Evidence against the Hypothesis that Endothelial Endothelin B Receptor Expression Is Regulated by Relaxin and Pregnancy
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内分泌学杂志 2005年第6期
Departments of Obstetrics, Gynecology, and Reproductive Sciences (L.J.K., J.N., K.H.-Y., K.D.D., K.P.C.) and Cell Biology and Physiology (K.P.C.), University of Pittsburgh School of Medicine and Magee Womens Research Institute, Pittsburgh, Pennsylvania 15213; and Department of Pathology (L.A.D.), University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
Address all correspondence and requests for reprints to: Kirk P. Conrad, M.D., Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213. E-mail: rsikpc@mwri.magee.edu.
Abstract
The endothelial endothelin B (ETB) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ETB receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 μg/h or vehicle for 5 d or 4–6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ETB expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ETB protein expression in arteries and cells was evaluated by Western analysis. No regulation of ETB expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ETB expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ETB receptor protein has little or no role in relaxin stimulation of the endothelial ETB/nitric oxide vasodilatory pathway.
Introduction
RELAXIN IS A 6-kDa protein hormone that is secreted by the corpus luteum during pregnancy in both human and rodent species (1). The hormone is a potent renal vasodilator when administered to conscious nonpregnant female and male rats and reduces the myogenic reactivity of small renal arteries ex vivo (2, 3, 4, 5). Elimination of relaxin from the circulation by ovariectomy or neutralization of circulating relaxin by antibodies abolishes the renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries in midterm pregnant rats (6). Up-regulation of vascular gelatinase activity contributes to the renal vasodilatory action of relaxin by processing big endothelin (ET) to ET1–32, thereby activating the endothelial ETB receptor and endothelial nitric oxide synthase (eNOS) (Ref. 7 ; and for review see Ref. 8).
Other potential loci in this vasodilatory pathway that may be regulated by relaxin include eNOS and the endothelial ETB receptor subtype. On the one hand, when renal vasodilation and hyperfiltration are at their peak in midterm pregnancy in the rat, immunoreactive levels of eNOS are not increased in renal tissues, isolated small renal arteries, or isolated and purified kidney microvasculature (9, 10), nor does relaxin infusion in nonpregnant rats increase immunoreactive nitric oxide (NO) synthase in isolated small renal arteries (9). On the other hand, Dschietzig et al. (11) recently reported that recombinant human relaxin (rhRLX) increased mRNA and protein expression of the ETB receptor in cultured human umbilical vein endothelial cells (HUVECs); increased the number of binding sites for radiolabeled ET-1 in cultured HUVECs; and increased the relaxation response to ET-3 in rat renal, mesenteric, and aortic strips in an endothelium-dependent fashion.
Because we showed that the endothelial ETB receptor subtype is critical to the renal vasodilatory response to relaxin in nonpregnant rats and during pregnancy (via endogenous circulating relaxin) (6), we also hypothesized that the endothelial ETB receptor subtype would be up-regulated by relaxin or pregnancy, thereby contributing to the renal vasodilatory response along with the augmentation of vascular gelatinase activity (3, 4, 7, 8, 12, 13). To our surprise, we found no evidence to support the concept.
Materials and Methods
Animal preparation
Long-Evans female rats were purchased from Harlan Sprague Dawley (Frederick, MD). They were provided PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds Inc., St. Louis, MO) and water ad libitum. The rats were maintained on a 12-h light, 12-h dark cycle. Those destined to become pregnant were housed with a male breeder and d 0 of pregnancy was documented by the presence of spermatozoa in the vaginal lavage. Pregnant rats were killed at midgestation (12–14 d). For short-term relaxin administration, Alzet model 2001D 1-d minipumps were inserted sc in the back of nonpregnant rats under isoflurane anesthesia. rhRLX (BAS Medical Inc., San Mateo, CA) or vehicle [20 mM sodium acetate (pH 5.0)] was delivered at a dose of 4.0 μg/h, which yields concentrations of circulating relaxin similar to those measured during midterm gestation in pregnant rats (1, 2, 3, 5, 7), when pregnancy-induced renal vasodilation is maximal in this species (14). Animals were killed after 4–6 h of rhRLX administration. For more chronic relaxin administration, rats were treated similarly, except that Alzet model 2001 7-d minipumps were used, and rats were killed after 4–7 d of infusion. All animal protocols were approved by the Institutional Animal Care and Use Committee of Magee-Womens Research Institute.
Measurement of rhRLX in rat serum
The levels of rhRLX in serum obtained from trunk blood were measured by a quantitative sandwich immunoassay as previously described (5). Briefly, wells of a 96-well microtiter plate (Maxisorp Immunomodules; Nalge Nunc International, Naperville, IL) were coated overnight with affinity-purified anti-rhRLX rabbit polyclonal antibody. Sera were diluted in PBS containing Tween 20, Thimerosal, BSA (Sigma Chemical Co., St. Louis, MO) and normal goat IgG (Organon Teknika-Cappel, Durham, NC), and 100 μl were added to each well in duplicate. After overnight incubation at 4 C, the wells were washed, and 100 μl of affinity-purified, peroxidase-conjugated anti-rhRLX rabbit polyclonal antibody were added to each well. After an appropriate incubation period at room temperature, the wells were washed again, and 100 μl of a tetramethylbenzidine solution were added to each well. After color development, the reaction was stopped, absorbances at 450/630 nm were measured, and rhRLX concentrations in the sera were determined by entering data into a four-parameter logistic curve-fitting program.
Tissue collection
After exposure of the kidneys through a ventral abdominal incision, they were gently lifted so as to not pull on the renal vessels. For Western blotting, the kidneys were then excised and placed immediately in ice-cold saline. For study of arterial functional behavior, the kidneys were placed in ice-cold HEPES-buffered physiological saline solution (H-PSS), a modified Kreb’s buffer composed of sodium chloride 142 nM, potassium chloride 4.7 nM, magnesium sulfate 1.17 nM, calcium chloride 2.5 nM, potassium phosphate 1.18 nM, HEPES 10 nM, and glucose 5.5 nM, maintained at pH 7.4. Small renal arteries were microdissected as previously described (4, 6, 7, 13). Briefly, kidneys were bisected and pinned to a Sylgard-coated dissection dish filled with chilled saline or H-PSS and kept on ice. The medulla was removed to reveal the underlying arterial tree, and using a dissecting microscope, small arteries with an inner diameter ranging from 50 to 400 μm were gently dissected from the kidney for Western blotting. For study of arterial functional behavior, small renal arteries with an inner diameter of 150–200 μm were used. Whole brain was harvested from a female Long Evans rat for use as a positive control for the ETB receptor by Western blotting (15).
Incubation of small renal arteries with rhRLX in vitro
Small renal arteries isolated from one rat were allocated equally into two tubes and incubated with either rhRLX diluted to 30 ng/ml in H-PSS buffer or a comparable volume of vehicle diluted in H-PSS buffer. Arteries were then incubated for 3 h at 37 C with gentle shaking. After incubation, arteries were removed from buffer, blotted on gauze, snap frozen in liquid nitrogen, and stored at –80 C.
