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Smooth Muscle Group, Department of Physiology, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK
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(Received 25 November 2003;
accepted after revision 3 February 2004;
first published online 6 February 2004)
Corresponding author K. D. Thornbury: Smooth Muscle Group, Department of Physiology, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK. Email: k.thornbury{at}qub.ac.uk
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Introduction |
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Recently, a role for a Ca2+-activated Cl– current in generating spontaneous myogenic tone in the cavernosum was proposed (Karkanis et al. 2003). Under voltage clamp conditions, it was shown that rat and human corpus cavernosum cells fired spontaneous transient inward currents (STICs) that appeared to be mediated by the Cl– current. Under physiological (non-voltage clamped) conditions, the STICs would be expected to depolarize the cells into the range where voltage-dependent Ca2+ channels open, thus increasing Ca2+ influx. One possible mode of action of the NO–cGMP pathway may therefore be to reduce either the frequency or amplitude of the STICs. Despite this, at present there have been no reports of the effect of nitric oxide donors on STICs in the cavernosum, and an attempt to demonstrate effects of the cell-permeant cGMP anologue 8-bromo-cGMP proved equivocal, with little effect observed unless it was combined with 8-bromo-cAMP (Karkanis et al. 2003). The purpose of the present study was firstly to characterize the Cl– current in isolated rabbit corpus cavernosum cells, secondly to study the mechanism underlying STICs in these cells, and thirdly to determine the effects of an NO donor and cGMP on STICs. Although Cl– currents have not been previously demonstrated in the rabbit corpus cavernosum, we chose this preparation because the rabbit is a widely used animal model in contractile studies (Ignarro et al. 1990; Holmquist et al. 1992; Cellek & Moncada, 1997) and its cavernosum is large enough to yield a reasonable number of viable cells for study with the patch clamp technique.
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Methods |
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All procedures were carried out in accordance with current UK legislation. Male New Zealand white rabbits (>2.5 kg) were humanely killed with a lethal injection of pentobarbitone (I.V.) and the penis was removed. The tunica albugenia was opened bilaterally to expose both corpora cavernosa and these were carefully removed, placed in Hanks' Ca2+-free solution (see below) and cut in to 1 mm3 pieces. The pieces were stored in Hanks' Ca2+-free solution for 30 min before they were incubated in an enzyme medium containing (per 5 ml of Hanks' Ca2+-free solution): collagenase 15 mg (Sigma type 1a), protease 1 mg (Sigma type XXIV), BSA 10 mg (Sigma) and trypsin inhibitor 10 mg (Sigma) for 5–10 min at 37°C. They were then placed in Hanks' Ca2+-free solution and stirred for a further 5–10 min to release cells. Several drops of the cell suspension were added to Petri dishes containing Hanks' solution (100 µM Ca2+) and stored at 4°C for use within 8 h. To minimize use of animals, some experiments utilized cells that were dispersed from whole corpus cavernosum tissue that had been stored at 4°C overnight. The results from these experiments were indistinguishable from those obtained from fresh tissue.
Electrophysiological recording
Recordings were made using the amphotericin B perforated patch method (Rae et al. 1991). After gigaseals were obtained the series resistance fell over a 10–15-min period to 10–15 M
and remained stable for up to 1 h. Although amphotericin pores are cation selective, the permeability for Cl– is not negligible (Kleinberg & Finkelstein, 1984), so the reversal potential of Cl– current is determined by the bath and pipette Cl– concentrations (Horn & Marty, 1988). Where stated, reversal potential data were corrected for junction potentials of –3 mV in PSS and +2 mV in low-chloride solution according to the method described by Neher (1992). Voltage-clamp commands were delivered with an Axopatch 1D patch-clamp amplifier (Axon Instruments) and currents were recorded by means of a 12 bit AD/DA converter (Labmaster, Scientific Solutions) interfaced to an Intel computer running pCLAMP software (Axon Instruments).
