[Ca2+]i Reduction Increases Cellular Proliferation and Apoptosis in Vascular Smooth Muscle Cells
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循环研究杂志 2005年第4期
the Division of Nephrology and Hypertension (S.N.K., Q.R., P.C.H., V.E.T., Q.Q.), Department of Physiology and Biomedical Engineering (L.W.H., G.C.S.), Mayo Clinic College of Medicine, Rochester, Minn
Section of Nephrology (S.S.), Department of Medicine, Yale University School of Medicine, New Haven, Conn.
Abstract
Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2+/eC vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca2+ ([Ca2+]i) regulation. We examined cAMP content in vascular smooth muscles from Pkd2+/eC mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2+/eC vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca2+]i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c, and activated caspase 9 and 3 analyses). The low [Ca2+]i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2+/eC VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca2+]i in wild-type VSMCs. The [Ca2+]i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.
Key Words: cAMP phosphodiesterase proliferation ERK apoptosis
Introduction
Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations to either PKD1 or PKD2 genes encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a plasma membrane receptoreClike protein that has functions in cell-cell or cell-extracellular matrix interactions. PC2 is a nonselective cation channel protein with a large single-channel conductance and high permeability to calcium (Ca2+). PC1 interacts with PC2. Their interaction can form a functional receptor-ion-channel-complex and regulate heterotrimeric G-proteineCmediated signaling cascades. The mutations of either gene cause a nearly identical clinical phenotype (see review1).
Cardiovascular complications are the leading cause of mortality and morbidity in ADPKD. The incidence of intracranial aneurysms/aneurysmal ruptures and thoracic aortic dissections in ADPKD is approximately 10-fold higher than in the general population.2 Even during the early stages of ADPKD, before the onset of hypertension or renal dysfunction, abnormal thickening of the intrarenal arteries and reduction in renal blood flows are evident.3,4
Although clinical and experimental evidence indicate a close relation between PKD/Pkd mutations and vascular complications, their pathogenesis is not understood. We have shown that when induced to develop hypertension, Pkd2+/eC mice have an increased susceptibility to vascular injury, manifested as premature death or developing prominent irregular thickening in the tunica media layer of intracranial vessels.5 The areas of irregular vessel wall thickening are correlated with abnormally increased or decreased number of VSMCs, indicating an imbalance between smooth muscle cellular proliferation and apoptosis.
Elevated rates of proliferation and apoptosis are the major phenotypic features of ADPKD cells; these abnormalities have been detected in multiple organ systems including kidneys, lungs, liver, heart, brain, spleen, thymus, and testis.6,7 Recent studies suggest that reduced basal intracellular Ca2+ concentration ([Ca2+]i) and elevated intracellular cyclic 3':5'-adenosine monophosphate (cAMP) play a role in this phenotype. Cystic renal tissues have elevated intracellular cAMP.8,9 In contrast to its growth inhibitory effect on wild-type renal epithelial cells, cAMP promotes proliferation in PKD/Pkd mutant renal epithelial cells.10eC12 This proliferative response to cAMP can also be induced in wild-type renal epithelial cells by reducing their basal [Ca2+]i,13 indicating a tight link between reduced [Ca2+]i and elevated cAMP to the abnormal proliferation in PKD/Pkd mutant epithelial cells. The mechanism underlying an increased rate of apoptosis is less understood. Studies have shown that a vasopressin (V2) receptor blocker, by blocking adenylyl cyclases (AC) and reducing renal cAMP content, inhibits cyst growth in animal models of PKD. This effect is accompanied by a marked reduction in apoptosis, suggesting that the elevated cellular cAMP contributes to the increased apoptosis in cystic renal epithelial cells.8,9
Information regarding cAMP content in Pkd mutant VSMCs and its effect on the VSMC phenotype is lacking. Previously, we have shown that Pkd2+/eC VSMCs have a significant reduction in PC2 expression and basal [Ca2+]i compared with those of wild-type VSMCs.5 However, the relationship between a reduced basal [Ca2+]i and their abnormal cellular phenotype is unknown. In this article, we tested the hypothesis that the [Ca2+]i reduction in VSMCs contributes to cAMP accumulation and the abnormalities in both [Ca2+]i and cAMP lead to an abnormal VSMC phenotype. We show that a reduction in [Ca2+]i in wild-type VSMCs causes an increase in intracellular cAMP, cellular proliferation, and apoptosis, mimicking PKD/Pkd mutant phenotype.
Materials and Methods
Genotyping, Isolation of the Thoracic Aortas from Adult Mice, and Generation of Cultured VSMCs
These methods were reported.5,14,15 All animal experiments were approved by the Institutional Animal Care and Use Committee. VSMCs were isolated using a commercial kit (Papain dissociation system, Worthington Biochemical) following the manufacturer protocol. Dissociated VSMCs were transferred into culture flasks containing DMEM (10% FCS), incubated at 37°C with 5% CO2, and the media changed every other day until the cells reached confluence. The purity of the VSMCs was confirmed both by morphological observation of the SMC-specific hill-and-valley growth pattern and homogeneous staining with smooth muscle -actin mAB that distinguishes SMCs from fibroblasts as described previously.15
cAMP Concentration in Tissues and Cells
Vascular smooth muscles or primary cultured VSMCs were lysed in 10 volumes of 5% TCA, pelleted by centrifugation (600g x10 minutes), and supernatants collected for cAMP determination using an enzyme immunoassay kit (Sigma). The results were expressed as pmol of cAMP/mg of protein.
Basal [Ca2+]i Measurements and Recording Techniques
VSMCs (treated or nontreated) grown on coverslides were loaded with fura-2 AM (Molecular Probes) for 30 minutes, rinsed with media, placed under an inverted Nikon Diaphot microscope, and excited at 340 and 380 nm. The emissions were collected by a 510-nm barrier filter, and images were acquired by a Photometric Cool Snap 12-bit digital camera (Roper Scientific). The ratio of fura-2 emissions was calculated every 0.7 seconds. The basal [Ca2+]i was acquired for 30 seconds. Post hoc analysis of [Ca2+]i was performed using the Metafluor software. [Ca2+]i levels in nmol/L were calculated using an in vitro calibration with known free Ca2+ (0 to 1.35 eol/L) and pentapotassium fura-2 (5 eol/L). The linear plot of the log of the [Ca2+] versus the log of the 340/380 fluorescence ratio [(ReCRmin)/(RmaxeCR)x(380min/380max)] was plotted following subtraction of unloaded cell and system background at each wavelength.
3H-Thymidine Incorporation
Primary cultured VSMCs (85% to 90% confluence, growth arrested x48 hours) were washed in PBS, pulsed with 1 e藽i 3H-Thymidine in media for 3 hours at 37°C, rinsed in ice-cold 10% TCA (3X) and in ddH20 (1X), and lysed with 0.2 mol/L NaOH containing 40 e/mL salmon sperm DNA. Radioactivities of lysates were quantified by liquid scintillation counting. Treated and nontreated VSMCs were studied in parallel and in triplicates.
Fluorescence-Activated Cell Sorting for S Phase and Apoptotic Cells
VSMCs were washed with PBS, reconstituted in PBS and equal amounts of 95% ethanol on ice for 1 hour, rehydrated in PBS, and resuspended in RNAse A solution (1 mg/mL in 0.1% Na+-citrate) at 37°C x 15 minutes. After addition of propidium iodide (100 e/mL of Na+-citrate), the S phase cells were detected by fluorescence activated cell sorting (FACS). An Annexin-V-FLUOS Staining kit (Roche) was used to stain the cell surface phosphotidylserine following the manufacturer’s protocol. The positive cells were detected by FACS (Becton Dickinson FAC Scanner, Mayo Core Facilities).
Cellular Protein Fractionation and Western Blot Analysis
Primary cultured VSMCs collected by scrapping were homogenized by a Polytron homogenizer (4°C) in buffer containing (in mmol/L): 10 Tris (pH7.5); 20 KCl; 1 EGTA; 250 sucrose; 1 PMSF and protease inhibitor cocktail (Roche). Post nuclear supernatants (1000g x15 minutes) were further centrifuged (15 000g x20 minutes), the resulting pellets were enriched with mitochondrial proteins, and the resulting supernatants were further centrifuged (100 000g x2 hours). The resulting supernatants represent cytosols. The lack of membrane proteins in cytosolic fraction was confirmed by immunoblotting with the membrane markers (Calreticulin and PC2). Protein content was determined by the Lowry assay (Bio-Rad). Proteins (20 to 40 e) were denatured in 1x sample buffer with 5% 2-mercaptoethanol at 75° to 95°C for 10 minutes, fractionated by SDS-PAGE (Invitrogen), electrotransferred to PVDF membrane, detected with specific antibody, and visualized by Luminal reagent for enhanced chemiluminescence (Santa Cruz).
