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Istituto Pasteur Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, 00161 Rome, Italy;
* Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK;
Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, and DIBIT S. Raffaele Scientific Institute, 20132 Milano, Italy; and
Department of Pharmacology, University of Cambridge, Cambridge, CB2 1QJ, UK
1Correspondence: Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, Via A. Scarpa, 14, 00161 Rome, Italy. E-mail: antonio.filippini{at}uniroma1.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ryanodine receptors • calcium signaling • ADP-ribosyl cyclase • seminiferous tubule
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. It is generally accepted that most hormone- or neurotransmitter-elicited intracellular Ca2+ release is mediated via inositol 1,4,5 trisphosphate (IP3) generation and activation of IP3 receptors in the endoplasmic reticulum (ER) (4)
. Although IP3 appears as a ubiquitous intracellular messenger for calcium mobilization, recent studies indicate that the Ca2+ release mechanism may also be regulated by a family of pyridine nucleotide metabolites. cADPR, a cyclic metabolite of NAD, was first shown to be active in releasing Ca2+ via RyRs from sea urchin egg microsomes (5
6
7)
; additional evidence has shown that cells responsive to cADPR are widespread and include cells from unicellular organisms to mammals, indicating the universality of cADPR as a Ca2+ signaling molecule (8)
.
In several mammalian cells, characterization of cADPR-induced calcium release is important for a variety of cell functions including secretion, cell proliferation, muscle contraction, and fertilization. A role for cADPR-mediated calcium mobilization through ryanodine receptors in different populations of smooth muscle cells was reported (9
10
11
12)
. However, little is known about the functional role of cADPR-mediated intracellular Ca2+ release in regulating agonist-stimulated contraction of smooth muscle cells.
ET-1 is a 21 amino acid peptide with vasoactive and mitogenic properties originally isolated from the supernatant of cultured porcine aortic endothelial cells (13)
. The three closely related endothelin isopeptides (ET-1, 2, and 3) and their two distinct subtypes of G-protein-coupled receptors, termed ETA and ETB, are expressed in numerous tissues and may mediate autocrine/paracrine actions (14
, 15)
. Increasing evidence suggests a biological role for ET-1 in the autocrine/paracrine regulation of testicular function (16)
. Myoid cells, recently classified as peritubular smooth muscle cells (PSMC), surround the seminiferous tubule, are responsible for peritubular contractility, and represent an interesting model of nonvascular smooth muscle cells (17
, 18)
. PSMC express 
-smooth muscle actin and desmin (19
, 20)
and respond specifically to endothelin undergoing cell contraction in both cell culture and peritubular tissue (21
22
23)
. The main biological function attributed to PSMC contractility is generation of impulses for the progression of spermatozoa toward the rete testis, a crucial function for male fertility. Since seminiferous tubules have been reported to undergo rhythmic contraction, in the apparent absence of nerve endings the fine regulation of contractility is presumably subject to paracrine control (16)
.
Recently, we demonstrated that ETA and ETB endothelin receptors are coexpressed on PSMC and that their activation by ET-1 results in intracellular Ca2+ mobilization and cell contraction through distinct Ca2+ signaling pathways. Activation of ETA receptors is coupled to rapid increases in phosphoinositide hydrolysis and subsequent calcium mobilization through IP3, whereas the selective stimulation of the ETB receptor induces mobilization of thapsigargin-sensitive intracellular Ca2+ pools independent of IP3 (23)
.
We tested the hypothesis that in ET-1-stimulated PSMC, a messenger besides IP3, could mobilize Ca2+ from intracellular stores. We first report that cADPR is involved in the ET-1-mediated regulation of calcium signaling and peritubular contractility. We have investigated whether cADPR as a Ca2+-mobilizing mediator induces Ca2+ release through an IP3R-insensitive mechanism. The role of RyR Ca2+ release channels, proposed targets of cADPR, was examined in ETB receptor-mediated contractions. We show that desensitization and inhibition of the cADPR/Ca2+ release system completely inhibited IP3 (through ETA receptors) or cADPR (through ETB receptors) -mediated calcium signaling, indicating a hierarchical organization of calcium signaling in PSMC. Most important, we observed that inhibition of cADPR-mediated Ca2+ release also completely blocked the peritubular contractility mediated by both subtypes of endothelin receptors, suggesting an essential role for cADPR-regulated Ca2+ release in the biological activity of PSMC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 25)
. Alkaline phosphatase is a marker for PSMC (26)
and is used routinely to detect PSMC purity, never evaluated at < 96%.