Solutions and drugs
The following solutions were used (concentrations mM): Hanks' Ca2+-free solution (solution 1; for cell dispersal): 141 Na+, 5.8 K+, 130.3 Cl–, 15.5 HCO3–, 0.34 HPO42–, 0.44 H2PO4–, 10 dextrose, 2.9 sucrose, 10 Hepes (Melford) (pH adjusted to 7.4 with NaOH); PSS (solution 2): 130 Na+, 5.8 K+, 135 Cl–, 4.16 HCO3–, 0.34 HPO32–, 0.44 H2PO4–, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42–, 10 dextrose, 2.9 sucrose, 10 Hepes (pH adjusted to 7.4 with NaOH); Cs+ pipette solution (solution 3): 133 Cs+, 1 Mg2+, 135 Cl–, 0.5 EGTA (Sigma), 10 Hepes (pH adjusted to 7.2 with CsOH); low-chloride solution (solution 4): similar to solution 2 but with 86 glutamate and 49 Cl–, instead of 135 Cl–; iodide-substituted solution (solution 5): similar to solution 2 but with 135 I–, instead of 135 Cl–.
S-Nitroso-L-cysteine (nitrosocysteine) was synthesized by reacting L-cysteine with sodium nitrite under acidic conditions, as previously described (Field et al. 1978; Kowaluk & Fung, 1990). Reactant solution contained 1 N HCl (5 ml; Reidel-deHaen), MeOH (5 ml; Laboratory-Scan), 37 N H2SO4 (0.5 ml; BDH), sodium nitrite (10 mM; BDH) and L-cysteine (5 mM; BDH). In control experiments, dilutions of the reactants (containing all of the reactants except L-cysteine) equivalent to nitrosocysteine (16 µM) had no effect on the spontaneous Cl– currents.
Other drugs were used were as follows: caffeine (Sigma), anthracene-9-carboxylic acid (A9C; Sigma), niflumic acid (Sigma), 2-nitro-4-carboxyl-N,N-diphenylcarbamate (NCDC; Sigma), 2-aminoethoxydiphenyl borate (2APB; Acros), cyclopiazonic acid (CPA; Calbiochem) (3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) and nifedipine (Bayer).
During experiments the dish containing the cells was superfused with PSS (solution 2). In addition, the cell under study was continuously superfused with PSS by means of a close delivery system consisting of a pipette (tip diameter 200 µm) placed approximately 300 µm away. This could be switched, with a dead space time of around 10 s, to a solution containing a drug. All experiments were carried out at 37°C. To deliver nitrosocysteine a variation in the normal procedure was necessary. This substance is stable in acidic stock solution, but breaks down with a half-life of 
10 s (Kowaluk & Fung, 1990) in physiological saline. To deliver it to a cell firing STICs, 2 µl of 160 mM stock was rapidly mixed with 20 ml of PSS in a primed channel of the drug delivery system, thus providing a nominal final concentration of 16 µM. The actual concentration seen by the cell will be somewhat lower, depending on the time to mix the solution and the dead space time of the delivery system. We estimate that when the solution first reaches the cell the concentration will probably be less than 8 µM and this will continue to fall throughout the period of drug perfusion.
Statistics and analysis
Data are presented as the mean ±S.E.M., and statistical differences were compared using Students paired t test, taking the P < 0.05 level as significant. Throughout, n refers to the number of cells in each experimental series. In each case n was obtained from a minimum of 3 animals.
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Results |
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The majority of (80–90%) dispersed cells observed with bright field microscopy were spindle shaped, unbranched and contracted on application of caffeine or deliberate disruption of the cell membrane with a patch pipette. These were typical of smooth muscle cells described elsewhere in other tissues (Sanders, 1989). In addition to smooth muscle cells, a second less abundant (10–20%) cell type was observed. These were highly branched and failed to contract under the above conditions, resembling the interstitial cells previously observed in the rabbit urethra (Sergeant et al. 2000). However, for the purposes of the present study, we confined our recordings to typical smooth muscle cells.
