Capsaicin-induced, capsazepine-insensitive relaxation of the guinea-pig ileum

The mechanisms underlying transient receptor potential vanilloid receptor type 1 (TRPV1)-independent relaxation elicited by capsaicin were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileum longitudinal smooth muscles of guinea-pigs. In acetylcholine-stimulated tissues, capsaicin (1-100 microM) and resiniferatoxin (10 nM-1 microM) produced a concentration-dependent relaxation. The relaxant response was attenuated by 4-aminopyridine and high-KCl solution, but not by capsazepine, tetraethylammonium, Ba(2+), glibenclamide, charybdotoxin plus apamin nor antagonists of cannabinoid receptor type 1 and calcitonin-gene related peptide. A RhoA kinase inhibitor reduced the relaxant effect of capsaicin at 30 microM. Capsaicin and resiniferatoxin reduced acetylcholine- and caffeine-induced transient contractions in a Ca(2+)-free, EGTA solution. Capsaicin at 30 microM for 20 min did not alter basal levels of MLC(20) phosphorylation, but abolished an increase by acetylcholine in MLC(20) phosphorylation. It is suggested that the relaxant effect of capsaicin at concentrations used is not mediated by TRPV1, but by 4-aminopyridine-sensitive K(+) channels, and that capsaicin inhibits contractile mechanisms involving Ca(2+) release from intracellular storage sites. The relaxation could be explained by a decrease in phosphorylation of MLC(20).     

The reactivity of serotonin, acetylcholine and kcl-induced contractions to relaxant agents in the rat gastric fundus.

The effects of nifedipine, cromakalim, diazoxide, caffeine and sodium nitroprusside (SNP) on acetylcholine, serotonin and KCl-induced contractions were studied in rat stomach fundus. Thus, we aimed to demonstrate how these contractions are modified by the substances acting on Ca (2+)influx and intracellular Ca (2+)stores. Serotonin (10(-9) - 10(-5) M) and KCl (20-80 mM) showed a similar contraction profile, which was slightly different from that of acetylcholine (10(-8)- 3 x 10(-3) M). In the experiments with the incubation of calcium-free/EGTA (0.5 mM) Krebs solution for 20 min, serotonin (3 x 10(-7)M) and KCl (40 mM) did not produce any contraction whereas, 10% of contraction to acetylcholine (3 x 10(-5) M) was still intact. Serotonin-induced contractions were readily reversed by nifedipine (10(-10) - 10(-4) M), cromakalim (10(-9) - 10(-4) M), diazoxide (10(-9) - 10(-4) M), caffeine (10(-5) - 10(-2) M) and SNP (10(-4) M) whereas, acetylcholine-induced contractions showed relative refractoriness to the above relaxant agents. 1 mM caffeine nearly fully inhibited serotonin-induced contraction, but not acetylcholine and high K-induced contractions whereas, 10 mM caffeine completely inhibited all the contractions. The relaxation pattern of nifedipine on serotonin and high K (+)-induced contractions was quite similar. Moreover, nifedipine and cromakalim showed equal dose effectiveness in relaxing acetylcholine and serotonin. The maximum relaxations induced by nifedipine and cromakalim in acetylcholine contractions were 61.51 +/- 6.92 % and 58.97 +/- 7.55 %, respectively. However their maximum relaxations in serotonin and high K (+)-induced contractions were almost 100%. The similarity in myorelaxants properties of cromakalim and nifedipine may relate to the similarity of their effects on calcium influx by a different mechanism of action in rat stomach fundus. In conclusion, acetylcholine-induced contraction is partially mediated both by calcium release from the intracellular Ca (2+) pool and calcium influx via L-type Ca (2+) channels. However, serotonin-induced contraction is possibly triggered by Ca (2+) release from sarcoplasmic reticulum and basically mediated by Ca (2+) influx via L-type Ca (2+)channels.     