Fluorescent microscopic analysis
The binding of Bodipy ryanodine (kindly provided by Dr. K. R. Bidasee) to its receptors was evaluated by confocal fluorescence microscopy using primary cultures of PSMC. The cells were fixed in 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, and incubated at 22°C for 1 h in Hanks’ buffered saline containing 25 µM Bodipy ryanodine. Specific high-affinity binding of Bodipy ryanodine was tested by pretreating cells with nonfluorescent ryanodine. Confocal imaging was then performed with a laser-scanning microscopy system configured with a Nikon microscope and a Krypton/argon laser (488 nM).
Western blots
Microsomes from PSMC were prepared as described (27)
. Cells were homogenized in ice-cold buffer A [320 mM sucrose, 5 mM Na-HEPES, pH 7.4, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] using a TeflonTM Potter. Homogenates were centrifuged at 7000 g for 5 min at 4°C. The supernatant obtained was centrifuged at 100,000 g for 1 h at 4°C. The microsomes were resuspended in buffer A and stored at -80°C. The protein concentration was quantified using the Bradford protein assay kit (Bio-Rad, Hercules, CA).
Microsomal proteins were separated by SDS-PAGE, as described (28
, 29)
. Proteins were then transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) by blotting gels for 5 h at 350 mA at 4°C in a transfer buffer containing 192 mM glycine, 25 mM Tris, 0.01% SDS, and 10% methanol. Membranes were incubated for 3 h in a blocking buffer containing 150 mM NaCl, 50 mM Tris-HCl-pH 7.4, 0.2% Tween-20, 5% nonfat dry milk. Primary antibodies (diluted 1:3000) were incubated with membranes overnight at room temperature. Detection was performed using amplified alkaline phosphatase immunoblot reagents obtained from Bio-Rad. Polyclonal rabbit antisera able to distinguish between the three RyRs were developed against purified GST fusion proteins corresponding to the region of low homology situated between transmembrane domains 4 and 5 (divergent region 1, or D1) of the RyR1, RyR2, and RyR3 proteins, as described previously (28)
, and have been shown not to cross-react.
To identify type-specific IP3Rs, cell lysates from isolated rat PSMC were subjected to electrophoresis and transferred to a PVDF membrane. The membrane was blocked with PBS + 7% nonfat dry milk and 0.2% Tween 20, then incubated with specific affinity-purified IP3R subtype I, II, and III antisera [kindly provided by Dr. C. W. Taylor, Cambridge (30)
]. The primary antibody reaction was carried out at 4°C overnight. After immunoreactivity was visualized using peroxidase-conjugated secondary antibody, the blot was processed for enhanced chemiluminescence and exposed to X-ray film.
ADP-ribosyl cyclase assay
ADP-ribosyl cyclase activity was measured in PSMC plasma membrane isolated as described (31)
. We measured cyclization of the NAD+ surrogate nicotinamide guanine nucleotide (NGD+) to its fluorescent and nonhydrolizable derivative cGDPR. Plasma membranes (25 µg) were incubated for 20 min at 37°C, in 20 mM Tris-HCl (pH 7.4) with 100 mM NGD+. The reaction was stopped with 5 µl of 100% (v/v) trichloroacetic acid. cGDPR formation was measured as fluorescence units using a high-sensitivity spectrofluorometer (
ex=300 nm; em=410 nm). The amount of cGDPR formed was determined by comparing the fluorescence intensity with that of cGDPR standards. To establish the specificity of the assay, we incubated membranes with antagonistic anti-CD38 antibody (1:1000) (SUN4B7 kindly provided by F. Malavasi) or NAD (400 µM) or 5 mM nicotinamide (NiAm). Mouse IgG2A was used as a control.
