide on GJIC in human osteoblasts exposed to oxygen deprivation. Human osteoblasts were quite resistant to hypoxia, but after 48 h of oxygen deprivation, a significant decrease in cell coupling was seen. Thus, human osteoblasts, like cardiomyocytes, respond to hypoxia with a decrease in GJIC. However, the most intriguing observation was that rotigaptide was able to reverse this decreased coupling completely. In addition to 48 h of hypoxia, we also examined the effect of rotigaptide during metabolic stress elicited by glucose deprivation + hypoxia. Again, the decreased GJIC during metabolic stress was reversed by rotigaptide, and the effect on cell coupling as measured in the parachute assay seemed to be more pronounced than under normoxic conditions. These findings concur with the fact that AAP10 and rotigaptide affects GJIC in cardiomyocytes only during conditions with metabolic stress (13, 14, 15).
Furthermore, GJIC has been reported to be involved in tumor control (30). DDT is an insecticide that has tumor-promoting properties and decreases GJIC (31, 32). The exact mechanism by which DDT affects GJIC is unclear, but some reports point to a DDT-induced increase in degradation of Cx43 via a proteasome and/or lysosome-dependent pathway (33). In this study, we examined the effect of rotigaptide on GJIC in human osteoblasts uncoupled by DDT, and in line with the previously discussed observations during metabolic stress, we found that rotigaptide also increased GJIC in cells that were partially uncoupled due to pretreatment with DDT. These findings suggest that the AAP analog rotigaptide could play a role in inhibiting tumor promotion induced by tumor promoting agents such as DDT.
Previous studies have shown that increased coupling among osteoblasts in vitro is followed by an increase of the synthesis of bone formation-specific proteins like alkaline phosphatase and osteocalcin (34). Accordingly, in vivo deletion of the Cx43 gene is followed by impaired osteoblast function in a mouse model in which Cx43 is knocked out (4). Furthermore, intercellular calcium signaling via Cx43 and purinergic receptors seems to be a way by which osteoblasts coordinate a number of activities as well as a way for osteocytes to translate mechanical forces to biological signals. Thus, intact cell-cell coupling seems to be important for a normal regenerating skeleton, and enhancing the signaling system might therefore be beneficial for bone. We therefore wanted to test whether the effects on cell coupling and calcium signaling were paralleled in vivo by changes in bone strength and BMD.
To examine whether the gap junction-modifying properties of rotigaptide could affect OVX-induced bone loss, a preventive treatment study was performed on old nullipara female rats exposed to OVX. This model is generally accepted as a predictive model of loss-of-estrogen-induced bone loss. Already 4 wk after OVX, we observed a significant 20% reduction of bone strength in the femoral head but not in the femoral body. Considering the fact that the femoral head is comprised of trabecular bone, which is far more metabolically active than the compact bone of the femoral body, it is not surprising that OVX-induced reduction in bone strength appeared earlier in the femoral head than in the femoral body. However, the most striking finding of this study was that both regimens of rotigaptide completely prevented OVX-induced reduction of bone strength in the femoral head. Interestingly, the effects of OVX on the femoral caput bone strength was also reflected in BMD measurements, although the relative change in bone strength (–20 ± 5%) appeared greater than the relative change in BMD in the femoral neck and caput region (–9.7 ± 2.2%). This observation may suggest that turnover of matrix protein is greater than turnover of minerals in the rat femoral caput and neck region during early osteoporosis or that the changes in biomechanical properties of trabecular bone after OVX are not linearly related to loss of bone mineral. Moreover, rotigaptide pulse therapy (high peak concentrations) prevented the OVX-induced decrease in BMD, whereas continuous ip infusion of rotigaptide had no effect on BMD (low constant concentrations). Further studies are warranted to examine whether the dose response relationship of rotigaptide on BMD is shifted to the right relative to the effect on bone strength in trabecular femoral bone.
In line with the consistent in vitro effects of 10 nM rotigaptide on gap junction intercellular communication, 4 wk infusion with rotigaptide at a dose level that produced a plasma concentration of 19 ± 2 nM was associated with marked in vivo effects. However, the present in vivo study was hampered by the relatively short period of OVX, and further studies are warranted to examine the long-term effects on bone formation and resorption markers. In absence of histomorphometry data and/or plasma concentrations of bone markers, the present study does not allow interpretations on the exact mechanism through which rotigaptide exerts its action on bone strength. Although the present study suggests that rotigaptide has direct effects on cell-to-cell coupling among osteoblasts, other mechanisms may be involved in the in vivo effect. Thus, further studies are warranted to investigate the effect AAPs on osteoblast and osteoclast activity.
In conclusion, we have for the first time demonstrated an effect of an AAP analog on human osteoblastic cells and bone strength in vivo. Rotigaptide produced only minor changes in GJIC and calcium signaling during normal physiological conditions, but during metabolic stress, rotigaptide elicited consistent stimulation of GJIC as demonstrated by three different techniques (Ca wave signaling, microinjected LY dye transfer, and noninvasive dye transfer of calcein). In vivo, rotigaptide prevented the 20% reduction of bone strength of the femoral head observed 4 wk after OVX. The effect of rotigaptide was seen in animals given continuous ip infusion by osmotic minipumps as well as in rats treated with sc injections twice daily. These findings suggest that AAP analogs may be an interesting new class of molecules to be used in studies on the role of GJIC in bone cells. Moreover, the in vivo results suggest that AAP analogs might be useful in the prevention of metabolic bone diseases; however, further studies are warranted to examine the potential therapeutic role of these compounds in cancer and bone disease.
Footnotes
Abbreviations: AAP, Antiarrhythmic peptide; AM, acetoxymethyl; BMAX, maximum binding; BMD, bone mineral density; Cx, connexin; DDT, dichlorodiphenyl trichloroethane; FACS, fluorescence activated cell sorting; GJIC, gap junction intercellular communication; hOB, human osteoblastic cell; KD, dissociation constant; LY, Lucifer Yellow; OVX, ovariectomy.