When cells were held at –80 mV and subjected to depolarizing potentials ranging from –70 to +50 mV, currents typical of those shown in Fig. 1A were evoked. These consisted of a transient inward current, similar in time course to the L-type Ca2+ current, followed by a large slowly developing inward current (Fig. 1A, inset) that reversed near 0 mV to give large, non-rectifying outward currents. In contrast to Cl– currents previously reported for rabbit and sheep urethra (Cotton et al. 1997; Sergeant et al. 2000), in most cells, these slow currents gradually decayed during the 500 ms depolarizing pulse to approximately 50% of their peak value. On repolarization to –80 mV inward tail currents were observed. A summary of the mean current–voltage (I–V) plot for the slow current in eight cells is shown in Fig. 1B. In these experiments the peak slow current was measured during the 500 ms sweep, excluding the first 30 ms to allow for inactivation of the L-type Ca2+ current. This produced a ‘U’ shaped I–V curve, similar to the Ca2+-activated Cl– current recorded in the urethra (Cotton et al. 1997; Sergeant et al. 2000). The mean inward current was maximal at –20 mV and reversed at near the chloride equilibrium potential (ECl= 0 mV).
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The Ca2+ dependence of the slow current was tested by using nifedipine to block Ca2+ influx during depolarization. An example is shown in Fig. 2A where 10 µM nifedipine completely blocked the initial Ca2+ current and all of the components of the slow current. A summary for four cells is presented in Fig. 2B where it is clear that nifedipine blocked the slow current at all potentials (P < 0.05).
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A characteristic of Ca2+-activated Cl– currents is that they can be activated by spontaneous Ca2+ release from stores to produce spontaneous transient inward currents (STICs). We found that the majority of myocytes (70%) in the corpus cavernosum exhibited this kind of activity. Sometimes (50%), the activity was irregular in amplitude and frequency, although more regular activity was also observed. An example is shown in Fig. 3A where both large (>400 pA) and small (<100 pA) STICs can be seen. Close inspection of this record reveals that the STICs were irregular in shape, often consisting of several phases. Usually, it was not possible to fit the decay phases of the STICs with simple exponential functions as described for the rabbit portal vein (Large & Wang, 1996). Figure 3B shows that STIC amplitudes were reduced with A9C (1 mM). For the purposes of summarizing these data, the average STIC amplitude in each cell was calculated for the 30 s period before application of the drug and again for a 30 s period after it had exerted its full effect. The average values were then used to calculate the means represented in Fig. 3C (n= 6). Overall, A9C reduced the amplitude of the STICs by >80% (P < 0.01), which is similar to its effects in the rabbit urethra (Sergeant et al. 2000). In contrast, niflumic acid was less effective than in the rabbit urethra. Figure 3D shows a typical example where 100 µM niflumic acid reduced STICs by approximately 50%, which is similar to its mean effect in five cells (Fig. 3E; P < 0.05). This contrasts with an 80% reduction achieved with only 10 µM of this blocker in the rabbit urethra (Sergeant et al. 2000).
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Mechanisms underlying STICs
The effect of cyclopiazonic acid (CPA, 10 µM), a blocker of the sarcoplasmic Ca2+-ATPase, was examined to test the role of internal Ca2+ stores in generating STICs. Figure 4A shows a typical example where STICs were reduced in amplitude, and then abolished in the presence of this drug. Six such experiments are summarized in Fig. 4B, where it is clear that the STICs were completely inhibited (P < 0.01). Next we examined the effects of two drugs that interfere with IP3 mechanisms, namely 2APB, a blocker of IP3 receptors (IP3Rs), and NCDC, a blocker of phospholipase C (PLC). Figure 4C shows an example where 2APB (100 µM) reversibly inhibited STICs. Following wash-out the STICs returned at a reduced frequency, although their amplitude recovered completely. A summary of seven cells is presented in Fig. 4D, where both STIC amplitude and frequency were reduced by 93% (P < 0.05) and 86% (P < 0.01), respectively. NCDC had a similar effect to 2APB (Fig. 4E, F; reductions in frequency and amplitude significant, P < 0.05, n= 5). Taken together, these results suggest that Ca2+ release from IP3Rs was a requirement for STICS in these cells.
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Since STICs are likely to cause cavernosum myocytes to contract (and therefore contribute to the detumescent state), it was of interest to find out if they were modulated by the NO–cGMP pathway. Firstly, the effect of the labile NO donor, nitrosocysteine, was examined. An example is shown in Fig. 5A, where nitrosocysteine completely inhibited STICs for 60 s, after which they returned. Interestingly, the STICs returned at a larger amplitude and lower frequency than control and this continued for about 10 min before control activity was restored. The effect of nitrosocysteine in five cells is shown in Fig. 5B. To summarize these data, a 30 s period of activity immediately before nitrosocysteine delivery was compared with the first 30 s after the onset of its action. During this period the activity was abolished in all of the cells. This suggests that NO can modulate the STIC activity of these cells.