Ca2+ Release experiments in permeabilized cells
PSMC were permeabilized in the presence of saponin (7 µg/ml) for 2 min in an intracellular buffer (20 mM HEPES, 110 mM KCl, 2 mM MgCl2, 5 mM KH2PO4, 10 nM NaCl, pH 7.2) at 37°C. Fluorescence was measured in a F-2000 fluorimeter with wavelength settings between 340 and 380 nm (excitation) and 510 nm (emission) at 37°C in the presence of Fura-2 free acid (1.5 µM). Reuptake of Ca2+ into the stores was achieved by addition of ATP (1 mM), creatine phosphate (20 mM), and creatine kinase (20 U/ml). At the end of each experiment, the free Ca2+ concentration was calibrated with Ca2+ ionomycin (5 µM) and then by addition of EGTA/Tris-HCl (60 mM, pH 10.5). The free Ca2+ concentration was calculated according to formulas in Thomas et al. (32)
.
Intact cell imaging and microinjection
PSMC were plated onto a polylysine-coated glass coverslip, which formed the bottom of a microincubation culture chamber (MS 200D, Medical Systems Corporation, NY), and loaded with Fura-2 by incubation with 1 µM of its acetoxymethyl ester for 40 min at room temperature. The chamber was mounted on the stage of an inverted epifluorescence microscope (Axiovert S 100, Zeiss) supported on a vibration-isolated table (Newport, Newbury, UK). Cells were viewed through a 40x, 1.3 numerical aperture, oil immersion objective lens (Fluar 40, Zeiss). Fura-2 was excited alternately at 340 and 380 nm; the resulting fluorescence at 510 nm was detected with a CCD camera (Orca, Hamamatsu). Images were captured and processed with the software OpenLab (Improvision, Covertry, UK). A background subtraction was applied before calculating the ratio image. Fura-2-loaded cells were pressure microinjected (Microinjector 5242, Eppendorf) with micropipettes pulled from borosilicate capillary tubes (1.2 mm ODx0.94 mm ID) on a horizontal puller (P-87, Sutter Instruments, Novato, CA). The injection buffer contained 150 mM KCl and 10 mM HEPES, pH 7.2 (KOH) as well as 10 mM 5/6-carboxyfluorescein, which served as a tracer to confirm the injection and label the injected cells. Carboxyfluorescein was excited at 480 nm; the resulting fluorescence was long-pass filtered (515 nm) and detected with a CCD camera.
Scanning electron microscopy
Testes from 2-month-old rats were decapsulated and digested under gentle shaking at room temperature in MEM containing 1 mg/ml collagenase. After dispersion of the interstitium, the tubular mass was rinsed in MEM, then stretches of tubules were dissected with sharp needles and carefully transferred to 35 mm culture dishes in 300 µl of medium. The tubules were incubated for 10 h at 32°C in a humidified chamber under an atmosphere containing 5% CO2. At the end of the incubation, the medium was replaced by 600 µl of medium to be tested at different times. Samples were fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, dehydrated and critical point dried in ethanol, coated with gold, and viewed in a Hitachi S-570 scanning electron microscope. PSMC in the tissue were permeabilized to allow cADPR access to their intracellular sites of action.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Competition experiments to determine binding specificity showed that the fluorescence signal was reduced to undetectable levels by treating rat PSMC with a combination of Bodipy ryanodine + excess unlabeled ryanodine (250 µM) (Fig. 1B
).
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Expression of ryanodine and IP3 receptors by Western blot analysis
Immunoblot analysis was used to test for the presence of RyRs isoforms in PSMC lysates. Blots probed with RyR2 antibody showed a single band of immunoreactivity with an apparent molecular mass of 565 kDa in microsomes prepared by PSMC. A single band of immunoreactivity with the same molecular mass was identified in lysates from cardiac muscle, which is known to heavily express RyR2 (Fig. 2
A). In contrast, blots probed with antibody against RyR1 or RyR3 showed no immunoreactivity in PSMC.