Arterial functional behavior
After isolation, small renal arteries were mounted in a pressurized arteriograph (Living Systems, Burlington, VT) and equilibrated to 37 C and an intralumenal pressure of 60 mm Hg. For each rat, a pair of vessels was mounted. One vessel was treated with 30 ng/ml rhRLX and the other with vehicle for 3 h before addition of ET-3 (Sigma). Two conditioning stretches (from 60 to 100 mm Hg) were performed during this incubation, one after the first 30 min and one at the beginning of the last 30 min. The vessels were then preconstricted with phenylephrine (Sigma) to 50% of the initial inner diameter. ET-3 was then added in a cumulative manner from 1 x 10–12 to 1 x 10–8 mol/liter, and inner diameters were measured at each concentration.
Preparation of tissues for Western blotting
After dissection, small renal arteries or brain tissue was snap frozen in liquid nitrogen then stored at –80 C. Frozen arteries from one rat were pooled and pulverized in a prechilled capsule with a steel ball for 8 sec using a Wig-L-Bug amalgamator (Crescent Dental Manufacturing. Co., Lyons, OH). Five volumes of homogenization buffer [10 mM Tris (pH 6.8), 1% sodium dodecyl sulfate, 10% glycerol, 7 M urea] containing 0.5 mM phenylmethyl sulfonyl fluoride and 10 μl/ml Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA) was added, and the contents were mixed by vigorous manual shaking followed by sonication for 10 sec. The homogenate was then centrifuged at 15,000 x g for 10 min at 4 C. Protein concentrations were determined on the supernatants by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), and 10 μg total protein per lane was used for the Western analysis.
Cell culture
All vascular cells and media were obtained from Cambrex (Walkersville, MD). Cells at passage 4–6 were grown to subconfluence in T-75 flasks in EBM phenol red-free medium supplemented with EGM-2MV singlequots and 5% fetal bovine serum. Fetal bovine serum was reduced to 2% for 3 h before treatment and during treatment with relaxin or vehicle. Relaxin (0.1, 1.0, or 5 ng/ml) or vehicle was added for 4, 8, or 24 h. After incubation, the media were removed, and flasks were rinsed with 10 ml cold Dulbecco’s PBS (MediaTech, Inc., Herndon, VA) and placed on ice. After decanting the wash, 1 ml of cold Dulbecco’s PBS was added and cells were scraped off with a rubber policeman. Cells were microcentrifuged at 4 C for 5 min at 2000 x g, and the pellets were snap frozen in liquid nitrogen then stored at –80 C.
Preparation of cell pellets for Western blotting
Cell pellets were kept on ice throughout the protein extraction protocol. Fifty microliters protein extraction buffer [50 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol containing 0.5 mM phenylmethyl sulfonyl fluoride, and 10 μl/ml Calbiochem Protease Inhibitor Cocktail Set III] was added to each cell pellet. Each pellet was sonicated for 10 sec and then centrifuged at 4 C for 10 min at 15,000 x g. Protein concentrations were determined on the supernatant by the Bio-Rad protein assay and10 μg total protein were used for the Western analysis.
Western blotting
For SDS-PAGE, samples were combined with an equal volume of double-strength sample buffer [2% sodium dodecyl sulfate, 10% glycerol, 5% ?-mercaptoethanol, 0.02% bromophenol blue in 0.05 M Tris (pH 6.8)], boiled for 4 min, and microcentrifuged briefly; then 20 μl were loaded onto a 10% gel (Invitrogen, Carlsbad, CA) and electrophoresed for 90 min at 100 V. The separated proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Bedford, MA) for 1 h at approximately 1 mA/cm2 using a semidry electrophoresis transfer system. After transfer, membranes were soaked briefly in methanol and dried completely. Membranes were rehydrated for 30 min in Tris-buffered saline [TBS; 10 mM Tris, 150 mM NaCl (ph7.4)] containing 0.5% Tween 20 before blocking for 1 h in 5% nonfat dried milk (NFDM) (Carnation, Solon, OH) diluted in TBS with 0.05% Tween 20 (TBST). Immunoblots were then incubated overnight at 4 C with rabbit antirat ETB receptor antibody (Alomone Labs, Jerusalem, Israel) diluted in NFDM to a final concentration of 1 μg/ml or with mouse monoclonal antibody directed against human ETB receptor (Institute of Immunology, Tokyo, Japan) diluted in NFDM to 3 μg/ml. A 1:1 mass ratio was employed for preabsorption of the rabbit antirat antibody with control peptide (Alomone Labs). Negative control blots were incubated with normal rabbit IgG (R&D Systems, Minneapolis, MN), or mouse IgG1 (Sigma) diluted in the same manner as the primary antibody. After three 10-min washes in TBST, blots were incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:15,000 in NFDM or with alkaline phosphatase-conjugated goat antimouse IgG secondary antibody (diluted 1:15,000; Cedarlane Labs, Hornby, Ontario, Canada). The blots were again washed in TBST as described above, followed by a quick rinse in TBS and equilibration in alkaline phosphatase detection buffer [100 mM Tris (pH 9.5), 150 mM NaCl, 5 mM MgCl2] for 10 min. The chemiluminescent substrate reagent CDP-Star (Roche Molecular Biochemicals, Indianapolis, IN) diluted 1:200 in alkaline phosphatase detection buffer was reacted with the blots for 5 min, and the membrane was then exposed to BioMax-MR film (Kodak, Rochester, NY) for signal detection.
The membranes were stripped with a buffer containing 62.5 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, and 100 mM ?-mercaptoethanol at 50 C for 30 min and reprobed with a monoclonal anti-?-actin antibody (clone AC-15, Sigma: 1:1000 dilution) to correct for loading variations.
Densitometry
The films were scanned using a Hewlett Packard laser scanner (Scanjet 7400C, Hewlett Packard, Palo Alto, CA), and densitometry was performed using automated digitizing software UN-SCAN-IT gel version 4.3 (Silk Scientific Inc., Orem, UT).
Data analysis
All data are expressed as mean ± SEM. For Western blots, the ratio of the densitometric values of the bands from pregnant/virgin rat small renal arteries, rhRLX-treated/vehicle-treated rat small renal arteries, or rhRLX-treated/vehicle-treated cultured human cells were calculated. These values were then compared with 1.0 using a one-sample t test. P < 0.05 was considered statistically significant.
Results
Figures 1 through 3 show validation studies for the antibodies used to detect the ETB receptor by Western analysis. Both the rabbit antirat polyclonal and mouse antihuman monoclonal antibodies directed against the ETB receptor showed an approximately 45-kDa band in the rat brain, the positive control tissue (15), although the mouse antihuman polyclonal produced a more diffuse band (Fig. 1). After preincubation of the rabbit antibody with the blocking peptide, the density of the approximately 45-kDa band was diminished, and substitution of either rabbit IgG or mouse IgG1k for their respective primary antibodies did not show nonspecific at binding at approximately 45-kDa.
FIG. 1. ETB receptor (ETBR) antibodies show a major band at 45 kDa in whole brain homogenates from Long Evans rats on Western analysis. The left panel (A) shows a blot that was incubated with rabbit antirat ETB receptor primary antibody. In the middle panel, the membrane was probed with the rabbit antirat ETB receptor primary antibody preincubated with the immunizing peptide. Normal rabbit IgG was substituted for the primary antibody in the right panel. The left and right panels (B) show rat brain homogenates probed with a mouse monoclonal antihuman ETB receptor antibody and with mouse IgG1, respectively.
FIG. 2. Western analysis reveals ETB receptor (ETBR) expression at 45 kDa in human coronary artery endothelial cell homogenates. A, Rat brain and human coronary artery endothelial cells (EC) homogenates were probed with rabbit antirat ETB receptor antibody (left panel) and normal rabbit IgG (right panel). B, Dose response of human coronary artery endothelial cell homogenates using the rabbit antirat ETB receptor antibody. C, The same protein preparations as in A probed with monoclonal antihuman ETB receptor antibody in the left panel and with mouse IgG1 in the right panel.