References
Kumar NM, Gilula NB 1996 The gap junction communication channel. Cell 84:381–388
Civitelli R, Beyer EC, Warlow PM, Robertson AJ, Geist ST, Steinberg TH 1993 Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 91:1888–1896
Steinberg TH, Civitelli R, Geist ST, Robertson AJ, Hick E, Veenstra RD, Wang HZ, Warlow PM, Westphale EM, Laing JG, Beyer EC 1994 Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J 13:744–750
Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R 2000 Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol 151:931–943
Chiba H, Sawada N, Oyamada M, Kojima T, Nomura S, Ishii S, Mori M 1993 Relationship between the expression of the gap junction protein and osteoblast phenotype in a human osteoblastic cell line during cell proliferation. Cell Struct Funct 18:419–426
Donahue HJ 1998 Gap junctional intercellular communication in bone: a cellular basis for the mechanostat set point. Calcif Tissue Int. 62:85–88 (Editorial)
Donahue HJ 2000 Gap junctions and biophysical regulation of bone cell differentiation. Bone 26:417–422
D’Andrea P, Vittur F 1996 Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes. Cell Calcium 20:389–397
Donahue HJ, Guilak F, Vander Molen MA, McLeod KJ, Rubin CT, Grande DA, Brink PR 1995 Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions. J Bone Miner Res 10:1359–1364
Jorgensen NR, Geist ST, Civitelli R, Steinberg TH 1997 ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells. J Cell Biol 139:497–506
Jorgensen NR, Henriksen Z, Brot C, Eriksen EF, Sorensen OH, Civitelli R, Steinberg TH 2000 Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms. J Bone Miner Res 15:1024–1032
Aonuma S, Kohama Y, Akai K, Komiyama Y, Nakajima S, Wakabayashi M, Makino T 1980 Studies on heart. XIX. Isolation of an atrial peptide that improves the rhythmicity of cultured myocardial cell clusters. Chem Pharm Bull (Tokyo) 28:3332–3339
Müller A, Gottwald M, Tudyka T, Linke W, Klaus W, Dhein S 1997 Increase in gap junction conductance by an antiarrhythmic peptide. Eur J Pharmacol 327:65–72
Daleau P 1998 Effects of antiarrhythmic agents on junctional resistance of guinea pig ventricular cell pairs. J Pharmacol Exp Ther 284:1174–1179
Dhein S, Manicone N, Muller A, Gerwin R, Ziskoven U, Irankhahi A, Minke C, Klaus W 1994 A new synthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval and diminishes alterations of epicardial activation patterns induced by regional ischemia. A mapping study. Naunyn Schmiedebergs Arch Pharmacol 350:174–184
Kjolbye AL, Knudsen CB, Jepsen T, Larsen BD, Petersen JS 2003 Pharmacological characterization of the new stable antiarrhythmic peptide analog Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2 (ZP123): in vivo and in vitro studies. J Pharmacol Exp Ther 306:1191–1199
Koenig JA 1999 Radioligand binding in intact cells. In: Keen M, ed. Methods in molecular biology: receptor binding techniques. Totowa, NJ: Humana Press Inc.; 89–118
Lowry OH, Rosebrough NJ, Farr AL, Randdall RJ 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Ziambaras K, Lecanda F, Steinberg TH, Civitelli R 1998 Cyclic stretch enhances gap junctional communication between osteoblastic cells. J Bone Miner Res 13:218–228
Goldberg GS, Bechberger JF, Naus CC 1995 A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer. Biotechniques 18:490–497
Xing D, Kjolbye AL, Nielsen MS, Petersen JS, Harlow KW, Holstein-Rathlou NH, Martins JB 2003 ZP123 increases gap junctional conductance and prevents reentrant ventricular tachycardia during myocardial ischemia in open chest dogs. J Cardiovasc Electrophysiol 14:510–520
Delmar M, Morley GE, Ek-Vitorin JF, Francis D, Homma N, Steriopoulos K, Lau A, Taffet S 2000 Intracellular regulation of the cardiac gap junction channel connexin43. In: Zipes DP, Jalife J, eds. Cardiac electrophysiology. From cell to bedside. Philadelphia: Saunders Co.
Duffy HS, Sorgen PL, Girvin ME, O’Donnell P, Coombs W, Taffet SM, Delmar M, Spray DC 2002 pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains. J Biol Chem 277:36706–36714
Tiemann U, Pohland R, Kuchenmeister U, Viergutz T 1998 Influence of organochlorine pesticides on transmembrane potential, oxidative activity, and ATP-induced calcium release in cultured bovine oviductal cells. Reprod Toxicol 12:551–557
Turusov V, Rakitsky V, Tomatis L 2002 Dichlorodiphenyltrichloroethane (DDT): ubiquity, persistence, and risks. Environ Health Perspect 110:125–128
Nielsen M, Ruch RJ, Vang O 2000 Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication. Biochem Biophys Res Commun 275:804–809
Lerner DL, Beardslee MA, Saffitz JE 2001 The role of altered intercellular coupling in arrhythmias induced by acute myocardial ischemia. Cardiovasc Res 50:263–269
Eloff BC, Gilat E, Wan X, Rosenbaum DS 2003 Pharmacological modulation of cardiac gap junctions to enhance cardiac conduction: evidence supporting a novel target for antiarrhythmic therapy. Circulation 108:3157–3163
Tuncay OC, Ho D, Barker MK 1994 Oxygen tension regulates osteoblast function. Am J Orthod Dentofacial Orthop 105:457–463
Yamasaki H, Naus CC 1996 Role of connexin genes in growth control. Carcinogenesis 17:1199–1213
Ruch RJ, Bonney WJ, Sigler K, Guan X, Matesic D, Schafer LD, Dupont E, Trosko JE 1994 Loss of gap junctions from DDT-treated rat liver epithelial cells. Carcinogenesis 15:301–306
Madhukar BV, Yoneyama M, Matsumura F, Trosko JE, Tsushimoto G 1983 Alteration of calcium transport by tumor promoters, 12-O-tetradecanoyl phorbol-13-acetate and p,p’-dichlorodiphenyltrichloroethane, in the Chinese hamster V79 fibroblast cell line. Cancer Lett 18:251–259
Guan X, Ruch RJ 1996 Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane. Carcinogenesis 17:1791–1798
Lecanda F, Towler DA, Ziambaras K, Cheng SL, Koval M, Steinberg TH, Civitelli R 1998 Gap junctional communication modulates gene expression in osteoblastic cells. Mol Biol Cell 9:2249–2258(Niklas Rye Jrgensen, Stef)
Zealand Pharma A/S (E.M., J.S.P.), DK-2600 Glostrup, Denmark
Abstract
Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body weight) or by continuous ip infusion (158 nmol per kilogram body weight per day). During metabolic stress, a high affinity-binding site (KD = 0.1 nM) with low density (15 fmol/mg protein) for [125I]di-I-AAP10 was demonstrated. During physiological conditions, specific binding sites for [125I]AAP10 could not be shown. Studies of the effects of rotigaptide on propagation of intercellular calcium waves and cell-to-cell coupling demonstrated that 10 nM rotigaptide produced a small increase in intercellular communication during physiological conditions (+4.5 ± 1.6% vs. vehicle; P < 0.05). During conditions with metabolic stress, 10 nM rotigaptide produced an increase in coupling measured by both methods. Four weeks after ovariectomy, bone strength of the femoral head was reduced by 20% in vehicle-treated ovariectomized rats, which was completely prevented in both rotigaptide-treated groups. Rotigaptide also prevented decreases in bone mineral. We conclude that the stable analog rotigaptide increases gap junctional communication in osteoblasts in vitro and preferably during conditions with metabolic stress. Rotigaptide further prevents ovariectomy-induced bone loss in vivo. Thus, gap junction modulation may be a promising new target for osteoporosis therapy.
