1Department of Physiology and Pharmacology and 3Department of Surgery, The University of Western Ontario, and 2Lawson Health Research Institute, London, Ontario, Canada
Submitted 27 September 2004 ; accepted in final form 6 November 2004
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ABSTRACT |
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calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor
Nitric oxide (NO), which is released by both endothelial cells and nonadrenergic, noncholinergic nerves, leads to relaxation of corpus cavernosum and is necessary for erection (6, 28). NO has a variety of cellular effects including direct effects on ion channels and activation of soluble guanylyl cyclase (sGC), which converts GTP to cGMP (4, 26). cGMP in turn activates cGMP-dependent ion channels, cGMP-dependent protein kinase (PKG), and cGMP-regulated phosphodiesterases (PDEs). It has been suggested that, in vascular smooth muscle, most of the cGMP effects are mediated by PKG, because in PKG-1-deficient mice, aortic and corpus cavernosum smooth muscles fail to relax upon activation of the NO/cGMP pathway (16, 27). PKG has a variety of effects in smooth muscle, including inhibition of the IP3 receptor (30) and regulation of the Ca2+ sensitivity of contractile proteins (23). Specific PKG mechanisms contributing to the regulation of SMC tone almost certainly vary from tissue to tissue, and the identity of specific targets involved in relaxation of corpus cavernosum SMCs remains uncertain.
PDE types 2, 3, 5, and 11 are expressed in corpus cavernosum SMCs, although the main PDE activity is due to PDE5, which hydrolyzes cGMP (5, 10, 25). Sildenafil citrate (Viagra) acts by enhancing NO-mediated smooth muscle relaxation by competitive inhibition of PDE5, thereby maintaining elevated intracellular cGMP levels (2, 5).
We investigated the effect of NO on adrenergically stimulated changes in intracellular Ca2+ concentration ([Ca2+]i) in freshly isolated corpus cavernosum SMCs and found that NO and cGMP act synergistically to reduce Ca2+ release from intracellular stores. We propose that dynamic regulation of Ca2+ release contributes to relaxation of the corpus cavernosum, which leads to penile erection.
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MATERIALS AND METHODS |
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Human tissue.
Tissue collection was carried out in accordance with guidelines of the University Review Board for Research Involving Human Subjects and conformed to the Helsinki Declaration. Fragments of corpus cavernosum were retrieved during reconstructive surgery and implant of penile prostheses. Tissue was obtained from patients with neurological damage following surgery for cancer or from patients with Peyronie's disease in which the corpus tissue is unaffected. Two samples were obtained from patients with diabetes. No differences in the responses to phenylephrine (PE) or S-nitroso-N-acetylpenicillamine (SNAP) and sildenafil were observed among the cells from different sources. Segments of cavernosal tissue (1 mm2) were dissociated as described previously (20).
Measurement of [Ca2+]i. Cells were loaded with 0.2 µM fura-2 acetoxymethyl ester (AM) for 2040 min at room temperature and allowed to settle on a glass perfusion chamber. The chamber was mounted on a Nikon inverted microscope and perfused with solution at 13 ml/min at room temperature. Cells were relaxed and contracted reversibly upon stimulation with phenylephrine. Cells were illuminated with alternating 345- and 380-nm light using a Deltascan system (Photon Technology International, London, ON, Canada), with the 510-nm emission detected using a photometer. [Ca2+]i was calibrated using the methods of Grynkiewicz et al. (14).
Solutions and chemicals. Solutions used for tissue retrieval and dissociation have been described previously (20). To minimize changes in cell membrane potential and reduce Ca2+ influx, we used a bath solution for [Ca2+]i measurements containing (in mM) 135 KCl, 20 HEPES, 10 D-glucose, 1 CaCl2, and 1 MgCl2 (pH 7.4 with KOH). For zero-Ca2+ bath solution, Ca2+ was replaced with 0.5 mM EGTA. Similar basal [Ca2+]i and Ca2+ transients were observed for cells bathed in Na-HEPES solution (NaCl replaced 130 mM KCl). Sodium nitroprusside (SNP) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) were obtained from Sigma (St. Louis, MO); SNAP, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and 8-bromo-cGMP (8-Br-cGMP) were from Calbiochem (San Diego, CA); and chemicals for the bath solutions were from BDH Limited (Toronto, ON, Canada). Sildenafil citrate was from Pfizer. Fura-2 AM (Molecular Probes, Eugene, OR) was prepared in dimethyl sulfoxide. PE was applied for 10 s at 10 µM unless otherwise stated. Mediators were applied focally to cells by pneumatic ejection from a micropipette attached to a Picospritzer (General Valve, Fairfield, NJ) while the bath was constantly perfused.
