Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Beatrice A. Williams,1 Caiqiong Liu,1 Ling DeYoung,2 Gerald B. Brock,2,3 and Stephen M. Sims1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca2+ concentration ([Ca2+]i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca2+]i in the presence and absence of extracellular Ca2+, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca2+]i, the PE-induced rise of [Ca2+]i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca2+]i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca2+ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca2+ release from intracellular stores. Reduction of intracellular Ca2+ release may contribute to relaxation of the corpus cavernosum, leading to erection.

calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor


THE TONE OF VASCULAR SMOOTH MUSCLE in the corpus cavernosum regulates penile tumescence: tonic contraction maintains the flaccid state, and relaxation leads to erection. Contractility of penile smooth muscle cells (SMCs) is modulated by various neurotransmitters and locally produced vasoactive substances. Considerable evidence suggests that norepinephrine is primarily responsible for tonic contraction of corpus cavernosum SMCs through activation of {alpha}-adrenergic receptors (1). Agonist binding to {alpha}-adrenergic receptors activates a G protein-coupled pathway increasing intracellular inositol 1,4,5-trisphosphate (IP3) and activation of IP3 receptors, followed by Ca2+ release (3).

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.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Rat cell isolation. Male Sprague-Dawley rats weighing 300–550 g were killed by injection of euthanyl (400 mg/kg ip). The penis was removed, and the smooth muscle tissue was isolated from the crura and dissociated using (in mg/ml) 1 papain, 2.4 BSA, 0.18 1,4-dithio-L-threitol, and 1.2 Sigma blend collagenase type F. Tissues were placed in a gently shaking water bath at 31°C for 30–60 min and dispersed by trituration for immediate use or stored overnight at 4°C. The following day, tissues were warmed to room temperature for 30 min and then dispersed. All cells were studied within 8 h. Procedures for animal handling were carried out in accordance with the guidelines of the Canadian Council on Animal Care. Experiments were carried out in rat corpus cavernosum cells unless specifically stated otherwise.

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 20–40 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 1–3 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PE induces release of Ca2+ from intracellular stores. Enzymatic dissociation of corpus cavernosum yielded spindle-shaped SMCs, which contracted reversibly in response to the {alpha}1-adrenergic agonist PE (see Fig. 8). [Ca2+]i was monitored in rat corpus cavernosum SMCs, and PE was applied at 5-min intervals to allow for recovery between stimulations. In bath solution containing 1 mM Ca2+, {alpha}1-adrenergic stimulation with PE (10 µM) induced a rapid and transient elevation in [Ca2+]i by 382 ± 19 nM (n = 109) from a basal level of 126 ± 3 nM (n = 109). The peak did not decrease significantly following repetitive stimulation; the fifth stimulation was 83 ± 20% (n = 5) of the first (Fig. 1, A and C). In Ca2+-free solution, the response to PE persisted, although there was gradual decline in the peak response, consistent with depletion of intracellular stores. The third stimulation in Ca2+-free solution was 46 ± 10% of control (n = 5, Fig. 1, B and C).



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Fig. 8. PE-induced contraction is reduced by treatment with SNAP and sildenafil. A: sequence of video frames illustrating reversible contraction in single cells. The relaxed cell (control) contracted by 33% of its initial length after application of 1 µM PE, with recovery evident 5 min later (wash). In contrast, after application of SNAP and sildenafil, 1 µM PE caused only a minor shortening of 8%. The second row of video frames is a continuation of the sequence, showing that after a recovery period, the cell was again responsive to PE. B: histogram of PE-induced contraction, measured as a reduction of cell length, showing that SNAP with sildenafil reduced the PE-induced contraction (n = 5). *P < 0.05.

 


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Fig. 1. Phenylephrine (PE) causes release of Ca2+ from intracellular stores in rat corpus cavernosum cells. A: there was no significant decline in the response to PE (100 µM) upon repetitive stimulation in solution containing 1 mM Ca2+. PE application is indicated by horizontal bars below the Ca2+ trace (n = 5–6 cells measured). B: response to PE gradually declined in Ca2+-free solution, consistent with a depletion of intracellular stores, and was partially reversible upon a return to 1 mM Ca2+ solution (n = 5–7). C: summarized data from A and B. [Ca2+]i, intracellular Ca2+ concentration. *P < 0.05 vs. stimulation 1.

 
We investigated the contribution of Ca2+ stores by using cyclopiazonic acid (CPA; 10 µM) to block the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Addition of CPA in Ca2+-free solution to eliminate store-operated Ca2+ influx led to an increase in basal [Ca2+]i and a rapid reduction in response to PE (10 µM), indicative of store depletion (n = 24, data not shown). Inhibition of SERCA by CPA was accompanied by a marked reduction in the rate of decline of [Ca2+]i. When the rate of [Ca2+]i decline was measured (31), the maximal slope of the decay under control conditions was 5 ± 2 nM/s and with CPA was 1.2 ± 0.5 nM/s (n = 7), indicating that SERCA contributes to the restoration of basal Ca2+ levels.

