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Journal of Physiology (2002), 538.2, pp. 573-581
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013049

Cellular mechanisms of nitric oxide-induced relaxation of corporeal smooth muscle in the guinea-pig

Hikaru Hashitani, Hiroyasu Fukuta, Emma J. Dickens * and Hikaru Suzuki

	ABSTRACT

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The cellular mechanism of nitric oxide (NO)-induced relaxation in corporeal smooth muscle (CSM) of the guinea-pig was investigated. Changes in the intracellular concentration of calcium ions ([Ca²⁺]_i), membrane potential and isometric tension were measured. CSM cells exhibited spontaneous depolarizations and transient increases in [Ca²⁺]_i (Ca²⁺ transients) which were accompanied by contractions. This spontaneous activity was abolished by nifedipine (10 µM). NO released by 3-morpholino-sydnonimine (SIN-1, 10 µM) hyperpolarized the membrane and prevented the generation of spontaneous depolarizations. SIN-1 also abolished Ca²⁺ transients and associated contractions. These effects of SIN-1 were blocked by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 µM), an inhibitor of guanylate cyclase. Noradrenaline (NA, 1 µM) increased [Ca²⁺]_i to levels similar to those produced by high potassium-containing solution (high K⁺ solution, [K⁺]_o = 40 mM), however, NA-induced contractions were three times greater in amplitude than those induced by high K⁺ solution. In NA precontracted preparations, SIN-1 inhibited 80 % of the contraction and decreased [Ca²⁺]_i by 20 %. In contrast, nifedipine reduced [Ca²⁺]_i by 80 %, while the level of contraction was decreased by only 20 %. SIN-1-induced reduction in [Ca²⁺]_i but not the tension effect, was abolished by pretreatment with cyclopiazonic acid (CPA, 10 µM). In high K⁺ precontracted preparations, SIN-1 inhibited 80 % of the contraction and reduced [Ca²⁺]_i by 20 %. Nifedipine, however, largely abolished increases in both [Ca²⁺]_i and tension under these circumstances. These results suggest that decreasing the sensitivity of contractile proteins to Ca²⁺ is probably the key mechanism of NO-induced relaxation in CSM of the guinea-pig.

(Received 26 July 2001; accepted after revision 28 September 2001)
Corresponding author H. Hashitani: Department of Physiology, Nagoya City University Medical School, Nagoya 467-8601, Japan.

	INTRODUCTION

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Penile erection results from relaxation of corporeal smooth muscle (CSM). The principal mediator of this relaxation has been shown to be nitric oxide (NO) which is released from both autonomic nerves and endothelium (Andersson & Wagner, 1995). Nitic oxide is thought to relax the CSM by activating soluble guanylate cyclase to increase cyclic GMP (cGMP) content (Ignarro et al. 1990; Holmquist et al. 1992), however, the cellular mechanism of NO/cGMP-induced relaxation in CSM has not been identified.

Observations on other types of smooth muscle preparations indicate that NO/cGMP may decrease [Ca²⁺]_i and reduce the sensitivity of contractile proteins to Ca²⁺, thereby resulting in the relaxation of smooth muscle (Lincoln & Cornwell, 1993; Carvajal et al. 2000). Several mechanisms have been suggested to underlie NO/cGMP-induced reduction in [Ca²⁺ ]_i. Cyclic GMP is thought to stimulate Ca²⁺-ATPase on intracellular Ca²⁺ stores and enhance calcium uptake into these stores (Cornwell et al. 1991). Inhibition of inositol 1,4,5-trisphosphate (InsP₃)-induced Ca²⁺ release from intracellular stores by NO has also been proposed (Komalavilas & Lincoln, 1994). Moreover, cGMP may decrease the influx of Ca²⁺ through L-type Ca²⁺ channels either by opening K⁺ channels which leads to hyperpolarization of the membrane (Archer et al. 1994; Murphy & Brayden, 1995; Koh et al. 1995) or by directly inhibiting Ca²⁺ channels (Ishikawa et al. 1993).