Comparative effects of niflumic acid and nifedipine on 5-hydroxytryptamine- and acetylcholine-induced contraction of the rat trachea

The effects of niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) (Cl((Ca))) channels, were compared with those of the voltage-dependent Ca(2+) channel (VDCC) blocker nifedipine on 5-hydroxytryptamine (5-HT)- and acetylcholine-induced contractions of the rat isolated trachea. Niflumic acid (3-100 microM) induced a concentration-dependent inhibition of 5-HT (10 microM)-induced contractions, with a reduction to 37.0+/-9.5% of the control at the highest concentration. One micromolar nifedipine, which completely blocked 60 mM KCl-induced contractions, reduced the response to 5-HT similarly to 39.2+/-11.5% of the control. The inhibition of the 5-HT response was not significantly different from that produced by the combined presence of nifedipine (1 microM) and niflumic acid (100 microM), suggesting that their effects were not additive. In contrast, neither niflumic acid (3-100 microM) nor nifedipine (1 microM) inhibited acetylcholine-induced contractions. The contraction to 5-HT (10 microM) in Cl(-)-free solution was decreased by more than approximately 85% of the control, whilst that of acetylcholine was reduced only by approximately 36%. Our data show that niflumic acid exerts selective inhibitory effects on 5-HT-induced contraction, and suggest that activation of Cl((Ca)) channels may be a mechanism whereby 5-HT (but not acetylcholine) induces Ca(2+) entry via VDCCs to elicit contraction.     

Effects of Y-27632 on acetylcholine-induced contraction of intact and permeabilized intrapulmonary bronchial smooth muscles in rats.

In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca(2+) sensitization of rat bronchial smooth muscle were examined. Intact and beta-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10(-6)-10(-4) M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10(-3) M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10(-4) M Y-27632, which had no effect on the resting tone. In beta-escin-permeabilized muscles, addition of acetylcholine (10(-5)-10(-3) M) plus GTP (100 microM) induced a further contraction, i.e., Ca(2+) sensitization at a constant Ca(2+) concentration of pCa=6.0. The acetylcholine-induced Ca(2+) sensitization was completely blocked in the presence of 10(-4) M Y-27632, whereas the Ca(2+)-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca(2+) sensitization.     

Selegiline, an MAO-B inhibitor, attenuates airway smooth muscle contraction in the rat trachea

Selegiline is widely used for Parkinson's disease and sometimes for Alzheimer's disease. It is reported to affect intracellular Ca(2+) concentration. Since intracellular Ca(2+) is partly regulated by phosphatidylinositol (PI) response and is important for smooth muscle contraction, selegiline may affect airway smooth muscle tension. We examined the effects of selegiline on acetylcholine (ACh)- and KCl-induced contractile and PI responses in rat trachea. The trachea was cut into 3-mm-wide ring segments or 1-mm-wide slices. ACh (3 microM, 50% effective dose) or KCl (40 mM) was added, and ring relaxation was induced by the addition of selegiline. Tracheal slices were incubated with [(3)H]myo-inositol and 3 microM ACh in the presence of selegiline, and [(3)H]inositol monophosphate (IP(1)) was measured. Selegiline dose-dependently attenuated ACh- and KCl-induced tracheal ring contractions. Fifty-percent inhibitory doses (ID50) of selegiline against ACh- and KCl-induced contraction were 120 +/- 30 microM and 80 +/- 20 microM, respectively. Basal and ACh-induced IP(1) accumulation were 2.20 +/- 0.20 Bq and 7.88 +/- 0.23 Bq, respectively, and selegiline at a dose of 1000 microM attenuated ACh-induced IP(1) accumulation (5.44 +/- 0.30 Bq). These results suggest that selegiline inhibits contractile responses through the inhibition of voltage-operated Ca(2+) channels and the PI response.     