Statistical Analysis
Data are expressed as mean±SE. Student t test and two-way ANOVA were used for comparisons between different groups.
Antibodies and Reagents
Antibodies were obtained from Santa Cruz for PCNA (sc-56), ERK1/2 (sc-93), pERK1/2 (sc7383), and B-Raf (sc-166); from Sigma-Aldrich for SM- actin (A2547); from BD Pharmagen for cytochrome c (556433), cleaved C9 (552036), and cleaved C3 (551150); and from R&D systems for Bax (AF820). Unless otherwise specified, all reagents were obtained from Sigma.
Results
Pkd2+/eC Vessels Have Increased Cellular cAMP Levels
Kidneys from Pkd2+/eC, pcy mice and PCK rats have increased intracellular cAMP. To examine whether this is true in Pkd2+/eC mutant arteries, we measured the cAMP content in the tunica media layer of individual aortas freshly isolated from sex-matched Pkd2+/eC and wild-type littermates. As shown in Figure 1A, Pkd2+/eC vascular smooth muscles have significantly higher levels of cAMP compared with the wild type (P<0.001), indicating an increased VSM cellular cAMP concentration.
Lowering the [Ca2+]i Raised cAMP Levels in Wild-Type VSMCs
Pkd2+/eC VSMCs have a reduced basal [Ca2+]i; a low [Ca2+]i could alter cAMP concentration by activating Ca2+-inhibitable adenylyl cyclases (especially AC5, 6) or by inhibiting Ca2+-stimulatable phosphodiesterases (PDEs).16eC18 To test whether a reduction of [Ca2+]i, as observed in Pkd2+/eC VSMCs, can lead to cAMP accumulation, we measured cAMP content in control (nontreated), Verapamil (20 eol/L), or BAPTA-AM (a membrane permeable Ca2+ chelator, 20 eol/L) treated wild-type cultured VSMCs (passage 4 to 6). The concentrations of Verapamil and BAPTA-AM were chosen because similar or higher concentrations have been applied to cultured cells without appreciable direct cytotoxicity.19,20 The basal [Ca2+]i was determined using a Ca2+ indicator Fura-2-AM; [Ca2+]i was modestly reduced (30% reduction) in treated VSMCs (P<0.0001), and this level was maintained throughout the duration of the experiments (Figure 1B). As shown in Figure 1C, a reduction of [Ca2+]i caused a small but highly significant increase in cellular cAMP (P=0.004 and P=0.0007, respectively).
To dissect whether the AC activation and/or the PDE inhibition is responsible for this effect, cAMP was measured after adding forskolin (a prototype-AC activator,21 10 eol/L) or IBMX (a pan-PDE inhibitor, 300 eol/L) to the VSMCs treated with Verapamil or BAPTA-AM. As shown in Figure 2, Forskolin further increased cAMP content (P<0.0001) up to 20-fold, whereas IBMX did not have a significant additive effect (P=0.98). These results suggest that cAMP accumulation is caused mainly by PDE inhibition. To further determine whether PDE4, the major PDE in VSMCs, is responsible for this effect, cells were treated with a combination of Verapamil and Rolipram (a selective PDE4 inhibitor, 30 eol/L) before cAMP content was measured. As shown in Figure 2 (black bars), the effect of Rolipram was similar to that of IBMX (Rolipram versus IBMX; P=0.67), suggesting that inhibition of PDE4 was mainly responsible for the low-[Ca2+]ieCinduced cAMP elevation.
Reduction in Basal [Ca2+]i Increased VSMC Proliferation
To study the relationship between the reduction of [Ca2+]i and the rate of proliferation, wild-type VSMCs were treated with Verapamil or BAPTA-AM at the concentration that reduced [Ca2+]i by 30%. The proliferative rates were measured by direct cell counting (data not shown); 3H-incorporation; FACS analysis for the number of S phase cells; quantitative Western analysis for PCNA, an indicator of active cellular proliferation; and phosphorylated ERK1/2. As shown in Figure 3A through 3D, under conditions of reduced [Ca2+]i, the rates of VSMC proliferation were significantly increased. Variations in the concentrations of Verapamil or BAPTA-AM between 5 to 15 eol/L did not change their effects on proliferation (Figure 3B) or on [Ca2+]i.
cAMP Inhibited VSMC Proliferation at Normal or Reduced Basal [Ca2+]i and Relation to the Expression Pattern of B-Raf Isoforms
cAMP is known to inhibit wild-type VSMC proliferation.22 Others have shown that in renal epithelial cells, Verapamil or EGTA (an extracellular Ca2+ chelator), which reduce [Ca2+]i, switch the effect of cAMP from growth inhibition into growth stimulation.10,12,13 To test whether this is true for VSMCs, we treated VSMCs with db-cAMP (membrane permeable active form of cAMP, 0.5 eol/L) in the presence or absence of Verapamil or BAPTA-AM. We found that the addition of cAMP not only inhibited the proliferation of VSMCs with normal [Ca2+]i, but also inhibited those with reduced [Ca2+]i (Figure 4A). To further determine whether this inhibitory effect is concentration-dependent, we repeated the experiments with combinations of db-cAMP and Verapamil or BAPTA-AM at various concentrations (0.002, 0.02, or 0.2 eol/L of db-cAMP combined with 1 or 2 eol/L of Verapamil or with 1 or 2 eol/L of BAPTA-AM). The basal [Ca2+]i concentrations were reduced by 10% to 25% (1 eol/L Verapamil or 1 or 2 eol/L BAPTA-AM reduced [Ca2+]i by 10% to 25%). The number of S phase cells was determined by FACS analyses after 12 and 24 hours of treatments; no increase in proliferation was detected by adding db-cAMP at any of these dosage combinations (see online Figure S1, in the online data supplement available at http://circres.ahajournals.org). To further determine whether the endogenous cAMP, induced by a low [Ca2+]i, contributes to growth inhibition, the proliferative rate was reassessed after the endogenous cAMP was blocked by Rp-cAMP. As shown in Figure 4B, the proliferation was further heightened by adding Rp-cAMP (P=0.017), indicating that endogenous cAMP is also growth inhibitory.
In Pkd mutant or low [Ca2+]i wild-type renal epithelial cells, the cAMP-induced proliferation is accompanied by an elevated B-Raf expression.11,13 Of the multiple B-Raf isoforms (62 to 95 kDa), only the 95-kDa isoform is known to be associated with cAMP-induced proliferation.13,23 To determine whether the lack of proliferation to cAMP is due to a cell type-specific B-Raf expression, total cell lysate from wild-type VSMCs was fractionated and blotted against a polyclonal B-Raf antibody. As shown in Figure 4C, the 95-kDa B-Raf isoform, which was readily detectable in the lysate from the brain, was not detected in VSMCs at baseline or after treatment with Verapamil or BAPTA-AM, with or without exogenous cAMP. Because the proliferative effect of cAMP is 95-kDa B-Raf dependent, the lack of VSMC proliferation to cAMP is likely due to the absence of the 95-kDa B-Raf isoform. The low [Ca2+]i-induced VSMC proliferation is therefore independent of cAMP/B-Raf signaling in this cellular system.
Basal [Ca2+]i Reduction Increased Apoptosis in VSMCs
Apoptosis can be triggered by [Ca2+]i reduction in certain cell types24,25; cystic epithelial cells are known to have elevated mitochondrial-mediated apoptosis.26 We hypothesized that the decreased [Ca2+]i in VSMCs leads to mitochondrial-mediated apoptosis. This hypothesis was tested by determining the apoptotic activities in wild-type VSMCs treated with Verapamil or BAPTA-AM (30% reduction in [Ca2+]i) and comparing them to nontreated VSMCs in parallel experiments. We found that the apoptosis was significantly increased by DAPI-nuclear staining (see online Figure S2) as well as the FACS analysis staining with Annexin-V for phosphotidylserine, an early cell-surface marker of apoptosis (Figure 5, top left, black bars). Three separate experiments, each in triplicate, with either Verapamil or BAPTA-AM, produced nearly identical results (P=0.0001 and P=0.0006, respectively). Quantitative Western analyses for the cellular apoptotic indicators were also performed. As shown in Figure 5, right panel, Bax, cytochrome c, and activated (cleaved) forms of caspase 9 and 3 were significantly elevated in VSMCs with reduced [Ca2+]i compared with those with normal [Ca2+]i, which showed no increase in apoptosis. Collectively, these data indicate that a deficiency of [Ca2+]i in VSMCs causes an increase in apoptosis, similar to that found in Pkd mutant epithelial cells.
