Effects of a newly synthesized K+ channel opener, Y-26763, on noradrenaline-induced Ca2+ mobilization in smooth muscle of the rabbit mesenteric artery.

1. The mechanisms underlying the vasodilatation induced by (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2, 2-dimethyl-2H-1-benzopyran-3-ol (Y-26763) were investigated by measuring membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and isometric force in smooth muscle cells of the rabbit mesenteric artery. 2. Y-26763 (0.03-1 microM) concentration-dependently hyperpolarized the membrane and glibenclamide (1-10 microM) inhibited this hyperpolarization. Noradrenaline (NA, 10 microM) depolarized the membrane and generated spike potentials. Y-26763 (1 microM) inhibited these NA-induced electrical responses. 3. In thin smooth muscle strips in 2.6 mM Ca2+ containing (Krebs) solution, 10 microM NA produced a large phasic, followed by a small tonic increase in [Ca2+]i and force with associated oscillations. In Ca(2+)-free solution (containing 2 mM EGTA), NA produced only phasic increases in [Ca2+]i and force. In ryanodine-treated strips, NA could not produce the phasic increases in [Ca2+]i and force even in the presence of 2.6 mM Ca2+, suggesting that ryanodine functionally removes the NA-sensitive intracellular storage sites. 4. Nicardipine (1 microM) partly inhibited the NA-induced tonic increases in [Ca2+]i and force but had no effect on either the resting [Ca2+]i or the NA-activated phasic increases in [Ca2+]i and force. By contrast, Y-26763 (10 microM) lowered the resting [Ca2+]i and also inhibited both the phasic and the tonic increases in [Ca2+]i and force induced by NA. All these actions of Y-26763 were inhibited by glibenclamide (10 microM). 5. In ryanodine-treated strips, nicardipine partly, but Y-26763 completely inhibited the NA-induced increases in [Ca2+]i, suggesting that Y-26763 inhibits both the nicardipine-sensitive and -insensitive Ca2+ influxes activated by NA. Y-26763 attenuated the phasic increase in [Ca2+]i and force in a Ca(2+)-free solution containing 5.9 mM K+, but not in one containing 50 mM K+, suggesting that Y-26763 inhibits NA-induced Ca2+ release, probably as a result of its membrane hyperpolarizing action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a water-soluble forskolin derivative (NKH477) and a membrane-permeable cyclic AMP analogue on noradrenaline-induced Ca2+ mobilization in smooth muscle of rabbit mesenteric artery.

1. Effects were studied of 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative and of dibutyryl-cyclic AMP, a membrane-permeable cyclic AMP analogue on noradrenaline (NA)-induced Ca2+ mobilization in smooth muscle strips of the rabbit mesenteric artery. The intracellular concentration of Ca2+ ([Ca2+]i), isometric force and cellular concentration of inositol 1,4,5-trisphosphate (InsP3) were measured. 2. NA (10 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force in a solution containing 2.6 mM Ca2+. NKH477 (0.01-0.3 microM) attenuated the phasic and the tonic increases in both [Ca2+]i and force induced by 10 microM NA, in a concentration-dependent manner. 3. In Ca(2+)-free solution containing 2 mM EGTA with 5.9 mM K+, NA (10 microM) produced only phasic increases in [Ca2+]i and force. NKH477 (0.01 microM) and dibutyryl-cyclic AMP (0.1 mM) each greatly inhibited these increases. 4. NA (10 microM) led to the production of InsP3 in intact smooth muscle strips and InsP3 (10 microM) increased Ca2+ in Ca(2+)-free solution after a brief application of Ca2+ in beta-escin-skinned smooth muscle strips. NKH477 (0.01 microM) or dibutyryl-cyclic AMP (0.1 mM) modified neither the NA-induced synthesis of InsP3 in intact muscle strips nor the InsP3-induced Ca2+ release in skinned strips. 5. In Ca(2+)-free solution, high K+ (40 and 128 mM) itself failed to increase [Ca2+]i but concentration-dependently enhanced the amplitude of the increase in [Ca2+]i induced by 10 microM NA with a parallel enhancement of the maximum rate of rise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of membrane hyperpolarization induced by a K+ channel opener on histamine-induced Ca2+ mobilization in rabbit arterial smooth muscle.