Direct stimulation of the sodium pump by NO has been suggested as a possible mechanism for decreasing [Ca²⁺]_i in several CSM preparations, including human corpus cavernosum (Gupta et al. 1995; Bivalacqua et al. 2000). A study by Hashitani (2000) demonstrated in the rat penile bulb that neurally released NO caused hyperpolarizations of the membrane that could be attributable to stimulation of the sodium pump. However, this study also showed that the majority of the NO-induced relaxations persisted after inhibiting these hyperpolarizations, indicating that NO relaxes CSM mainly through other mechanisms.

Although there have been extensive studies investigating the contractile responses of CSM (Andersson & Wagner, 1995), only a limited number of electrophysiological studies have been performed (Christ et al. 1993; Hashitani, 2000). To date, information regarding changes in [Ca²⁺]_i of CSM has arisen from studies using cultured cells (Christ et al. 1992). As we do not have information about calcium homeostasis in intact CSM preparations, the cellular mechanisms of the NO/cGMP-induced relaxation, particularly the calcium-force relationship, under physiological conditions remain unidentified.

The present study employed simultaneous measurements of changes in [Ca²⁺]_i and isometric tension in intact CSM preparations of the guinea-pig. Intracellular microelectrode recordings were also made at the same time to characterize the associated membrane potential changes.

	METHODS

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The procedures described were approved by the Animal Experimentation Ethics Committee of Nagoya City University Medical School. Male guinea-pigs, weighing 250-350 g, were rapidly killed by a blow to the head followed by cervical exsanguination and the penises removed immediately. The bulbospongiosum muscle tunica albuginea, which covers the bottom of the penile shaft, was dissected free and slit open with a lateral incision. The corpus spongeosum was then dissected from the tunica albuginea and the urethra. The corpus spongeosum preparations were pinned out in a dissecting chamber and several layers of smooth muscle were removed leaving an underlying single layer of CSM.

For the measurement of changes in [Ca²⁺]_i, preparations were pinned out on a Sylgard plate (silicone elastomer, Dow Corning Corporation, Midland, MI, USA) that formed the bottom of a recording chamber (volume, ~1 ml) which was mounted on the stage of an inverted microscope. After 1 h incubation with warmed (35 °C) physiological saline, spontaneous movements of the tissues were observed. Subsequently preparations were loaded with the fluorescent Ca²⁺ indicator, fura-2, by incubation in nominally Ca²⁺-free solution containing fura-2 AM (10 µM), p-(dipropylsulfamoyl) benzoic acid (probenecid, 3 mM) and cromphor EL (0.01 %) for 1 h at room temperature. After loading, preparations were superfused with dye-free warmed (35 °C) physiological saline at a constant flow (about 2 ml min^-1) for 30 min. Preparations loaded with fura-2 were illuminated with ultraviolet light, wavelengths 340 and 380 nm, alternating at a frequency higher than 40 Hz. The ratio of the emission fluorescences (R_340/380) in a desired size of rectangular window was measured through a barrier filter (peak transmission 510 nm; sampling time 80-200 ms), using a microphotoluminescence measurement system (ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan).

In most experiments, isometric tension recordings were taken simultaneously with measurements of [Ca²⁺]_i. To detect isometric tension changes of the preparations, an L-shaped fine needle connected to a force transducer was inserted into the cavernosal space, and then carefully pulled towards surrounding muscle bundles.

For the recording membrane potential changes, preparations were pinned out in the same type of recording chamber as used for the calcium experiments and were superfused with warmed (35 °C) physiological saline at a constant flow rate (2 ml min^-1). After 1 h equilibration, individual CSM cells were impaled with glass capillary microelectrodes, filled with 0.5 M KCl (tip resistance 150-250 M). Membrane potential changes were recorded using a high input impedance amplifier (Axoclamp-2B, Axon Instruments, Inc., Foster City, CA, USA), and displayed on a cathode ray oscilloscope (SS-9622, Iwatsu, Tokyo, Japan). After low-pass filtering (cut-off frequency, 1 kHz), membrane potential changes were digitized with a Digidata 1200 interface (Axon Instruments, Inc., Union City, CA, USA), and stored on a personal computer for later analysis.