FIG. 3. Western analysis reveals ETB receptor (ETBR) expression at 45 kDa in rat small renal artery homogenates. A, Dose response of small renal artery (SRA) homogenates pooled from three female Long Evans rats as well as whole brain and renal inner medulla homogenates from one female Long Evans rat probed with the rabbit antirat ETB receptor antibody. The same protein preparations were probed with monoclonal antihuman ETB receptor antibody (B) and mouse IgG1 (C).
In Fig. 2, A and B, the rabbit antirat polyclonal antibody was used to detect the ETB receptor in cultured human coronary artery endothelial cells. An approximately 45-kDa band aligning with the positive control rat brain was observed. In addition, a robust dose response was observed between the amount of protein loaded per lane from 5 to 15 μg and the signal intensity. Similar results were obtained for the mouse antihuman ETB receptor antibody (Fig. 2C), although the rabbit antirat polyclonal antibody yielded a sharper and more intense band. Again, substitution of rabbit IgG or mouse IgG1k for their respective primary antibodies did not demonstrate nonspecific binding at approximately 45 kDa.
In Fig. 3A, the rabbit antirat polyclonal was employed to detect ETB receptor in rat small renal arteries. An approximately 45-kDa band aligning with the positive control rat brain as well as the renal inner medulla (16) was noted. A robust dose response was observed between the amount of protein loaded per lane from 2.5 to 40 μg and the signal intensity. Similar findings were obtained for the mouse antihuman ETB receptor antibody (Fig. 3B). However, the rabbit antirat polyclonal antibody again yielded a superior signal. Substitution of mouse IgG1k for the primary antibody yielded a clean blot (Fig. 3C).
The rabbit antirat polyclonal antibody was used for detection of the ETB receptor in small renal arteries isolated from eight pairs of virgin and midterm pregnant rats. A representative blot is shown in Fig. 4A. Normalization was carried out using ?-actin as a housekeeping protein. A graphical summary of the data is shown in Fig. 4B. The overall densitometric ratio of pregnant to virgin was 0.89 ± 0.10 (p = NS vs. 1.0).
FIG. 4. ETB receptor protein is not up-regulated in small renal arteries at midgestation in rats. P, Midgestation; V, virgin. A representative Western blot is shown (A). Rat brain was used as a positive control for ETB receptor protein expression. B, Mean ± SEM (n = 8 pairs of V and P rats) of the ratio of the densitometric values for ETB receptor bands normalized to ?-actin. A ratio equal to 1.0 indicates no difference in ETB receptor protein expression between small renal arteries from midterm pregnant and virgin rats.
The rabbit antirat polyclonal antibody was also used to detect the ETB receptor in small renal arteries isolated from nonpregnant rats administered rhRLX or vehicle for 5 d. A representative blot is shown in Fig. 5A. Normalization was carried out using ?-actin as a housekeeping protein. A graphical summary of the data for nine pairs of rhRLX- and vehicle-treated nonpregnant rats is shown in Fig. 5B. The overall densitometric ratio of relaxin to virgin was 1.11 ± 0.12 (p = NS vs. 1.0). In addition, nonpregnant rats were treated with rhRLX or vehicle for 4–6 h before isolation of small renal arteries, or small renal arteries were harvested from nonpregnant rats and subsequently incubated with 30 ng/ml rhRLX in vitro for 3 h. Neither protocol resulted in significant alterations in ETB receptor expression: densitometry ratios of 0.89 ± 0.08 and 0.99 ± 0.14, respectively, both p = NS vs. 1.0 (Fig. 5B). In the nonpregnant rats treated with rhRLX for 4–6 h, serum concentrations were 26.7 ± 1.4 ng/ml.
FIG. 5. ETB receptor (ETBR) protein is not up-regulated in small renal arteries isolated from nonpregnant rats treated with rhRLX. A representative Western blot is shown (A) of small renal arteries isolated from rats treated 5 d with rhRLX (Rlx) or vehicle (veh). Rat brain was used as a positive control for ETB receptor protein expression. B, Mean ± SEM of the ratio of the densitometric values for ETB receptor bands normalized to ?-actin (n = 9 rat pairs treated for 5 d, n = 7 rat pairs treated for 4–6 h, and n = 7 rats used for 3 h in vitro treatment of arteries). A ratio equal to 1.0 indicates no difference in ETB receptor protein expression between treatment groups.
Human coronary artery endothelial cells in culture were incubated with rhRLX or vehicle at various doses and times. The rabbit antirat polyclonal antibody was used to detect the ETB receptor in homogenates of the cell pellets and the results were normalized to ?-actin. A representative Western blot is shown in Fig. 6A, and a graphical summary of several experiments is depicted in Fig. 6B. The results for cultured human aortic and microvascular as well as umbilical vein endothelial cells treated with rhRLX or vehicle for various doses and times are shown in Table 1. Except for a few sporadic, significant increases or decreases, there was no consistent alteration in ETB receptor expression. To be certain that we were not missing an effect of rhRLX on ETB receptor expression, we tested cells of both genders, various confluencies ranging from 50 to 100%, passage numbers 4–6, and different quiescent protocols (serum deprivation overnight or 2% serum for 3 h before addition of rhRLX or vehicle (11) as well as medium with and without phenol red, which possesses weak estrogenic activity.
FIG. 6. ETB receptor (ETBR) protein is inconsistently regulated in human coronary artery endothelial cells treated with rhRLX. Rlx, rhRLX; veh, vehicle. A representative Western blot is shown (A). B, Mean ± SEM of the ratio of the densitometric values for ETB receptor bands normalized to ?-actin. See Table 1 for further details. A ratio equal to 1.0 indicates no difference in ETB receptor protein expression between rhRLX- and vehicle-treated cells.
TABLE 1. ETB receptor protein expression is inconsistently regulated by treatment with rhRLX in human vascular endothelial cells
Figure 7 portrays functional behavior of small renal arteries preconstricted with phenylephrine and then stimulated with ET-3 after incubation with rhRLX or vehicle for 3 h in vitro. The relaxation response to ET-3 was not significantly enhanced in arteries pretreated with rhRLX. We have previously shown that such pretreatment significantly decreases the myogenic reactivity of small renal arteries (Novak, J., and K. P. Conrad, unpublished observations).
FIG. 7. Small renal arteries from rats treated for 3 h in vitro with rhRLX (RLX) or vehicle (VEH) show no difference in their relaxation response to ET-3. Percent relaxation was calculated as follows: 100 x [(final diameter at each concentration of ET-3 – diameter at 50% constriction)/diameter at 50% constriction]. Values expressed are mean ± SEM.
Discussion
In previous work we identified the endothelial ETB receptor subtype (via NO) to be a critical step in the renal vasodilatory pathway induced by endogenous relaxin that circulates during pregnancy or by relaxin administration to nonpregnant rats (reviewed in Refs. 8 , 17). Therefore, it was logical to test whether relaxin might stimulate this vasodilatory pathway by up-regulating the expression of the endothelial ETB receptor. However, the findings of the current work do not support this hypothesis. The immunoreactive mass of the ETB receptor is not increased in small renal arteries isolated from midterm pregnant rats, nonpregnant rats treated with rhRLX for various periods of time, or small renal arteries treated with rhRLX in vitro, all situations that profoundly vasodilate the renal circulation and/or induce marked reduction in myogenic reactivity of small renal arteries (e.g. Refs. 2 , 4 , 5 , 13 , 14). Furthermore, the failure of small renal arteries treated with rhRLX in vitro to show enhanced relaxation responses to ET-3 is a functional corroboration of the negative molecular analyses. Finally, rhRLX treatment of cultured human endothelial cells of various types failed to show consistent regulation of the ETB receptor by the hormone.