Analysis of pharmacological effects of drugs used for treatment of urinary disturbance based on anticholinergic and smooth muscle-relaxing effects

Propiverine hydrochloride, oxybutynin hydrochloride and terodiline hydrochloride have both anticholinergic and antispasmodic effects, and are used for the management of urinary frequency and incontinence. The average standard therapeutic doses of these drugs differ greatly. We retrospectively analyzed their pharmacological effects with consideration given to muscarinic acetylcholine receptor binding affinities, anticholinergic activities, and inhibitory effects on KCl-induced contraction. Muscarinic acetylcholine receptor occupancies and the inhibitory ratios of the drugs for both acetylcholine-induced and KCl-induced contraction in a steady state after oral administration of standard doses were calculated based on pharmacokinetics and the receptor occupancy theory. The average muscarinic acetylcholine receptor occupancy and inhibitory ratio of acetylcholine-induced contraction were estimated to be 12.6+/-1.06% and 3.27+/-0.74%, respectively, with no significant differences found between the drugs for those parameters. A significant linear relationship was found between muscarinic acetylcholine receptor occupancy and the maximum ratio of increase in bladder urinary capacity. On the other hand, the inhibitory ratios of KCl-induced contraction varied from 0.01 to 0.48%. The present results suggest that muscarinic acetylcholine receptor occupancy is a principal determinant of the therapeutic effect of a drug used for treatment of urinary disturbance.     

Stimulated calcium entry and constitutive RhoA kinase activity cause stretch-induced detrusor contraction

Urinary bladder wall muscle (i.e., detrusor smooth muscle; DSM) contracts in response to a quick-stretch, but this response is neither fully characterized, nor completely understood at the subcellular level. Strips of rabbit DSM were quick-stretched (5 ms) and held isometric for 10 s to measure the resulting peak quick-stretch contractile response (PQSR). The ability of selective Ca(2+) channel blockers and kinase inhibitors to alter the PQSR was measured, and the phosphorylation levels of myosin light chain (MLC) and myosin phosphatase targeting regulatory subunit (MYPT1) were recorded. DSM responded to a quick-stretch with a biphasic response consisting of an initial contraction peaking at 0.24+/-0.02-fold the maximum KCl-induced contraction (F(o)) by 1.48+/-0.17 s (PQSR) before falling to a weaker tonic (10 s) level (0.12+/-0.03-fold F(o)). The PQSR was dependent on the rate and degree of muscle stretch, displayed a refractory period, and was converted to a sustained response in the presence of muscarinic receptor stimulation. The PQSR was inhibited by nifedipine, 2-aminoethoxydiphenyl borate (2-APB), 100 microM gadolinium and Y-27632, but not by atropine, 10 microM gadolinium, LOE-908, cyclopiazonic acid, or GF-109203X. Y-27632 and nifedipine abolished the increase in MLC phosphorylation induced by a quick-stretch. Y-27632, but not nifedipine, inhibited basal MYPT1 phosphorylation, and a quick-stretch failed to increase phosphorylation of this rhoA kinase (ROCK) substrate above the basal level. These data support the hypothesis that constitutive ROCK activity is required for a quick-stretch to activate Ca(2+) entry and cause a myogenic contraction of DSM.     

An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery

1. The possibility that stimulation of smooth muscle alpha(1)-adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries. 2. N(omega)-nitro-L-arginine methyl ester, (L-NAME, 100 microM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1 - 3 microM) by approximately 2 fold (from a control level of 14.2+/-3.0 to 34. 1+/-4.2% of the maximum contraction of the artery, n=20). The action of L-NAME was abolished by disrupting the endothelium. 3. The subsequent addition of apamin (to inhibit small conductance Ca(2+)-activated K(+) channels, 50 nM) further augmented phenylephrine contractions, in an endothelium-dependent manner, to more than 3 fold above control (50.4+/-5.3% of the maximum contraction, n=11). 4.Charybdotoxin (non-selective inhibitor of large conductance Ca(2+)-activated K(+) channels, BK(Ca), 50 nM) plus L-NAME augmented the level of phenylephrine contraction to 4 - 5-fold above control (64.1+/-3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BK(Ca) inhibitor, iberiotoxin,. 5. Apamin together with L-NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5 - 6-fold, to 79.9+/-3.5% of the maximum contraction of the artery (n=13). 6. Phenylephrine failed directly to increase the intracellular Ca(2+) concentration in endothelial cells freshly isolated from the small mesenteric artery. 7. Stimulation of smooth muscle alpha(1)-adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K(+) from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium-derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.     

Effects of calcium antagonists on rat normal and skinned fundus.