The ionic composition of physiological saline was as follows (mM): NaCl, 119; KCl, 5.0; CaCl₂, 2.5; MgCl₂, 2.0; NaHCO₃, 25.0; NaH₂PO₄, 1.0 and glucose, 11.0. The solution was bubbled with 95 % O₂-5 % CO₂ to maintain pH 7.4. High potassium-containing solution was prepared by replacing sodium chloride with an isosmotic amount of potassium chloride. Nominally calcium-free solutions were prepared by omitting CaCl₂.

Drugs used were 8-bromo cyclic GMP (8Br-cGMP) sodium salt, cromphor EL, p-(dipropylsulfamoyl) benzoic acid (probenecid), 3-morpholino-sydnonimine (SIN-1) hydrochloride, N-nitro-L-arginine (LNA), cyclopiazonic acid (CPA), nifedipine, 1H-[1,2,4]-oxadiazole[4,3-a]quinoxalin-1-one (ODQ), noradrenaline hydrochloride (all from Sigma, St Louis, MI, USA) and fura-2 AM (from Dojindo, Kumamoto, Japan). These drugs were dissolved in distilled water except nifedipine which was dissolved in 100 % ethanol and CPA, fura-2 and ODQ which were dissolved in dimethyl sulphoxide (DMSO). Cromphor EL and probenecid were directly dissolved into nominally calcium-free solution. The final concentration of these solvents in physiological saline did not exceed 1:1000.

Measured values are expressed as means ± S.D. Statistical significance was tested using Student's t test, and probabilities of less than 5 % were considered significant.

	RESULTS

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General observations

Visual observations indicated that CSM of the guinea-pig underwent spontaneous contractions. When intracellular recordings were made from the smooth muscle, spontaneous depolarizations which were similar to those recorded from the smooth muscle of the rat penile bulb were generated (Hashitani, 2000; Fig. 1A and Ba). Spontaneous depolarizations were characterized in 17 preparations; they occurred either continuously or in bursting patterns with frequencies between 10 and 40 min^-1 (mean 21 ± 7.9 min^-1, n = 17) and had peak amplitudes between 7 and 40 mV (mean 22.1 ± 7.4 mV, n = 17). For all the experiments, resting membrane potentials, which were defined as the most negative potential level between spontaneous depolarizations, varied between -40 and -54 mV (mean -46.3 ± 4.3 mV, n = 28).

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Figure 1. Effects of SIN-1 and nifedipine on spontaneous depolarizations recorded from corporeal smooth muscle of the guinea-pig Corporeal smooth muscle cells exhibited spontaneous action potentials (A). SIN-1 (10 µM) hyperpolarized the membrane by some 5 mV and prevented the generation of spontaneous action potentials. In another preparation, smooth muscle cells exhibited spontaneous action potentials (Ba). Nifedipine (10 µM) abolished spontaneous action potentials without detectable change in the resting membrane potential (Bb). Resting membrane potentials were -43 mV in A and -50 mV in B. Scale bars (bottom right) apply to whole figure.

When measurements of changes in [Ca²⁺]_i were made simultaneously with isometric tension recordings, smooth muscle cells exhibited spontaneous increases in [Ca²⁺]_i (Ca²⁺ transients) and associated contractions (see Fig. 2). Spontaneous calcium transients were characterized in 10 preparations; they occurred periodically with frequencies between 9 and 21 min^-1(mean 16.7 ± 3.6 min^-1, n = 10) and had peak amplitudes between 0.05 and 0.13R_340/380 (mean 0.086 ± 0.027R_340/380, n = 10).