1. The role of membrane hyperpolarization on agonist-induced contraction was investigated in intact and alpha-toxin-skinned smooth muscles of rabbit mesenteric artery by use of the ATP-sensitive K+ channel opener, (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2- dimethyl-2H-1-benzopyran-3-ol (Y-26763), and either histamine (Hist) or noradrenaline (NA). 2. Hist (3 microM) and NA (10 microM) both produced a phasic, followed by a tonic increase in intracellular Ca2+ concentration ([Ca2+]i) and force. Y-26763 (10 microM) potently inhibited the NA-induced phasic and tonic increase in [Ca2+]i and force. In contrast, Y-26763 attenuated the Hist-induced phasic increase in [Ca2+]i and force but had almost no effect on the tonic response. However, ryanodine-treatment of muscles in order to inhibit the function of intracellular Ca2+ storage sites altered the action of Y-26763 which now attenuated the Hist-induced tonic increase in [Ca2+]i and force in a concentration-dependent manner (at concentrations > 1 microM). Glibenclamide (10 microM) attenuated the inhibitory action of Y-26763. 3. Hist (3 microM) depolarized the smooth muscle cells to the same extent as NA (10 microM). In the absence of either agonist, Y-26763 (over 30 nM) hyperpolarized the membrane and glibenclamide inhibited this hyperpolarization. Y-26763 (10 microM) almost abolished the NA-induced membrane depolarization, but only slightly attenuated the Hist-induced membrane depolarization in which the delta (delta) value (the difference before and after application of Hist) was not modified by any concentration of Y-26763. In ryanodine-treated smooth muscle cells, Y-26763 hyperpolarized the membrane and potently inhibited the membrane depolarization induced by Hist. 4. In ryanodine-treated muscle, Y-26763 had no measurable effect on the Hist-induced [Ca2+]i-force relationship. Y-26763 also had no apparent effect on the myofilament Ca(2+)-sensitivity in the presence of Hist in alpha-toxin-skinned smooth muscles. 5. It is concluded that the membrane hyperpolarization induced by Y-26763 may not be enough to inhibit the Hist-activated Ca2+ influx. It is also suggested that Hist prevents the membrane hyperpolarization induced by Y-26763, activating an unknown mechanism which is thought to depend on the function of intracellular Ca2+ storage sites.     

Mechanism underlying H2O2-induced inhibition of acetylcholine-induced contraction in rabbit tracheal smooth muscle

The mechanism underlying the inhibition by H2O2 of acetylcholine-induced contraction was investigated in epithelium-denuded strips of rabbit trachea. Acetylcholine (10 microM) generated a phasic, followed by a tonic increase in both the intracellular Ca2+ concentration ([Ca2+]i) and force. Although the acetylcholine-induced tonic contraction was around 9 times the high K+ (80 mM)-induced one, the two stimulants induced similar [Ca2+]i increases (around 0.2 microM), indicating that acetylcholine generates tonic contraction via increases in both [Ca2+]i and myofilament Ca2+-sensitivity. H2O2 (30 microM) (a) enhanced the acetylcholine-induced tonic (not phasic) increase in [Ca2+]i but attenuated both phases of the acetylcholine-induced contraction and (b) enhanced the high K+-induced increase in [Ca2+]i but did not modify the high K+-induced contraction. In beta-escin-skinned strips, application of acetylcholine in the presence of GTP enhanced the contraction induced by 0.3 microM Ca2+ so that its amplitude became similar to that induced by 1 microM Ca2+. H2O2 (30 microM) attenuated the contraction induced by 0.3 microM Ca2+ (alone or in the presence of acetylcholine) but not those induced by higher concentrations of Ca2+ alone (0.5 microM and 1 microM). These results indicate that H2O2 acts directly on contractile proteins in rabbit tracheal smooth muscle to inhibit the contraction induced by low concentrations of Ca2+ (<0.5 microM). An action of H2O2 that increases [Ca2+]i (and thereby masks this reactive-oxygen-induced inhibition of myofilament Ca2+-sensitivity) is apparent in the presence of high K+ but not of acetylcholine. Thus, in rabbit tracheal smooth muscle H2O2 downregulates myofilament Ca2+-sensitivity more potently during acetylcholine-induced contraction than during high-K+-induced contraction, leading to an effective inhibition of the former contraction.     