In the current work, we invested considerable effort in validating the antibodies used to assess ETB receptor expression by Western analysis (Fig. 1). First, we used two different antibodies, a rabbit antirat polyclonal and a mouse antihuman monoclonal. The former recognizes a 16-amino acid sequence in the third cytoplasmic loop (manufacturer’s specification sheet), and the latter is directed against most of the cytoplasmic tail (18) that is homologous in rat and human except for one amino acid. The full-length human and rat receptor is 442 amino acids corresponding to a calculated molecular weight of 49–50 kDa. After subtracting the 26-amino acid signal peptide, the ETB receptor has a calculated molecular mass of 46–47 kDa, which is similar to the approximately 45-kDa band recognized by both antibodies in rat brain or renal inner medullary tissue, positive control tissues for the ETB receptor subtype (15, 16). Moreover, we were able to greatly diminish the signal intensity by preincubating the rabbit antirat antibody with immunizing peptide, and substitution of the primary antibodies with IgG did not reveal nonspecific binding in the region of approximately 45 kDa. Thus, we are reasonably confident in our detection of the ETB receptor by Western blotting.
Using these two antibodies, we detected a band of approximately 45 kDa in cultured human endothelial cells (Fig. 2) and small renal arteries (Fig. 3) that aligned with the positive control tissue(s). However, the rabbit antirat polyclonal antibody yielded sharper and more intense bands. Therefore, this antibody was used for subsequent determinations of ETB receptor expression.
We previously showed that renal vasodilation and hyperfiltration is a consistent finding in midterm rat pregnancy and during relaxin administration to nonpregnant rats (e.g.2, 5, 14, 19). Moreover, the small renal arteries harvested from these animals show profound decreases in myogenic reactivity consistent with the changes in the renal circulation observed in the intact rat (4, 13). Because these vasodilatory phenomena are mediated by the endothelial ETB receptor through NO (8, 17), we reasoned that relaxin or pregnancy (via endogenous circulating relaxin) (6) would most likely accentuate this vasodilatory pathway by up-regulating endothelial ETB receptor expression. While we were investigating this hypothesis, Dschietzig et al. (11) reported that rhRLX increased mRNA and protein expression of the ETB receptor in cultured HUVECs; the number of binding sites for radiolabeled ET-1 in cultured HUVECs; and the relaxation response to ET-3 in rat renal, mesenteric, and aortic strips in an endothelium-dependent fashion. Therefore, we were additionally surprised that our findings were unsupportive.
There are potential explanations for the apparent discrepancies in the work of Dschietzig et al. and our own. First, our studies focused on the physiology of renal vasodilation produced by pregnancy per se (via endogenous circulating relaxin) and rhRLX administration to nonpregnant rats. In these conditions, we showed that the ETB receptor is not up-regulated in small renal arteries isolated from midterm pregnant rats or from rhRLX infused nonpregnant rats after either 4–6 h or 5 d of hormone administration (Figs. 4 and 5). Yet we know that these conditions are typified by marked renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries ex vivo (8, 17). Moreover, we know that these vasodilatory phenomena can be prevented by ETB receptor antagonists or by using the ETB receptor-deficient rat indicating the critical nature of the endothelial ETB receptor in this vasodilatory pathway (3, 4, 7, 8, 12, 13, 17). Thus, we reasoned that the findings of Dschietzig et al. may have been different from our own because their conclusions were based on the treatment of cultured cells and isolated arteries with rhRLX in vitro.
Therefore, we continued our investigation using in vitro approaches in an attempt to corroborate the findings of Dscheitzig et al. However, by treating small renal arteries in vitro with rhRLX, we were unable to augment ETB receptor expression (Fig. 5B) or enhance the relaxation response to ET-3 (Fig. 7), the latter serving as a functional confirmation of the molecular analyses. Yet we know that this treatment leads to reduction in myogenic reactivity (Novak, J. and K. P. Conrad, unpublished data) as previously reported in small renal arteries harvested from nonpregnant rats treated with rhRLX in vivo via the endothelial ETB receptor/NO vasodilatory pathway (4). Possible differences in experimental conditions could explain the discrepancies between our results. For example, the preparations of rhRLX were from different sources. Ours was manufactured by Connetics Corp. (now licensed by BAS Medical) and has consistently yielded the expected physiological responses attesting to its identity and bioactivity (8, 17). In addition, we added purified porcine relaxin instead of rhRLX to cultured HUVECs in a few experiments but again failed to observe regulation of the ETB receptor. Dscheitzig et al. used rhRLX manufactured by Immunodiagnostik AG, (Bensheim, Germany), but we are unaware of publications in which this preparation was shown to elicit physiological effects such as reducing plasma osmolality when administered to nonpregnant rats. As another example, although both Dschietzig et al. and we treated rat arteries with rhRLX in vitro and assessed the functional response to ET-3, both the arterial segments and strain of rat used were different.
Both Dschietzig et al. and we treated cultured HUVECs with rhRLX. Whereas they found up-regulation of ETB receptor expression, we did not (Table 1). Possibly, cultured HUVECs may not all be the same. For example, ours were purchased from Cambrex and they were of mixed male and female origin, and we studied them during passages 5 and 6. Dscheitzig et al. isolated their own HUVECs and studied them in passages 3 and 4. They did not report the gender of these cells. However, we did match their cell quiescence protocol in many experiments, and we tested a wide range of cell confluency from 50 to 100% but all to no avail. Whereas Dscheitzig et al. further investigated bovine endothelial cells with positive results, we tested a variety of other human endothelial cells of adult origin and both genders, but again, our results were inconsistent (Fig. 6 and Table 1). Possibly the numerous growth factors used to maintain the various endothelial cells purchased from Cambrex masked any additional effect of rhRLX. However, our negative results using cultured human endothelial cells are consistent with those using arteries isolated from midterm pregnant or rhRLX-treated nonpregnant rats (supra vide).
In previous work, we and other investigators demonstrated that eNOS expression is not up-regulated in renal tissues and arteries isolated from midterm pregnant or relaxin-infused nonpregnant rats (9, 10). However, another critical and proximal element in this vasodilatory pathway is increased, i.e. vascular gelatinase activity, which processes big ET-1 to ET1–32, thereby stimulating the endothelial ETB receptor and NO production (7). Because the endothelial ETB receptor subtype is not regulated by relaxin, at least in our hands, perhaps vascular gelatinase is the major locus of regulation by relaxin in this vasodilatory pathway.
Acknowledgments
We thank Julie Matthews for technical assistance and Elaine Unemori, Ph.D. (BAS Medical) for the generous gift of recombinant human relaxin.
