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)     

Cyclopiazonic acid, an inhibitor of Ca(2+)-ATPase in sarcoplasmic reticulum, increases excitability in ileal smooth muscle.

1. Effects of cyclopiazonic acid (CPA), a specific inhibitor of Ca(2+)-ATPase in endo- and sarcoplasmic reticulum (ER/SR), on contractile responses, cytosolic Ca2+ concentration and spontaneous electrical activity were examined in ileal longitudinal smooth muscle strips. 2. After intracellular stored Ca2+ in intact ileal strips was depleted by application of 25 mM caffeine in Ca(2+)-free solution, Ca(2+)-loading was performed in the absence or presence of 10 microns CPA in a standard solution containing 2.2 mM Ca2+. Subsequent application of caffeine in Ca(2+)-free solution induced a phasic contraction which was significantly smaller in the strip pretreated with CPA than that in the control. 3. Spontaneous and 20 mM K(+)-induced contractions in the presence of 1 microM atropine were markedly enhanced by 1-30 microM CPA, whereas that induced by 80 mM K+ was not. The magnitude of repetitive transient elevation of cytosolic Ca2+ concentration ([Ca2+])i) and concomitant phasic contractions were markedly enhanced by CPA. The effects were abolished by 10 microM verapamil and restored by 10 microM Bay K 8644. 4. Application of 10 microM CPA depolarized the cell by about 5 mV, decreased the action potential (AP) afterhyperpolarization and markedly increased the frequency of spontaneous AP. These effects were mimicked by 100 nM charybdotoxin. 5. The rate of decay of [Ca2+]i and tension after the bathing solution was changed from one containing 140 mM K+ and 2.2 mM Ca2+ to one containing 5.9 mM K+ and 0 mM Ca2+ was significantly slowed when 10 microM CPA was added to the latter solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile and relaxant effects of phorbol ester in the intestinal smooth muscle of guinea-pig taenia caeci.

1. Effects of phorbol esters on the cytosolic Ca2+ level ([Ca2+]i) and muscle tension in the intestinal smooth muscle of guinea-pig taenia caeci were examined. 2. 12-Deoxyphorbol 13-isobutyrate (DPB, 1 microM) did not change the [Ca2+]i and tension in resting muscle. 3. In high K(+)-stimulated muscle, 1 microM DPB transiently augmented the contraction and decreased [Ca2+]i. 12-Deoxyphorbol 13-isobutyrate 20-acetate (1 microM) and phorbol 12, 13-dibutyrate (1 microM) showed similar effects to DPB whereas phorbol 12-myristate 13-acetate (1 microM) and phorbol 12, 13-didecanoate (1 microM) were ineffective. 4. DPB (1 microM) inhibited both [Ca2+]i and tension stimulated by 300 nM carbachol or 3 microM histamine. In the presence of a higher concentration of carbachol (1 microM), DPB decreased [Ca2+]i and transiently increased muscle tension. 5. In the muscle strips permeabilized with bacterial alpha-toxin, 1 microM DPB shifted the Ca(2+)-tension curve to the left. An inhibitor of protein kinase C, H-7 (30 microM), inhibited the effect of DPB. 6. DPB did not change the high K(+)-induced contraction in the muscle strips pretreated with 3 microM phorbol 12-myristate 13-acetate for 24 h. 7. These results suggest that activation of protein kinase C has dual effects; it augments contraction by increasing the Ca2+ sensitivity of the contractile elements and it inhibits contraction by decreasing [Ca2+]i.     

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.     

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) in vascular smooth muscle

The inhibitory effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca2+ level ([Ca2+]i) were examined using isolated rabbit aorta loaded with a fluorescent Ca2+ indicator, fura-2. TMB-8 (100 microM) decreased the high K(+)-induced increase in muscle tension, and [Ca2+]i and 45Ca2+ influx to their respective resting levels. TMB-8 (100 microM) almost completely inhibited the increase in [Ca2+]i and 45Ca2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 microM) inhibited the remaining portion of the contraction without additional decrease in [Ca2+]i. The inhibitory effect of TMB-8 on high K(+)-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca2+ concentrations or by the Ca2+ channel activators, CGP 28,392 and by Bay K8644. In Ca(2+)-free solution, norepinephrine-induced transient increases in [Ca2+]i and muscle tension and 100 microM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca2+]i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 microM) did not inhibit the Ca(2+)-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

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)     

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.     

Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.

1. The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 microM, respectively. 2. Atropine (10 microM) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3. AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 119 and high affinity for 4-DAMP in antagonizing this response. 4. The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mM EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mM) in the continued presence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-images of smooth muscle cells and endothelial cells in one confocal plane in femoral artery segments of the rat.

Simultaneous recording of Ca2+-images in one confocal plane from vascular smooth muscle cells (SMCs) and endothelial cells (ECs) of an intact rat femoral artery segment was performed using indo-1 and a confocal microscope. During application of 10 microM acetylcholine (ACh), elevation and oscillation of intracellular Ca2+ concentration ([Ca2+]i) were observed in ECs but not in SMCs. Sequential conduction of Ca2+ oscillation from an EC to the neighboring ECs in one longitudinal direction was often observed in the presence of ACh. On the other hand, the activation of voltage-dependent Ca2+ channels by external 30 mM K+ resulted in the elevation of [Ca2+]i only in SMCs. When 10 microM ACh was added in the presence of 30 mM K+, it was observed in one confocal plane that [Ca2+]i in ECs and SMCs was almost simultaneously increased and decreased, respectively. The simultaneous recording method in this intact preparation will provide a line of valuable information about the interactions between SMCs and ECs, based on spatio-temporal analyses of absolute values of [Ca2+]i in individual cells.     