Sevoflurane and bradykinin-induced calcium mobilization in pulmonary arterial valvular endothelial cells in situ: sevoflurane stimulates plasmalemmal calcium influx into endothelial cells

Kinins locally synthesized in the cardiovascular tissue are believed to contribute to the regulation of cardiovascular homeostasis by stimulating the endothelial cells to release nitric oxide, prostacyclin, or a hyperpolarizing factor via autocrine-paracrine mechanisms. This study was designed to investigate the action of sevoflurane on bradykinin-induced Ca2+ mobilization in endothelial cells in situ. Utilizing fura-2-loaded rat pulmonary arterial valve leaflets, the effects of sevoflurane were examined on bradykinin-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ. In the presence of extracellular Ca2+ (1.5 mM), bradykinin (3-30 microM) produced an initial phasic and a subsequent tonic increase in [Ca2+]i in a concentration-dependent manner. However, it produced only the phasic increase in [Ca2+]i in the absence of extracellular Ca2+. Sevoflurane (5%, 0.67 mM) inhibited both the phasic and tonic responses to bradykinin. In these experiments, sevoflurane (3-5%) generated sustained increases (approximately 20-40% of the bradykinin-induced maximal increase in [Ca2+]i) in the resting [Ca2+]i level. Sevoflurane still increased [Ca2+]i after depletion of the intracellular Ca stores with ionomycin (0.1 microM ). However, the sevoflurane-induced increase in [Ca2+]i was eliminated by removal of the extracellular Ca and attenuated by NiCl (1-3 mM). In conclusion, in the pulmonary arterial valvular endothelial cells, sevoflurane inhibits both bradykinin-induced Ca2+ release from the intracellular stores and bradykinin-induced plasmalemmal Ca2+ influx. In addition, sevoflurane appears to stimulate the plasmalemmal Ca2+ influx and thereby increase the endothelial [Ca2+]i level. Sevoflurane might influence the pulmonary vascular tone through its direct action on the pulmonary arterial valvular endothelial cells.     

Contractile 5-HT1 receptors in human isolated pial arterioles: correlation with 5-HT1D binding sites.

1. Effects of (-)-cromakalim (lemakalim) on tension and Ca2+ mobilization induced by noradrenaline (NA) were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) isometric tension and production of inositol-1,4,5-trisphosphate (IP3) in smooth muscle strips of the rabbit mesenteric artery. 2. In thin smooth muscle strips, 10 microM NA produced a large phasic, followed by a small tonic increase in [Ca2+]i, which correlated well with the evoked phasic and tonic contractions, respectively. Lemakalim (0.1-10 microM) lowered the resting [Ca2+]i without a decrease in the resting tension, and also inhibited the increased [Ca2+]i and tension induced by 10 microM NA, all in a concentration-dependent manner. Glibenclamide (1 microM) inhibited these actions of lemakalim. 3. In Ca(2+)-free solution containing 2 mM EGTA, NA (10 microM) transiently increased [Ca2+]i, tension and synthesis of IP3. Lemakalim (over 0.01 microM) inhibited these actions of NA in Ca(2+)-free solution containing 5.9 mM K+, but not in Ca(2+)-free solution containing 128 mM K+. These actions of lemakalim were prevented by glibenclamide (1 microM). Lemakalim (1 microM) did not modify the increases in [Ca2+]i and tension induced by 10 mM caffeine. 4. In beta-escin-skinned strips, 10 microM NA increased [Ca2+]i in Ca(2+)-free solution containing 50 microM EGTA, 3 microM guanosine triphosphate (GTP) and 2 microM Fura 2 after the storage sites were loaded by application of 0.3 microM Ca2+ for 2 min, suggesting that Ca2+ is released from intracellular storage sites following activation of the alpha-adrenoceptor. Lemakalim (1 microM) did not inhibit the Ca2+ release from storage sites induced by NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pinacidil on contractile proteins in high K(+)-treated intact, and in beta-escin-treated skinned smooth muscle of the rabbit mesenteric artery.