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Conrad KP 1984 Renal hemodynamics during pregnancy in chronically catheterized, conscious rats. Kidney Int 26:24–29
Cheng HF, Su YM, Yeh JR, Chang KJ 1993 Alternative transcript of the nonselective-type endothelin receptor from rat brain. Mol Pharmacol 44:533–538
Gellai M, DeWolf R, Pullen M, Nambi P 1994 Distribution and functional role of renal ET receptor subtypes in normotensive and hypertensive rats. Kidney Int 46:1287–1294
Conrad KP 2004 Mechanisms of renal vasodilation and hyperfiltration during pregnancy. J Soc Gynecol Investig 11:438–448
Satoh M, Miyamoto C, Terashima H, Tachibana Y, Wada K, Watanabe T, Hayes AE, Gentz R, Furuichi Y 1997 Human endothelin receptors ETA and ETB expressed in baculovirus-infected insect cells: direct application for signal transduction analysis. Eur J Biochem 249:803–811
Danielson LA, Conrad KP 1995 Acute blockage of nitric oxide synthase inhibits renal vasodilation and hyperfiltration during pregnancy in chronically instrumented conscious rats. J Clin Invest 96:482–490
Address all correspondence and requests for reprints to: Kirk P. Conrad, M.D., Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213. E-mail: rsikpc@mwri.magee.edu.
Abstract
The endothelial endothelin B (ETB) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ETB receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 μg/h or vehicle for 5 d or 4–6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ETB expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ETB protein expression in arteries and cells was evaluated by Western analysis. No regulation of ETB expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ETB expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ETB receptor protein has little or no role in relaxin stimulation of the endothelial ETB/nitric oxide vasodilatory pathway.
Introduction
RELAXIN IS A 6-kDa protein hormone that is secreted by the corpus luteum during pregnancy in both human and rodent species (1). The hormone is a potent renal vasodilator when administered to conscious nonpregnant female and male rats and reduces the myogenic reactivity of small renal arteries ex vivo (2, 3, 4, 5). Elimination of relaxin from the circulation by ovariectomy or neutralization of circulating relaxin by antibodies abolishes the renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries in midterm pregnant rats (6). Up-regulation of vascular gelatinase activity contributes to the renal vasodilatory action of relaxin by processing big endothelin (ET) to ET1–32, thereby activating the endothelial ETB receptor and endothelial nitric oxide synthase (eNOS) (Ref. 7 ; and for review see Ref. 8).
Other potential loci in this vasodilatory pathway that may be regulated by relaxin include eNOS and the endothelial ETB receptor subtype. On the one hand, when renal vasodilation and hyperfiltration are at their peak in midterm pregnancy in the rat, immunoreactive levels of eNOS are not increased in renal tissues, isolated small renal arteries, or isolated and purified kidney microvasculature (9, 10), nor does relaxin infusion in nonpregnant rats increase immunoreactive nitric oxide (NO) synthase in isolated small renal arteries (9). On the other hand, Dschietzig et al. (11) recently reported that recombinant human relaxin (rhRLX) increased mRNA and protein expression of the ETB receptor in cultured human umbilical vein endothelial cells (HUVECs); increased the number of binding sites for radiolabeled ET-1 in cultured HUVECs; and increased the relaxation response to ET-3 in rat renal, mesenteric, and aortic strips in an endothelium-dependent fashion.
Because we showed that the endothelial ETB receptor subtype is critical to the renal vasodilatory response to relaxin in nonpregnant rats and during pregnancy (via endogenous circulating relaxin) (6), we also hypothesized that the endothelial ETB receptor subtype would be up-regulated by relaxin or pregnancy, thereby contributing to the renal vasodilatory response along with the augmentation of vascular gelatinase activity (3, 4, 7, 8, 12, 13). To our surprise, we found no evidence to support the concept.
Materials and Methods
Animal preparation
Long-Evans female rats were purchased from Harlan Sprague Dawley (Frederick, MD). They were provided PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds Inc., St. Louis, MO) and water ad libitum. The rats were maintained on a 12-h light, 12-h dark cycle. Those destined to become pregnant were housed with a male breeder and d 0 of pregnancy was documented by the presence of spermatozoa in the vaginal lavage. Pregnant rats were killed at midgestation (12–14 d). For short-term relaxin administration, Alzet model 2001D 1-d minipumps were inserted sc in the back of nonpregnant rats under isoflurane anesthesia. rhRLX (BAS Medical Inc., San Mateo, CA) or vehicle [20 mM sodium acetate (pH 5.0)] was delivered at a dose of 4.0 μg/h, which yields concentrations of circulating relaxin similar to those measured during midterm gestation in pregnant rats (1, 2, 3, 5, 7), when pregnancy-induced renal vasodilation is maximal in this species (14). Animals were killed after 4–6 h of rhRLX administration. For more chronic relaxin administration, rats were treated similarly, except that Alzet model 2001 7-d minipumps were used, and rats were killed after 4–7 d of infusion. All animal protocols were approved by the Institutional Animal Care and Use Committee of Magee-Womens Research Institute.
Measurement of rhRLX in rat serum
The levels of rhRLX in serum obtained from trunk blood were measured by a quantitative sandwich immunoassay as previously described (5). Briefly, wells of a 96-well microtiter plate (Maxisorp Immunomodules; Nalge Nunc International, Naperville, IL) were coated overnight with affinity-purified anti-rhRLX rabbit polyclonal antibody. Sera were diluted in PBS containing Tween 20, Thimerosal, BSA (Sigma Chemical Co., St. Louis, MO) and normal goat IgG (Organon Teknika-Cappel, Durham, NC), and 100 μl were added to each well in duplicate. After overnight incubation at 4 C, the wells were washed, and 100 μl of affinity-purified, peroxidase-conjugated anti-rhRLX rabbit polyclonal antibody were added to each well. After an appropriate incubation period at room temperature, the wells were washed again, and 100 μl of a tetramethylbenzidine solution were added to each well. After color development, the reaction was stopped, absorbances at 450/630 nm were measured, and rhRLX concentrations in the sera were determined by entering data into a four-parameter logistic curve-fitting program.
Tissue collection
After exposure of the kidneys through a ventral abdominal incision, they were gently lifted so as to not pull on the renal vessels. For Western blotting, the kidneys were then excised and placed immediately in ice-cold saline. For study of arterial functional behavior, the kidneys were placed in ice-cold HEPES-buffered physiological saline solution (H-PSS), a modified Kreb’s buffer composed of sodium chloride 142 nM, potassium chloride 4.7 nM, magnesium sulfate 1.17 nM, calcium chloride 2.5 nM, potassium phosphate 1.18 nM, HEPES 10 nM, and glucose 5.5 nM, maintained at pH 7.4. Small renal arteries were microdissected as previously described (4, 6, 7, 13). Briefly, kidneys were bisected and pinned to a Sylgard-coated dissection dish filled with chilled saline or H-PSS and kept on ice. The medulla was removed to reveal the underlying arterial tree, and using a dissecting microscope, small arteries with an inner diameter ranging from 50 to 400 μm were gently dissected from the kidney for Western blotting. For study of arterial functional behavior, small renal arteries with an inner diameter of 150–200 μm were used. Whole brain was harvested from a female Long Evans rat for use as a positive control for the ETB receptor by Western blotting (15).
Incubation of small renal arteries with rhRLX in vitro
Small renal arteries isolated from one rat were allocated equally into two tubes and incubated with either rhRLX diluted to 30 ng/ml in H-PSS buffer or a comparable volume of vehicle diluted in H-PSS buffer. Arteries were then incubated for 3 h at 37 C with gentle shaking. After incubation, arteries were removed from buffer, blotted on gauze, snap frozen in liquid nitrogen, and stored at –80 C.