Introduction
GAP JUNCTION CHANNELS are specialized protein pores through which cells communicate and coordinate intercellular signals and responses to hormones. Each gap junction channel is comprised of six connexin (Cx) proteins that are arranged as a hemichannel called the connexon. Placed in the plasma membrane, connexons from one cell form gap junction channels in conjunction with connexons located in membranes of apposing cells (1). Multiple gap junction channels are held together in a plaque of channels called the gap junction.
Twenty different Cxs have been identified with Cx43 being the most abundant Cx in cardiomyocytes and in bone (2, 3). Gap junctions are particularly well studied in the heart in which they are responsible for the electrical coupling of cardiomyocytes and synchronization of contraction of the myocardium. In bone, the functions of gap junctions are less well understood, but they seem to be important in embryonic bone development (4) and differentiation and maturation of the bone-forming cells, i.e. the osteoblasts (5). They also serve to link osteocytes together in a network throughout the bone, enabling them to propagate responses to mechanical forces to the osteoblasts (6, 7). Intracellular calcium is considered a major signaling molecule in bone, and in vitro studies have demonstrated that the intercellular calcium signal travels partly through gap junctions in cultures of chondrocytes (8, 9), osteoblastic cell lines (10), and human osteoblast-like cells (11). We previously reported that human osteoblast-like cells possess the ability to propagate calcium transients by two mechanisms, one involving purinergic receptors and the other involving passage of a messenger through gap junction channels (11). By desensitizing purinergic receptors with ATP, the gap junction wave is uncovered (11), and the number of cells in the gap junction-mediated wave can be determined. Intercellular calcium signaling might be a way for cells involved in regulation of bone turnover to communicate signals throughout the network of cells in bone. Both osteoblasts and osteocytes are coupled through gap junctional plaques between cells. Thus, improving intercellular signaling among these cells might affect the regulation of bone turnover.
The pathophysiological role of altered gap junction function has been hampered by the lack of selective pharmacological tools. Therefore, we found it interesting that the family of antiarrhythmic peptides (AAPs) has been reported to stimulate gap junction intercellular communication (GJIC) between cardiomyocytes, but effects in noncardiac and human tissue are unknown (12, 13, 14, 15). However, both the endogenous AAP (H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH) and the synthetic derivative AAP10 (H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH2) are enzymatically unstable, which render the use of these compounds as pharmacological tools difficult. Therefore, we developed a highly stable AAP analog called rotigaptide (Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2; formerly known as ZP123) that is closely related structurally to AAP10 but with a half-life in human plasma in vitro of 14 d relative to 3–4 min for AAP10 (16). Thus, in the present experiments, we used the stable AAP analog rotigaptide to examine the hypothesis that AAPs could affect calcium signaling and cell-to-cell coupling in primary human osteoblasts. In addition, we wanted to examine the in vivo effects of rotigaptide on bone strength and bone mass in both compact bone (femoral body) and trabecular bone (femoral head/neck). The effect was examined 4 wk after ovariectomy (OVX) in groups of rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body weight), or continuous ip infusion (158 nmol per kilogram body weight per day).
Materials and Methods
Cell culture
Human osteoblastic cells (hOBs) were isolated from human bone marrow obtained by puncture of the posterior iliac spine of healthy volunteers (aged 20–36 yr): 10–15 ml marrow material were collected in 15 ml PBS+Ca,Mg (Life Technologies, Grand Island, NY) with 100 U/ml Heparin (catalog no. H-3149; Sigma, St. Louis, MO). The mononuclear fraction of the marrow was isolated on a Lymphoprep gradient (Nycomed Pharma, Oslo, Norway) by centrifugation at 750 x g for 30 min. After harvesting, the mononuclear fraction was washed once with culture medium and centrifuged at 400 x g for 10 min. Subsequently cells were counted and plated in culture medium at 8 x 106 cells per 100-mm dish. hOB culture medium (all reagents obtained from Life Technologies) was composed of the following: MEM without Phenol Red with Glutamax supplemented with 10% heat-inactivated fetal calf serum and 0.1% penicillin/streptomycin. Culture medium was changed the following day, and the cells were cultured at 37 C in 5% CO2 with medium change every 7 d. After 3–4 wk of culture, the cells had reached 70% confluence. The medium was then supplemented with 100 nM dexamethasone (Sigma) for 7 d. Then cells were plated for video imaging experiments on a 25-mm no. 1 glass coverslip that was placed in a 35-mm dish (or in each well of a six-well multidish). Cells were plated at 2.5 x 105 cells/coverslip and cultured for 2–3 d before use.
The study complies with the Declaration of Helsinki, and was approved by the Danish Ethics Committee System.
Binding of [125I]di-I-AAP10 to hOB cells
hOB cells grown for 3–4 wk were replated in 24-multiwell dishes at a density of at 2.1 x 105 cells/cm2 (4 x 104 cells/well) and grown for 3 d in vitro in 0.50 ml/well of F-12K nutrient (Gibco BRL, Gaithersburg, MD), mixture supplemented with 10% fetal calf serum and 1000 U penicillin per 1000 μg streptomycin (Gibco BRL) in a normal atmosphere adjusted to 5% CO2 and 100% humidity at 37 C. [125I]di-I-AAP10 (2000 Ci/mmol) was custom synthesized by Amersham Pharmacia Biotech (Little Chalfont, Wales, UK).
In situ binding experiments with [125I]di-I-AAP10 were done in triplicate and performed essentially as described by Koenig (17). On the day of analysis, growth medium was removed and washed twice with 2 ml Dulbecco’s PBS (Gibco BRL). Increasing concentrations (0.05, 0.1, and 0.5 nM) of [125I]di-I-AAP10 solution (0.50 ml) with or without unlabeled 1 mM di-I-AAP10 (to estimate nonspecific binding) was added to the cell cultures and incubated for 2 h at 21 C. The binding reaction was stopped by rapidly rinsing each well, one at a time, with 2 x 1 ml ice-cold (0 C) Dulbecco’s PBS and left to dry. The cellular material with bound [125I]di-I-AAP10 was collected by adding 0.25 ml of 0.5% Triton X-100 (vol/vol) to each well and incubated for at least 1 h at room temperature to solubilize the cells. The extract was then transferred to counting vials, the wells rinsed with 0.25 ml water, and the rinse extract added to the corresponding vials. The vials were counted in a -counter. The protein contents of test samples were measured according to Lowry et al. (18). Data on specific binding were fitted to the equation:
where BMAX is maximum binding, s is the ligand concentration, and KD is the dissociation constant.