1. The effects of pinacidil were investigated on changes in cellular Ca2+ concentration ([Ca2+]i) and tension in intact and chemically skinned smooth muscle strips of the rabbit mesenteric artery. 2. High K+ (128 mM) produced a large phasic followed by a tonic increase in [Ca2+]i and tension in intact muscle strips. Pinacidil at 10 microM but not 1 microM, inhibited the phasic and tonic contractions induced by 128 mM K+ without a corresponding change in [Ca2+]i. 3. In beta-escin-treated skinned smooth muscle, the minimum Ca2+ concentration that produced contraction was 0.1 microM and the maximum contraction was obtained at 10 microM. Pinacidil at 10 microM but not 1 microM, shifted the pCa-tension relation curve to the right and also inhibited the maximum contraction induced by Ca2+. The concentrations of Ca2+ required for half maximal tension were 0.9 microM in control and 1.5 microM in the presence of 10 microM pinacidil. Calmodulin (2 microM) increased the contraction induced by 0.3 microM Ca2+ (but not by 10 microM Ca2+) in the skinned strips. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM or 10 microM Ca2+ in the presence of 2 microM calmodulin. 4. Noradrenaline (NA, 10 microM) with guanosine triphosphate (GTP, 3 microM), guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 3 microM) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) all enhanced the contraction induced by 0.3 microM Ca2+. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM Ca2+ more strongly in the presence of the above agents than in their absence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible mechanisms underlying the vasodilatation induced by olprinone, a phosphodiesterase III inhibitor, in rabbit coronary artery

The possible mechanisms underlying the vasodilatation induced by olprinone, a phosphodiesterase type III inhibitor, were investigated in smooth muscle of the rabbit coronary artery. Isometric force and membrane potential were measured simultaneously using endothelium-denuded smooth muscle strips. Acetylcholine (ACh, 3 microM) produced a contraction with a membrane depolarization (15. 2+/-1.1 mV). In a solution containing 5.9 mM K(+), olprinone (100 microM) hyperpolarized the resting membrane and (i) caused the absolute membrane potential level reached with ACh to be more negative (but did not reduce the delta membrane potential seen with ACh, 15.2+/-1.8 mV) and (ii) attenuated the ACh-induced contraction. In a solution containing 30 mM K(+), these effects were not seen with olprinone. Glibenclamide (10 microM) blocked the olprinone-induced membrane hyperpolarization. 4-AP (0.1 mM) significantly attenuated the olprinone-induced resting membrane hyperpolarization but TEA (1 mM) had no such effect. Glibenclamide (10 +microM), TEA (1 mM) and 4-AP (0.1 mM), given separately, all failed to modify the inhibitory actions of olprinone on (i) the absolute membrane potential level seen with ACh and (ii) the ACh-induced contraction. It is suggested that olprinone inhibits the ACh-induced contraction through an effect on the absolute level of membrane potential achieved with ACh in smooth muscle of the rabbit coronary artery. It is also suggested that glibenclamide-sensitive, ATP-sensitive K(+) channels do not play an important role in the olprinone-induced inhibition of the ACh-induced contraction.     

Effects of semotiadil fumarate, a novel Ca2+ antagonist, on cytosolic Ca2+ level and force of contraction in porcine coronary arteries.

1. The mechanisms of action of semotiadil fumarate, a novel Ca2+ antagonist, were examined by measuring the cytosolic Ca2+ level ([Ca2+]i) and force of contraction in porcine coronary arteries, and by determining [3H]-pyrilamine binding to bovine cerebellar membranes. 2. Semotiadil or verapamil (0.1 and 1 microM) inhibited both the high KCl-induced increases in [Ca2+]i and force in a concentration-dependent manner. 3. Histamine (30 microM) produced transient increases followed by sustained increases in [Ca2+]i and force, which were inhibited by semotiadil and verapamil (1 and 10 microM). The agents were different in that semotiadil reduced the maximum [Ca2+]i and force responses to histamine, but not pD2 values, whereas verapamil did reduce the pD2 values for histamine, but not the maximum responses. 4. Verapamil (10 microM), but not semotiadil, inhibited histamine-induced increases in [Ca2+]i and force in Ca(2+)-free solution. Neither semotiadil nor verapamil affected the increases in [Ca2+]i and force induced by caffeine. Semotiadil even at the higher concentration (10 microM) did not displace specific binding of [3H]-pyrilamine to bovine cerebellar membranes. 5. These results suggest that semotiadil inhibits both KCl- and histamine-induced contractions mainly by blocking voltage-dependent L-type Ca2+ channels.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions.

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of some organic calcium antagonists and other procedures affecting Ca2+ Translocation on KCl-induced contractions in the rat vas deferens.