To determine whether the cAMP elevation contributes to apoptosis, we reexamined low-[Ca2+]ieCinduced apoptosis in the presence of cAMP inhibitor (Rp-cAMP, 100 eol/L) or exogenous cAMP (db-cAMP, 0.5 eol/L). As shown in Figure 5 (top left, white bars), Rp-cAMP largely rescued the low-[Ca2+]ieCinduced apoptosis. Conversely, adding cAMP further heightened the rate of apoptosis (hatched bars). Similar results were obtained regardless of whether the reduction in [Ca2+]i was induced by Verapamil or BAPTA-AM. These observations indicate that cellular cAMP accumulation contributes to the increased apoptosis in low [Ca2+]i VSMCs.
Discussion
Heterozygous Pkd2+/eC VSMCs have an abnormal phenotype, manifested by an imbalance in proliferation and apoptosis, and defects in their intracellular Ca2+ regulation, manifested as reductions in basal [Ca2+]i and in the SR Ca2+ store. In this article, we have extended these findings by defining the relationship between basal [Ca2+]i and cellular cAMP concentration and by determining their roles in controlling the VSMC phenotype.
Our first set of experiments show that Pkd2+/eC mutant vascular smooth muscles contain a higher level of cAMP compared with that of wild-type ones from their littermates (Figure 1A). This finding is consistent with the observations in Pkd mutant renal epithelial cells, indicating a commonality in the underlying abnormality associated with Pkd mutations in both systems. Cellular cAMP is synthesized from ATP by ACs (AC1eC9) and degraded to 5'-ATP by PDEs (PDE1eC7). Because the activities of both can be directly or indirectly affected by diverse factors such as [Ca2+]i, Gs/Gi signaling, and ERK signaling,16,27,28 the elevated cAMP content might be a downstream consequence of the [Ca2+]i dysregulation associated with Pkd mutations. This hypothesis was tested by determining the changes of cellular cAMP content in wild-type VSMCs after a reduction of [Ca2+]i. We found that a modest reduction in basal [Ca2+]i (to 70% of normal) inhibits the activity of PDEs, mainly PDE4 in this primary cultured mice VSMC system, and results in cAMP accumulation (Figure 1B and 1C).
The PDE4 family of PDEs is one of the predominant PDEs in VSMCs.29 They are cAMP-specific and encoded by four genes (PDE4A, PDE4B, PDE4C, and PDE4D); each gene has multiple isoforms resulting from either alternative mRNA splicing or different promoter utilization. Although their activities are not directly modulated by [Ca2+]i,16,30 there is a complex circuitry of cross talks between ERK and cAMP that affects primarily the PDE4 activities.28 All four PDE4 (A through D) genes possess FQF and KIM motifs onto which ERK-2 can dock. Furthermore, many widely expressed members of PDE4s, specifically the long forms, possess a C-terminal SPS ERK-consensus motif; the Ser within this motif is specifically phosphorylated by ERK-2. This C-terminal Ser phosphorylation can profoundly inhibit PDE activities and lead to cAMP accumulation.27,31,32 We observed a marked increase in ERK-2 signaling in low [Ca2+]i VSMCs accompanying the accelerated cellular proliferation (Figure 3D). Therefore, it is conceivable that the PDE4 inhibition under this condition might be due to an ERK-2eCmediated PDE4 phosphorylation. Further experiments to confirm this possible mechanism will be a focus of future study.
Cytosolic [Ca2+] and cAMP are the two major intracellular second messengers and defects of either can cause abnormal cellular proliferation and/or apoptosis.25,33 Our next set of experiments was designed to address whether [Ca2+]i reduction leads to an appreciable change in VSMC proliferation. We show that lowering [Ca2+]i (to 70% of normal) by either Verapamil or BAPTA-AM induced a significant increase in VSMC proliferation demonstrated by multiple experimental methods (Figure 3). This effect was maintained even when the cells were treated with lower concentrations of Verapamil or BAPTA-AM (5 to 15 eol/L, Figure 3B), suggesting that a mild to moderate disturbance in [Ca2+]i can have a strong effect on the VSMC phenotype. These observations are compatible with report by others showing that ketamine (an anesthetic agent that inhibits the SR Ca2+ release and lowers the [Ca2+]i) causes human aortic VSMC proliferation accompanied by elevations of MAP kinase and pERK1/2.34 Contrary to these observations, others have reported that L-type Ca2+ channel blockers can inhibit VSMC proliferation induced by insulin or growth factors (IGF-1 or PDGF or FGF), while having no inhibitory effect when applied alone.35,36 We believe that the failure to detect a proliferative effect of Ca2+ channel blockers is likely due to the fact that these studies did not investigate or take into account the coexisting apoptosis. The net cell number would not be expected to change significantly if both the proliferative and apoptotic processes are simultaneously activated, as we have shown to be the case when VSMCs were treated with Verapamil. In fact, their results are similar to ours if only the net cell numbers are considered without considering the significantly different rates of apoptosis between the cells with normal and low [Ca2+]i.
We next determined the effects of cAMP on VSMC proliferation. cAMP is known to inhibit VSMC proliferation and to promote cellular differentiation.22 Consistent with this observation, we found that cAMP inhibited proliferation in primary cultured wild-type VSMCs (Figure 4A). However, in contrast to a proliferative effect observed in low [Ca2+]i wild-type and Pkd mutant renal epithelial cells, exogenous cAMP inhibited proliferation of VSMCs with low [Ca2+]i. Furthermore, when the endogenous cAMP, induced by PDE inhibition, was blocked by Rp-cAMP, the rate of proliferation was further heightened (Figure 4B). These findings indicate that a higher level of cAMP, endogenous or exogenous, is growth inhibitory to VSMCs regardless of their [Ca2+]i level. This opposite cAMP response might be explained by the different patterns of B-Raf expression in VSMCs compared with that of renal epithelial cells.
B-Raf is one of the three kinases (c-Raf-1, B-Raf, and A-Raf) that belong to a cytoplasmic serine/threonine kinase family. It has multiple isoforms, 95-kDa and 62 to 77 kDa (splice variants of 95-kDa form lacking the N-terminus).37 Only the 95-kDa isoform is known to mediate cAMP induced proliferation, in some cells through Rap-1/B-Raf signaling pathway.23 Although Rap-1 is ubiquitously expressed, B-Raf isoforms have a more cell typeeCrestricted pattern of expression.37 Although renal epithelial cells express 95-kDa B-Raf,13,37 the B-Raf isoform expression in VSMCs has not been defined. We show that VSMCs express only the 68-kDa B-Raf; the 95-kDa isoform cannot be detected, nor be induced by decreasing [Ca2+]i or increasing cellular cAMP concentration (Figure 4C). This finding, compounded with the observation that the proliferative response to cAMP in the Pkd mutant or low [Ca2+]i wild-type renal epithelial cells is accompanied by a simultaneous rise in 95-kDa B-Raf isoform,11,13 indicates that the lack of 95-kDa B-raf in VSMCs likely accounts for the lack of proliferative response to cAMP.
We have shown that Pkd2+/eC VSMCs accumulate cAMP; in certain cell types, cAMP triggers apoptosis.22,38,39 Our last set of experiments examined whether the elevated apoptosis can be caused by cellular cAMP accumulation or a low [Ca2+]i in VSMCs. We found that cAMP triggers apoptosis in VSMCs with either normal or low [Ca2+]i; this apoptotic process appears mainly via a mitochondrial-mediated mechanism, demonstrated by a concordant increase in the mitochondrial-cytochrome c release and the recruitment of Bax into the mitochondrial fraction. This apoptotic effect was further confirmed by data showing that when endogenous cAMP was blocked, the rate of apoptosis was reverted to near normal range despite the fact that these VSMCs were still maintained in low [Ca2+]i condition (Figure 5, top left). These results indicate that the elevated cellular cAMP, not the low [Ca2+]i, is the main determinant of an accelerated apoptosis. Our findings are in accord with a recent study showing that the reduction in cellular cAMP by upregulating PDE4B in diffuse large B-cell lymphomas causes resistance to chemotherapy-induced apoptosis. This resistance is mediated by a low cAMP concentration, which leads to an increase in PI3 kinase activity and its downstream AKT signaling.40 Our results show, conversely, a higher VSMC cAMP level triggers apoptosis, likely via the same mechanism, but turning down instead of turning up, the PI3 kinase and AKT-mediated survival signaling.