Intercellular calcium signaling
The cells cultured on coverslips were loaded with 5 μM fura 2-acetoxymethyl (AM) (Molecular Probes, Eugene, OR) for 30 min at 37 C and incubated in fresh medium for 20 min. Coverslips were then affixed to a PDMI-2 culture chamber (Medical Systems Corp., Greenvale, NY), maintained at 37 C with superfused CO2, on a Axiovert microscope (Zeiss, New York, NY). Intercellular calcium waves were induced by mechanical stimulation of a single cell using a borosilicate glass micropipette affixed to an Eppendorf 5171 micromanipulator (Eppendorf, Netheler, Germany). Imaging was performed using a MetaMorph imaging system (Universal Imaging, Downingtown, PA). The excitation light (340 and 380 nm) was provided by a monochromator (T.I.L.L. Photonics GmbH, Planegg, Germany). Images were acquired with an intensified CCD camera (Dage MTI, Michigan City, IN) and digitized with a Matrox MVP image processing board (Matrox, Dorval, Canada). The number of cells in the wave was counted, and triple determinations were performed for each experiment. The n denotes the number of experiments.
To suppress purinergic receptor-mediated calcium waves, cells were pretreated with 100 μM ATP immediately before the experiment. We have previously shown that this protocol effectively desensitizes purinergic receptors in hOB cells (10, 11).
Cell coupling
Microinjections were performed in monolayer of cells cultured on glass coverslips as described above using the Eppendorf 5171 micromanipulator and the Eppendorf Transjector 5346 system. A micropipette was loaded with a 10 mM Lucifer Yellow (LY) solution (Sigma), and one single cell was carefully injected with LY for 30 sec. The micropipette was removed from the cell and after 30 sec the number of cells that showed dye transfer were counted. The excitation light for LY was 430 nm, and images were acquired as described above.
The parachute assay is a noninvasive technique to monitor the diffusion of fluorescent dyes through gap junctions (19, 20). In this assay, cells are preloaded with an AM-ester of a dye that is gap junction permeant and the cells are parachuted down on an unloaded monolayer of cells. With this method, the cell membrane is not disrupted as it is in microinjection experiments. Within a few hours, gap junction channels are formed by docking of hemichannels from adjacent cells, which allows the cytoplasmatic fluorescent dye to pass through gap junction channels from the donor cell to a number of neighboring acceptor cells.
In these experiments, acceptor cells were prelabeled with the nontransferable dye PKH-26 (Sigma) 2 d before the parachute assay. Cells were labeled with PKH-26 according to the manufacturer’s instructions in a solution with a total PKH-26 concentration of 2 μM. To terminate the reaction, heat-inactivated fetal bovine serum was added. Cells were centrifuged, resuspended in medium, and replated in 35-mm plates with approximately 2.5 x 105 cells/plate. Donor cells were exposed to the membrane permeant dye calcein-AM (1 μM; Molecular Probes) for 45 min at 37 C. Cells were washed with PBS and harvested in a single cell suspension using trypsin digestion. Then 1000 donor cells for each plate were parachuted on the acceptor cells and incubated for 150 min at 37 C. After washing the cells with PBS, cells were harvested using trypsin digestion, and the cell suspension was diluted to a cell concentration of 1.5–2 x 105 cells/ml. The ratio of cells possessing both dyes relative to cells labeled with PKH-26 alone was used as a measure of GJIC. Fluorescent signals were measured by fluorescence activated cell sorting (FACS) using a model FACSCalibur machine (Becton Dickinson, Franklin Lakes, NJ).
In vivo study
Forty-six female nullipara Wistar rats, 8 months old (264 ± 5 g), were randomly allocated to four groups of treatment on d 0. Rats were anesthetized with a sc injection of 2 ml/kg of a 1:1 mixture of Hypnorm (fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml, Janssen, Beerse, Belgium) and midazolam (5 mg/ml, Hameln Pharmaceuticals, Hameln, Germany) diluted 1:1 in distilled water. When surgical anesthesia was established, a midline incision was made that allowed bilateral OVX or exposure of the ovaries without removal (sham-OVX). In addition, all rats had an Alzet osmotic minipump (flow rate 2.6 μl/h) inserted into the ip cavity for chronic administration of vehicle or rotigaptide. The pump was primed with vehicle or rotigaptide for 24 h before the operation. To relieve postoperative pain, the rats were treated with Temgesic (buprenorphine 20 μg/100 g sc twice daily, Schering-Plough, Brussels, Belgium) and Metacam (meloxicam 0.1 mg/100 g sc once daily, Boehringer Ingelheim, Mannheim, Germany) for 3 d after surgery.
Four groups of rats were studied: 1) sham + vehicle (n = 11); 2) OXV + vehicle (n = 11); 3) OVX + rotigaptide by continuous ip infusion (158 nmol per kilogram body weight per day) (n = 12); and 4) OVX + rotigaptide by sc injection (300 nmol per kilogram body weight twice daily) (n = 12).
On d 30, animals were killed, and femurs were collected for bone strength measurements and stored at –20 C. Furthermore, the opposite femur was collected for bone mineral density (BMD) measurements and stored in 70% ethanol at 5 C. For measurement of plasma concentrations of rotigaptide, blood samples were collected 2–4 h after the last dosing in rats treated with sc injection of rotigaptide. Plasma was handled and analyzed for rotigaptide as previously described in detail (16, 21). Bone strength measurements were performed on a compression device (Lloyd Instruments, Fareham, UK). BMD was performed as duplicate determinations on a PIXImus dual-energy x-ray densitometer (Lunar Corp., Madison, WI).
Drugs
Rotigaptide (proposed INN name for ZP123) and di-I-AAP10 were synthetized at Zealand Pharma A/S (Glostrup, Denmark). Tritiation of di-I-AAP10 was performed by Amersham (Aylesbury, UK).
Statistics
The nonparametric Wilcoxon test was used to examine statistical differences for data collected on a discontinuous scale. For data collected on a continuous scale, overall comparison among groups was performed using a one-way ANOVA followed by post hoc analysis using Fisher’s least significant difference test. Differences were considered significant at the 5% level.
Results
Binding of [125I]di-I-AAP10 to human osteoblastic cells during normoxia and hypoxia
When cells were deprived of oxygen and glucose and exposed to low Ca2+ (<5 μM), specific high-affinity binding sites for [125I]di-I-AAP10 could be demonstrated on intact hOB cells. The binding site had a KD = 0.4 nM but displayed a very low density with BMAX = 15 ± 3 fmol/mg protein. However, we were unable to demonstrate specific binding sites for [125I]di-I-AAP10 when hOB cells were exposed to physiological Krebs-buffer with normal concentrations of Ca2+ (1.8 mM) and glucose (6 mM) at atmospheric O2 tension (21%).