1 Both phasic and tonic responses to KCl 160 mM were reduced by Ca2+ deprivation. After 90 min, the phasic response was abolished but 13 +/- 1.5% of the tonic response remained. This resistant component was still present if the Ca2+-free solution contained EGTA 0.1 mM. The tonic response was more resistant to deprivation in the prostatic half, while the phasic was more resistant in the epididymal half. KCl-induced contractions were completely restored 5 min after readmission of Ca2+. 2 Both the phasic and the tonic responses were reduced on lowering, and increased on raising [Ca2+]0. In 0.1 mM Ca2+, the phasic response was abolished, but 23 +/- 4% of the tonic response remained (mainly attributable to the prostatic half). These resistant contractions indicate that some of the extracellular Ca2+, especially in the prostatic half, is bound with high affinity, probably to the plasma membrane. 3 Incubation with LaCl3 (0.3-10 mM) for 15 min inhibited the phasic response more than the tonic. After incubation for 1 h, 3 mM LaCl3 abolished both phases. It is concluded that La3+ blocks Ca2+ channels most readily when they are opened during the spike. Hydralazine (0.76-5.1 mM) resembled LaCl3 in that it reduced the phasic response with little effect on the tonic. 4 MnCl2 (0.3-10mM) reduced the phasic but increased the tonic response at all concentrations. The augmenting effect may be due to release of intracellular Ca2+ or to inhibition of Ca2+ efflux. 5 The tonic response was inhibited more than the phasic response by nifedipine (0.002-0.01 microM), methoxyverapamil (0.06-2 microM), verapamil (0.2-1 microM), flunarizine (0.2-100 microM) and diazoxide (22-650 microM). With higher concentrations, only flunarizine, remained selective for the tonic response. It is concluded that flunarizine blocks Ca2+ channels most readily when opened during sustained spike-free depolarization. 6 Methoxyverapamil 48 microM and verapamil 100 microM virtually abolished both phases of the contraction to KCl 160 mM, but no more than 80% inhibition could be produced with nifedipine. It is concluded that voltage-sensitive Ca2+ channels exist in two sub-types, one of which is blocked by nifedipine, and both are blocked by verapamil, methoxyverapamil and flunarizine. Nitroprusside 17 microM had no effect on the phasic response but inhibited the nifedipine-resistant component of the tonic response. 7 Increasing [ca2+]0 reversed the effects of verapamil, methoxyverapamil, nifedipine and MnCl2, but not the effects of LaCl3. 8 Dantrolene sodium (1.25-25 microM) had no effect on KCl-induced contractions.     

The effects of ATP and alpha,beta-methylene-ATP on cytosolic Ca2+ level and force in rat isolated aorta.

1. The effects of a non-selective P2-receptor agonist ATP and a selective P2x-receptor agonist alpha,beta-methylene-ATP on intracellular free Ca2+ level ([Ca2+]i) and force were examined in rat isolated aorta without endothelium. 2. Both ATP (1-1000 microM) and alpha,beta-methylene-ATP (0.1-100 microM) induced transient increase followed by small sustained increase in [Ca2+]i in a concentration-dependent manner. Compared with the force induced by a high concentration of KCl, the force induced by alpha,beta-methylene-ATP was smaller and that induced by ATP was much smaller at a given [Ca2+]i. 3. An L-type Ca2+ channel blocker, verapamil (10 microM), completely inhibited the high K(+)-stimulated [Ca2+]i and force. Verapamil partially inhibited the transient and sustained increases in [Ca2+]i induced by 10 microM alpha,beta-methylene-ATP and the sustained increase but not the transient increase induced by 1 mM ATP. 4. In the absence of extracellular Ca2+ (with 0.5 mM EGTA) 1 mM ATP caused transient increase in [Ca2+]i while 10 microM alpha,beta-methylene-ATP was ineffective 5. ATP, but not alpha,beta-methylene-ATP, increased the tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) level. 6. These data suggest that ATP and alpha,beta-methylene-ATP increase [Ca2+]i by an activation of both L-type and non-L-type Ca2+ channels. In addition, ATP, but not alpha,beta-methylene-ATP, increases [Ca2+]i by a release of Ca2+ from an intracellular Ca2+ store. Possible reasons are discussed as to why the increase in [Ca2+]i due to ATP and alpha,beta-methylene-ATP resulted in only a small contraction.     