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Figure 2. Effects of SIN-1, nifedipine and 8Br-cGMP on spontaneous increases in [Ca2+]i and tension Spontaneous changes in [Ca2+]i (upper traces) were measured simultaneously with tension changes (lower traces) in CSM preparations. SIN-1 (10 µM) prevented the generation of Ca2+ transients and reduced the resting level of [Ca2+]i (Aa). SIN-1 also abolished spontaneous contractions and lowered the resting muscle tone (Ab). In another preparation, nifedipine (10 µM) prevented the generation of spontaneous increases in [Ca2+]i and reduced the resting level of [Ca2+]i (Ba). Nifedipine also abolished spontaneous contractions and lowered the resting tone (Bb). In a different preparation, 8Br-cGMP (1 mM) abolished Ca2+ transients and reduced the resting level of [Ca2+]i (Ca). 8Br-cGMP also prevented spontaneous contractions and lowered the resting muscle tone (Cb). Scale bar to the right of Ca applies to all calcium traces and the scale bar to the right of Cb applies to all tension traces, the time calibration applies to the whole figure.

Effects of SIN-1 and nifedipine on spontaneous changes in membrane potential, [Ca²⁺]_i and tension

SIN-1 (10 µM), a NO donor, hyperpolarized the membrane and abolished spontaneous action potentials (Fig. 1A). The amplitude of SIN-1-induced hyperpolarizations ranged between 5 and 12.3 mV (mean of 8.2 ± 2.5 mV, n = 12). In a separate series of experiments, SIN-1 (10 µM) prevented spontaneous Ca²⁺ transients and reduced the resting calcium level by about 0.06R_340/380 (mean 0.064 ± 0.028R_340/380, n = 6; Fig. 2Aa). SIN-1 also abolished associated contractions and reduced the resting tension (Fig. 2Ab).

The possible involvement of cGMP in SIN-1-induced inhibition of spontaneous increases in [Ca²⁺]_i and tension was examined. 8-Bromo cyclic GMP (1 mM), an activator of protein kinase G, inhibited both calcium responses and associated contractile responses (n = 3, Fig. 2Ca and b). Furthermore, the inhibitory effects of SIN-1 on both [Ca²⁺]_i and tension were blocked by ODQ (10 µM, n = 4), an inhibitor of guanylate cyclase, suggesting that SIN-1 inhibited spontaneous activity by increasing cGMP.

Nifedipine (10 µM) abolished spontaneous depolarizations without changing the resting membrane potential (n = 5, Fig. 1Bb). In a separate series of experiments, nifedipine abolished spontaneous Ca²⁺ transients and reduced the resting [Ca²⁺]_i level by about 0.05R_340/380 (mean 0.053 ± 0.029R_340/380, n = 5; Fig. 2Ba). Nifedipine also abolished associated contractions and reduced the resting tension (Fig. 2Bb), suggesting that spontaneous depolarizations were initiated by the opening of L-type Ca²⁺ channels and that the Ca²⁺ influx through these channels increased [Ca²⁺]_i to initiate contractions.

To examine the contribution of the calcium uptake into intracellular stores, the effect of CPA, an inhibitor of intracellular Ca²⁺ store Ca²⁺-ATPase, on SIN-1-induced inhibitions was studied. CPA (10 µM) caused transient increases in both [Ca²⁺]_i and tension which gradually returned to control levels over 20 min. In preparations which had been exposed to CPA (10 µM) for 20 min, SIN-1 still prevented the occurrence of Ca²⁺ transients and associated contractions (n = 3). SIN-1 continued to abolish spontaneous action potentials and to cause membrane hyperpolarizations of similar amplitude to those recorded in control solution (n = 3). These results indicated that stimulation of Ca²⁺-ATPase is probably not involved in SIN-1-induced inhibition of spontaneous increases in [Ca²⁺]_i and tension.

Comparison between the effects of NA and high potassium containing solution on [Ca²⁺]_i and tensions

In preliminary experiments, it was found that pretreatment with LNA, an inhibitor of NO synthase, increased the amplitude of NA- and high K⁺-induced contractions, suggesting spontaneous production of NO occurred in the absence of obvious stimulation (Ozaki et al. 1992). This was consistent with a previous report in the rat penile bulb which showed either LNA or ODQ depolarized the membrane by about 5 mV (Hashitani, 2000). To exclude the influence of background release of NO, all the following experiments were carried out in the presence of LNA (0.1 mM).