Effects of rotigaptide on intercellular calcium signaling in osteoblastic cells during normoxia
To assess the ability of rotigaptide to increase gap junction-mediated intercellular calcium signals, monolayers of human osteoblastic cells on glass coverslips were loaded with the calcium indicator dye fura 2. During real-time imaging, a mechanical stimulation with a glass micropipette was performed on hOB cells desensitized with ATP. After mechanical stimulation an increase in the intracellular calcium appeared with subsequent spreading of the signal to the surrounding cells (Fig. 1).
As illustrated in Fig. 2, vehicle and 0.1 nM rotigaptide had no effect on the spreading of intercellular calcium waves in hOB cells cultured during physiological conditions. However, 10 nM produced a consistent increase in the spreading of intercellular waves (P < 0.001), whereas the effect obtained after incubation with the highest concentration of rotigaptide (1 μM) was not statistically increased (P = 0.09). To compare responses during different conditions using different techniques, we used the 10 nM concentration of rotigaptide in the following studies.
Effects of rotigaptide on cell coupling in osteoblasts during normoxia
To assess whether the effect of rotigaptide on intercellular calcium signaling is a result of a direct effect on cellular coupling between osteoblastic cells, microinjection experiments were performed. The gap junction permeant dye LY was injected into a single human osteoblast in a monolayer. After 60 sec, the number of cells containing dye was assessed. During normoxia, the dye spread to a median of 10.4 cells (Fig. 3). Then rotigaptide was added to the bathing solution in a final concentration of 10 nM, and the microinjection with LY was repeated after 10 min. In this case the dye was distributed to a median of 9.2 cells, which was not significantly different from before adding the compound. Thus, during normoxic conditions rotigaptide did not affect the basic cell-to-cell gap junctional coupling in human osteoblastic cells as assessed by the microinjection technique (Fig. 3).
We also wanted to assess whether we could see an effect of rotigaptide on cell coupling in human osteoblasts using a noninvasive technique, the parachute assay. Parallel cultures were exposed to vehicle, rotigaptide (10 nM), or the gap junction inhibitor heptanol (3.5 mM). Two populations were identified: one positive for PKH-26 only and one positive for both calcein and PKH-26. The ratio between cells that were labeled with calcein (acceptor cells, positive for both calcein and PKH-26) and the total number of potential acceptor cells (all cells positive for PKH-26) was used as a measure for cell coupling. The parachute assay appeared to be more sensitive than LY dye transfer for detecting small increases in cell coupling because using this technique, an increase of 4.5 ± 1.6% (mean ± SEM) in cell-to-cell coupling was detected in cell populations treated with 10 nM rotigaptide for 48 h (Fig. 3). As expected, heptanol decreased coupling to 39.0 ± 2.7% of the coupling in control populations. Thus, during normoxia, a small rotigaptide-induced increase in cell-to-cell coupling in human osteoblasts could be detected but only with the noninvasive parachute assay.
Effects of rotigaptide on impaired cell coupling and signaling induced by different types of metabolic stress in bone cells
The degree of coupling between Cx43-expressing cells is regulated by intracellular pH (22, 23). Thus, acidification-induced closure of gap junctions during myocardial ischemia is considered an important substrate for lethal ventricular arrhythmias (24, 25). In this study, we examined the effect of rotigaptide on cellular uncoupling in bone cells exposed to metabolic stress. First, we investigated the effect of removal of glucose from medium on the propagation of intercellular calcium signaling in monolayers of human osteoblast-like cells. One single cell was stimulated mechanically. The stimulated cell showed an increase in intracellular calcium concentration, which subsequently spread to the neighboring cells, with a median wave propagation to 10.0 cells. ATP was added in a final concentration of 100 μM to desensitize purinergic receptors on the cell membrane. All cells in the field of view showed increases in intracellular calcium concentrations, which returned to resting calcium levels within 1–2 min. Another mechanical stimulus was applied, now with a median wave propagation to 3.0 cells. To induce metabolic stress, the medium was changed to a medium without glucose, and after 8 min of aglycemia, one single cell was poked. Again, an increase in intracellular calcium was observed in the stimulated cell, but in absence of glucose, the wave propagation decreased to a median of only 1.2 cells in the wave (P = 0.027). In vehicle-treated, time-control experiments, the uncoupling was sustained throughout the experiment (Fig. 4, top panel). In another group of cultures, rotigaptide was added in a concentration of 10 nM, and after 5 min of equilibration, the mechanical stimulation was repeated, still under glucose-free conditions. Now the wave was almost completely restored, with median wave propagation to 2.8 cells (P = 0.012) (Fig. 4). Thus, metabolic stress induced by removal of glucose decreases intercellular calcium signaling in human osteoblastic cells; however, calcium signaling is completely restored by adding 10 nM rotigaptide.
In addition, we investigated the effect of hypoxia on the propagation of intercellular calcium signaling in monolayers of fura 2-loaded hOB cells. Cells were incubated for approximately 48 h in a humidified atmosphere containing 3–6% O2, 5% CO2, and 89–92% N2. On the microscope, one single cell was stimulated mechanically, and the wave propagation was recorded. However, after 48 h of hypoxia, the intracellular calcium concentration was too high to determine the exact wave propagation properties.
To examine whether hypoxia affected the cellular coupling as assessed by LY dye transfer, we microinjected LY in single human osteoblastic cells grown in a monolayer. After 60 sec we counted the number of coupled cells. The osteoblasts were quite resistant to hypoxia in terms of cell coupling. Thus, after 24 h no difference was seen in coupling between cells cultured during hypoxia and normoxia. But after 48 h, cells cultured under hypoxic conditions showed a decreased dye transfer to a median of 8.2 cells (P = 0.01), compared with 11.7 in control cells cultured during normoxic conditions. After adding 10 nM rotigaptide, the cell-to-cell coupling was restored to a median of 10.5 cells, although this did not reach statistical significance (P = 0.09 vs. before rotigaptide) (Fig. 3).
Moreover, we tested the effect of rotigaptide on intercellular coupling using the parachute assay. Cells were kept under hypoxic conditions for 48 h and treated with either vehicle or 10 nM rotigaptide (Fig. 3). Rotigaptide-treated cells showed an increase of 34.4 ± 15.6% in coupling ratio, compared with vehicle (P = 0.06). Again cell coupling could be inhibited by heptanol, with a coupling ratio of only 38.7 ± 20.1% (P < 0.05 vs. vehicle).