Effects of trimebutine on cytosolic Ca2+ and force transitions in intestinal smooth muscle

The effects of trimebutine maleate on cytosolic free Ca2+ and force transitions in the guinea-pig taenia cecum were studied by fura-2 fluorometry and tension recording. The addition of 80 mM K+ induced a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) and tension, followed by a sustained increase. Trimebutine (10 microM) suppressed both [Ca2+]i elevation and tension development. The tonic responses were more potently inhibited than the phasic responses. Phasic components gradually increased as the added K+ increased (10-40 mM). The relationship between the peak increases in [Ca2+]i and tension was not affected by trimebutine (10 microM). This means that trimebutine does not affect the Ca2+ sensitivity of contractile elements. In a high K+ and Ca(2+)-free medium, carbachol (10 microM) or caffeine (30 mM) caused transient [Ca2+]i elevation and tension development in the smooth muscle. Trimebutine (10 microM) decreased the amplitude of both responses. Trimebutine (10 microM) inhibited the spontaneous fluctuations in [Ca2+]i and motility of taenia cecum in the presence of tetrodotoxin (TTX; 0.3 microM). These results suggest that trimebutine has two types of inhibitory actions on intestinal smooth muscle; one, the inhibition of Ca2+ influx through voltage-dependent calcium channels, and the other, the inhibition of Ca2+ release from intracellular storage sites.     

Dissociation of P2 purinoceptor-mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta.

1. The effects of P2 agonists, adenosine-5'-triphosphate (ATP), alpha, beta-methylene-adenosine-5'-triphosphate (alpha, beta-me-ATP) and adenosine 5-O-(3-thiotriphosphate) (ATP gamma S), on the intracellular free Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction were examined in vascular smooth muscle of rat aorta. 2. ATP (0.1 microM-1 mM), alpha, beta-me-ATP (0.1-100 microM) and ATP gamma S (1-100 microM) induced transient increases followed by sustained increase in [Ca2+]i. The effects of these agonists were concentration-dependent. Compared with the effects of a high concentration of KCl (17.5-72.4 mM), the contractions induced by these P2 purinoceptor agonists were smaller at a given [Ca2+]i. 3. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), ATP gamma S (10 microM) induced large transient increase in [Ca2+]i with only small contraction in Ca(2+)-free solution. In contrast, alpha, beta-me-ATP (10 microM) induced only a very small increase in [Ca2+]i and contraction. 4. ATP (1 mM), alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), added during stimulation with 0.1 microM noradrenaline, induced additional and transient increases in [Ca2+]i which were also not associated with contraction. 5. High K+ (72.4 mM) increased MLC phosphorylation with a similar time course to that of the increase in [Ca2+]i (peak phosphorylation was 56% when [Ca2+]i increased to 100%). In contrast, the time course of the increase in MLC phosphorylation due to ATP (1 mM) did not coincide with that of the large increases in [Ca2+]i; MLC phosphorylation increased to only 31% when [Ca2+]i increased to 163%. The MLC phosphorylation due to alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), measured at peak [Ca2+]i, were only 19% and 14%, respectively, irrespective of a large increase in [Ca2+]i (138% and 188%, respectively). 6. The absence of a clear relationship between P2-purinoceptor-mediated increase in [Ca2+]i (either by Ca2+ influx or Ca2+ release) and MLC phosphorylation or force generation appears to imply that elevation in [Ca2+]i does not contribute to these responses.     

Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the Gq/11 protein

BACKGROUND AND PURPOSE: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca2+ provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M3 muscarinic receptors. We examined effects of YM-254890 (YM), a Gq/11-specific inhibitor, on contraction, NSCC currents and [Ca2+]i elevation induced by carbachol (CCh). EXPERIMENTAL APPROACH: Isometric tension was recorded from ciliary muscle bundles excised from bovine eyes. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp and the intracellular free Ca2+ concentration [Ca2+]i was monitored using the Fluo-4 fluorophore. Existence and localization of M3 receptors and the alpha subunit of Gq/11 (Galpha(q/11)) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies. KEY RESULTS: Both phasic and tonic components of contractions evoked by 2 microM CCh were inhibited by YM (3-10 microM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 microM) evoked an NSCC current as well as an elevation of the [Ca2+]i. Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 microM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M3 receptors and Galpha(q/11). CONCLUSIONS AND IMPLICATIONS: The tonic as well as phasic component of the ciliary muscle contraction appears to be under control of signals conveyed by a G(q/11)-coupled pathway. YM is a useful tool to assess whether Gq/11 is involved in a signal transduction system.     