To characterize the mechanisms of NA-induced contractions, the effects of NA (1 µM) on [Ca²⁺]_i and tension were compared with those of high K⁺ solution. NA increased [Ca²⁺]_i to a level similar to that produced by high K⁺ solution ([K⁺]_o = 40 mM, Fig. 3Aa and Ba; NA 0.35 ± 0.14R_340/380, high K⁺ 0.31 ± 0.11R_340/380, n = 12, P > 0.05). When isometric tension was measured simultaneously with changes in [Ca²⁺]_i, NA caused contractions that were three times larger in amplitude than those produced by high K⁺ (Fig. 3Ab and Bb; NA 3.7 ± 1.1 mN, high K⁺ 1.5 ± 0.6 mN, n = 12, P < 0.05). These results are summarized in Fig. 3Ca and b, and suggest that NA may increase the sensitivity of contractile proteins to Ca²⁺.

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Figure 3. Effects of increased [K+]o and bath-applied NA on changes in [Ca2+]i and tension In a CSM preparation, high K+ solution ([K+]o = 40 mM) increased [Ca2+]i by some 0.25R340/380 (Aa). In the same preparation, NA (1 µM) increased [Ca2+]i to a similar level (Ba). When changes in tension were measured simultaneously with changes in [Ca2+]i, high K+ solution caused a contraction with amplitude of about 1.5 mN (Ab), while NA increased the tension by about 4 mN (Bb). The results obtained from seven preparations are summarized in Ca ([Ca2+]i) and Cb (tension). All experiments were carried out in the presence of LNA (0.1 mM). The scale bar to the right of Ba applies to both [Ca2+]i traces and the scale bar to the right of Bb applies to both tension traces.

Effects of SIN-1 and nifedipine on NA-induced increases in [Ca²⁺]_i and tension

To investigate the mechanism of NO-induced relaxation in NA precontracted CSM, the effects of SIN-1 and nifedipine on tension and [Ca²⁺]_i were studied in preparations which had been precontracted with NA (1 µM). SIN-1 lowered [Ca²⁺]_i by about 20 % (17 ± 5.7 %, n = 9), while nifedipine reduced [Ca²⁺]_i by about 80 % (77.7 ± 5.3 %, n = 9; Fig. 4Aa and Ba). These results suggest that NA-induced increases in [Ca²⁺]_i mainly resulted from calcium entry through L-type calcium channels. They also indicate that SIN-1 did not inhibit this type of channel in the presence of NA. Indeed, when intracellular recordings were made, NA (1 µM) depolarized the membrane to around -20 mV (-22.7 ± 4 mV; Fig. 5A and Bb) and subsequent applications of SIN-1 repolarized the membrane by about 5 mV but failed to prevent oscillation of the membrane potential (Fig. 5Bc).

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Figure 4. Effects of SIN-1 and nifedipine on NA-induced increases in [Ca2+]i and tension In a CSM preparation which had been exposed to NA (1 µM), SIN-1 (10 µM) reduced the increased [Ca2+]i level by about 15 % (Aa). In the same preparation, nifedipine (10 µM) reduced the [Ca2+]i level by some 75 % (Ba). When changes in tension were measured simultaneously with changes in [Ca2+]i, SIN-1 reduced the increased tension by about 80 % (Ab), while nifedipine reduced the tension by only 20 % (Bb). The results obtained from seven preparations are summarized in Ca ([Ca2+]i) and Cb (tension). All experiments were carried out in the presence of LNA (0.1 mM). The scale bar to the right of Ba applies to both [Ca2+]i traces and the scale bar to the right of Bb applies to both tension traces.