Calcium chloride (CaCl2) (0.1-25 mM, in K(+)-depolarized tissue), KCl (10-112 mM) and acetylcholine (1 x 10(-9) M-1 mM) produced concentration-dependent contractions of rat isolated fundus. Verapamil (0.01-100 microM), cinnarizine (1-100 microM), trifluoperazine (10-500 microM) and dantrolene (50-250 microM) each produced a concentration-related rightward and downward shift of the log concentration-effect curve for CaCl2. The rank order of potencies of these antagonists, measured as the IC50 against Ca2+ (25 mM)-induced contraction of depolarized fundus, was verapamil (2.5 microM) greater than cinnarizine (8.7 microM) greater than trifluoperazine (85.1 microM) greater than dantrolene (greater than 250 microM). Cinnarizine (0.5 mM) and trifluoperazine (0.5 mM), but neither verapamil nor dantrolene depressed Ca2+ (20 microM)-evoked contraction of rat skinned fundus preparations. In intact preparations of rat fundus, verapamil had greater inhibitory effects on contractions produced by KCl than against those elicited by acetylcholine while trifluoperazine depressed to the same extent the responses to these two spasmogens. Dantrolene was without effect on contractions elicited by KCl or acetylcholine. Cinnarizine inhibited acetylcholine-induced responses but enhanced contractions to KCl. Augmentation of KCl-induced responses by cinnarizine is resistant to verapamil (1 microM). This enhancing effect of cinnarizine was not observed for KCl-induced contraction of guinea-pig fundus or rat gastro-oesophageal sphincter. In the rat fundus, cinnarizine (1-100 microM) produced an additional and concentration-related contraction when added on the plateau contraction to KCl (100 mM). The enhancing effect and the direct contraction produced by cinnarizine are at least partly dependent on extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of non-selective Ca2+ channels in the contraction induced by alkalinization of rat anococcygeus muscle cells

Intracellular pH is a modulator of cellular functions such as smooth muscle contraction. Changes in cytosolic Ca(2+) concentration ([Ca(2+)](c)) associated with contraction are brought about by Ca(2+) influx and release from the sarcoplasmic reticulum, and alterations in the intracellular pH can affect both processes. In this work, therefore, we have investigated the Ca(2+) influx pathway that contributes to the contraction induced by the alkalinizing agent NH(4)Cl in the rat anococcygeus smooth muscle. For this purpose, we measured the isometric tension in muscle preparations, and [Ca(2+)](c) was measured on isolated cells loaded with 5 micromol/l FURA2/AM by using the ratio 340/380 nm. NH(4)Cl (10 mmol/l) induced a larger increase in [Ca(2+)](c) (100%) when compared with the [Ca(2+)](c) increase induced by 0.1 micromol/l phenylephrine (57.0+/-12.3% n=4). Incubation of the muscle preparations for 1 min in Ca(2+)-free medium reduced the contractions induced by 10 mmol/l NH(4)Cl to 11.5+/-5.1% (n=5), when compared with the contractions induced in 2.5 mmol/l Ca(2+) solution (100%). After 3 min in Ca(2+) free medium, contractions stimulated with NH(4)Cl were almost abolished (0.6+/-0.4%, n=5). In the same way, incubation with 10 micromol/l 1-[beta-[3[(4-methoxyphenyl)propoxyl]-4-methoxy-phenetyl]-1H-imidazole hydrochloride (SKF96365), a non-selective Ca(2+) channels, reduced the contractions stimulated with NH(4)Cl to 47.6+/-6.7% (n=7). On the other hand, 1 micromol/l verapamil, a voltage-operated Ca(2+) channel blocker and 0.05 micromol/l calphostin C, a protein kinase-C inhibitor, did not alter the contractions induced by NH(4)Cl. On isolated cells, [Ca(2+)](c) was reduced to 72.2+/-1.7% (n=4) by 10 micromol/l SKF96365. Taken together, our results suggest that NH(4)Cl induces contraction of rat anococcygeus smooth muscle cells, as well as [Ca(2+)](c) increase due to Ca(2+) influx through non-selective Ca(2+) channels.     