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To characterize the expression of each subtype of IP3Rs, immunoblot analysis was carried out on PSMC lysates. Three immunoreactive bands were obtained by antibodies specific for type I, II, III receptors, respectively (Fig. 2B
). These bands comigrated with those obtained using authentic type I, II, III standards (not shown).
Cytosolic Ca2+ signals mediated by cADPR
In permeabilized PSMC, microfluorimetric analysis demonstrated that stimulation with cADPR (10 µM) induces Ca2+ release from intracellular stores. Permeabilized cells became progressively less sensitive to subsequent additions of cADPR, suggesting that desensitization of a cADPR-sensitive calcium release mechanism occurs (Fig. 3
A). However, subsequent application of IP3 was still able to induce a large Ca2+ release. Similarly, sequential additions of IP3 showing progressively reduced Ca2+ release (Fig. 3B
) indicate homologous desensitization of IP3Rs. Likewise, PSMC desensitized to IP3 could still fully respond to stimulation with cADPR. These data show the presence of two separate Ca2+ release mechanisms, each selectively activated by IP3 and cADPR, respectively.
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To further investigate the specificity of the response to cADPR, we used 8Br-cADPR, a selective antagonist of cADPR-mediated Ca2+ release that has been found to antagonize the Ca2+-releasing effect of cADPR. As shown in Fig. 4
A, 8Br-cADPR (50 µM) blocked the cADPR-induced Ca2+ release, whereas IP3-induced Ca2+ release remained unchanged. In contrast, heparin (10 µg/ml), a competitive antagonist of IP3 receptor, did not affect cADPR-induced Ca2+ release in PSMC (Fig. 4B
), strongly suggesting that cADPR does not release Ca2+ via IP3 receptors. The quantitative effects of the different treatments on [Ca2+]i increments are statistically compared in Fig. 4C
.
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We also tested the effects of ryanodine on the Ca2+ responses elicited by cADPR. In general, ryanodine has complex effects on RyRs, since low concentrations tend to open the channels and higher concentrations are more likely to evoke closure. When PSMC were preloaded and desensitized by sequential stimulations with low ryanodine (5 µM), a subsequent application of cADPR was ineffective in eliciting any change (Fig. 5
). These results indicate that the cADPR-induced changes are mediated by RyR Ca2+ release channels. To estimate the potential involvement of cADPR in calcium mobilization mediated through the ETB receptor in the ET-1-induced contraction, we tested the effect of 8Br-cADPR on the response to the selective agonist of ETB receptor, IRL1620 (100 nM). We confirmed that rapid changes in intracellular calcium levels occur after stimulation with either ET-1 (100 nM) or IRL1620 (100 nM) in permeabilized PSMC (Fig. 6
A), suggesting that coupling to signal transduction mechanisms is preserved. Under these conditions, 8Br-cADPR blocked the Ca2+ response to a subsequent exposure to IRL1620 and to ET-1 (Fig. 6B
).
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Upon pretreatment with heparin (10 µg/ml), PSMC exposed to 100 nM of IRL1620 were still responsive; these results clearly demonstrated that Ca2+ signaling via ETB receptor is not dependent on IP3Rs. When cells were desensitized to repetitive additions of IRL1620, ET-1 could still elicit Ca2+-responses, albeit lower than controls (Fig. 6C
). Our previous studies demonstrated that selective stimulation of ETA receptor with a combination of the ETB receptor antagonist BQ-788 and the general ET receptor agonist ET-1 induced a rapid transient Ca2+ release that was also observed in the presence of a phospholipase C (PLC) blocker, which prevents IP3 production (23)
. In the present experiments, we found that stimulation of ETA receptors by ET-1 in the presence of BQ-788 (10 µM) was abolished by 50 µM 8Br-cADPR. This suggests that although ETA receptors are coupled to IP3 production, Ca2+ mobilization mediated by this receptor subtype has an absolute requirement for cADPR-induced Ca2+ release (Fig. 6D
). We observed that after selective stimulation of ETA receptor by ET-1 in the presence of BQ-788, the cells were still responsive to ET-1 even though IP3Rs had been blocked by heparin (Fig. 6E
). In agreement with Ca2+ measurement using permeabilized PSMC, microinjection of 8NH2-cADPR completely abolished subsequent stimulation of Ca2+ signaling via ETA and ETB receptors, indicating that a functional cADPR Ca2+ release system is essential for PSMC Ca2+ signaling (Fig. 7
). These results indicate that cADPR plays an obligatory role for Ca2+ signaling by ETA and ETB receptors.