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We also carried out experiments to examine the interaction of the cGMP pathway with the effect of noradrenaline on these cells. In general, noradrenaline (10 µM) caused a large inward Cl– current, followed by a burst of smaller currents at a higher frequency than the basal rate. Its effects were easiest to isolate in examples like that shown in Fig. 6A, where the cell was quiescent or had a low firing rate. In this experiment two applications of noradrenaline applied during control conditions evoked almost identical bursts of inward currents. YC-1 (30 µM) abolished these responses, although they could be repeated several minutes after wash-out. Summary data for six cells are presented in Fig. 6B, where the data were quantified by measuring the amplitude of the first large current evoked by noradrenaline. The amplitude of this response was reduced by > 60% in the presence of YC-1 (P < 0.05). To test whether the effect of YC-1 could be due to either direct blockade of Cl– channels, or a non-specific effect on Ca2+ stores, we also examined its effects on caffeine-evoked currents. As caffeine is known to evoke Ca2+ release by acting on ryanodine receptors (RyRs), rather than IP3Rs, its mode of action is different to that of noradrenaline. An example is shown in Fig. 6C, where it clear that YC-1 failed to reduce caffeine-evoked Cl– currents. Summary data in eight cells confirm that no reduction in caffeine-evoked currents occurred in the presence of YC-1 (indeed, a small but statistically significant increase in current was found, P < 0.01). These results suggest that the actions of cGMP are mediated by an interaction with IP3-dependent Ca2+ release.
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Discussion |
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The current in the rabbit cavernosum was similar, although not identical, to the Cl– current previously described in rabbit and sheep urethra (Cotton et al. 1997; Sergeant et al. 2000). One difference was that in the urethra the current did not decay during depolarizing steps, while a decline of 50% was observed during 500 ms test pulses in the present study. Although we cannot exclude the possibility that this reflects a difference in Ca2+ handling between the two cell types, it may indicate that an inactivation mechanism is present in the cavernosum, but lacking in the urethra. Such a difference has also been found in other smooth muscle cell types. For example, a Ca2+–calmodulin-dependent kinase II (CaMKII)-mediated inactivation of Ca2+-activated Cl– currents has been demonstrated in trachea (Wang & Kotlikoff, 1997) and confirmed in aortic and pulmonary artery myocytes, but was less prominent in portal vein smooth muscle cells (Greenwood et al. 2001). At present we do not know why the current declines in the cavernosum, or what physiological significance, if any, this may have.
Another unusual feature of the Cl– current in the cavernosum was its insensitivity to blockade with niflumic acid. In the sheep and rabbit urethra as little as 10 µM of niflumic acid was sufficient to almost abolish the current (Cotton et al. 1997; Sergeant et al. 2000), while in the cavernosum up to 100 µM was necessary to produce only a modest block. Indeed, the sustained component of current was not reduced at all by this drug, while the amplitudes of the tail currents were even enhanced by it (Fig. 1C). Anomalous effects of niflumic acid on Ca2+-activated Cl– currents have been studied in more detail in rabbit pulmonary artery where the intracellular Ca2+ was clamped at 500 nM (Piper et al. 2002). Under these conditions niflumic acid was found to block the outward Cl– current elicited on stepping from –50 to +70 mV, but to enhance the inward current observed on stepping back to –50 mV. Ion substitution experiments performed to shift the reversal potential showed that these inhibitory and enhancing effects depended on the direction of current flow, rather than the potential at which the cell was held. Piper et al. (2002) therefore proposed that niflumic acid had a dual effect, acting as a blocker while simultaneously increasing the open probability of the channel. Since it was also a poorer blocker of outward Cl– current than inward Cl– current, the resultant effects were blockade of the outward current, but enhancement of the inward current. The enhancing effect had not previously been observed, and was only seen when the cells had been exposed to sustained increases in internal Ca2+. Thus the enhanced tail amplitudes during niflumic acid exposure in the present study might suggest that the corpus cavernosum cells have a higher resting Ca2+ level than many other smooth muscle cell types. Niflumic acid also markedly prolonged the tail currents in the present study, an effect also noted on the decay of STICs in the rabbit portal vein (Hogg et al. 1994). These authors suggested that this result could be explained if niflumic acid acted as an open channel blocker, which remained in the channel for a short time relative to the mean open duration of the channel. Their model assumed that a STIC was activated by a single Ca2+‘spark’ that decayed more rapidly than the STIC, so that the rate of STIC decay was determined by the kinetics of Cl– channel, rather than the rate of Ca2+ removal from the internal side of the cell membrane. The effect of the open channel blocker in these circumstances would be to prevent the channel from entering the normal closed state, causing prolonged ‘bursting’ activity between the open state and the blocked state. While this mechanism might contribute to prolongation of the tails under some circumstances, it is possible that the tails in the present study (especially longer ones, e.g. Fig. 2) reflect the rate of Ca2+ removal from the cell as well as simple channel kinetics. Also, the fact that the initial amplitude of the tails was enhanced, rather than reduced by niflumic acid, makes open channel block seem a less feasible explanation in this case. Therefore more study is required before the effects of niflumic acid in corpus cavernosum cells are fully understood.