Calcium-independent activation of the contractile apparatus in smooth muscle of mouse aorta by protein phosphatase inhibition

Smooth muscle contraction is primarily regulated by reversible phosphorylation of the 20-kDa light chains of myosin (MLC(20)) involving Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and serine/threonine protein phosphatases (PP).The aim of this study was to investigate the effects of the protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) inhibitor cantharidin (Cant), its structural analogue endothall (ETA) and microcystin LR (MC) on force of contraction and MLC(20)-phosphorylation in arterial smooth muscle of mouse aorta. Cant increased force of contraction and MLC(20)-phosphorylation in intact arterial rings of mouse aorta in the presence of Ca(2+) whereas ETA and MC were ineffective under the same experimental conditions. In contrast, all compounds induced contraction and led to enhanced MLC(20)-phosphorylation in nominally Ca(2+)-free solution in fibers of mouse aorta permeabilised (skinned) with Triton X-100.In addition, Western blot analysis revealed that skinning of mouse aorta did not result in a loss of PP1 and PP2A compared to intact rings. Thus, both PP must be tightly bound to structural proteins, e.g. myosin. The findings indicate a Ca(2+)-independent mechanism of smooth muscle contraction involving inhibition of PP1- and/or PP2A-activities leading to enhanced force and MLC(20)-phosphorylation of arterial smooth muscle.     

Endothelium-derived factors and hyperpolarization of the carotid artery of the guinea-pig.

1. Transmembrane potentials were recorded from isolated carotid arteries of the guinea-pig superfused with modified Krebs-Ringer bicarbonate solution. Smooth muscle cells were impaled from the adventitial side with intracellular glass microelectrodes filled with KCl (30-80 M omega). 2. Acetylcholine (1 microM) in the presence of inhibitors of nitric oxide synthase, (N omega-nitro-L-arginine (L-NOARG) 100 microM) and cyclo-oxygenase, (indomethacin 5 microM) induced an endothelium-dependent hyperpolarization (-18.9 +/- 1.6 mV, n = 15). 3. In the presence of these two inhibitors, S-nitroso-L-glutathione (10 microM), sodium nitroprusside (10 microM), 3-morpholinosydnonimine (SIN-1, 10 microM) and iloprost (0.1 microM) induced endothelium-independent hyperpolarizations of the smooth muscle cells (respectively: -16.0 +/- 2.3, -16.3 +/- 3.4, -12.8 +/- 2.0 and -14.5 +/- 1.5 mV, n = 4-6). 4. The addition of glibenclamide (1 microM) did not influence the acetylcholine-induced L-NOARG/ indomethacin-resistant hyperpolarization (-18.0 +/- 1.8 mV, n = 10). In contrast, the responses induced by S-nitroso-L-glutathione, sodium nitroprusside, SIN-1 and iloprost were abolished (changes in membrane potential: -0.8 +/- 1.1, 1.3 +/- 3.9, 4.5 +/- 4.6 and 0.3 +/- 0.8 mV respectively, n = 4-5). 5. In the presence of NO synthase and cyclo-oxygenase inhibitors, charybdotoxin (0.1 microM) or apamin (0.5 microM) did not influence the hyperpolarization produced by acetylcholine. However, in the presence of the combination of charybdotoxin and apamin, the acetylcholine-induced L-NOARG/indomethacin-resistant hyperpolarization was converted to a depolarization (4.4 +/- 1.2 mV, n = 20) while the endothelium-independent hyperpolarizations induced by S-nitroso-L-glutathione, sodium nitroprusside, SIN-1 and iloprost were not affected significantly (respectively: -20.4 +/- 3.4, -22.5 +/- 4.9, -14.5 +/- 4.7 and -14.5 +/- 0.5 mV, n = 4-5). 6. In the presence of the combination of charybdotoxin and apamin and in the absence of L-NOARG and indomethacin, acetylcholine induced a hyperpolarization (-19.5 +/- 3.7 mV, n = 4). This hyperpolarization induced by acetylcholine was not affected by the addition of indomethacin (-18.3 +/- 4.6 mV, n = 3). In the presence of the combination of charybdotoxin, apamin and L-NOARG (in the absence of indomethacin), acetylcholine, in 5 out of 7 vessels, still produced hyperpolarization which was not significantly smaller (-9.1 +/- 5.6 mV, n = 7) than the one observed in the absence of L-NOARG. 7. These findings suggest that, in the guinea-pig isolated carotid artery, the endothelium-independent hyperpolarizations induced by NO donors and iloprost involve the opening of KATP channels while the acetylcholine-induced endothelium-dependent hyperpolarization (resistant to the inhibition of NO-synthase and cyclo-oxygenase) involves the opening of Ca(2+)-activated potassium channel(s). Furthermore, in this tissue, acetylcholine induces the simultaneous release of various factors from endothelial origin: hyperpolarizing factors (NO, endothelium derived hyperpolarizing factor (EDHF) and prostaglandins) and possibly a depolarizing factor.     