Effects of azelastine on contraction of guinea pig tracheal smooth muscle

Azelastine [4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepine-4-yl)-1(2H)-phthalazinone hydrochloride] is a new anti-asthmatic drug. We examined the mechanism of its inhibitory action on guinea pig tracheal smooth muscle contraction by measuring membrane potential and isometric force using intracellular microelectrodes and a micro-force transducer. The mean resting membrane potential of guinea pig tracheal muscle cells was -54 mV. Perfusion with 20 mM tetraethylammonium (TEA) caused membrane depolarization and elicited spontaneous action potentials. Azelastine (1-100 microM) suppressed both the amplitude and maximal rate of rise of the action potentials in a concentration-dependent manner. Complete abolition occurred at 100 microM. Similarly, azelastine (0.1-100 microM) inhibited and abolished 50 mM KCl-induced contractions. These results suggest that azelastine may inhibit voltage-dependent Ca2+ channels. Next, pretreatment of tracheal muscle (for 15 min) with azelastine (0.01-100 microM) inhibited subsequent acetylcholine (ACh) (0.01-100 microM)-induced contractions. Azelastine, 100 microM, completely abolished the ACh-induced contractions. In contrast, high concentrations of Ca2+ channel antagonists diltiazem (10-100 microM) or nifedipine (20 microM), and Ca2(+)-free solution, only partially depressed the ACh contractions suggesting that azelastine has an additional effect on intracellular Ca2+ release. In Ca2(+)-free solution (containing 0.5 mM EGTA), azelastine (1-100 microM) depressed and abolished the transient contractions induced by 10 microM ACh. We conclude that azelastine inhibits airway constriction by inhibiting both voltage-sensitive Ca2+ slow channels on the cell membrane and Ca2+ release from a intracellular storage site.     

The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells.

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.     

Effect of thimerosal, a preservative in vaccines, on intracellular Ca2+ concentration of rat cerebellar neurons

The effect of thimerosal, an organomercurial preservative in vaccines, on cerebellar neurons dissociated from 2-week-old rats was compared with those of methylmercury using a flow cytometer with appropriate fluorescent dyes. Thimerosal and methylmercury at concentrations ranging from 0.3 to 10 microM increased the intracellular concentration of Ca2+ ([Ca2+]i) in a concentration-dependent manner. The potency of 10 microM thimerosal to increase the [Ca2+]i was less than that of 10 microM methylmercury. Their effects on the [Ca2+]i were greatly attenuated, but not completely suppressed, under external Ca(2+)-free condition, suggesting a possibility that both agents increase membrane Ca2+ permeability and release Ca2+ from intracellular calcium stores. The effect of 10 microM thimerosal was not affected by simultaneous application of 30 microM L-cysteine whereas that of 10 microM methylmercury was significantly suppressed. The potency of thimerosal was similar to that of methylmercury in the presence of L-cysteine. Both agents at 1 microM or more similarly decreased the cellular content of glutathione in a concentration-dependent manner, suggesting an increase in oxidative stress. Results indicate that thimerosal exerts some cytotoxic actions on cerebellar granule neurons dissociated from 2-week-old rats and its potency is almost similar to that of methylmercury.     

The effect of L-type calcium channel modulators on the mobilization of intracellular calcium stores in guinea-pig intestinal smooth muscle.