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Seminiferous tubule contractility mediated by cADPR
In the adult testis, PSMC appear to be arranged in a continuous monolayer of epithelioid polygonal cells, particularly flat and wide, and with an apparent bulging central nucleus. Figure 8
A shows the scanning electron micrographs of a control sample of freshly isolated seminiferous tubules from adult rats. After addition of 100 nM ET-1, we observed a dramatic contraction of PSMC, displaying enhanced bulging of the central area and reduced distance between cell centers in most areas (Fig. 8B
). When we stimulated permeabilized seminiferous tubules with cADPR (10 µM), we observed a contractile response of PSMC comparable to that observed with ET-1 stimulation, although lower (Fig. 8C
). After prior exposure of a seminiferous tubule to 8Br-cADPR (50 µM), we instead observed that cADPR-mediated contractile response was inhibited (Fig. 8D
). In accordance with Ca2+ release experiments, we also found that pretreatment with 8Br-cADPR inhibited the contractile effect of ET-1 (Fig. 8E
). Taken together, these data demonstrate that cADPR is involved in regulating ET-mediated contraction of seminiferous tubule.
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ADP-ribosyl-cyclase activity in isolated plasma membranes
Measurement of ADP-ribosyl cyclase activity is a key assay for cADPR production. To investigate ADP-ribosyl cyclase activity in PSMC isolated membranes, we used a well-characterized NGD+ assay first described by Graeff et al. (33)
, which depends on the cyclization of NGD+, an NAD+ surrogate, to its hydrolysis-resistant fluorescent derivative, cGDPR. This is important since many ADP-ribosyl cyclases, including CD38, are bifunctional enzymes expressing ADP-ribosyl cyclase and cADPR hydrolase activity. CD38, the best-characterized protein involved in mammalian cell cADPR metabolism, thus cyclizes NAD+ to cADPR but also hydrolyses cADPR to ADPR. In contrast, cGDPR is nonhydrolizable, so its measurement as a single reaction product represents a distinct advantage over measuring cADPR. Membranes (100 µg protein/ml) from PSMC were incubated with 40 µM NGD+ for 20 min and fluorescence was monitored. The fluorescent intensity was converted to cGDPR concentration by calibrating with authentic cGDPR standards. Our results clearly show significant ADP-ribosyl cyclase activity in plasma membranes from PSMC (Fig. 9
). We found that the antagonistic -CD38 antibody inhibited cGDPR formation, indicating that the cyclase activity detected probably was attributable to catalytic function of membrane-bound CD38. Preimmune mouse IgG2A failed to attenuate cGDPR formation. cGDPR production was also inhibited competitively by NAD+ (400 µM), indicating that NGD+ and NAD+ likely bind the same catalytic site on the CD38-like molecule. Most important, in membranes derived from ET-1 and IRL1620 pretreated intact PSMC, a consistent increase in cGDPR production was detected over those obtained from unstimulated cells. These data indicate that intracellular ET-induced Ca2+ release is regulated by cADPR formation through activation of the CD38/ADP-ribosyl cyclase pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Seminiferous tubule contractility is fundamental for sperm progression toward the rete testis and its control therefore represents a key regulatory process in male fertility. We have investigated the contractile response of PSMC to ET-1 in cell culture and tubular explants and analyzed the Ca2+ signaling mechanisms involved. We previously reported that treatment of PSMC in culture with ET-1 resulted in marked increases in intracellular Ca2+ concentration, mediated in part by activation of the phosphoinositide pathway (23
, 24)
. Moreover, we demonstrated that two subtypes of receptors account for the actions of ET-1 on contractile activity of seminiferous tubule: 1) an ETA receptor coupled to PLC, IP3 production, and Ca2+ mobilization; and 2) an ETB receptor that induces mobilization of a thapsigargin-sensitive intracellular Ca2+ pool independent of IP3-mediated Ca2+ signaling.