Organic Ca(2+)-antagonist-resistant response to FMRF-NH2 on the molluscan smooth muscle.

1. FMRF-amide (10(-7)-10(-5) M) contracted molluscan anterior byssus retractor muscle in a concentration-dependent fashion. 2. The concentration-response curve of FMRF-amide was shifted rightward by an analogue of FMRF-amide, FMRf-amide ([D-Phe4]FMRF-amide, putative FMRF-amide receptor antagonist) in a parallel manner (pA2 = 4.87 +/- 0.04). 3. Although a contractile response to KCl was reduced by the organic Ca2+ antagonists (verapamil, diltiazem and high concentration of nifedipine and nicardipine). FMRF-amide-induced contraction was not markedly reduced by them. 4. In the Ca(2+)-free medium, FMRF-amide-induced contraction was diminished. The response was also reduced by TMB-8 (10(-4) M), suggesting that FMRF-amide-induced contraction might be partly dependent on intracellular Ca2+. 5. An inorganic Ca(2+)-antagonist, MnCl2, markedly reduced the FMRF-amide- and KCl-induced contraction. The results show that FMRF-amide-induced contraction might be dependent on extracellular Ca2+. 6. These findings suggest that FMRF-amide-induced contraction might be mediated through an action on FMRF-amide receptors and not through the activation of organic Ca2+ antagonist-sensitive Ca2+ channels.     

Mechanism of human urotensin II-induced contraction in rat aorta

Urotensin II induced sustained contraction with an EC(50) value of 2.29 +/- 0.12 nM in rat aorta. Urotensin II (100 nM) transiently increased cytosolic Ca(2+) level ([Ca(2+)](i)), followed by a small sustained phase superimposed with rhythmic oscillatory change. In the presence of verapamil and La(3+), the [Ca(2+)](i) oscillation was completely inhibited, although a small transient increase in [Ca(2+)](i) remained. The urotensin II-induced contraction was also partially inhibited by verapamil and La(3+). Combined application of verapamil, La(3+), and thapsigargin completely inhibited the increase in [Ca(2+)](i) with only partial inhibition of the contraction elicited by urotensin II. Urotensin II increased myosin light chain (MLC) phosphorylation to a level greater than that induced by 72.7 mM KCl (high K(+)). Pretreatment with Go6983 (PKC inhibitor), U0126 (MEK inhibitor), or SB203580 (p38MARK inhibitor) partially inhibited the urotensin II-induced contraction with no effects on the high K(+)-induced contractions. Wortmannin (MLC kinase inhibitor) only partially inhibited urotensin II-induced contraction, although it completely inhibited the high K(+)-induced contraction. These results suggest that urotensin II-induced contraction is mediated by the Ca(2+)/calmodulin/MLC kinase system and modulated by the Ca(2+) sensitization mechanisms to increase MLC phosphorylation. In addition, activations of PKC, p38MAPK, and ERK1/2 modulate the contractility mediated by urotensin II in rat aorta.     