Arterial functional behavior
After isolation, small renal arteries were mounted in a pressurized arteriograph (Living Systems, Burlington, VT) and equilibrated to 37 C and an intralumenal pressure of 60 mm Hg. For each rat, a pair of vessels was mounted. One vessel was treated with 30 ng/ml rhRLX and the other with vehicle for 3 h before addition of ET-3 (Sigma). Two conditioning stretches (from 60 to 100 mm Hg) were performed during this incubation, one after the first 30 min and one at the beginning of the last 30 min. The vessels were then preconstricted with phenylephrine (Sigma) to 50% of the initial inner diameter. ET-3 was then added in a cumulative manner from 1 x 10–12 to 1 x 10–8 mol/liter, and inner diameters were measured at each concentration.
Preparation of tissues for Western blotting
After dissection, small renal arteries or brain tissue was snap frozen in liquid nitrogen then stored at –80 C. Frozen arteries from one rat were pooled and pulverized in a prechilled capsule with a steel ball for 8 sec using a Wig-L-Bug amalgamator (Crescent Dental Manufacturing. Co., Lyons, OH). Five volumes of homogenization buffer [10 mM Tris (pH 6.8), 1% sodium dodecyl sulfate, 10% glycerol, 7 M urea] containing 0.5 mM phenylmethyl sulfonyl fluoride and 10 μl/ml Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA) was added, and the contents were mixed by vigorous manual shaking followed by sonication for 10 sec. The homogenate was then centrifuged at 15,000 x g for 10 min at 4 C. Protein concentrations were determined on the supernatants by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), and 10 μg total protein per lane was used for the Western analysis.
Cell culture
All vascular cells and media were obtained from Cambrex (Walkersville, MD). Cells at passage 4–6 were grown to subconfluence in T-75 flasks in EBM phenol red-free medium supplemented with EGM-2MV singlequots and 5% fetal bovine serum. Fetal bovine serum was reduced to 2% for 3 h before treatment and during treatment with relaxin or vehicle. Relaxin (0.1, 1.0, or 5 ng/ml) or vehicle was added for 4, 8, or 24 h. After incubation, the media were removed, and flasks were rinsed with 10 ml cold Dulbecco’s PBS (MediaTech, Inc., Herndon, VA) and placed on ice. After decanting the wash, 1 ml of cold Dulbecco’s PBS was added and cells were scraped off with a rubber policeman. Cells were microcentrifuged at 4 C for 5 min at 2000 x g, and the pellets were snap frozen in liquid nitrogen then stored at –80 C.
Preparation of cell pellets for Western blotting
Cell pellets were kept on ice throughout the protein extraction protocol. Fifty microliters protein extraction buffer [50 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol containing 0.5 mM phenylmethyl sulfonyl fluoride, and 10 μl/ml Calbiochem Protease Inhibitor Cocktail Set III] was added to each cell pellet. Each pellet was sonicated for 10 sec and then centrifuged at 4 C for 10 min at 15,000 x g. Protein concentrations were determined on the supernatant by the Bio-Rad protein assay and10 μg total protein were used for the Western analysis.
Western blotting
For SDS-PAGE, samples were combined with an equal volume of double-strength sample buffer [2% sodium dodecyl sulfate, 10% glycerol, 5% ?-mercaptoethanol, 0.02% bromophenol blue in 0.05 M Tris (pH 6.8)], boiled for 4 min, and microcentrifuged briefly; then 20 μl were loaded onto a 10% gel (Invitrogen, Carlsbad, CA) and electrophoresed for 90 min at 100 V. The separated proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Bedford, MA) for 1 h at approximately 1 mA/cm2 using a semidry electrophoresis transfer system. After transfer, membranes were soaked briefly in methanol and dried completely. Membranes were rehydrated for 30 min in Tris-buffered saline [TBS; 10 mM Tris, 150 mM NaCl (ph7.4)] containing 0.5% Tween 20 before blocking for 1 h in 5% nonfat dried milk (NFDM) (Carnation, Solon, OH) diluted in TBS with 0.05% Tween 20 (TBST). Immunoblots were then incubated overnight at 4 C with rabbit antirat ETB receptor antibody (Alomone Labs, Jerusalem, Israel) diluted in NFDM to a final concentration of 1 μg/ml or with mouse monoclonal antibody directed against human ETB receptor (Institute of Immunology, Tokyo, Japan) diluted in NFDM to 3 μg/ml. A 1:1 mass ratio was employed for preabsorption of the rabbit antirat antibody with control peptide (Alomone Labs). Negative control blots were incubated with normal rabbit IgG (R&D Systems, Minneapolis, MN), or mouse IgG1 (Sigma) diluted in the same manner as the primary antibody. After three 10-min washes in TBST, blots were incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:15,000 in NFDM or with alkaline phosphatase-conjugated goat antimouse IgG secondary antibody (diluted 1:15,000; Cedarlane Labs, Hornby, Ontario, Canada). The blots were again washed in TBST as described above, followed by a quick rinse in TBS and equilibration in alkaline phosphatase detection buffer [100 mM Tris (pH 9.5), 150 mM NaCl, 5 mM MgCl2] for 10 min. The chemiluminescent substrate reagent CDP-Star (Roche Molecular Biochemicals, Indianapolis, IN) diluted 1:200 in alkaline phosphatase detection buffer was reacted with the blots for 5 min, and the membrane was then exposed to BioMax-MR film (Kodak, Rochester, NY) for signal detection.
The membranes were stripped with a buffer containing 62.5 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, and 100 mM ?-mercaptoethanol at 50 C for 30 min and reprobed with a monoclonal anti-?-actin antibody (clone AC-15, Sigma: 1:1000 dilution) to correct for loading variations.
Densitometry
The films were scanned using a Hewlett Packard laser scanner (Scanjet 7400C, Hewlett Packard, Palo Alto, CA), and densitometry was performed using automated digitizing software UN-SCAN-IT gel version 4.3 (Silk Scientific Inc., Orem, UT).
Data analysis
All data are expressed as mean ± SEM. For Western blots, the ratio of the densitometric values of the bands from pregnant/virgin rat small renal arteries, rhRLX-treated/vehicle-treated rat small renal arteries, or rhRLX-treated/vehicle-treated cultured human cells were calculated. These values were then compared with 1.0 using a one-sample t test. P < 0.05 was considered statistically significant.
Results
Figures 1 through 3 show validation studies for the antibodies used to detect the ETB receptor by Western analysis. Both the rabbit antirat polyclonal and mouse antihuman monoclonal antibodies directed against the ETB receptor showed an approximately 45-kDa band in the rat brain, the positive control tissue (15), although the mouse antihuman polyclonal produced a more diffuse band (Fig. 1). After preincubation of the rabbit antibody with the blocking peptide, the density of the approximately 45-kDa band was diminished, and substitution of either rabbit IgG or mouse IgG1k for their respective primary antibodies did not show nonspecific at binding at approximately 45-kDa.
FIG. 1. ETB receptor (ETBR) antibodies show a major band at 45 kDa in whole brain homogenates from Long Evans rats on Western analysis. The left panel (A) shows a blot that was incubated with rabbit antirat ETB receptor primary antibody. In the middle panel, the membrane was probed with the rabbit antirat ETB receptor primary antibody preincubated with the immunizing peptide. Normal rabbit IgG was substituted for the primary antibody in the right panel. The left and right panels (B) show rat brain homogenates probed with a mouse monoclonal antihuman ETB receptor antibody and with mouse IgG1, respectively.