Statistical analysis. Values are provided as means ± SE, with error bars in the figures representing SE and with n indicating the number of cells studied. For each treatment group, cells were obtained from at least three rats. Statistical comparisons were made using either repeated-measures ANOVA with the Tukey-Kramer post hoc analysis or paired Student's t-test. P < 0.05 indicates significance.
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RESULTS |
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Attenuation of the PE-induced [Ca2+]i transient by NO and sildenafil. NO mediates relaxation of corporal smooth muscle necessary for erection (6, 28). Sildenafil citrate enhances NO-mediated smooth muscle relaxation by inhibiting hydrolysis of cGMP by PDE5 (5). We therefore examined effects of NO and sildenafil on intracellular Ca2+ levels. The NO donor SNAP (10 µM), applied with sildenafil (10 µM) for 3 min, had no effect on basal Ca2+ levels. In contrast, SNAP and sildenafil significantly reduced the PE-induced Ca2+ transient. From a resting level of 98 ± 5 nM, PE elicited a rise of 350 ± 49 nM, compared with 252 ± 43 nM after SNAP and sildenafil application (27 ± 7% reduction; n = 21, P < 0.005). Recovery to 356 ± 48 nM was evident within 5 min (n = 21, Fig. 2). SNAP and sildenafil markedly delayed the rise of Ca2+ (Fig. 2C). By contrast, vehicle treatment had no effect on the PE-induced transients (Fig. 2B). In addition, another NO donor, SNP (100 µM), applied with sildenafil (10 µM), also significantly attenuated the Ca2+ transient, indicating a common effect of SNAP and SNP. The PE-induced peak under control conditions was 298 ± 42 nM compared with 166 ± 48 nM after SNP and sildenafil application and recovered to 286 ± 49 nM (46 ± 8% reduction; n = 16, P < 0.05).
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To examine whether SNAP and sildenafil regulate Ca2+ influx from the bath or Ca2+ release from intracellular stores, we repeated experiments in Ca2+-free solution. Results were essentially the same as those obtained in 1 mM Ca2+ bath solution. SNAP and sildenafil reduced the PE-induced transient by 79 ± 8% (1 µM PE; n = 10, P < 0.01) of control measured in zero-Ca2+ solution (Fig. 5E). By comparison, in Ca2+-containing solution, SNAP and sildenafil reduced the PE-induced transient by 54 ± 8% (1 µM PE; n = 24, P < 0.01). The time to peak in zero-Ca2+ solution was increased by 200 ± 46% after SNAP and sildenafil (n = 10, P < 0.01, Fig. 5F). Thus, even in the absence of extracellular Ca2+, SNAP and sildenafil inhibited the Ca2+ transient, indicating regulation of Ca2+ release from intracellular stores.
Role of cGMP in attenuation of PE-induced [Ca2+]i transient. To understand the signaling cascade mediating the reduction of agonist-induced Ca2+ release, we applied sildenafil, cGMP analogs with sildenafil, and an sGC inhibitor to the cells. As expected, sildenafil alone had no effect on basal Ca2+ or on the PE-induced transient (n = 10, data not shown). To verify the involvement of sGC, we tested the effect of ODQ, a selective sGC inhibitor. SNAP and sildenafil reduced the PE-induced transient (1 µM PE) by 63 ± 12% of control, whereas in the presence of 10 µM ODQ in the same cells, SNAP and sildenafil reduced the transient by only 35 ± 15% of control (n = 7, P < 0.05, Fig. 6). To control for nonspecific effects, we tested ODQ in the absence of SNAP and sildenafil and found that ODQ alone did not affect the transient (n = 11).