1. The action of Ca2+ channel modulators has been examined on the intracellular Ca2+ signal in the longitudinal smooth muscle cells of the guinea-pig intestine after exposure to histamine and to agents known to affect intracellular Ca2+ stores. Isometric contraction has been measured simultaneously with front-surface fluorometry of fura 2-loaded preparations. 2. Histamine (10 microM) evoked a phasic and tonic increase in [Ca2+]i and contraction which were both sensitive to the Ca2+ channel blockers, nimodipine and D600. 3. Caffeine (10 mM) evoked in rapid increase in [Ca2+]i which was sustained as long as the preparation was exposed to the drug, whereas the contractile response was only phasic. In the presence of nimodipine 1 microM, the phasic contraction was absent although the fura 2-Ca2+ signal amounted to 32% of the control. 4. Ryanodine (10 microM) evoked a slow increase in [Ca2+]i and a contraction, both of which were reversed after exposure to nimodipine (1 microM) or D600 (10 microM). In the presence of diazoxide (500 microM), a hyperpolarizing agent, the ryanodine-evoked increase in [Ca2+]i and in muscle tone were inhibited. 5. Thapsigargin (1 microM) also produced an increase in [Ca2+]i and a contraction both of which were blocked by nimodipine (1 microM). 6. In Ca2+-free solution, histamine 10 microM evoked non-reproducible phasic Ca2+ signal and contraction. This response was recovered after refilling in Ca2+ containing solution. The recovery was blocked by nimodipine, D600 or diazoxide and was facilitated by the Ca2+ channel activator, Bay K 8644. When the refilling medium was supplemented with thapsigargin, the recovered response was significantly reduced, but Bay K 8644 still had some action. 7. The present results show that blockage of L-type Ca2+ channels inhibited changes in [Ca2+]i evoked by histamine, caffeine and ryanodine which are generally attributed to Ca2+ mobilization from intracellular stores. They also show that when the tissue was exposed to nimodipine, D600 and diazoxide during the procedure of refilling after depletion of intracellular stores, the action of histamine on [Ca2+]i and contraction was blocked. Bay K 8644 had an opposite effect even when the Ca2+ pumping activity of the sarcoplasmic reticulum was reduced by thapsigargin. This indicates that refilling of intracellular Ca2+ stores depleted by histamine in guinea-pig intestine mainly occurred through L-type Ca2+ channels.     

Electrical and mechanical properties of the capsular smooth muscles of the rabbit prostate in relation to the actions of the alpha 1-adrenoceptor blocker, YM-12617.

1. Electrical and mechanical properties of smooth muscle cells of the rabbit prostate capsule and the actions of the alpha 1-adrenoceptor blocker, YM-12617, were investigated using microelectrode and isometric tension recording methods. 2. The capsular muscles comprised thick and thin muscle bundles. In the former, noradrenaline (NA; 0.1-10 microM) provoked the phasic and tonic mechanical responses, with twitch contractions superimposed on the tonic response. YM-12617, in concentrations over 1 nM inhibited the contraction evoked by any given concentration of NA. Yohimbine (up to 10 microM) slightly inhibited the NA-induced contraction whilst clonidine (up to 10 microM) and acetylcholine (ACh; up to 10 microM) produced no mechanical response. 3. In thin muscle bundles, NA (0.1-10 microM) produced a contraction but the phasic response was small and the tonic response was negligible. These changes were blocked by YM-12617. In contrast, ACh (0.1-10 microM) produced atropine-sensitive, large phasic and tonic responses similar to those observed on application of NA to thick muscle bundles. 4. In thin and thick muscle bundles, the mean resting membrane potentials were -54 and -56 mV, respectively, values which were not statistically different. However, in thick muscle bundles, NA (over 0.1 microM) depolarized the membrane in a concentration-dependent manner and produced repetitive spike generation; ACh (up to 1 microM) did not modify the membrane potential. In thin muscle bundles, the above concentrations of NA hyperpolarized the membrane but ACh produced a large depolarization with repetitive spike generation. 5. In thick muscle bundles, nifedipine (0.3 microM) blocked twitch contractions generated spontaneously or provoked by application of NA with no effect on phasic and tonic responses. The NA-induced depolarization persisted after superfusion with nifedipine up to a concentration of 1.0 microM. In a Ca-free solution containing 2 mM EGTA, NA produced only the phasic responses, and re-addition of Ca (2.6 mM) restored the generation of a tonic response. 6. After application of 0.3 microM nifedipine, the effects of YM-12617 and prazosin were observed on the tonic component of the NA-induced contraction of thick muscle bundles. The ID50 values for YM-12617 and prazosin were 1 nM and 15 nM, respectively (n = 4). YM-12617 shifted the NA concentration-response curve to the right in a concentration-dependent and parallel manner. The Schild plot yielded a straight line with slope of 0.97 +/- 0.05, (n = 4). The pA2 value for YM-12617 was 10.4 +/- 0.05, (n = 4).(ABSTRACT TRUNCATED AT 400 WORDS)