FIG. 2. Western analysis reveals ETB receptor (ETBR) expression at 45 kDa in human coronary artery endothelial cell homogenates. A, Rat brain and human coronary artery endothelial cells (EC) homogenates were probed with rabbit antirat ETB receptor antibody (left panel) and normal rabbit IgG (right panel). B, Dose response of human coronary artery endothelial cell homogenates using the rabbit antirat ETB receptor antibody. C, The same protein preparations as in A probed with monoclonal antihuman ETB receptor antibody in the left panel and with mouse IgG1 in the right panel.
FIG. 3. Western analysis reveals ETB receptor (ETBR) expression at 45 kDa in rat small renal artery homogenates. A, Dose response of small renal artery (SRA) homogenates pooled from three female Long Evans rats as well as whole brain and renal inner medulla homogenates from one female Long Evans rat probed with the rabbit antirat ETB receptor antibody. The same protein preparations were probed with monoclonal antihuman ETB receptor antibody (B) and mouse IgG1 (C).
In Fig. 2, A and B, the rabbit antirat polyclonal antibody was used to detect the ETB receptor in cultured human coronary artery endothelial cells. An approximately 45-kDa band aligning with the positive control rat brain was observed. In addition, a robust dose response was observed between the amount of protein loaded per lane from 5 to 15 μg and the signal intensity. Similar results were obtained for the mouse antihuman ETB receptor antibody (Fig. 2C), although the rabbit antirat polyclonal antibody yielded a sharper and more intense band. Again, substitution of rabbit IgG or mouse IgG1k for their respective primary antibodies did not demonstrate nonspecific binding at approximately 45 kDa.
In Fig. 3A, the rabbit antirat polyclonal was employed to detect ETB receptor in rat small renal arteries. An approximately 45-kDa band aligning with the positive control rat brain as well as the renal inner medulla (16) was noted. A robust dose response was observed between the amount of protein loaded per lane from 2.5 to 40 μg and the signal intensity. Similar findings were obtained for the mouse antihuman ETB receptor antibody (Fig. 3B). However, the rabbit antirat polyclonal antibody again yielded a superior signal. Substitution of mouse IgG1k for the primary antibody yielded a clean blot (Fig. 3C).
The rabbit antirat polyclonal antibody was used for detection of the ETB receptor in small renal arteries isolated from eight pairs of virgin and midterm pregnant rats. A representative blot is shown in Fig. 4A. Normalization was carried out using ?-actin as a housekeeping protein. A graphical summary of the data is shown in Fig. 4B. The overall densitometric ratio of pregnant to virgin was 0.89 ± 0.10 (p = NS vs. 1.0).
FIG. 4. ETB receptor protein is not up-regulated in small renal arteries at midgestation in rats. P, Midgestation; V, virgin. A representative Western blot is shown (A). Rat brain was used as a positive control for ETB receptor protein expression. B, Mean ± SEM (n = 8 pairs of V and P rats) of the ratio of the densitometric values for ETB receptor bands normalized to ?-actin. A ratio equal to 1.0 indicates no difference in ETB receptor protein expression between small renal arteries from midterm pregnant and virgin rats.
The rabbit antirat polyclonal antibody was also used to detect the ETB receptor in small renal arteries isolated from nonpregnant rats administered rhRLX or vehicle for 5 d. A representative blot is shown in Fig. 5A. Normalization was carried out using ?-actin as a housekeeping protein. A graphical summary of the data for nine pairs of rhRLX- and vehicle-treated nonpregnant rats is shown in Fig. 5B. The overall densitometric ratio of relaxin to virgin was 1.11 ± 0.12 (p = NS vs. 1.0). In addition, nonpregnant rats were treated with rhRLX or vehicle for 4–6 h before isolation of small renal arteries, or small renal arteries were harvested from nonpregnant rats and subsequently incubated with 30 ng/ml rhRLX in vitro for 3 h. Neither protocol resulted in significant alterations in ETB receptor expression: densitometry ratios of 0.89 ± 0.08 and 0.99 ± 0.14, respectively, both p = NS vs. 1.0 (Fig. 5B). In the nonpregnant rats treated with rhRLX for 4–6 h, serum concentrations were 26.7 ± 1.4 ng/ml.
FIG. 5. ETB receptor (ETBR) protein is not up-regulated in small renal arteries isolated from nonpregnant rats treated with rhRLX. A representative Western blot is shown (A) of small renal arteries isolated from rats treated 5 d with rhRLX (Rlx) or vehicle (veh). Rat brain was used as a positive control for ETB receptor protein expression. B, Mean ± SEM of the ratio of the densitometric values for ETB receptor bands normalized to ?-actin (n = 9 rat pairs treated for 5 d, n = 7 rat pairs treated for 4–6 h, and n = 7 rats used for 3 h in vitro treatment of arteries). A ratio equal to 1.0 indicates no difference in ETB receptor protein expression between treatment groups.
Human coronary artery endothelial cells in culture were incubated with rhRLX or vehicle at various doses and times. The rabbit antirat polyclonal antibody was used to detect the ETB receptor in homogenates of the cell pellets and the results were normalized to ?-actin. A representative Western blot is shown in Fig. 6A, and a graphical summary of several experiments is depicted in Fig. 6B. The results for cultured human aortic and microvascular as well as umbilical vein endothelial cells treated with rhRLX or vehicle for various doses and times are shown in Table 1. Except for a few sporadic, significant increases or decreases, there was no consistent alteration in ETB receptor expression. To be certain that we were not missing an effect of rhRLX on ETB receptor expression, we tested cells of both genders, various confluencies ranging from 50 to 100%, passage numbers 4–6, and different quiescent protocols (serum deprivation overnight or 2% serum for 3 h before addition of rhRLX or vehicle (11) as well as medium with and without phenol red, which possesses weak estrogenic activity.
FIG. 6. ETB receptor (ETBR) protein is inconsistently regulated in human coronary artery endothelial cells treated with rhRLX. Rlx, rhRLX; veh, vehicle. A representative Western blot is shown (A). B, Mean ± SEM of the ratio of the densitometric values for ETB receptor bands normalized to ?-actin. See Table 1 for further details. A ratio equal to 1.0 indicates no difference in ETB receptor protein expression between rhRLX- and vehicle-treated cells.
TABLE 1. ETB receptor protein expression is inconsistently regulated by treatment with rhRLX in human vascular endothelial cells
Figure 7 portrays functional behavior of small renal arteries preconstricted with phenylephrine and then stimulated with ET-3 after incubation with rhRLX or vehicle for 3 h in vitro. The relaxation response to ET-3 was not significantly enhanced in arteries pretreated with rhRLX. We have previously shown that such pretreatment significantly decreases the myogenic reactivity of small renal arteries (Novak, J., and K. P. Conrad, unpublished observations).
FIG. 7. Small renal arteries from rats treated for 3 h in vitro with rhRLX (RLX) or vehicle (VEH) show no difference in their relaxation response to ET-3. Percent relaxation was calculated as follows: 100 x [(final diameter at each concentration of ET-3 – diameter at 50% constriction)/diameter at 50% constriction]. Values expressed are mean ± SEM.