In summary, we have found that (1) Pkd2+/eC VSMCs have cAMP elevation; (2) a reduction of [Ca2+]i in wild-type VSMCs causes an increase in the cellular cAMP by PDE inhibition; (3) a reduction in [Ca2+]i stimulates VSMC proliferation, which appears to be independent of cellular cAMP/B-Raf signaling; and (4) an elevated cAMP inhibits VSMC proliferation and induces mainly a mitochondrial-mediated apoptosis. We recognize the inherent limitations of these experiments: the degree of [Ca2+]i reduction might not precisely match that of Pkd2+/eC VSMCs at their in vivo state; also, it is possible that our experimental conditions might exaggerate the defects observed in Pkd mutant VSMCs. However, in all experiments, [Ca2+]i was reduced to only a mild to moderate degree and without any observable evidence of direct cytotoxicity. Therefore, the data presented here are sufficient to establish a relationship between [Ca2+]i and VSM cellular phenotype. The fact that a reduction in the [Ca2+]i, induced by two different mechanisms (Verapamil or BAPTA-AM), produced nearly identical phenotypic effects in VSMCs further strengthens our hypothesis and indicates that [Ca2+]i likely plays a central role in regulating VSMC phenotype. Although speculative, these in vitro data suggest that the reduction in [Ca2+]i observed in Pkd mutant VSMCs could be a key factor leading to their defective cellular phenotype and vascular complications in ADPKD.
Acknowledgments
This study was supported by NIH DK63064 (Q.Q.), NIH GM56686 (G.C.S.), PKD Foundation 41A2R (Q.Q.), NIH DK44863 (V.E.T.), and Mayo Clinic School of Medicine (Q.Q.).
References
Torres VE, Harris PC. Autosomal dominant polycystic kidney disease. Nefrologia. 2003; 23 (suppl 1): 14eC22.
Torres V, Holley K, Offord K. Epidemiology. In: Grantham J, Gardner K, eds. Problems in Diagnosis and Management of Polycystic Kidney Disease. Kansas City: PKR Foundation; 1985: 346eC349.
Zeier M, Fehrenbach P, Geberth S, Mohring K, Waldherr R, Ritz E. Renal histology in polycystic kidney disease with incipient and advanced renal failure. Kidney Int. 1992; 42: 1259eC1265.
King BF, Torres VE, Brummer ME, Chapman AB, Bae KT, Glockner JF, Arya K, Felmlee JP, Grantham JJ, Guay-Woodford LM, Bennett WM, Klahr S, Hirschman GH, Kimmel PL, Thompson PA, Miller JP. Magnetic resonance measurements of renal blood flow as a marker of disease severity in autosomal-dominant polycystic kidney disease. Kidney Int. 2003; 64: 2214eC2221.
Qian Q, Hunter LW, Li M, Marin-Padilla M, Prakash YS, Somlo S, Harris PC, Torres VE, Sieck GC. Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells. Hum Mol Genet. 2003; 12: 1875eC1880.
Woo D. Apoptosis and loss of renal tissue in polycystic kidney diseases. N Engl J Med. 1995; 333: 18eC25.
Winyard PJ, Nauta J, Lirenman DS, Hardman P, Sams VR, Risdon RA, Woolf AS. Deregulation of cell survival in cystic and dysplastic renal development. Kidney Int. 1996; 49: 135eC146.
Gattone VH2nd, Wang X, Harris PC, Torres VE. Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist. Nat Med. 2003; 9: 1323eC1326.
Torres VE, Wang X, Qian Q, Somlo S, Harris PC, Gattone VH2nd. Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease. Nat Med. 2004; 10: 363eC364.
Yamaguchi T, Pelling JC, Ramaswamy NT, Eppler JW, Wallace DP, Nagao S, Rome LA, Sullivan LP, Grantham JJ. cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway. Kidney Int. 2000; 57: 1460eC1471.
Yamaguchi T, Nagao S, Wallace DP, Belibi FA, Cowley BD, Pelling JC, Grantham JJ. Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. Kidney Int. 2003; 63: 1983eC1994.
Belibi FA, Reif G, Wallace DP, Yamaguchi T, Olsen L, Li H, Helmkamp GM Jr, Grantham JJ. Cyclic AMP promotes growth and secretion in human polycystic kidney epithelial cells. Kidney Int. 2004; 66: 964eC973.
Yamaguchi T, Wallace DP, Magenheimer BS, Hempson SJ, Grantham JJ, Calvet JP. Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype. J Biol Chem. 2004; 279: 40419eC40430.
Wu G, D’Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, Maeda Y, Le TC, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S. Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell. 1998; 93: 177eC188.
Qian Q, Li M, Cai Y, Ward CJ, Somlo S, Harris PC, Torres VE. Analysis of the polycystins in aortic vascular smooth muscle cells. J Am Soc Nephrol. 2003; 14: 2280eC2287.
Houslay MD, Milligan G. Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem Sci. 1997; 22: 217eC224.
Chabardes D, Imbert-Teboul M, Elalouf JM. Functional properties of Ca2+-inhibitable type 5 and type 6 adenylyl cyclases and role of Ca2+ increase in the inhibition of intracellular cAMP content. Cell Signal. 1999; 11: 651eC663.
Ostrom RS, Liu X, Head BP, Gregorian C, Seasholtz TM, Insel PA. Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains. Mol Pharmacol. 2002; 62: 983eC992.
Collett A, Tanianis-Hughes J, Warhurst G. Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions Biochem Pharmacol. 2004; 68: 783eC790.
Garnovskaya MN, Mukhin YV, Turner JH, Vlasova TM, Ullian ME, Raymond JR. Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin. Biochemistry. 2003; 42: 7178eC7187.
Hanoune J, Defer N. Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol. 2001; 41: 145eC174.
Hayashi S, Morishita R, Matsushita H, Nakagami H, Taniyama Y, Nakamura T, Aoki M, Yamamoto K, Higaki J, Ogihara T. Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells, accompanied by induction of p53 and p21. Hypertension. 2000; 35: 237eC243.
Takahashi H, Honma M, Miyauchi Y, Nakamura S, Ishida-Yamamoto A, Iizuka H. Cyclic AMP differentially regulates cell proliferation of normal human keratinocytes through ERK activation depending on the expression pattern of B-Raf. Arch Dermatol Res. 2004; 296: 74eC82.
Yano S, Tokumitsu H, Soderling TR. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature. 1998; 396: 584eC587.
Yoon WJ, Won SJ, Ryu BR, Gwag BJ. Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax-cytochrome C-caspase pathway: the causative role of Ca2+ deficiency. J Neurochem. 2003; 85: 525eC533.
Ecder T, Melnikov VY, Stanley M, Korular D, Lucia MS, Schrier RW, Edelstein CL. Caspases, Bcl-2 proteins and apoptosis in autosomal-dominant polycystic kidney disease. Kidney Int. 2002; 61: 1220eC1230.
Conti M, Richter W, Mehats C, Livera G, Park JY, Jin C. Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J Biol Chem. 2003; 278: 5493eC5496.
Houslay MD, Adams DR. PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J. 2003; 370: 1eC18.
Jackson EK, Mi Z, Carcillo JA, Gillespie DG, Dubey RK. Phosphodiesterases in the rat renal vasculature. J Cardiovasc Pharmacol. 1997; 30: 798eC801.
Ang KL, Antoni FA. Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation. J Neurochem. 2002; 81: 422eC433.
Hoffmann R, Baillie GS, MacKenzie SJ, Yarwood SJ, Houslay MD. The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. EMBO J. 1999; 18: 893eC903.
MacKenzie SJ, Baillie GS, McPhee I, Bolger GB, Houslay MD. ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions. J Biol Chem. 2000; 275: 16609eC16617.
Sand TE, Thoresen GH, Refsnes M, Christoffersen T. Growth-regulatory effects of glucagon, insulin, and epidermal growth factor in cultured hepatocytes. Temporal aspects and evidence for bidirectional control by cyclic AMP. Dig Dis Sci. 1992; 37: 84eC92.
Boulom V, Lee HW, Zhao L, Eghbali-Webb M. Stimulation of DNA synthesis, activation of mitogen-activated protein kinase ERK2 and nuclear accumulation of c-fos in human aortic smooth muscle cells by ketamine. Cell Prolif. 2002; 35: 155eC165.
Ruiz-Torres A, Lozano R, Melon J, Carraro R. L-calcium channel blockade induced by diltiazem inhibits proliferation, migration and F-actin membrane rearrangements in human vascular smooth muscle cells stimulated with insulin and IGF-1. Int J Clin Pharmacol Ther. 2003; 41: 386eC391.
Marche P, Stepien O. Calcium antagonists and vascular smooth muscle cell reactivity. Z Kardiol. 2000; 89 (suppl 2): 140eC144.
Barnier JV, Papin C, Eychene A, Lecoq O, Calothy G. The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. J Biol Chem. 1995; 270: 23381eC23389.
Ogawa R, Streiff MB, Bugayenko A, Kato GJ. Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells. Blood. 2002; 99: 3390eC3397.
Zakaria N, Knisely A, Portmann B, Mieli-Vergani G, Wendon J, Arya R, Devlin J. Acute sickle cell hepatopathy represents a potential contraindication for percutaneous liver biopsy. Blood. 2003; 101: 101eC103.