The aim of the present study was to characterize the signaling pathway activated through the ETB receptor and to investigate possible cross-talk between ETA and ETB receptor-mediated calcium signaling pathways in this experimental model of smooth muscle cells. We focused first on the possibility that cADPR acts as trigger of Ca2+ release via ryanodine-sensitive, but IP3-insensitive, Ca2+ release channels. We have shown that in addition to IP3, cADPR can mobilize Ca2+ from the internal stores. In agreement with the hypothesis that cADPR releases Ca2+ via ryanodine receptors, we found that cADPR responses were blocked by ryanodine at high concentrations and by 8Br-cADPR, but not by heparin. IP3 and cADPR display homologous but not heterologous desensitization in these cells. It is likely that ryanodine receptor 2 is responsible for conferring the sensitivity of PSMC to cADPR, since it was the only subtype of RyR we identified in this cell type, though different IP3 receptor isoforms were detectable in these cells. We have also shown that ETB receptor activation increases cADPR levels and that 8Br-cADPR blocks the Ca2+ response to a subsequent exposure to IRL1620 and ET-1, the latter needed for ETA receptor response. Accordingly, pretreatment of PSMC with a combination of BQ-788, an ETB receptor antagonist, and 8Br-cADPR completely abolished the ability of the cells to respond to ET-1. This result is in accordance with contractility experiments showing that the ET-1 mechanical response is also mediated by cADPR. In fact, we examined the causal relation and biological significance of the cADPR-calcium signaling pathway in the contraction of PSMC. In previous studies we reported that BQ-123 and BQ-788 (ETA and ETB selective antagonists, respectively) completely inhibited ET-1-induced contraction only when in combination (23)
. From calcium measurements, we observed that 8Br-cADPR inhibits the actions of IRL1620 and of ET-1. Our data provide the first evidence for the involvement of cADPR via an ETB receptor-dependent Ca2+ increase in regulation of ET-mediated contraction, as supported by the finding that 8Br-cADPR strongly inhibited the contractile effect of ET-1 in the seminiferous tubule. The present work demonstrates that the effect of ET-1 on PSMC is mediated via two subtypes of ET receptors that produce Ca2+ signals mediated by cADPR. We also demonstrate that endothelin responses are mediated by the concerted action of cADPR and IP3 on ryanodine receptors and IP3Rs, respectively. When the cADPR/Ca2+ release system was inhibited by pretreatment with 8Br-cADPR or 8NH2-cADPR, the selective stimulation of ETB receptors failed to elicit Ca2+ signaling. Subsequent stimulation of the same cells with ET-1 (acting through ETA and ETB receptors) was surprisingly ineffective, indicating a hierarchical organization of calcium signaling in which cADPR appears to act in temporal sequence, providing the calcium trigger needed for the IP3 Ca2+ release system. ETA signals through IP3, which may be amplified by cADPR, whereas ETB signals through cADPR alone. A similar amplification effect has been described for T lymphocytes after stimulation of the T cell receptor/CD3 (34)
and in pancreatic acinar cells stimulated with acetylcholine and cholecystokinin (35)
. The intriguing question of why cells possess two different Ca2+ signaling mechanisms converging for ET-mediated contraction remains to be explored. Study of the potential functional role of cADPR-mediated Ca2+ signaling could be of considerable value in characterizing specific control mechanism of contractile response by ET-1; in fact, actin filaments in PSMC are arranged to form an orthogonal meshwork (22)
. It is possible that in each cell, either direction of contraction could be operated through either ETA or ETB stimulation via a different spatio-temporal regulation of intracellular signaling pathways.
In this model, convincing evidence suggests that two Ca2+ signaling pathways, cADPR and IP3-sensitive, regulate temporal patterns of Ca2+ release elicited by ET-1 stimulation in PSMC. In summary, these results indicate a novel and complex interplay of Ca2+ signaling pathways for ET-induced smooth muscle contraction.
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ACKNOWLEDGMENTS |
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Received for publication October 15, 2001.
Revision received January 14, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50 µM. After recovery from the injection (