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Attenuation of PE-induced contraction by NO and sildenafil. The functional significance of attenuating the PE-induced [Ca2+]i transient was tested by monitoring contraction of single cells. PE was applied at 5-min intervals as before, and cell length was measured from video images of the cells (Fig. 8). Spindle-shaped cells contracted briskly, reducing cell length by 26 ± 6 µm (32 ± 7%; n = 5) 1520 s after application of PE. Upon washout of PE, the cells relaxed to 90 ± 3% of their original length. We attribute this degree of relaxation to a lack of restorative tension, as would occur in intact tissue. SNAP and sildenafil were applied for 3 min and had no effect on the resting cell length; the subsequent PE application led to only minimal shortening (12 ± 6 µm, 17 ± 5%; n = 5). This effect was significant and reversible (Fig. 8B). Similar experiments were performed with SNAP alone (no sildenafil), and no significant effect of SNAP was observed. Under control conditions, PE led to a contraction of 17 ± 3%, whereas after SNAP, PE led to a contraction of 14 ± 2% (n = 13). Thus the reduction of the PE-induced Ca2+ transient by SNAP and sildenafil is reflected in a reduction of the contractile response.
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DISCUSSION |
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Synergism of NO and cGMP. In other smooth muscles, NO or cGMP alone has been shown to regulate Ca2+ release. In aortic SMCs, NO inhibits vasopressin- and angiotensin-induced rise of intracellular Ca2+ level, and the effect is mimicked by the cGMP analog 8-Br-cGMP (9, 11, 15). Similarly, in tracheal SMCs, SNAP inhibits Ca2+ release induced by acetylcholine, and 8-Br-cGMP mimics the response (18). In corpus cavernosum, PDE5 has been shown to be abundant and to be the predominant PDE enzyme in this tissue (5, 10). One possible explanation for our results is that in an isolated cell preparation, the cGMP generated is degraded too quickly by PDE5 to exert its full effect. There is support for this possibility from the studies of Kim et al. (21). They did not observe any significant changes in cGMP content compared with control after treatment with 10 µM SNP alone on their cultured corpus cavernosum cells, whereas SNP with sildenafil increased cGMP levels 2.8-fold (21). Thus the difference between our results and those previously published may be due to NO producing sufficient cGMP in other SMCs, whereas in corpus cavernosum, the effect of PDE5 cannot be overcome.
Although NO has been shown by many groups to relax intact tissue from corpus cavernosum (8, 16, 28), few studies have investigated the mechanism of NO on acutely isolated corpus cavernosum cells. Tissue strips, in contrast to isolated cells, contain other cell types, including neuronal and endothelial cells (for review, see Ref. 1). It is possible that these other cell types contribute to the tissue response (1, 12). Escrig et al. (12) demonstrated that after electrical stimulation of cavernosal nerves in anesthetized rats, the rise in NO outlived the increase in intercavernosal pressure by several minutes. This finding led those authors to suggest that NO is necessary but not sufficient for the maintenance of penile erection and that some additional factor may be involved in mediating relaxation. In this regard, there are several vasodilators that act through cAMP, and interactions between cGMP- and cAMP-mediated mechanisms have been demonstrated in vascular as well as corporal smooth muscle (21, 22). Thus sildenafil may substitute for a vasodilator co-mediator not present in our isolated single-cell preparation.
Unexpectedly, we found that membrane-permeant cGMP analogs were ineffective in attenuating the PE-induced rise in Ca2+. However, we did find that 8-Br-cGMP or YC-1, when combined with an NO donor, decreased the PE-induced transient. The failure to observe reversibility of these combinations probably reflects kinetic parameters different from those of sildenafil, which is readily reversible (2, 13). Our results demonstrate an interesting feature of corpus cavernosum cells, whereby NO and cGMP act synergistically to reduce PE-induced Ca2+ release from stores.
Mechanism of NO and cGMP inhibition of Ca2+ release. Studies of PKG-1-deficient mice have revealed that a major target of cGMP in corpus cavernosum is PKG. The corpus cavernosum of these mice fails to relax upon activation of the NO/cGMP pathway, resulting in erectile dysfunction (16). Our results using the sGC inhibitor ODQ confirmed that activation of sGC is necessary for SNAP and sildenafil to reduce Ca2+ release. We suggest, therefore, that cGMP most likely acts through PKG to regulate PE-induced Ca2+ release through IP3 receptors. Both IP3 and Ca2+ modulate the release of Ca2+ through the IP3 receptor, and moreover, it has been shown that decreasing IP3 or Ca2+ levels increases the response time to the IP3 stimulus (35). Thus the increased latency to peak Ca2+ is consistent with either reduced concentration of IP3 or reduced sensitivity of the IP3 receptor to IP3 or Ca2+. Phosphorylation by PKG regulates several targets in the G protein-coupled pathway affecting the sensitivity of the IP3 receptor. Schlossman et al. (30) demonstrated that PKG phosphorylates the IP3 receptor-associated-cGMP kinase substrate IRAG, leading to a reduction in Ca2+ release.