Smith PG, Wang F, Wilkinson KN, Savage KJ, Klein U, Neuberg DS, Bollag G, Shipp MA, Aguiar RC. The phosphodiesterase PDE4B limits cAMP associated, PI3K/AKT-dependent, apoptosis in diffuse large B-cell lymphoma. Blood. 2005; 105: 308eC316.(Sertac N. Kip, Larry W. H)
Section of Nephrology (S.S.), Department of Medicine, Yale University School of Medicine, New Haven, Conn.
Abstract
Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2+/eC vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca2+ ([Ca2+]i) regulation. We examined cAMP content in vascular smooth muscles from Pkd2+/eC mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2+/eC vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca2+]i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c, and activated caspase 9 and 3 analyses). The low [Ca2+]i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2+/eC VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca2+]i in wild-type VSMCs. The [Ca2+]i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.
Key Words: cAMP phosphodiesterase proliferation ERK apoptosis
Introduction
Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations to either PKD1 or PKD2 genes encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a plasma membrane receptoreClike protein that has functions in cell-cell or cell-extracellular matrix interactions. PC2 is a nonselective cation channel protein with a large single-channel conductance and high permeability to calcium (Ca2+). PC1 interacts with PC2. Their interaction can form a functional receptor-ion-channel-complex and regulate heterotrimeric G-proteineCmediated signaling cascades. The mutations of either gene cause a nearly identical clinical phenotype (see review1).
Cardiovascular complications are the leading cause of mortality and morbidity in ADPKD. The incidence of intracranial aneurysms/aneurysmal ruptures and thoracic aortic dissections in ADPKD is approximately 10-fold higher than in the general population.2 Even during the early stages of ADPKD, before the onset of hypertension or renal dysfunction, abnormal thickening of the intrarenal arteries and reduction in renal blood flows are evident.3,4
Although clinical and experimental evidence indicate a close relation between PKD/Pkd mutations and vascular complications, their pathogenesis is not understood. We have shown that when induced to develop hypertension, Pkd2+/eC mice have an increased susceptibility to vascular injury, manifested as premature death or developing prominent irregular thickening in the tunica media layer of intracranial vessels.5 The areas of irregular vessel wall thickening are correlated with abnormally increased or decreased number of VSMCs, indicating an imbalance between smooth muscle cellular proliferation and apoptosis.
Elevated rates of proliferation and apoptosis are the major phenotypic features of ADPKD cells; these abnormalities have been detected in multiple organ systems including kidneys, lungs, liver, heart, brain, spleen, thymus, and testis.6,7 Recent studies suggest that reduced basal intracellular Ca2+ concentration ([Ca2+]i) and elevated intracellular cyclic 3':5'-adenosine monophosphate (cAMP) play a role in this phenotype. Cystic renal tissues have elevated intracellular cAMP.8,9 In contrast to its growth inhibitory effect on wild-type renal epithelial cells, cAMP promotes proliferation in PKD/Pkd mutant renal epithelial cells.10eC12 This proliferative response to cAMP can also be induced in wild-type renal epithelial cells by reducing their basal [Ca2+]i,13 indicating a tight link between reduced [Ca2+]i and elevated cAMP to the abnormal proliferation in PKD/Pkd mutant epithelial cells. The mechanism underlying an increased rate of apoptosis is less understood. Studies have shown that a vasopressin (V2) receptor blocker, by blocking adenylyl cyclases (AC) and reducing renal cAMP content, inhibits cyst growth in animal models of PKD. This effect is accompanied by a marked reduction in apoptosis, suggesting that the elevated cellular cAMP contributes to the increased apoptosis in cystic renal epithelial cells.8,9
Information regarding cAMP content in Pkd mutant VSMCs and its effect on the VSMC phenotype is lacking. Previously, we have shown that Pkd2+/eC VSMCs have a significant reduction in PC2 expression and basal [Ca2+]i compared with those of wild-type VSMCs.5 However, the relationship between a reduced basal [Ca2+]i and their abnormal cellular phenotype is unknown. In this article, we tested the hypothesis that the [Ca2+]i reduction in VSMCs contributes to cAMP accumulation and the abnormalities in both [Ca2+]i and cAMP lead to an abnormal VSMC phenotype. We show that a reduction in [Ca2+]i in wild-type VSMCs causes an increase in intracellular cAMP, cellular proliferation, and apoptosis, mimicking PKD/Pkd mutant phenotype.
Materials and Methods
Genotyping, Isolation of the Thoracic Aortas from Adult Mice, and Generation of Cultured VSMCs
These methods were reported.5,14,15 All animal experiments were approved by the Institutional Animal Care and Use Committee. VSMCs were isolated using a commercial kit (Papain dissociation system, Worthington Biochemical) following the manufacturer protocol. Dissociated VSMCs were transferred into culture flasks containing DMEM (10% FCS), incubated at 37°C with 5% CO2, and the media changed every other day until the cells reached confluence. The purity of the VSMCs was confirmed both by morphological observation of the SMC-specific hill-and-valley growth pattern and homogeneous staining with smooth muscle -actin mAB that distinguishes SMCs from fibroblasts as described previously.15
cAMP Concentration in Tissues and Cells
Vascular smooth muscles or primary cultured VSMCs were lysed in 10 volumes of 5% TCA, pelleted by centrifugation (600g x10 minutes), and supernatants collected for cAMP determination using an enzyme immunoassay kit (Sigma). The results were expressed as pmol of cAMP/mg of protein.
Basal [Ca2+]i Measurements and Recording Techniques
VSMCs (treated or nontreated) grown on coverslides were loaded with fura-2 AM (Molecular Probes) for 30 minutes, rinsed with media, placed under an inverted Nikon Diaphot microscope, and excited at 340 and 380 nm. The emissions were collected by a 510-nm barrier filter, and images were acquired by a Photometric Cool Snap 12-bit digital camera (Roper Scientific). The ratio of fura-2 emissions was calculated every 0.7 seconds. The basal [Ca2+]i was acquired for 30 seconds. Post hoc analysis of [Ca2+]i was performed using the Metafluor software. [Ca2+]i levels in nmol/L were calculated using an in vitro calibration with known free Ca2+ (0 to 1.35 eol/L) and pentapotassium fura-2 (5 eol/L). The linear plot of the log of the [Ca2+] versus the log of the 340/380 fluorescence ratio [(ReCRmin)/(RmaxeCR)x(380min/380max)] was plotted following subtraction of unloaded cell and system background at each wavelength.
3H-Thymidine Incorporation
Primary cultured VSMCs (85% to 90% confluence, growth arrested x48 hours) were washed in PBS, pulsed with 1 e藽i 3H-Thymidine in media for 3 hours at 37°C, rinsed in ice-cold 10% TCA (3X) and in ddH20 (1X), and lysed with 0.2 mol/L NaOH containing 40 e/mL salmon sperm DNA. Radioactivities of lysates were quantified by liquid scintillation counting. Treated and nontreated VSMCs were studied in parallel and in triplicates.
Fluorescence-Activated Cell Sorting for S Phase and Apoptotic Cells
VSMCs were washed with PBS, reconstituted in PBS and equal amounts of 95% ethanol on ice for 1 hour, rehydrated in PBS, and resuspended in RNAse A solution (1 mg/mL in 0.1% Na+-citrate) at 37°C x 15 minutes. After addition of propidium iodide (100 e/mL of Na+-citrate), the S phase cells were detected by fluorescence activated cell sorting (FACS). An Annexin-V-FLUOS Staining kit (Roche) was used to stain the cell surface phosphotidylserine following the manufacturer’s protocol. The positive cells were detected by FACS (Becton Dickinson FAC Scanner, Mayo Core Facilities).
Cellular Protein Fractionation and Western Blot Analysis
Primary cultured VSMCs collected by scrapping were homogenized by a Polytron homogenizer (4°C) in buffer containing (in mmol/L): 10 Tris (pH7.5); 20 KCl; 1 EGTA; 250 sucrose; 1 PMSF and protease inhibitor cocktail (Roche). Post nuclear supernatants (1000g x15 minutes) were further centrifuged (15 000g x20 minutes), the resulting pellets were enriched with mitochondrial proteins, and the resulting supernatants were further centrifuged (100 000g x2 hours). The resulting supernatants represent cytosols. The lack of membrane proteins in cytosolic fraction was confirmed by immunoblotting with the membrane markers (Calreticulin and PC2). Protein content was determined by the Lowry assay (Bio-Rad). Proteins (20 to 40 e) were denatured in 1x sample buffer with 5% 2-mercaptoethanol at 75° to 95°C for 10 minutes, fractionated by SDS-PAGE (Invitrogen), electrotransferred to PVDF membrane, detected with specific antibody, and visualized by Luminal reagent for enhanced chemiluminescence (Santa Cruz).
Statistical Analysis
Data are expressed as mean±SE. Student t test and two-way ANOVA were used for comparisons between different groups.