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Figure 5. Effects of NA and SIN-1 on changes in membrane potential recorded from corporeal smooth muscle cells In CSM cells which exhibited spontaneous action potentials, NA (1 µM) depolarized the membrane by about 25 mV and increased the frequency of action potentials (A). Spontaneous action potentials occurred with a frequency of 25 min-1 and had amplitudes ranging between 10 and 30 mV in control conditions (Ba). In the presence of NA, action potentials occurred with a frequency of 50 min-1 and had amplitudes of 7 mV (Bb). Subsequent application of SIN-1 (10 µM) repolarized the membrane by some 5 mV. In the presence of NA and SIN-1, action potentials occurred with a frequency of 40 min-1 and had an amplitude of about 15 mV (Bc). Resting membrane potentials were -50 mV. The scale bar to the right of A applies to the trace in A. The scale bar to the right of Bc applies to all traces in B.

In the same preparations, isometric tension recordings were made simultaneously with measurements of [Ca²⁺]_i. SIN-1 reduced tension by about 80 % (77.5 ± 7.1 %, n = 9; Fig. 4Ab), while nifedipine reduced tension by only 20 % (18.7 ± 4.2 %, n = 9; Fig. 4Bb). These results are summarized in Fig. 4Ca and b.

To examine the role of calcium uptake into intracellular calcium stores, the effects of CPA on SIN-1-induced inhibitions were also studied. As described above, CPA (10 µM) caused transient increases in both [Ca²⁺]_i and tension. After 20 min exposure to CPA, NA increased [Ca²⁺]_i to about 60 % of control (64.8 ± 8 %, n = 5; Fig. 6Aa and Ba) and evoked a contraction about 60 % as large as in control conditions (55.8 ± 9.4 %, n = 5; Fig. 6Ab and Bb). In such precontracted preparations, SIN-1 failed to reduce [Ca²⁺]_i (Fig. 6Ba) but caused relaxations which were similar to those without CPA pretreatment (77.5 ± 7.1 %, n = 7; Fig. 6Bb). Taken together, these results suggest that SIN-1 relaxed precontracted CSM mainly by reducing the sensitivity of contractile proteins to Ca²⁺.

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Figure 6. Effects of CPA on SIN-1-induced reductions in both tension and [Ca2+]i In control conditions, NA (1 µM) increased [Ca2+]i (Aa) and contracted CSM preparations (Ab). Subsequent application of SIN-1 (10 µM) reduced [Ca2+]i by some 20 % (Aa) and reduced NA contractions by about 80 % (Ab). In the same preparations following exposure to CPA (10 µM) for over 20 min, NA caused smaller increases in both [Ca2+]i (Ba) and tension (Bb). SIN-1 failed to reduce [Ca2+]i (Ba) but still reduced NA contractions by some 80 % (Bb). All experiments were carried out in the presence of LNA (0.1 mM). The scale bar to the right of Ba applies to all [Ca2+]i traces and the scale bar on the right of Bb applies to all tension traces.

The possible involvement of cGMP in NO-induced relaxation of precontracted CSM was examined. In preparations which had been exposed to NA (1 µM), 8-Br-cGMP (1 mM) reduced the increased [Ca²⁺]_i level by some 15 % (13.5 ± 6.3 %, n = 3) and suppressed about 70 % of the contraction (65.8 ± 8.4 %, n = 3). Furthermore, ODQ (10 µM) prevented the inhibitory effects of SIN-1 on both [Ca²⁺]_i and tension (n = 5), suggesting that SIN-1 relaxes NA precontracted preparations through the activation of guanylate cyclase.

The effects of SIN-1 and nifedipine on high K⁺ solution-induced increases in [Ca²⁺]_i and tension

To further clarify the mechanism of SIN-1-induced relaxation of CSM, the effects of SIN-1 and nifedipine on high K⁺ solution-induced increases in [Ca²⁺]_i and tension were examined. High K⁺ solution-induced increases in [Ca²⁺]_i and tension were largely abolished by nifedipine (10 µM; n = 5; Fig. 7Ba and b), indicating that high K⁺ solution caused contraction almost solely through the influx of Ca²⁺ through L-type calcium channels. SIN-1 reduced the increased calcium level by about 20 % (17.2 ± 3.7 %, n = 8, Fig. 7Aa), while tension was reduced by some 80 % (77.5 ± 2.9 %, n = 8; Fig. 7Ab). These results are summarized in Fig. 7Ca and b and they suggest that SIN-1 relaxes high K⁺ precontacted preparations presumably by reducing the calcium sensitivity of contractile proteins. When intracellular recordings were made separately, high K⁺ solution depolarized the membrane to around -20 mV (-21 ± 2 mV, n = 5). The subsequent application of SIN-1 caused only a small hyperpolarization (3.5 ± 1.5 mV, n = 4) and failed to prevent small oscillations in membrane potential.