We have also attempted to examine the mechanism underlying STICs in the corpus cavernosum. Blockade with CPA reversibly abolished them, suggesting that they were activated by cyclical Ca2+ release from the sarcoplasmic reticulum. STICs were also reduced by substances that interfered with IP3-mediated Ca2+ release, namely 2APB, an IP3 receptor blocker, and by NCDC, an inhibitor of PLC. These effects were similar to our findings in the rabbit urethra, where it was proposed that large STICs were evoked by large intracellular Ca2+ oscillations that required co-operative Ca2+ release from IP3 and ryanodine receptors (Sergeant et al. 2000, 2001). Strong evidence also exists for a central role for IP3-mediated Ca2+ release in generating slow waves in the gastrointestinal system (Suzuki et al. 2000; van Helden et al. 2000; Hirst & Edwards, 2001; Hirst et al. 2002).
To investigate modulation of STICs by the NO–cGMP pathway, we examined the effects of nitrosocysteine (an NO donor) and 8-bromo-cGMP (a cell-permeant analogue of cGMP). We also examined the effect of YC-1, which can activate soluble guanylate cyclase independently of NO by binding to a site different from the haem group, and also potentiates the stimulatory effect of NO on the enzyme (Friebe & Koesling, 2003). All three compounds had broadly similar effects, profoundly depressing both the frequency and amplitude of STICs. The effects with YC-1 were achieved with a similar concentration to that required to relax isolated strips of rabbit corpus cavernosum and in vitro (Hsieh et al. 2003). The inhibition by YC-1 was more long lasting than nitrosocysteine, possibly due its well-documented ability to inhibit the phosphodiesterases that break down cGMP (Galle et al. 1999). On wash-out the STIC frequency was slowed for up to 10 min and, sometimes, the amplitude was initially larger than in the control. We do not yet understand these wash-out effects, but speculate that the increased amplitude may be due to the reduced frequency, which would allow more time for store loading. Increased store loading may also have been due to increased sascoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump activity due to inhibition of phospholamban by cGMP (Raeymaekers et al. 1988).