Antibodies and Reagents
Antibodies were obtained from Santa Cruz for PCNA (sc-56), ERK1/2 (sc-93), pERK1/2 (sc7383), and B-Raf (sc-166); from Sigma-Aldrich for SM- actin (A2547); from BD Pharmagen for cytochrome c (556433), cleaved C9 (552036), and cleaved C3 (551150); and from R&D systems for Bax (AF820). Unless otherwise specified, all reagents were obtained from Sigma.
Results
Pkd2+/eC Vessels Have Increased Cellular cAMP Levels
Kidneys from Pkd2+/eC, pcy mice and PCK rats have increased intracellular cAMP. To examine whether this is true in Pkd2+/eC mutant arteries, we measured the cAMP content in the tunica media layer of individual aortas freshly isolated from sex-matched Pkd2+/eC and wild-type littermates. As shown in Figure 1A, Pkd2+/eC vascular smooth muscles have significantly higher levels of cAMP compared with the wild type (P<0.001), indicating an increased VSM cellular cAMP concentration.
Lowering the [Ca2+]i Raised cAMP Levels in Wild-Type VSMCs
Pkd2+/eC VSMCs have a reduced basal [Ca2+]i; a low [Ca2+]i could alter cAMP concentration by activating Ca2+-inhibitable adenylyl cyclases (especially AC5, 6) or by inhibiting Ca2+-stimulatable phosphodiesterases (PDEs).16eC18 To test whether a reduction of [Ca2+]i, as observed in Pkd2+/eC VSMCs, can lead to cAMP accumulation, we measured cAMP content in control (nontreated), Verapamil (20 eol/L), or BAPTA-AM (a membrane permeable Ca2+ chelator, 20 eol/L) treated wild-type cultured VSMCs (passage 4 to 6). The concentrations of Verapamil and BAPTA-AM were chosen because similar or higher concentrations have been applied to cultured cells without appreciable direct cytotoxicity.19,20 The basal [Ca2+]i was determined using a Ca2+ indicator Fura-2-AM; [Ca2+]i was modestly reduced (30% reduction) in treated VSMCs (P<0.0001), and this level was maintained throughout the duration of the experiments (Figure 1B). As shown in Figure 1C, a reduction of [Ca2+]i caused a small but highly significant increase in cellular cAMP (P=0.004 and P=0.0007, respectively).
To dissect whether the AC activation and/or the PDE inhibition is responsible for this effect, cAMP was measured after adding forskolin (a prototype-AC activator,21 10 eol/L) or IBMX (a pan-PDE inhibitor, 300 eol/L) to the VSMCs treated with Verapamil or BAPTA-AM. As shown in Figure 2, Forskolin further increased cAMP content (P<0.0001) up to 20-fold, whereas IBMX did not have a significant additive effect (P=0.98). These results suggest that cAMP accumulation is caused mainly by PDE inhibition. To further determine whether PDE4, the major PDE in VSMCs, is responsible for this effect, cells were treated with a combination of Verapamil and Rolipram (a selective PDE4 inhibitor, 30 eol/L) before cAMP content was measured. As shown in Figure 2 (black bars), the effect of Rolipram was similar to that of IBMX (Rolipram versus IBMX; P=0.67), suggesting that inhibition of PDE4 was mainly responsible for the low-[Ca2+]ieCinduced cAMP elevation.
Reduction in Basal [Ca2+]i Increased VSMC Proliferation
To study the relationship between the reduction of [Ca2+]i and the rate of proliferation, wild-type VSMCs were treated with Verapamil or BAPTA-AM at the concentration that reduced [Ca2+]i by 30%. The proliferative rates were measured by direct cell counting (data not shown); 3H-incorporation; FACS analysis for the number of S phase cells; quantitative Western analysis for PCNA, an indicator of active cellular proliferation; and phosphorylated ERK1/2. As shown in Figure 3A through 3D, under conditions of reduced [Ca2+]i, the rates of VSMC proliferation were significantly increased. Variations in the concentrations of Verapamil or BAPTA-AM between 5 to 15 eol/L did not change their effects on proliferation (Figure 3B) or on [Ca2+]i.
cAMP Inhibited VSMC Proliferation at Normal or Reduced Basal [Ca2+]i and Relation to the Expression Pattern of B-Raf Isoforms
cAMP is known to inhibit wild-type VSMC proliferation.22 Others have shown that in renal epithelial cells, Verapamil or EGTA (an extracellular Ca2+ chelator), which reduce [Ca2+]i, switch the effect of cAMP from growth inhibition into growth stimulation.10,12,13 To test whether this is true for VSMCs, we treated VSMCs with db-cAMP (membrane permeable active form of cAMP, 0.5 eol/L) in the presence or absence of Verapamil or BAPTA-AM. We found that the addition of cAMP not only inhibited the proliferation of VSMCs with normal [Ca2+]i, but also inhibited those with reduced [Ca2+]i (Figure 4A). To further determine whether this inhibitory effect is concentration-dependent, we repeated the experiments with combinations of db-cAMP and Verapamil or BAPTA-AM at various concentrations (0.002, 0.02, or 0.2 eol/L of db-cAMP combined with 1 or 2 eol/L of Verapamil or with 1 or 2 eol/L of BAPTA-AM). The basal [Ca2+]i concentrations were reduced by 10% to 25% (1 eol/L Verapamil or 1 or 2 eol/L BAPTA-AM reduced [Ca2+]i by 10% to 25%). The number of S phase cells was determined by FACS analyses after 12 and 24 hours of treatments; no increase in proliferation was detected by adding db-cAMP at any of these dosage combinations (see online Figure S1, in the online data supplement available at http://circres.ahajournals.org). To further determine whether the endogenous cAMP, induced by a low [Ca2+]i, contributes to growth inhibition, the proliferative rate was reassessed after the endogenous cAMP was blocked by Rp-cAMP. As shown in Figure 4B, the proliferation was further heightened by adding Rp-cAMP (P=0.017), indicating that endogenous cAMP is also growth inhibitory.
In Pkd mutant or low [Ca2+]i wild-type renal epithelial cells, the cAMP-induced proliferation is accompanied by an elevated B-Raf expression.11,13 Of the multiple B-Raf isoforms (62 to 95 kDa), only the 95-kDa isoform is known to be associated with cAMP-induced proliferation.13,23 To determine whether the lack of proliferation to cAMP is due to a cell type-specific B-Raf expression, total cell lysate from wild-type VSMCs was fractionated and blotted against a polyclonal B-Raf antibody. As shown in Figure 4C, the 95-kDa B-Raf isoform, which was readily detectable in the lysate from the brain, was not detected in VSMCs at baseline or after treatment with Verapamil or BAPTA-AM, with or without exogenous cAMP. Because the proliferative effect of cAMP is 95-kDa B-Raf dependent, the lack of VSMC proliferation to cAMP is likely due to the absence of the 95-kDa B-Raf isoform. The low [Ca2+]i-induced VSMC proliferation is therefore independent of cAMP/B-Raf signaling in this cellular system.
Basal [Ca2+]i Reduction Increased Apoptosis in VSMCs
Apoptosis can be triggered by [Ca2+]i reduction in certain cell types24,25; cystic epithelial cells are known to have elevated mitochondrial-mediated apoptosis.26 We hypothesized that the decreased [Ca2+]i in VSMCs leads to mitochondrial-mediated apoptosis. This hypothesis was tested by determining the apoptotic activities in wild-type VSMCs treated with Verapamil or BAPTA-AM (30% reduction in [Ca2+]i) and comparing them to nontreated VSMCs in parallel experiments. We found that the apoptosis was significantly increased by DAPI-nuclear staining (see online Figure S2) as well as the FACS analysis staining with Annexin-V for phosphotidylserine, an early cell-surface marker of apoptosis (Figure 5, top left, black bars). Three separate experiments, each in triplicate, with either Verapamil or BAPTA-AM, produced nearly identical results (P=0.0001 and P=0.0006, respectively). Quantitative Western analyses for the cellular apoptotic indicators were also performed. As shown in Figure 5, right panel, Bax, cytochrome c, and activated (cleaved) forms of caspase 9 and 3 were significantly elevated in VSMCs with reduced [Ca2+]i compared with those with normal [Ca2+]i, which showed no increase in apoptosis. Collectively, these data indicate that a deficiency of [Ca2+]i in VSMCs causes an increase in apoptosis, similar to that found in Pkd mutant epithelial cells.
To determine whether the cAMP elevation contributes to apoptosis, we reexamined low-[Ca2+]ieCinduced apoptosis in the presence of cAMP inhibitor (Rp-cAMP, 100 eol/L) or exogenous cAMP (db-cAMP, 0.5 eol/L). As shown in Figure 5 (top left, white bars), Rp-cAMP largely rescued the low-[Ca2+]ieCinduced apoptosis. Conversely, adding cAMP further heightened the rate of apoptosis (hatched bars). Similar results were obtained regardless of whether the reduction in [Ca2+]i was induced by Verapamil or BAPTA-AM. These observations indicate that cellular cAMP accumulation contributes to the increased apoptosis in low [Ca2+]i VSMCs.