Discussion
In previous work we identified the endothelial ETB receptor subtype (via NO) to be a critical step in the renal vasodilatory pathway induced by endogenous relaxin that circulates during pregnancy or by relaxin administration to nonpregnant rats (reviewed in Refs. 8 , 17). Therefore, it was logical to test whether relaxin might stimulate this vasodilatory pathway by up-regulating the expression of the endothelial ETB receptor. However, the findings of the current work do not support this hypothesis. The immunoreactive mass of the ETB receptor is not increased in small renal arteries isolated from midterm pregnant rats, nonpregnant rats treated with rhRLX for various periods of time, or small renal arteries treated with rhRLX in vitro, all situations that profoundly vasodilate the renal circulation and/or induce marked reduction in myogenic reactivity of small renal arteries (e.g. Refs. 2 , 4 , 5 , 13 , 14). Furthermore, the failure of small renal arteries treated with rhRLX in vitro to show enhanced relaxation responses to ET-3 is a functional corroboration of the negative molecular analyses. Finally, rhRLX treatment of cultured human endothelial cells of various types failed to show consistent regulation of the ETB receptor by the hormone.
In the current work, we invested considerable effort in validating the antibodies used to assess ETB receptor expression by Western analysis (Fig. 1). First, we used two different antibodies, a rabbit antirat polyclonal and a mouse antihuman monoclonal. The former recognizes a 16-amino acid sequence in the third cytoplasmic loop (manufacturer’s specification sheet), and the latter is directed against most of the cytoplasmic tail (18) that is homologous in rat and human except for one amino acid. The full-length human and rat receptor is 442 amino acids corresponding to a calculated molecular weight of 49–50 kDa. After subtracting the 26-amino acid signal peptide, the ETB receptor has a calculated molecular mass of 46–47 kDa, which is similar to the approximately 45-kDa band recognized by both antibodies in rat brain or renal inner medullary tissue, positive control tissues for the ETB receptor subtype (15, 16). Moreover, we were able to greatly diminish the signal intensity by preincubating the rabbit antirat antibody with immunizing peptide, and substitution of the primary antibodies with IgG did not reveal nonspecific binding in the region of approximately 45 kDa. Thus, we are reasonably confident in our detection of the ETB receptor by Western blotting.
Using these two antibodies, we detected a band of approximately 45 kDa in cultured human endothelial cells (Fig. 2) and small renal arteries (Fig. 3) that aligned with the positive control tissue(s). However, the rabbit antirat polyclonal antibody yielded sharper and more intense bands. Therefore, this antibody was used for subsequent determinations of ETB receptor expression.
We previously showed that renal vasodilation and hyperfiltration is a consistent finding in midterm rat pregnancy and during relaxin administration to nonpregnant rats (e.g.2, 5, 14, 19). Moreover, the small renal arteries harvested from these animals show profound decreases in myogenic reactivity consistent with the changes in the renal circulation observed in the intact rat (4, 13). Because these vasodilatory phenomena are mediated by the endothelial ETB receptor through NO (8, 17), we reasoned that relaxin or pregnancy (via endogenous circulating relaxin) (6) would most likely accentuate this vasodilatory pathway by up-regulating endothelial ETB receptor expression. While we were investigating this hypothesis, Dschietzig et al. (11) reported that rhRLX increased mRNA and protein expression of the ETB receptor in cultured HUVECs; the number of binding sites for radiolabeled ET-1 in cultured HUVECs; and the relaxation response to ET-3 in rat renal, mesenteric, and aortic strips in an endothelium-dependent fashion. Therefore, we were additionally surprised that our findings were unsupportive.
There are potential explanations for the apparent discrepancies in the work of Dschietzig et al. and our own. First, our studies focused on the physiology of renal vasodilation produced by pregnancy per se (via endogenous circulating relaxin) and rhRLX administration to nonpregnant rats. In these conditions, we showed that the ETB receptor is not up-regulated in small renal arteries isolated from midterm pregnant rats or from rhRLX infused nonpregnant rats after either 4–6 h or 5 d of hormone administration (Figs. 4 and 5). Yet we know that these conditions are typified by marked renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries ex vivo (8, 17). Moreover, we know that these vasodilatory phenomena can be prevented by ETB receptor antagonists or by using the ETB receptor-deficient rat indicating the critical nature of the endothelial ETB receptor in this vasodilatory pathway (3, 4, 7, 8, 12, 13, 17). Thus, we reasoned that the findings of Dschietzig et al. may have been different from our own because their conclusions were based on the treatment of cultured cells and isolated arteries with rhRLX in vitro.
Therefore, we continued our investigation using in vitro approaches in an attempt to corroborate the findings of Dscheitzig et al. However, by treating small renal arteries in vitro with rhRLX, we were unable to augment ETB receptor expression (Fig. 5B) or enhance the relaxation response to ET-3 (Fig. 7), the latter serving as a functional confirmation of the molecular analyses. Yet we know that this treatment leads to reduction in myogenic reactivity (Novak, J. and K. P. Conrad, unpublished data) as previously reported in small renal arteries harvested from nonpregnant rats treated with rhRLX in vivo via the endothelial ETB receptor/NO vasodilatory pathway (4). Possible differences in experimental conditions could explain the discrepancies between our results. For example, the preparations of rhRLX were from different sources. Ours was manufactured by Connetics Corp. (now licensed by BAS Medical) and has consistently yielded the expected physiological responses attesting to its identity and bioactivity (8, 17). In addition, we added purified porcine relaxin instead of rhRLX to cultured HUVECs in a few experiments but again failed to observe regulation of the ETB receptor. Dscheitzig et al. used rhRLX manufactured by Immunodiagnostik AG, (Bensheim, Germany), but we are unaware of publications in which this preparation was shown to elicit physiological effects such as reducing plasma osmolality when administered to nonpregnant rats. As another example, although both Dschietzig et al. and we treated rat arteries with rhRLX in vitro and assessed the functional response to ET-3, both the arterial segments and strain of rat used were different.
Both Dschietzig et al. and we treated cultured HUVECs with rhRLX. Whereas they found up-regulation of ETB receptor expression, we did not (Table 1). Possibly, cultured HUVECs may not all be the same. For example, ours were purchased from Cambrex and they were of mixed male and female origin, and we studied them during passages 5 and 6. Dscheitzig et al. isolated their own HUVECs and studied them in passages 3 and 4. They did not report the gender of these cells. However, we did match their cell quiescence protocol in many experiments, and we tested a wide range of cell confluency from 50 to 100% but all to no avail. Whereas Dscheitzig et al. further investigated bovine endothelial cells with positive results, we tested a variety of other human endothelial cells of adult origin and both genders, but again, our results were inconsistent (Fig. 6 and Table 1). Possibly the numerous growth factors used to maintain the various endothelial cells purchased from Cambrex masked any additional effect of rhRLX. However, our negative results using cultured human endothelial cells are consistent with those using arteries isolated from midterm pregnant or rhRLX-treated nonpregnant rats (supra vide).
In previous work, we and other investigators demonstrated that eNOS expression is not up-regulated in renal tissues and arteries isolated from midterm pregnant or relaxin-infused nonpregnant rats (9, 10). However, another critical and proximal element in this vasodilatory pathway is increased, i.e. vascular gelatinase activity, which processes big ET-1 to ET1–32, thereby stimulating the endothelial ETB receptor and NO production (7). Because the endothelial ETB receptor subtype is not regulated by relaxin, at least in our hands, perhaps vascular gelatinase is the major locus of regulation by relaxin in this vasodilatory pathway.
Acknowledgments
We thank Julie Matthews for technical assistance and Elaine Unemori, Ph.D. (BAS Medical) for the generous gift of recombinant human relaxin.
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