Myosin light chain phosphorylation and Mn2+-dependent acetylcholine-induced contractions in guinea pig vas deferens

We have reported that noradrenaline but not K+ induced a sustained and dose-dependent contraction without extracellular Ca2+ and Mn2+ in Ca2+-depleted Mn2+loaded vas deferens from the guinea pig. The Mn2+dependent noradrenaline-induced contractions developed without an increase in phosphorylation of 20-kDa myosin light chain. To clarify whether such an unusual Mn2+dependent contraction was induced only by noradrenaline or not, we examined effects of acetylcholine on Ca2+-depleted Mn2+-loaded guinea pig vas deferens. Acetylcholine (1 microM(-1) mM) induced a sustained dose-dependent contraction without extracellular Ca2+ or Mn2+. W-7 (10 microM or 100 microM) and wortmannin (1 microM) both reduced the Mn2+dependent acetylcholine-induced contractions similarly to Ca2+-dependent acetylcholine-induced contractions in isolated vas deferens without either Ca2+ depletion or Mn2+ loading. However, the Mn2+-dependent acetylcholine-induced contractions developed without a significant increase in the phosphorylation of the myosin light chain determined by urea-glycerol polyacrylamide gel electrophoresis and immunoblotting. These results indicate that acetylcholine as well as noradrenaline induces Mn2+-dependent contraction and are consistent with our previous assumption that Mn2+ may preferentially support receptor-mediated contractions in the guinea pig isolated vas deferens. The results also suggest that the activation of myosin light chain kinase is essential for the development of Mn2+-dependent acetylcholine-induced contractions, and that Mn2+ may accelerate formation of non-phosphorylated attached cross-bridges during receptor activation.     

Pharmacological comparison of the vasorelaxant action displayed by kaurenoic acid and pimaradienoic acid

The vascular effects of two natural occurring diterpenes from the kaurane and pimarane classes were compared. The diterpenes ent-kaur-16-en-19-oic acid (kaurenoic acid; KA) and ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) were tested for their antispasmodic activity on isolated rat aorta. Vascular reactivity experiments, using standard muscle bath procedures, showed that KA and PA (both at 50 and 100 microM) inhibited phenylephrine and KCl-induced contraction in both endothelium-intact and endothelium-denuded rat aortic rings, with PA being more effective than KA. These compounds also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mm). Again, PA produced a greater reduction in CaCl(2)-induced contraction than KA. PA (1-300 microM) and KA (1-450 microM) concentration dependently relaxed endothelium-denuded aortic rings pre-contracted with KCl (maximum relaxation 102.31+/-6.94% and 82.71+/-1.40%, respectively). Similarly, the relaxation induced by KA on aortic rings pre-contracted with phenylephrine (73.06+/-3.68%) was less pronounced than that found for PA (102.21+/-3.64%). Incubation of endothelium-denuded rings for different periods showed that at 50 microM, KA and PA achieved maximum inhibitory activity on KCl-induced contraction after incubation for 60 (53.48+/-5.83%) and 30 min (83.89+/-2.12%), respectively. At 100 microM, KA and PA inhibited KCl-induced contraction, with a maximum after incubation for 30 min (73.58+/-5.30% and 92.07+/-1.20%, respectively). The maximum inhibition induced by PA at both concentrations tested was greater than that induced by KA. The results provide evidence that structural differences between diterpenes, independent of the C-19 carboxylic acid site, influence selectivity for voltage-operated Ca2+ channels and rate of equilibrium with the target site for their vasorelaxant action in rat aortic rings.     

Augmentation of SR Ca(2+) release by rapamycin and FK506 causes K(+)-channel activation and membrane hyperpolarization in bladder smooth muscle