Discussion
Heterozygous Pkd2+/eC VSMCs have an abnormal phenotype, manifested by an imbalance in proliferation and apoptosis, and defects in their intracellular Ca2+ regulation, manifested as reductions in basal [Ca2+]i and in the SR Ca2+ store. In this article, we have extended these findings by defining the relationship between basal [Ca2+]i and cellular cAMP concentration and by determining their roles in controlling the VSMC phenotype.
Our first set of experiments show that Pkd2+/eC mutant vascular smooth muscles contain a higher level of cAMP compared with that of wild-type ones from their littermates (Figure 1A). This finding is consistent with the observations in Pkd mutant renal epithelial cells, indicating a commonality in the underlying abnormality associated with Pkd mutations in both systems. Cellular cAMP is synthesized from ATP by ACs (AC1eC9) and degraded to 5'-ATP by PDEs (PDE1eC7). Because the activities of both can be directly or indirectly affected by diverse factors such as [Ca2+]i, Gs/Gi signaling, and ERK signaling,16,27,28 the elevated cAMP content might be a downstream consequence of the [Ca2+]i dysregulation associated with Pkd mutations. This hypothesis was tested by determining the changes of cellular cAMP content in wild-type VSMCs after a reduction of [Ca2+]i. We found that a modest reduction in basal [Ca2+]i (to 70% of normal) inhibits the activity of PDEs, mainly PDE4 in this primary cultured mice VSMC system, and results in cAMP accumulation (Figure 1B and 1C).
The PDE4 family of PDEs is one of the predominant PDEs in VSMCs.29 They are cAMP-specific and encoded by four genes (PDE4A, PDE4B, PDE4C, and PDE4D); each gene has multiple isoforms resulting from either alternative mRNA splicing or different promoter utilization. Although their activities are not directly modulated by [Ca2+]i,16,30 there is a complex circuitry of cross talks between ERK and cAMP that affects primarily the PDE4 activities.28 All four PDE4 (A through D) genes possess FQF and KIM motifs onto which ERK-2 can dock. Furthermore, many widely expressed members of PDE4s, specifically the long forms, possess a C-terminal SPS ERK-consensus motif; the Ser within this motif is specifically phosphorylated by ERK-2. This C-terminal Ser phosphorylation can profoundly inhibit PDE activities and lead to cAMP accumulation.27,31,32 We observed a marked increase in ERK-2 signaling in low [Ca2+]i VSMCs accompanying the accelerated cellular proliferation (Figure 3D). Therefore, it is conceivable that the PDE4 inhibition under this condition might be due to an ERK-2eCmediated PDE4 phosphorylation. Further experiments to confirm this possible mechanism will be a focus of future study.
Cytosolic [Ca2+] and cAMP are the two major intracellular second messengers and defects of either can cause abnormal cellular proliferation and/or apoptosis.25,33 Our next set of experiments was designed to address whether [Ca2+]i reduction leads to an appreciable change in VSMC proliferation. We show that lowering [Ca2+]i (to 70% of normal) by either Verapamil or BAPTA-AM induced a significant increase in VSMC proliferation demonstrated by multiple experimental methods (Figure 3). This effect was maintained even when the cells were treated with lower concentrations of Verapamil or BAPTA-AM (5 to 15 eol/L, Figure 3B), suggesting that a mild to moderate disturbance in [Ca2+]i can have a strong effect on the VSMC phenotype. These observations are compatible with report by others showing that ketamine (an anesthetic agent that inhibits the SR Ca2+ release and lowers the [Ca2+]i) causes human aortic VSMC proliferation accompanied by elevations of MAP kinase and pERK1/2.34 Contrary to these observations, others have reported that L-type Ca2+ channel blockers can inhibit VSMC proliferation induced by insulin or growth factors (IGF-1 or PDGF or FGF), while having no inhibitory effect when applied alone.35,36 We believe that the failure to detect a proliferative effect of Ca2+ channel blockers is likely due to the fact that these studies did not investigate or take into account the coexisting apoptosis. The net cell number would not be expected to change significantly if both the proliferative and apoptotic processes are simultaneously activated, as we have shown to be the case when VSMCs were treated with Verapamil. In fact, their results are similar to ours if only the net cell numbers are considered without considering the significantly different rates of apoptosis between the cells with normal and low [Ca2+]i.
We next determined the effects of cAMP on VSMC proliferation. cAMP is known to inhibit VSMC proliferation and to promote cellular differentiation.22 Consistent with this observation, we found that cAMP inhibited proliferation in primary cultured wild-type VSMCs (Figure 4A). However, in contrast to a proliferative effect observed in low [Ca2+]i wild-type and Pkd mutant renal epithelial cells, exogenous cAMP inhibited proliferation of VSMCs with low [Ca2+]i. Furthermore, when the endogenous cAMP, induced by PDE inhibition, was blocked by Rp-cAMP, the rate of proliferation was further heightened (Figure 4B). These findings indicate that a higher level of cAMP, endogenous or exogenous, is growth inhibitory to VSMCs regardless of their [Ca2+]i level. This opposite cAMP response might be explained by the different patterns of B-Raf expression in VSMCs compared with that of renal epithelial cells.
B-Raf is one of the three kinases (c-Raf-1, B-Raf, and A-Raf) that belong to a cytoplasmic serine/threonine kinase family. It has multiple isoforms, 95-kDa and 62 to 77 kDa (splice variants of 95-kDa form lacking the N-terminus).37 Only the 95-kDa isoform is known to mediate cAMP induced proliferation, in some cells through Rap-1/B-Raf signaling pathway.23 Although Rap-1 is ubiquitously expressed, B-Raf isoforms have a more cell typeeCrestricted pattern of expression.37 Although renal epithelial cells express 95-kDa B-Raf,13,37 the B-Raf isoform expression in VSMCs has not been defined. We show that VSMCs express only the 68-kDa B-Raf; the 95-kDa isoform cannot be detected, nor be induced by decreasing [Ca2+]i or increasing cellular cAMP concentration (Figure 4C). This finding, compounded with the observation that the proliferative response to cAMP in the Pkd mutant or low [Ca2+]i wild-type renal epithelial cells is accompanied by a simultaneous rise in 95-kDa B-Raf isoform,11,13 indicates that the lack of 95-kDa B-raf in VSMCs likely accounts for the lack of proliferative response to cAMP.
We have shown that Pkd2+/eC VSMCs accumulate cAMP; in certain cell types, cAMP triggers apoptosis.22,38,39 Our last set of experiments examined whether the elevated apoptosis can be caused by cellular cAMP accumulation or a low [Ca2+]i in VSMCs. We found that cAMP triggers apoptosis in VSMCs with either normal or low [Ca2+]i; this apoptotic process appears mainly via a mitochondrial-mediated mechanism, demonstrated by a concordant increase in the mitochondrial-cytochrome c release and the recruitment of Bax into the mitochondrial fraction. This apoptotic effect was further confirmed by data showing that when endogenous cAMP was blocked, the rate of apoptosis was reverted to near normal range despite the fact that these VSMCs were still maintained in low [Ca2+]i condition (Figure 5, top left). These results indicate that the elevated cellular cAMP, not the low [Ca2+]i, is the main determinant of an accelerated apoptosis. Our findings are in accord with a recent study showing that the reduction in cellular cAMP by upregulating PDE4B in diffuse large B-cell lymphomas causes resistance to chemotherapy-induced apoptosis. This resistance is mediated by a low cAMP concentration, which leads to an increase in PI3 kinase activity and its downstream AKT signaling.40 Our results show, conversely, a higher VSMC cAMP level triggers apoptosis, likely via the same mechanism, but turning down instead of turning up, the PI3 kinase and AKT-mediated survival signaling.
In summary, we have found that (1) Pkd2+/eC VSMCs have cAMP elevation; (2) a reduction of [Ca2+]i in wild-type VSMCs causes an increase in the cellular cAMP by PDE inhibition; (3) a reduction in [Ca2+]i stimulates VSMC proliferation, which appears to be independent of cellular cAMP/B-Raf signaling; and (4) an elevated cAMP inhibits VSMC proliferation and induces mainly a mitochondrial-mediated apoptosis. We recognize the inherent limitations of these experiments: the degree of [Ca2+]i reduction might not precisely match that of Pkd2+/eC VSMCs at their in vivo state; also, it is possible that our experimental conditions might exaggerate the defects observed in Pkd mutant VSMCs. However, in all experiments, [Ca2+]i was reduced to only a mild to moderate degree and without any observable evidence of direct cytotoxicity. Therefore, the data presented here are sufficient to establish a relationship between [Ca2+]i and VSM cellular phenotype. The fact that a reduction in the [Ca2+]i, induced by two different mechanisms (Verapamil or BAPTA-AM), produced nearly identical phenotypic effects in VSMCs further strengthens our hypothesis and indicates that [Ca2+]i likely plays a central role in regulating VSMC phenotype. Although speculative, these in vitro data suggest that the reduction in [Ca2+]i observed in Pkd mutant VSMCs could be a key factor leading to their defective cellular phenotype and vascular complications in ADPKD.