Effects of rotigaptide on dichlorodiphenyl trichloroethane (DDT)-induced cellular uncoupling
The insecticide DDT is a well-known carcinogen and uncoupler of gap junctions, and it has been proposed that this effect may account for some of its oncogenic properties (26). To examine whether rotigaptide was able to counteract the DDT-induced effect on cell-to-cell coupling, microinjection experiments were performed on human osteoblastic cells grown in monolayers. The cells were incubated for 1 h with DDT at a concentration of 13 μM, which is considered a relevant concentration because it has been shown to affect depolarization and oxidative control of cells (24). Furthermore, concentrations of approximately one sixtieth of the ones used in this study have been detected in blood from healthy nonsymptomatic women (25), and because DDT is accumulated in human tissues, the concentrations used in the present study may be of relevance in human toxicology. Microinjection experiments were performed as described earlier, and the number of coupled cells was recorded. In hOB cells that were pretreated with DDT, 10 min of incubation with 10 nM rotigaptide increased the median number of coupled cells (Fig. 5), whereas cell coupling was unchanged throughout the experiment in pair-treated cells that received vehicle instead of rotigaptide. Thus, rotigaptide was able to reverse DDT-induced uncoupling in human bone cells.
Effect of rotigaptide on bone strength and BMD in OVX rats
The in vivo effect of rotigaptide on bone was examined 4 wk after OVX. BMD is usually used as a noninvasive indicator of bone status and bone strength, but the ultimate parameter for assessing bone quality is directly measured mechanical bone strength. Bone strength was determined at two different sites of the femur: the femoral bone shaft (i.e. femoral body), which is primarily comprised of compact bone, and the femoral head, which is mainly constructed by trabecular bone. Thus, the architecture of the femoral head is similar to the architecture of the femoral neck, in which hip fractures most often appear. Bone strength of the femoral body was performed using the three point bending test, and as shown in Fig. 6, no differences in bone strength of the femoral body were observed 4 wk following OVX. Bone strength of the femoral head was performed using a compression test, and as illustrated in Fig. 6, bone strength of the femoral head was significantly reduced in vehicle-treated OVX rats, compared with vehicle-treated, sham-operated control rats (167 ± 11 vs. 208 ± 13 N; P < 0.05). Interestingly, the OVX-induced decreased in bone strength of the femoral head was completely prevented in both rotigaptide-treated groups. To examine BMD in areas that were used for determination of femoral trabecular and cortical bone strength, respectively, BMD was measured at the femoral head and neck region and at the middle third of the femoral shaft. BMD measurements confirmed that the reduction in bone density 4 wk after OVX was greater in the femoral neck and head (–9.7 ± 2.2%; P < 0.01 vs. sham + vehicle) than in the femoral shaft (–1.1 ± 1.1%; n.s. vs. sham + vehicle). In agreement with bone strength data, pulse treatment with rotigaptide prevented the OVX-induced bone loss, whereas continuous ip infusion of rotigaptide had no significant effect on BMD in the femoral neck and head region (Fig. 7). At the time of killing, the plasma concentration of rotigaptide was 19 ± 2 nM in rats treated with continuous ip infusion and 635 ± 52 nM in rats treated with sc injections (2–4 h after the last sc injection).
Discussion
The purpose of this study was to examine the effects of AAPs on bone in vitro and in vivo. First, we wanted to establish whether we could show specific binding of the AAPs to primary hOB cells and investigate whether the stable AAP analog rotigaptide was able to affect gap junction intercellular coupling in these cells. Furthermore, we wanted to test the in vitro results in an in vivo setting because it has been shown that coupling among osteoblasts is related to production of bone-related proteins. Thus, a compound capable of increasing osteoblastic cell coupling in vitro might be able to affect bone turnover in vivo.
During metabolic stress (no oxygen, no glucose, low calcium), a high-affinity binding site for [125I]di-I-AAP10 was demonstrated (KD = 0.1 nM), which displayed a very low density (BMAX = 0.5 fmol/mg protein). However, during physiological conditions, specific binding sites for [125I]di-I-AAP10 could not be shown. Functional studies of the effects of rotigaptide on propagation of intercellular calcium waves and cell-to-cell calcein transfer (using the noninvasive parachute assay) demonstrated that 10 nM rotigaptide produced a small increase in cell-to-cell coupling during physiological conditions. Moreover, during physiological conditions, 10 nM rotigaptide did not affect gap junctional diffusion of microinjected LY. This is possibly related to the poor sensitivity (low number of observations) of this method relative to the noninvasive parachute assay (FACS recording of more than 20.000 cells/observation). However, during conditions with metabolic stress in hOB cells, 10 nM rotigaptide in most cases produced an increase in coupling measured by intercellular calcium waves (P < 0.05), LY dye coupling (P < 0.05), and in the noninvasive parachute assay (P = 0.06). Furthermore, 10 nM rotigaptide were able to revert cell-to-cell uncoupling induced by the insecticide DDT (P < 0.001). These findings suggest that hOB cells express binding sites for AAPs that are dormant during physiological conditions but appear during metabolic stress. Furthermore, the stable AAP analog rotigaptide increases gap junctional coupling in primary human osteoblasts.
However, no obvious differences between the parachute assay and the microinjection experiments were seen. In contrast, more convincing effects were seen when using the calcium wave assay. Using this technique, we found significant effects, even under normoxic conditions. Calcium wave propagation among osteoblasts is a far more complicated process than simple cell-cell coupling via gap junctions. First, it involves purinergic P2Y receptors, which we have desensitized in the current study, and second, it involves the passage of a soluble agent through the gap junctions, inducing opening of voltage-operated calcium channels in the neighboring cells, which subsequently leads to influx of calcium from the extracellular space. The nature of this soluble agent has not yet been clarified, but it could be an electrical current. Gating of gap junctions is also a complex process, and the opening of these intercellular junctions is highly regulated and specific. These basic differences between dye coupling/parachute assay and the calcium wave assay might be the explanation for the different responses we see of the compound. Furthermore, other explanations could also underlie the missing effect of the compound in some of the assays applied to the bone cells. The cells used are phenotypically mature osteoblastic cells at the end of the differentiation process. At this stage cells are highly coupled and might not be able to increase coupling more, at least at detectable levels. Thus, it might have been interesting to test the effect of the drug on cells earlier in the osteoblastic lineage, during which gap junctional communication/coupling is not so abundant as in later stages. This explanation is also supported by the fact that the most dramatic effects of the compound were seen under circumstances in which coupling was inhibited by DDT treatment or removal of glucose. These conditions reduced cell coupling and calcium wave propagation significantly, and to a higher degree than hypoxia alone, and the reduced coupling could be at least partly reversed by rotigaptide.