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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Fig. 2. A: S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (both 10 µM) had no effect on basal Ca2+, but the subsequent PE-induced transient was reversibly reduced (10 µM PE). B: there was no decline in the response to PE after vehicle treatment, whereas there was a clear reduction in response to PE after SNAP and sildenafil in the same cell. C: transients from B, indicated as i, ii, and iii, are superimposed to compare responses. D: histogram showing that the Ca2+ transient was significantly reduced by SNAP and sildenafil (n = 21). Sil, sildenafil citrate (Viagra). *P < 0.05.

 
NO donors alone do not attenuate the PE-induced transient. NO has been shown by many groups to relax corpus cavernosum tissue (8, 16, 28). We therefore tested the effect of SNAP and SNP in the absence of sildenafil citrate on our isolated cells. Surprisingly, application of the NO donors SNAP (10 µM, n = 6) and SNP (100 µM, n = 6) without sildenafil had no significant effect on basal Ca2+ levels or PE-induced Ca2+ transients (Fig. 3). To confirm these results, we examined the effect of SNAP in experiments in which we first established that the cell was responsive to SNAP and sildenafil. The PE transient following SNAP alone (10 µM) was 96 ± 5% of control, compared with 72 ± 10% of control following SNAP and sildenafil (both 10 µM; n = 17, Fig. 3). Increasing the concentration of SNAP to 250 µM also had no significant effect (n = 5, Fig. 3C). One possible explanation for these results is that the cGMP generated in our isolated cell model is degraded too quickly by PDE5 to effect a change, in line with previous reports on human corpus cavernosum (21), as considered in DISCUSSION.



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Fig. 3. Nitric oxide (NO) donors alone have no effect on the height of the PE-induced Ca2+ transient. A: application of 10 µM SNAP alone had no effect on the subsequent Ca2+ transient. Data are representative of 6 cells. B: SNAP with sildenafil (both 10 µM) reduced the Ca2+ transient, whereas in the same cell, 10 µM SNAP alone had no effect. Data are representative of 17 cells. C: summary of experiments shown in B, as well as individual experiments with 250 µM SNAP and 100 µM sodium nitroprusside (SNP). Numbers in parentheses indicate number of cells measured. *P < 0.05.

 
Human corpus cavernosum cells. The effect of NO donors and sildenafil was examined on freshly isolated human corpus cavernosum SMCs. Following the protocol developed for rat cells, we applied PE at 5-min intervals in the presence and absence of extracellular Ca2+. The response to PE persisted in Ca2+-free solution, consistent with release from intracellular stores (n = 3, Fig. 4A). Furthermore, SNAP and sildenafil attenuated the PE-induced Ca2+ transient by 55 ± 15% without affecting basal [Ca2+]i (n = 9, P < 0.05, Fig. 4). Thus the PE-induced transient is primarily due to Ca2+ release from intracellular stores in both human and rat SMCs. The average elevation in Ca2+ in response to PE in human cells was 380 ± 77 nM (n = 12), which was not significantly different from that in rat cells. Furthermore, both human and rat SMCs respond in a similar manner to NO donors by decreasing release of Ca2+ from intracellular stores.



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Fig. 4. PE-induced Ca2+ release in human corpus cavernosum. A: repetitive stimulation with 10 µM PE in Ca2+-free solution continued to elicit Ca2+ transients, indicating release from intracellular stores. Data are representative of 3 cells. B: SNAP with sildenafil (both 10 µM) caused no change in basal Ca2+ but attenuated the transient. C: histogram showing that SNAP and sildenafil reduced Ca2+ transients in human corpus cavernosum cells (n = 9). *P < 0.05.

 
NO and sildenafil regulate release from intracellular Ca2+ stores. To characterize the mechanism of inhibition, we quantified the rates of rise and decay of the PE-induced transient. To minimize cell-to-cell variability, we applied SNAP and sildenafil as well as SNAP alone to the same cell. Under control conditions, the average time to peak [Ca2+]i was significantly increased by 144 ± 39% after treatment with SNAP and sildenafil (n = 17, P < 0.01). In contrast, the time to peak was not different after SNAP alone (Fig. 5A). The rise to peak after PE application was also significantly delayed in the presence of another NO donor, SNP, with sildenafil (n = 16, data not shown).



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Fig. 5. A: PE-induced Ca2+ transients are superimposed, illustrating that SNAP and sildenafil (both 10 µM; shaded line) reduced the peak compared with control (solid line) and increased time to peak [Ca2+]i (10 µM PE, 1 mM Ca2+ in bath). B: histogram of time to peak showing a significant increase after application of SNAP with sildenafil but no effect of SNAP alone applied to the same cell (n = 17). *P < 0.001. C: the decay phase of the transients was fit with a monoexponential (dashed line). Decay time constants from the fit of control (solid line) and SNAP with sildenafil (shaded line) are shown in a representative trace. Average values in D reveal no significant differences (n = 16). E: SNAP with sildenafil (shaded line) reduced the peak and increased the time to peak Ca2+ compared with control (solid line) in Ca2+-free solution (1 µM PE) similar to results in A, confirming that SNAP with sildenafil alters the release of Ca2+ from stores. F: histogram showing that time to peak was increased by SNAP and sildenafil in zero-Ca2+ solution (n = 10). *P < 0.001. Upon switching from 1 mM Ca2+ solution to zero-Ca2+ solution (control PE vs. PE), there was no significant change.