In an attempt to understand how cGMP could inhibit STICs, we examined the effect of YC-1 on both caffeine- and noradrenaline-evoked Cl– currents. Noradrenaline is thought to stimulate IP3 production (and therefore stimulate IP3-dependent Ca2+ release) following binding to 
1 adrenoceptors (Kobayashi et al. 1989), which are the predominant post-junctional subtype in the corpus cavernosum (Andersson, 2001). Caffeine, on the other hand, is thought to block IP3 receptors (Parker & Ivorra, 1991), but also to cause Ca2+ release from the SR by opening RyR (Zucchi & Ronca-Testoni, 1997). Caffeine therefore was utilized to examine the effect of YC-1 on IP3-independent Ca2+ release. The fact that the caffeine-induced responses were not blocked by YC-1 suggests that the modulation of STICs by cGMP occured at a site upstream from the activation of the Cl– channels by Ca2+. Also, this result makes it less likely that the YC-1 acted by causing direct Cl– channel blockade, or by non-specific interference with the SR Ca2+ store. In contrast to the caffeine responses, noradrenaline-induced Cl– currents were reduced by YC-1, suggesting that the NO–cGMP pathway could interfere with IP3-mediated Ca2+ release. These results also help to explain the observation by Cellek & Moncada (1997) that nitrergic inhibition appears to be specifically directed at the adrenergic-induced contraction in the cavernosum. They argued that this interaction took place predominantly at the post-junctional level and later suggested that this might occur at the level of Ca2+ signalling in the smooth muscle cells (Cellek, 2000). Although we have not measured intracellular Ca2+ directly, the fact that YC-1 blocked noradrenaline-induced Cl– currents suggests that a specific interaction did indeed occur at this level. Moreover, there is substantial evidence in the literature that cGMP modulates Ca2+ mobilization by inhibiting IP3 generation (Murthy et al. 1993; Ruth et al. 1993; Ding & Abdel-Latif, 1997) or by inhibiting Ca2+ release from IP3Rs (Komalavilas & Lincoln, 1996; Schlossmann et al. 2000; Feil et al. 2002; Murthy & Zhou, 2003). These effects appear to be mediated by cGMP-dependent protein kinase 1 (GK-1), absence of which in ‘knock out’ mice leads to low fecundity and reduced sensitivity to the relaxant action of NO in the corpus cavernosum (Hedlund et al. 2000).
Although the corpus cavernosum is often considered to be a tonic smooth muscle, it also readily develops spontaneous phasic contractions in vitro (Christ et al. 1990; Hoppner et al. 1996) and electromyographic (EMG) recordings suggest that it is a well synchronized muscle (Jiang et al. 2003). Implicit in our observations of STICs is the fact that the cytosolic Ca2+ levels spontaneously oscillate. Phasic contraction could therefore conceivably occur as a direct result of the oscillations, or indirectly due to activation of Cl– channels, followed by depolarization and Ca2+ influx via L-type Ca2+ channels. Although we know of no reports of intracellular microelectrode recordings in intact corpus cavernosum, spontaneous depolarizations similar to urethral slow waves have been reported in the corpus spongiosum of the guinea-pig (Hashitani et al. 2002). A physiological role for the Cl– current in the corpus cavernosum was suggested by Karkanis et al. (2003) when they showed that intracavernosal pressure in rats was increased by Cl– channel blockers, arguing that depolarization due to Cl– current activated voltage-dependent Ca2+ influx and caused contraction. This would be consistent with the findings that L-type Ca2+ channel antagonists reduce spontaneous phasic contractions, spontaneous tone and noradrenaline-induced tone in the cavernosum (Christ et al. 1990; Hoppner et al. 1996). The question, naturally, arises as to how an essentially oscillatory mechanism can often produce tone. The answer to this may lie in the recent discovery that the RhoA–Rho-kinase system is very highly expressed in the cavernosum (Wang et al. 2002). This system sensitizes the contractile proteins to Ca2+, enhancing and prolonging contraction, and would therefore increase the likelihood of temporal summation of the phasic contractions.
In conclusion, we have demonstrated that rabbit corpus cavernosum myocytes express a Ca2+-activated Cl– current and a high proportion of these cells also fire spontaneous Cl– currents. Our results suggest that oscillatory Ca2+ release from the SR underlies these events. Moreover, the currents are enhanced by noradrenaline and suppressed by NO–cGMP, suggesting that they are under the control of the normal physiological mechanisms that regulate tone in this tissue. We propose that detumescent tone is maintained by a combination of the underlying spontaneous mechanism and its enhancement by noradrenaline-induced Ca2+ release. To achieve penile erection, cGMP both suppresses the spontaneous mechanism and specifically inhibits the effect of noradrenaline, possibly at the level of either IP3 production or Ca2+ release from the IP3 receptor. These effects are likely to work in conjunction with additional cGMP-dependent mechanisms such as inhibition of RhoA-induced Ca2+ sensitization (Sauzeau et al. 2000; Mills et al. 2002), activation of myosin light chain phosphatase (Lee et al. 1997) and opening of K+ channels (Lee & Kang, 2001).
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