Another possible site of action is upstream of the Ca2+ release site. Phosphorylation of a regulator of G protein signaling, RGS-2, by PKG leads to inhibition of IP3 production, and consequently, RGS-2-deficient mice develop hypertension and decreased cGMP-mediated relaxation (34). The rate of decay of IP3-mediated Ca2+ transients in these RGS-2-deficient mice were significantly slowed (17). Our finding that NO and sildenafil did not affect the rate of decay of the PE-induced Ca2+ transient would argue against the involvement of RGS-2 in mediating the effects reported in the present study in corpus cavernosum cells.
The role of NO, apart from activating sGC, in reducing the PE-induced Ca2+ transient is unclear. However, NO is known to have diverse effects on proteins, interacting with metal and thiol groups, and conceivably could directly modulate signaling molecules of the Ca2+ release pathway (33). Cohen et al. (9) suggested that NO leads to increased Ca2+ uptake via activation of SERCA because the rate of decay of the IP3-mediated Ca2+ rise in aortic SMCs was increased; however, it was not determined whether this was a direct effect of NO or occurred via cGMP. Others have shown that phosphorylation of the regulatory protein phospholamban by PKG is associated with activation of SERCA (19). Because NO and sildenafil did not affect baseline Ca2+ levels or the rate of decay of the Ca2+ transient, our data also do not support the involvement of SERCA in mediating the effects in corpus cavernosum.
Effects of NO and cGMP on PE-induced contraction. The functional significance of the NO and cGMP effect was demonstrated in single corpus cavernosum cells by measuring PE-induced contraction after application of SNAP with sildenafil. SNAP alone had no effect on PE-induced contraction, whereas SNAP with sildenafil reduced PE-induced contraction in addition to reducing PE-induced Ca2+ release. Both the concentration of intracellular Ca2+ and the sensitivity of the contractile proteins to Ca2+, called Ca2+ sensitization, regulate contraction in vascular smooth muscle. Ca2+ sensitization is a result of phosphorylation of the myosin light chain, leading to increased muscle tension for a given Ca2+ concentration (32). In this regard, RhoA/Rho-kinase and PKG both have been shown to regulate Ca2+ sensitization/desensitization in smooth muscles (23, 32, 36).
The NO/cGMP pathway has been suggested to control relaxation of corpus cavernosum by acting in two ways: by lowering intracellular Ca2+ and by inhibiting Rho-kinase (24). RhoA is highly expressed in corpus cavernosum, and several groups have shown that inhibition of Rho-kinase promotes relaxation of corpus cavernosum tissue and erection (7, 29, 36). From studies on Ca2+ sensitization of corpus cavernosum tissue, however, the role cGMP/PKG remains unclear. Wang et al. (36) demonstrated that whereas the Rho-kinase inhibitor Y-27632 caused almost complete relaxation, 8-Br-cGMP led to only partial relaxation of Ca2+-sensitized corpus cavernosum tissue. In contrast, Chuang et al. (8) found that SNP in the presence of PE increased cGMP levels and relaxed tissue strips, but without an associated decrease in myosin light chain phosphorylation. Thus inhibition of contraction of single cells by NO/cGMP could be due to regulation of several signaling pathways.
We have demonstrated that the NO/cGMP pathway indeed lowers intracellular Ca2+ release after PE stimulation. Our experiments did not address the issue of Ca2+ sensitization, and we cannot rule out the possibility that Ca2+ desensitization by NO/cGMP may be involved in reducing PE-induced contraction. However, a reduction in contraction of single cells occurred under the same conditions as a reduction in intracellular Ca2+ release, supporting the notion that the reduction in the PE-induced release of intracellular Ca2+ by NO and cGMP contributes to relaxation in corpus cavernosum.
In summary, we have shown that NO and sildenafil regulate 1-adrenergically induced Ca2+ release from intracellular stores in corpus cavernosum SMCs. This regulation appears to require synergistic actions of NO and cGMP, because neither NO donors alone nor stable cGMP analogs were effective. We suggest that the reduction in agonist-induced release of Ca2+ from stores by NO and cGMP may contribute to relaxation in corpus cavernosum.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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