Acknowledgments
This study was supported by NIH DK63064 (Q.Q.), NIH GM56686 (G.C.S.), PKD Foundation 41A2R (Q.Q.), NIH DK44863 (V.E.T.), and Mayo Clinic School of Medicine (Q.Q.).
References
Torres VE, Harris PC. Autosomal dominant polycystic kidney disease. Nefrologia. 2003; 23 (suppl 1): 14eC22.
Torres V, Holley K, Offord K. Epidemiology. In: Grantham J, Gardner K, eds. Problems in Diagnosis and Management of Polycystic Kidney Disease. Kansas City: PKR Foundation; 1985: 346eC349.
Zeier M, Fehrenbach P, Geberth S, Mohring K, Waldherr R, Ritz E. Renal histology in polycystic kidney disease with incipient and advanced renal failure. Kidney Int. 1992; 42: 1259eC1265.
King BF, Torres VE, Brummer ME, Chapman AB, Bae KT, Glockner JF, Arya K, Felmlee JP, Grantham JJ, Guay-Woodford LM, Bennett WM, Klahr S, Hirschman GH, Kimmel PL, Thompson PA, Miller JP. Magnetic resonance measurements of renal blood flow as a marker of disease severity in autosomal-dominant polycystic kidney disease. Kidney Int. 2003; 64: 2214eC2221.
Qian Q, Hunter LW, Li M, Marin-Padilla M, Prakash YS, Somlo S, Harris PC, Torres VE, Sieck GC. Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells. Hum Mol Genet. 2003; 12: 1875eC1880.
Woo D. Apoptosis and loss of renal tissue in polycystic kidney diseases. N Engl J Med. 1995; 333: 18eC25.
Winyard PJ, Nauta J, Lirenman DS, Hardman P, Sams VR, Risdon RA, Woolf AS. Deregulation of cell survival in cystic and dysplastic renal development. Kidney Int. 1996; 49: 135eC146.
Gattone VH2nd, Wang X, Harris PC, Torres VE. Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist. Nat Med. 2003; 9: 1323eC1326.
Torres VE, Wang X, Qian Q, Somlo S, Harris PC, Gattone VH2nd. Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease. Nat Med. 2004; 10: 363eC364.
Yamaguchi T, Pelling JC, Ramaswamy NT, Eppler JW, Wallace DP, Nagao S, Rome LA, Sullivan LP, Grantham JJ. cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway. Kidney Int. 2000; 57: 1460eC1471.
Yamaguchi T, Nagao S, Wallace DP, Belibi FA, Cowley BD, Pelling JC, Grantham JJ. Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. Kidney Int. 2003; 63: 1983eC1994.
Belibi FA, Reif G, Wallace DP, Yamaguchi T, Olsen L, Li H, Helmkamp GM Jr, Grantham JJ. Cyclic AMP promotes growth and secretion in human polycystic kidney epithelial cells. Kidney Int. 2004; 66: 964eC973.
Yamaguchi T, Wallace DP, Magenheimer BS, Hempson SJ, Grantham JJ, Calvet JP. Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype. J Biol Chem. 2004; 279: 40419eC40430.
Wu G, D’Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, Maeda Y, Le TC, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S. Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell. 1998; 93: 177eC188.
Qian Q, Li M, Cai Y, Ward CJ, Somlo S, Harris PC, Torres VE. Analysis of the polycystins in aortic vascular smooth muscle cells. J Am Soc Nephrol. 2003; 14: 2280eC2287.
Houslay MD, Milligan G. Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem Sci. 1997; 22: 217eC224.
Chabardes D, Imbert-Teboul M, Elalouf JM. Functional properties of Ca2+-inhibitable type 5 and type 6 adenylyl cyclases and role of Ca2+ increase in the inhibition of intracellular cAMP content. Cell Signal. 1999; 11: 651eC663.
Ostrom RS, Liu X, Head BP, Gregorian C, Seasholtz TM, Insel PA. Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains. Mol Pharmacol. 2002; 62: 983eC992.
Collett A, Tanianis-Hughes J, Warhurst G. Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions Biochem Pharmacol. 2004; 68: 783eC790.
Garnovskaya MN, Mukhin YV, Turner JH, Vlasova TM, Ullian ME, Raymond JR. Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin. Biochemistry. 2003; 42: 7178eC7187.
Hanoune J, Defer N. Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol. 2001; 41: 145eC174.
Hayashi S, Morishita R, Matsushita H, Nakagami H, Taniyama Y, Nakamura T, Aoki M, Yamamoto K, Higaki J, Ogihara T. Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells, accompanied by induction of p53 and p21. Hypertension. 2000; 35: 237eC243.
Takahashi H, Honma M, Miyauchi Y, Nakamura S, Ishida-Yamamoto A, Iizuka H. Cyclic AMP differentially regulates cell proliferation of normal human keratinocytes through ERK activation depending on the expression pattern of B-Raf. Arch Dermatol Res. 2004; 296: 74eC82.
Yano S, Tokumitsu H, Soderling TR. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature. 1998; 396: 584eC587.
Yoon WJ, Won SJ, Ryu BR, Gwag BJ. Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax-cytochrome C-caspase pathway: the causative role of Ca2+ deficiency. J Neurochem. 2003; 85: 525eC533.
Ecder T, Melnikov VY, Stanley M, Korular D, Lucia MS, Schrier RW, Edelstein CL. Caspases, Bcl-2 proteins and apoptosis in autosomal-dominant polycystic kidney disease. Kidney Int. 2002; 61: 1220eC1230.
Conti M, Richter W, Mehats C, Livera G, Park JY, Jin C. Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J Biol Chem. 2003; 278: 5493eC5496.
Houslay MD, Adams DR. PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J. 2003; 370: 1eC18.
Jackson EK, Mi Z, Carcillo JA, Gillespie DG, Dubey RK. Phosphodiesterases in the rat renal vasculature. J Cardiovasc Pharmacol. 1997; 30: 798eC801.
Ang KL, Antoni FA. Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation. J Neurochem. 2002; 81: 422eC433.
Hoffmann R, Baillie GS, MacKenzie SJ, Yarwood SJ, Houslay MD. The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. EMBO J. 1999; 18: 893eC903.
MacKenzie SJ, Baillie GS, McPhee I, Bolger GB, Houslay MD. ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions. J Biol Chem. 2000; 275: 16609eC16617.
Sand TE, Thoresen GH, Refsnes M, Christoffersen T. Growth-regulatory effects of glucagon, insulin, and epidermal growth factor in cultured hepatocytes. Temporal aspects and evidence for bidirectional control by cyclic AMP. Dig Dis Sci. 1992; 37: 84eC92.
Boulom V, Lee HW, Zhao L, Eghbali-Webb M. Stimulation of DNA synthesis, activation of mitogen-activated protein kinase ERK2 and nuclear accumulation of c-fos in human aortic smooth muscle cells by ketamine. Cell Prolif. 2002; 35: 155eC165.
Ruiz-Torres A, Lozano R, Melon J, Carraro R. L-calcium channel blockade induced by diltiazem inhibits proliferation, migration and F-actin membrane rearrangements in human vascular smooth muscle cells stimulated with insulin and IGF-1. Int J Clin Pharmacol Ther. 2003; 41: 386eC391.
Marche P, Stepien O. Calcium antagonists and vascular smooth muscle cell reactivity. Z Kardiol. 2000; 89 (suppl 2): 140eC144.
Barnier JV, Papin C, Eychene A, Lecoq O, Calothy G. The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. J Biol Chem. 1995; 270: 23381eC23389.
Ogawa R, Streiff MB, Bugayenko A, Kato GJ. Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells. Blood. 2002; 99: 3390eC3397.
Zakaria N, Knisely A, Portmann B, Mieli-Vergani G, Wendon J, Arya R, Devlin J. Acute sickle cell hepatopathy represents a potential contraindication for percutaneous liver biopsy. Blood. 2003; 101: 101eC103.
Smith PG, Wang F, Wilkinson KN, Savage KJ, Klein U, Neuberg DS, Bollag G, Shipp MA, Aguiar RC. The phosphodiesterase PDE4B limits cAMP associated, PI3K/AKT-dependent, apoptosis in diffuse large B-cell lymphoma. Blood. 2005; 105: 308eC316.(Sertac N. Kip, Larry W. H)