A large amount of work has demonstrated that in cardiomyocytes, hypoxia leads to uncoupling of GJIC, which is an important substrate for the development of cardiac arrhythmias (27). In a series of pioneering studies, Dhein and colleagues (13, 15) and Daleau (14) demonstrated that the synthetic AAP analog AAP10 prevented GJIC uncoupling in cardiomyocytes during hypoxia, but during normal physiological conditions AAP10 had no effect on GJIC or the electrical properties of the heart. Recent publications on rotigaptide (16, 21, 28) indicate that this novel stable compound has similar electrophysiological properties and that the effect of rotigaptide on cardiac GJIC is most pronounced during metabolic stress. In bone, the effect of oxygen deprivation on GJIC is less well studied, but it has been demonstrated that during hypoxia, osteoblast proliferation and differentiation are decreased (29). Thus, impaired osteoblast GJIC during hypoxia may be an important pathophysiological mechanism in metabolic bone diseases that involve poor vascularization. Therefore, we examined the effects of rotigaptide on GJIC in human osteoblasts exposed to oxygen deprivation. Human osteoblasts were quite resistant to hypoxia, but after 48 h of oxygen deprivation, a significant decrease in cell coupling was seen. Thus, human osteoblasts, like cardiomyocytes, respond to hypoxia with a decrease in GJIC. However, the most intriguing observation was that rotigaptide was able to reverse this decreased coupling completely. In addition to 48 h of hypoxia, we also examined the effect of rotigaptide during metabolic stress elicited by glucose deprivation + hypoxia. Again, the decreased GJIC during metabolic stress was reversed by rotigaptide, and the effect on cell coupling as measured in the parachute assay seemed to be more pronounced than under normoxic conditions. These findings concur with the fact that AAP10 and rotigaptide affects GJIC in cardiomyocytes only during conditions with metabolic stress (13, 14, 15).
Furthermore, GJIC has been reported to be involved in tumor control (30). DDT is an insecticide that has tumor-promoting properties and decreases GJIC (31, 32). The exact mechanism by which DDT affects GJIC is unclear, but some reports point to a DDT-induced increase in degradation of Cx43 via a proteasome and/or lysosome-dependent pathway (33). In this study, we examined the effect of rotigaptide on GJIC in human osteoblasts uncoupled by DDT, and in line with the previously discussed observations during metabolic stress, we found that rotigaptide also increased GJIC in cells that were partially uncoupled due to pretreatment with DDT. These findings suggest that the AAP analog rotigaptide could play a role in inhibiting tumor promotion induced by tumor promoting agents such as DDT.
Previous studies have shown that increased coupling among osteoblasts in vitro is followed by an increase of the synthesis of bone formation-specific proteins like alkaline phosphatase and osteocalcin (34). Accordingly, in vivo deletion of the Cx43 gene is followed by impaired osteoblast function in a mouse model in which Cx43 is knocked out (4). Furthermore, intercellular calcium signaling via Cx43 and purinergic receptors seems to be a way by which osteoblasts coordinate a number of activities as well as a way for osteocytes to translate mechanical forces to biological signals. Thus, intact cell-cell coupling seems to be important for a normal regenerating skeleton, and enhancing the signaling system might therefore be beneficial for bone. We therefore wanted to test whether the effects on cell coupling and calcium signaling were paralleled in vivo by changes in bone strength and BMD.
To examine whether the gap junction-modifying properties of rotigaptide could affect OVX-induced bone loss, a preventive treatment study was performed on old nullipara female rats exposed to OVX. This model is generally accepted as a predictive model of loss-of-estrogen-induced bone loss. Already 4 wk after OVX, we observed a significant 20% reduction of bone strength in the femoral head but not in the femoral body. Considering the fact that the femoral head is comprised of trabecular bone, which is far more metabolically active than the compact bone of the femoral body, it is not surprising that OVX-induced reduction in bone strength appeared earlier in the femoral head than in the femoral body. However, the most striking finding of this study was that both regimens of rotigaptide completely prevented OVX-induced reduction of bone strength in the femoral head. Interestingly, the effects of OVX on the femoral caput bone strength was also reflected in BMD measurements, although the relative change in bone strength (–20 ± 5%) appeared greater than the relative change in BMD in the femoral neck and caput region (–9.7 ± 2.2%). This observation may suggest that turnover of matrix protein is greater than turnover of minerals in the rat femoral caput and neck region during early osteoporosis or that the changes in biomechanical properties of trabecular bone after OVX are not linearly related to loss of bone mineral. Moreover, rotigaptide pulse therapy (high peak concentrations) prevented the OVX-induced decrease in BMD, whereas continuous ip infusion of rotigaptide had no effect on BMD (low constant concentrations). Further studies are warranted to examine whether the dose response relationship of rotigaptide on BMD is shifted to the right relative to the effect on bone strength in trabecular femoral bone.
In line with the consistent in vitro effects of 10 nM rotigaptide on gap junction intercellular communication, 4 wk infusion with rotigaptide at a dose level that produced a plasma concentration of 19 ± 2 nM was associated with marked in vivo effects. However, the present in vivo study was hampered by the relatively short period of OVX, and further studies are warranted to examine the long-term effects on bone formation and resorption markers. In absence of histomorphometry data and/or plasma concentrations of bone markers, the present study does not allow interpretations on the exact mechanism through which rotigaptide exerts its action on bone strength. Although the present study suggests that rotigaptide has direct effects on cell-to-cell coupling among osteoblasts, other mechanisms may be involved in the in vivo effect. Thus, further studies are warranted to investigate the effect AAPs on osteoblast and osteoclast activity.
In conclusion, we have for the first time demonstrated an effect of an AAP analog on human osteoblastic cells and bone strength in vivo. Rotigaptide produced only minor changes in GJIC and calcium signaling during normal physiological conditions, but during metabolic stress, rotigaptide elicited consistent stimulation of GJIC as demonstrated by three different techniques (Ca wave signaling, microinjected LY dye transfer, and noninvasive dye transfer of calcein). In vivo, rotigaptide prevented the 20% reduction of bone strength of the femoral head observed 4 wk after OVX. The effect of rotigaptide was seen in animals given continuous ip infusion by osmotic minipumps as well as in rats treated with sc injections twice daily. These findings suggest that AAP analogs may be an interesting new class of molecules to be used in studies on the role of GJIC in bone cells. Moreover, the in vivo results suggest that AAP analogs might be useful in the prevention of metabolic bone diseases; however, further studies are warranted to examine the potential therapeutic role of these compounds in cancer and bone disease.
Footnotes
Abbreviations: AAP, Antiarrhythmic peptide; AM, acetoxymethyl; BMAX, maximum binding; BMD, bone mineral density; Cx, connexin; DDT, dichlorodiphenyl trichloroethane; FACS, fluorescence activated cell sorting; GJIC, gap junction intercellular communication; hOB, human osteoblastic cell; KD, dissociation constant; LY, Lucifer Yellow; OVX, ovariectomy.