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Figure 7. Effects of SIN-1 and nifedipine on high K+ solution-induced increases in [Ca2+]i and tension In high K+ solution ([K+]o = 40 mM), SIN-1 (10 µM) reduced the increased [Ca2+]i level by some 20 % (Aa). In the same preparation, nifedipine (10 µM) reduced the [Ca2+]i level by over 95 % (Ba). In these preparations, SIN-1 reduced the increased tension by about 80 % (Ab), while nifedipine almost completely inhibited the increased tension (Bb). The results obtained from seven preparations are summarized in Ca ([Ca2+]i) and Cb (tension). All experiments were carried out in the presence of LNA (0.1 mM). The scale bar to the right of Ba refers to both [Ca2+]i traces and the scale bar to the right of Bb refers to both tension traces.

	DISCUSSION

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In CSM of the guinea-pig, changes in [Ca²⁺]_i and tension were measured simultaneously. In control conditions, both SIN-1 and nifedipine prevented the generation of spontaneous action potentials, and abolished Ca²⁺ transients and associated contractions. In contrast, in NA precontracted preparations, SIN-1 caused an 80 % reduction in tension with only a corresponding 20 % reduction in [Ca²⁺]_i. Under the same conditions nifedipine reduced [Ca²⁺]_i by about 80 % and tension by 20 %. These results suggest that SIN-1 relaxes CSM mainly by reducing the sensitivity of contractile proteins to Ca²⁺ and not by lowering [Ca²⁺]_i levels.

Spontaneous contractions have been recorded from CSM obtained from various mammals, including humans, and may contribute to the maintenance of detumescence (Andersson & Wagner, 1995). In the present study, spontaneous increases in [Ca²⁺]_i were associated with contractions, and both events were abolished by nifedipine. Nifedipine also prevented the generation of spontaneous action potentials as observed in the rat penile bulb (Hashitani, 2000), suggesting that the opening of L-type Ca²⁺ channels may be the common mechanism of spontaneous activity in CSM. Since the activation of L-type calcium channels occurs in a voltage-dependent manner, SIN-1 presumably prevents the generation of spontaneous action potentials by hyperpolarizing the membrane. Thus SIN-1 fails to reduce [Ca²⁺]_i in both NA and high K⁺ precontracted preparations, where the membrane potential oscillates around -20 mV, because SIN-1 induced hyperpolarizations (5 mV) are of insufficient amplitude to close L-type calcium channels. In contrast, nifedipine, which directly blocks L-type Ca²⁺ channels, causes large decreases in [Ca²⁺]_i in both NA and high K⁺ pretreated preparations. These results indicate that NO does not directly inhibit L-type calcium channels as reported for other smooth muscle tissues (Ishikawa et al. 1993).

Generally, NO relaxes many types of smooth muscle by stimulating soluble guanylate cyclase to increase cGMP (Lincoln & Cornwell, 1993; Carvajal et al. 2000). In CSM of the guinea-pig, 8Br-cGMP mimicked the effects of SIN-1; 8Br-cGMP abolished spontaneous increases in [Ca²⁺]_i and associated contractions, and also relaxed NA precontracted preparations with a small reduction in [Ca²⁺]_i. Furthermore, the inhibitory effects of SIN-1 on both spontaneous activity and NA-mediated excitation were abolished by ODQ, an inhibitor of soluble guanylate cyclase, indicating the involvement of cGMP in SIN-1-induced inhibitory effects.