1. The immunosuppressants rapamycin and FK506 are known to relax smooth muscle despite facilitating Ca(2+) release through ryanodine-receptors of the sarcoplasmic reticulum (SR). The apparent contradiction was studied in isolated guinea-pig urinary bladder myocytes. 2. Modulation of spontaneous SR Ca(2+) release was monitored by means of spontaneous transient outward currents (or STOCs) in isolated smooth muscle cells voltage-clamped to -20 mV. Rapamycin (10 microM, n=18) significantly increased amplitude (50+/-12%, mean+/-s.d.), life time (77+/-19%), and time integral of STOCs (113+/-22%), and it reduced the interval between STOCs (20+/-7%). FK506 (20 microM, n=24) increased amplitude (15+/-7%), life time (50+/-7%), time integral (104+/-26%). Cyclosporin A (20 microM, n=18) had no significant effects on STOCs. 3. The basal cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) measured by Indo1-fluorescence was insensitive to rapamycin or FK506. Pretreatment with rapamycin (20 microM, 2 min) did not impair the SR Ca(2+) load as can be concluded from caffeine-induced Ca(2+)-transients. 4. As it was expected from the enhanced STOC activity, the non-clamped membrane was hyperpolarized by rapamycin (15+/-2 mV) or by FK506 (15+/-3 mV). 5. The data are consistent with the idea that rapamycin and FK506 augment spontaneous SR Ca(2+) release by removal of FK-binding proteins from the RyR-complex. Smooth muscle relaxation is interpreted as negative Ca(2+) feedback: augmented Ca(2+) activation of STOCs induces membrane hyperpolarization that reduces Ca(2+) influx through voltage gated channels.     

Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones

1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.     

Contraction of isolated guinea-pig ileum by urotensin II via activation of ganglionic cholinergic neurons and acetylcholine release

Urotensin II and its receptor are expressed in the gastrointestinal tract of mice, but the effects of urotensin II on the gastrointestinal functions have not been established. In the present study, we investigated the effects of human urotensin II on a segment of the guinea-pig ileum. The addition of urotensin II induced contraction of the ileum in concentration-manner (-log EC(50) value was 8.13+/-0.21). The response by urotensin II was extracellular CaCl(2)-dependent and easily desensitized. Like nicotine, the contraction induced by 100 nM urotensin II was inhibited by treatment with atropine, hexamethonium, D-tubocurarine, tetrodotoxin or hemicholinium-3, and enhanced by physostigmine. Treatment with omega-conotoxin GVIA (an inhibitor of N-type Ca(2+) channels, 300 nM) inhibited 100 nM urotensin II- and 4 microM nicotine-, but not 3 microM acetylcholine-, induced contraction. Both urotensin II and nicotine stimulated [(3)H]choline release in a tetrodotoxin-sensitive manner from the prelabeled slices of the ileum. These findings suggest that urotensin II stimulated acetylcholine release from the ganglionic cholinergic neurons and thus stimulated contraction via muscarinic acetylcholine receptors in the guinea-pig ileum. Urotensin II receptor system in the myenteric neurons may regulate the gastrointestinal functions.     

Alteration of the [Ca(2+)](i)-force relationship during the vasorelaxation induced by a Ca(2+) channel blocker SR33805 in the porcine coronary artery

The mechanism of vasorelaxation induced by SR33805 was investigated by simultaneously monitoring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force, and by determining level of myosin light chain (MLC) phosphorylation in the medial strip of the porcine coronary artery. SR33805 inhibited the sustained increases in [Ca(2+)](i) and force (IC(50); 3.2+/-1.0 and 49.4+/-27.5 nM, respectively) induced by 118 mM K(+)-depolarization. There was about a 10 fold difference in the inhibitory potency between [Ca(2+)](i) and force. SR33805 completely inhibited the [Ca(2+)](i) elevation induced by a thromboxane A(2) analogue, U46619 and histamine, at concentrations (1 microM) higher than those required for the complete inhibition of K(+)-depolarization induced [Ca(2+)](i) elevation. SR33805 had no effect on the [Ca(2+)](i) elevation induced by histamine or caffeine in the absence of extracellular Ca(2+). SR33805 caused a leftward shift of the [Ca(2+)](i)-force relationship of the contraction induced by cumulative application of extracellular Ca(2+) during 118 mM K(+)-depolarization. The relationship between [Ca(2+)](i) and MLC phosphorylation also shifted to the left by SR33805, while the relationship between MLC phosphorylation and force remained unaffected. In conclusion, SR33805 caused an apparent leftward shift of the [Ca(2+)](i)-force relationship, accompanied by a greater degree of MLC phosphorylation for a given level of [Ca(2+)](i). The mechanism of this leftward shift, however, still remains to be elucidated.