References
Kumar NM, Gilula NB 1996 The gap junction communication channel. Cell 84:381–388
Civitelli R, Beyer EC, Warlow PM, Robertson AJ, Geist ST, Steinberg TH 1993 Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 91:1888–1896
Steinberg TH, Civitelli R, Geist ST, Robertson AJ, Hick E, Veenstra RD, Wang HZ, Warlow PM, Westphale EM, Laing JG, Beyer EC 1994 Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J 13:744–750
Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R 2000 Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol 151:931–943
Chiba H, Sawada N, Oyamada M, Kojima T, Nomura S, Ishii S, Mori M 1993 Relationship between the expression of the gap junction protein and osteoblast phenotype in a human osteoblastic cell line during cell proliferation. Cell Struct Funct 18:419–426
Donahue HJ 1998 Gap junctional intercellular communication in bone: a cellular basis for the mechanostat set point. Calcif Tissue Int. 62:85–88 (Editorial)
Donahue HJ 2000 Gap junctions and biophysical regulation of bone cell differentiation. Bone 26:417–422
D’Andrea P, Vittur F 1996 Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes. Cell Calcium 20:389–397
Donahue HJ, Guilak F, Vander Molen MA, McLeod KJ, Rubin CT, Grande DA, Brink PR 1995 Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions. J Bone Miner Res 10:1359–1364
Jorgensen NR, Geist ST, Civitelli R, Steinberg TH 1997 ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells. J Cell Biol 139:497–506
Jorgensen NR, Henriksen Z, Brot C, Eriksen EF, Sorensen OH, Civitelli R, Steinberg TH 2000 Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms. J Bone Miner Res 15:1024–1032
Aonuma S, Kohama Y, Akai K, Komiyama Y, Nakajima S, Wakabayashi M, Makino T 1980 Studies on heart. XIX. Isolation of an atrial peptide that improves the rhythmicity of cultured myocardial cell clusters. Chem Pharm Bull (Tokyo) 28:3332–3339
Müller A, Gottwald M, Tudyka T, Linke W, Klaus W, Dhein S 1997 Increase in gap junction conductance by an antiarrhythmic peptide. Eur J Pharmacol 327:65–72
Daleau P 1998 Effects of antiarrhythmic agents on junctional resistance of guinea pig ventricular cell pairs. J Pharmacol Exp Ther 284:1174–1179
Dhein S, Manicone N, Muller A, Gerwin R, Ziskoven U, Irankhahi A, Minke C, Klaus W 1994 A new synthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval and diminishes alterations of epicardial activation patterns induced by regional ischemia. A mapping study. Naunyn Schmiedebergs Arch Pharmacol 350:174–184
Kjolbye AL, Knudsen CB, Jepsen T, Larsen BD, Petersen JS 2003 Pharmacological characterization of the new stable antiarrhythmic peptide analog Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2 (ZP123): in vivo and in vitro studies. J Pharmacol Exp Ther 306:1191–1199
Koenig JA 1999 Radioligand binding in intact cells. In: Keen M, ed. Methods in molecular biology: receptor binding techniques. Totowa, NJ: Humana Press Inc.; 89–118
Lowry OH, Rosebrough NJ, Farr AL, Randdall RJ 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Ziambaras K, Lecanda F, Steinberg TH, Civitelli R 1998 Cyclic stretch enhances gap junctional communication between osteoblastic cells. J Bone Miner Res 13:218–228
Goldberg GS, Bechberger JF, Naus CC 1995 A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer. Biotechniques 18:490–497
Xing D, Kjolbye AL, Nielsen MS, Petersen JS, Harlow KW, Holstein-Rathlou NH, Martins JB 2003 ZP123 increases gap junctional conductance and prevents reentrant ventricular tachycardia during myocardial ischemia in open chest dogs. J Cardiovasc Electrophysiol 14:510–520
Delmar M, Morley GE, Ek-Vitorin JF, Francis D, Homma N, Steriopoulos K, Lau A, Taffet S 2000 Intracellular regulation of the cardiac gap junction channel connexin43. In: Zipes DP, Jalife J, eds. Cardiac electrophysiology. From cell to bedside. Philadelphia: Saunders Co.
Duffy HS, Sorgen PL, Girvin ME, O’Donnell P, Coombs W, Taffet SM, Delmar M, Spray DC 2002 pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains. J Biol Chem 277:36706–36714
Tiemann U, Pohland R, Kuchenmeister U, Viergutz T 1998 Influence of organochlorine pesticides on transmembrane potential, oxidative activity, and ATP-induced calcium release in cultured bovine oviductal cells. Reprod Toxicol 12:551–557
Turusov V, Rakitsky V, Tomatis L 2002 Dichlorodiphenyltrichloroethane (DDT): ubiquity, persistence, and risks. Environ Health Perspect 110:125–128
Nielsen M, Ruch RJ, Vang O 2000 Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication. Biochem Biophys Res Commun 275:804–809
Lerner DL, Beardslee MA, Saffitz JE 2001 The role of altered intercellular coupling in arrhythmias induced by acute myocardial ischemia. Cardiovasc Res 50:263–269
Eloff BC, Gilat E, Wan X, Rosenbaum DS 2003 Pharmacological modulation of cardiac gap junctions to enhance cardiac conduction: evidence supporting a novel target for antiarrhythmic therapy. Circulation 108:3157–3163
Tuncay OC, Ho D, Barker MK 1994 Oxygen tension regulates osteoblast function. Am J Orthod Dentofacial Orthop 105:457–463
Yamasaki H, Naus CC 1996 Role of connexin genes in growth control. Carcinogenesis 17:1199–1213
Ruch RJ, Bonney WJ, Sigler K, Guan X, Matesic D, Schafer LD, Dupont E, Trosko JE 1994 Loss of gap junctions from DDT-treated rat liver epithelial cells. Carcinogenesis 15:301–306
Madhukar BV, Yoneyama M, Matsumura F, Trosko JE, Tsushimoto G 1983 Alteration of calcium transport by tumor promoters, 12-O-tetradecanoyl phorbol-13-acetate and p,p’-dichlorodiphenyltrichloroethane, in the Chinese hamster V79 fibroblast cell line. Cancer Lett 18:251–259
Guan X, Ruch RJ 1996 Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane. Carcinogenesis 17:1791–1798
Lecanda F, Towler DA, Ziambaras K, Cheng SL, Koval M, Steinberg TH, Civitelli R 1998 Gap junctional communication modulates gene expression in osteoblastic cells. Mol Biol Cell 9:2249–2258(Niklas Rye Jrgensen, Stef)