Contractions of CSM predominantly result from neurally released NA which activates -adrenoceptors (Andersson & Wagner, 1995). The -adrenoceptor mediated contractions are thought to result from both calcium release from intracellular stores following InsP₃ production and calcium influx through L-type calcium channels (Fovaeus et al. 1987; Hashitani, 2000). In vascular smooth muscle preparations, NA has also been reported to increase the sensitivity of contractile proteins to calcium (Kitazawa et al. 1988, 1991). In the present study, NA increased internal calcium to levels similar to those produced by high K⁺ solution, while NA-induced contractions were three times larger in amplitude than those generated by high K⁺ solution. In addition, NA-induced increases in [Ca²⁺]_i were largely reversed by nifedipine, while much of the contraction persisted in the presence of nifedipine. The simplest explanation for these observations is that NA increases the sensitivity of contractile proteins to calcium as reported in vascular smooth muscles (Kitazawa et al. 1988, 1991). Alternatively, calcium released from intracellular stores has better access to contractile proteins, and thus triggers contraction more readily than calcium which enters via calcium channels as proposed for other smooth muscles (Morgan & Morgan, 1984; Bramich & Hirst, 1999).

In the present study, SIN-1 inhibited approximately 80 % of the NA-induced contraction with only a 20 % reduction in [Ca²⁺]_i. On the other hand, nifedipine reduced NA-induced increases in calcium by 80 % but inhibited contraction by only 20 %. Since we measured global changes in [Ca²⁺]_i, our experiments were unable to detect localized calcium changes. Calcium compartmentalisation has been reported (Abe et al. 1995) and a 'contractile calcium compartment' described which is coupled with contractile proteins. Other 'non-contractile calcium compartments' are described which are mainly accessed by calcium entry mechanisms. Therefore, SIN-1 might effectively reduce calcium in 'contractile compartments' possibly by stimulating Ca²⁺-ATPase. However, this is unlikely because in preparations that had been treated with CPA, SIN-1 failed to reduce [Ca²⁺]_i but caused reductions in tension similar to those observed without CPA. Therefore, the most likely explanation of this observation is that SIN-1 reduced the sensitivity of contractile proteins to calcium ions. This mechanism has been reported in several smooth muscle tissues (Dickens et al. 2000) and is thought to be mediated by activating myosin light chain phosphatase with protein kinase G (Lee & Kitazawa, 1997; Tran et al. 1998), although thin filament-linked regulatory mechanisms cannot be excluded.

It has been reported that cGMP inhibits the activity of either phospholipase C (PLC) or protein kinase C (PKC) (Cornwell & Lincoln, 1993; Kumar et al. 1997). The activation of -adrenoceptors generally stimulates PLC to produce InsP₃ which releases Ca²⁺ from intracellular stores. The stimulation of PLC also activates PKC which increases the sensitivity of contractile proteins to calcium ions (Ruzycky & Morgan, 1989). Therefore, SIN-1 might interrupt NA-induced contractions by inhibiting the -adrenoceptor-mediated pathway. However, in preparations precontracted with high K⁺ solution, SIN-1 caused an 80 % reduction in the contraction with only a 20 % reduction in [Ca²⁺]_i. Therefore, SIN-1/cGMP may directly reduce the calcium sensitivity of contractile proteins.

In conclusion, NO/cGMP relaxes the CSM of the guinea-pig mainly by reducing the sensitivity of the contractile proteins to Ca²⁺. NO/cGMP also lowers [Ca²⁺]_i by stimulating Ca²⁺-ATPase and by inhibiting L-type calcium channels through hyperpolarization of the membrane. Spontaneous contractions depend principally on calcium entry through calcium channels, while NA may cause contractions by increasing the sensitivity of contractile proteins. Thus modulation of calcium sensitivity may be a key mechanism for determining the tone of CSM and so would be a novel target for pharmacological treatment of erectile dysfunction.

	REFERENCES

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Acknowledgements

The authors wish to thank Dr F. R. Edwards for his critical reading of the manuscript. This project was supported by a research grant from Mitsui Life Social Welfare Foundation.

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