 
NO is reported to activate SERCA in other vascular muscles, reducing the peak and increasing the rate of decay of the Ca2+ transient (9, 19). In our experiments, the rapid decay phase began 2.5 ± 0.2 s (n = 112) from the end of PE application and was well fit with a single exponential (Fig. 5C). Neither SNAP alone nor SNAP with sildenafil had any significant effect on the rate of decay (n = 16, Fig. 5D). Our data therefore are not consistent with NO activating SERCA in corpus cavernosum.

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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Fig. 6. A: the effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) on the reduction of the PE-induced transient by SNAP and sildenafil was examined in experiments in which we first established that the cell was responsive to SNAP and sildenafil (both 10 µM). In the representative experiment, SNAP and sildenafil reduced the PE-induced transient by 84% of control, whereas with ODQ, SNAP and sildenafil reduced the transient by only 37%. B: responses in A, indicated by i, ii, iii, and iv, are superimposed to show that ODQ diminished the response to SNAP and sildenafil. C: rise of [Ca2+]i as a percentage of the preceding control (PE alone). ODQ significantly inhibited the effect of SNAP and sildenafil on the peak (n = 7). *P < 0.05. ODQ on its own had no effect (n = 11).

 
The effect of membrane-permeant cGMP analogs on PE-induced transients was then examined. Because the results from NO donors suggested that PDE5 was very active, sildenafil was included with the cGMP analogs to exclude any possibility of degradation. Application of either 8-Br-cGMP (n = 19, Fig. 7A) or 8-pCPT-cGMP (n = 11) in the presence of sildenafil had no significant effect on basal Ca2+ or PE-induced transients. We next investigated whether application of YC-1 (100 µM), a nitric oxide-independent activator of sGC, could mimic the effect of NO donors. YC-1 with sildenafil caused a slight rise in basal Ca2+ but had no significant effect on PE-induced transients (n = 13, Fig. 7A). These findings demonstrate that neither NO nor cGMP alone was sufficient to reduce the PE-induced Ca2+-transient.



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Fig. 7. A: histograms showing that the PE-induced Ca2+ transient was unaffected by 2 mM 8-bromo-cGMP (8-Br-cGMP) with 10 µM sildenafil (n = 19) or by 100 µM 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) with 10 µM sildenafil (10 µM PE; n = 13). B: representative trace showing the reduction in the PE-induced transient by 10 µM SNAP with 2 mM 8-Br-cGMP. C: histograms showing that the PE-induced transient was reduced by 10 µM SNAP with 2 mM 8-Br-cGMP (n = 22) and by SNAP with 30 µM YC-1 (n = 5). *P < 0.05 vs. PE.

 
The ineffectiveness of cGMP analogs to mimic SNAP and sildenafil suggested that both NO and cGMP were necessary to mediate the reduction of Ca2+ release. We confirmed that SNAP with 8-Br-cGMP but without sildenafil significantly reduced the rise of Ca2+, although this effect was not readily reversible. After application of SNAP and 8-Br-cGMP, the PE-induced transient was reduced by 42 ± 9% of control (1 µM PE; n = 22, P < 0.01, Fig. 7B). This was not significantly different from the reduction by SNAP and sildenafil (54 ± 8% reduction, 1 µM PE; n = 24). We also applied 10 µM SNAP with 30 µM YC-1, which also significantly reduced the PE-induced transient (by 96 ± 8%; n = 5, P < 0.01, Fig. 7C).

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) 15–20 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.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NO has a variety of cellular effects, including direct modulation of ion channels and activation of sGC (4, 26). In smooth muscle, NO is believed to mediate relaxation primarily by activation of sGC, increasing intracellular cGMP level (26). We have demonstrated that NO donors in the presence of sildenafil dramatically reduce PE-induced Ca2+ release from intracellular stores. This was reflected functionally as a reduction in PE-induced contraction. We suggest that relaxation of the corpus cavernosum, leading to erection, may involve downregulation of agonist-induced contraction by inhibition of Ca2+ release from stores.

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 {alpha}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.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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We gratefully acknowledge support of the Heart and Stroke Foundation of Ontario (NA4944) and The Canadian Institutes of Health Research (MOP 10019) (to S. M. Sims) and The Canadian Male Sexual Health Council (to G. B. Brock) for support of these studies.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. A. Williams, Dept. of Physiology and Pharmacology, The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: bwilli8{at}uwo.ca)

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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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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