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)     

Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium

Resveratrol causes endothelium dependent and independent relaxation of vascular smooth muscle. This study investigated the mechanisms behind the effect of resveratrol on vascular tone. Resveratrol (0.1 mM) inhibited KCl-stimulated contractions in endothelium-denuded rat aorta and this inhibition was not reversed by tetraethylammonium (TEA) (5 mM), glyburide (3 microM), ouabain (0.1 mM), thapsigargin (1 microM), or indomethacin (10 microM). KCl (90 mM) increased the intracellular free calcium concentration ([Ca2+]i) in the isolated smooth muscle cells from the rat aorta and resveratrol (0.1 mM) did not inhibit the KCl-stimulated [Ca2+]i increase. The CaCl2 (0.1-100 microM) stimulated contractions were inhibited by resveratrol (0.1 mM) in the Triton X-100 skinned smooth muscle of the aorta. In heart valve endothelium, resveratrol (0.1 mM) augmented the acetylcholine (10 microM) stimulated [Ca2+]i increase. Resveratrol-induced augmentation of the acetylcholine-stimulated [Ca2+]i elevation was reversed by glyburide (3 microM), but not by TEA (5 mM). The present study indicated that resveratrol affected vascular smooth muscle and endothelium in different ways. Resveratrol decreased the Ca2+ sensitivity but did not affect the KCl-stimulated [Ca2+]i increase in the vascular smooth muscle. In the endothelial cells, resveratrol enhanced the agonist-stimulated [Ca2+]i increase that might trigger nitric oxide synthesis from endothelial cells.     

Mechanisms underlying the inhibitory effect of dibutyryl cyclic AMP in vascular smooth muscle.

The mechanism by which dibutyryl cyclic AMP (db-cAMP) induces vasodilatation was examined in isolated rat aorta. The contraction induced by norepinephrine (NE) was more sensitive to the inhibitory effect of db-cAMP than that induced by high K+, and the contraction induced by lower concentrations of each stimulant was more sensitive to db-cAMP than that induced by higher concentrations. Db-cAMP at 10 microM inhibited the increases in muscle tension and cytosolic Ca2+ level ([Ca2+]i) without changing the [Ca2+]i-tension relationship, suggesting that the inhibitory effect is mainly due to a decrease in [Ca2+]i. A higher concentration (300 microM) of db-cAMP inhibited muscle tension more strongly than [Ca2+]i suggesting that db-cAMP decreases Ca2+ sensitivity of contractile elements. In contrast, 10 microM verapamil inhibited the NE-stimulated [Ca2+]i more strongly than the NE-induced contraction. The verapamil-insensitive portion of the NE-stimulated [Ca2+]i and contraction was inhibited by db-cAMP, suggesting that db-cAMP and verapamil act by different mechanisms. In Ca(2+)-free solution, 1 microM NE induced transient increases in muscle tension and [Ca2+]i. The transient contraction was inhibited by 1 mM db-cAMP more strongly than [Ca2+]i. An activator of adenylate cyclase, forskolin, showed inhibitory effects similar to those of db-cAMP. The inhibitory effects of db-cAMP and forskolin were inversely proportional to [Ca2+]i before the addition of these inhibitors. These results suggest that db-cAMP inhibits smooth muscle contraction by decreasing [Ca2+]i and the Ca2+ sensitivity of contractile elements, and that both of these effects are stronger when [Ca2+]i is lower.     

The mechanism of the decrease in cytosolic Ca2+ concentrations induced by angiotensin II in the high K(+)-depolarized rabbit femoral artery

1. Using front-surface fluorometry of fura-2-loaded strips, and measuring the transmembrane 45Ca2+ fluxes of ring preparations of the rabbit femoral artery, the mechanism underlying a sustained decrease in the cytosolic Ca2+ concentration ([Ca2+]i) induced by angiotensin II (AT-II) was investigated. 2. The application of AT-II during steady-state 118 mM K(+)-induced contractions caused a sustained decrease in [Ca2+]i following a rapid and transient increase in [Ca2+]i, while the tension was transiently enhanced. 3. When the intracellular Ca2+ stores were depleted by thapsigargin, the initial rapid and transient increase in [Ca2+]i was abolished, however, neither the sustained decrease in [Ca2+]i nor the enhancement of tension were affected. 4. Depolarization with 118 mM K+ physiological salt solution containing 1.25 mM Ba2+ induced a sustained increase in both the cytosolic Ba2+ concentration ([Ba2+]i) level and tension. However, the application of 10(-6) M AT-II during sustained Ba(2+)-contractions was found to have no effect on [Ba2+]i, but it did enhance tension. 5. After thapsigargin treatment, AT-II neither decreased nor increased the enhanced Ca2+ efflux rate induced by 118 mM K(+)-depolarization, whereas AT-II did increase the enhanced 45Ca2+ influx and the 45Ca2+ net uptake induced by 118 mM K(+)-depolarization. 6. Pretreatment with calphostin-C, partially, but significantly inhibited the decrease in [Ca2+]i induced by AT-II. 7. These findings therefore suggest that AT-II stimulates Ca2+ sequestration into the thapsigargin-insensitive Ca2+ stores, and thus induces a decrease in [Ca2+]i in the high external K(+)-stimulated rabbit femoral artery.     

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)     

Antagonism of calcium by zinc in guinea-pig isolated taenia caeci and trachealis muscle.

1 In guinea-pig isolated taenia caeci and trachealis bathed in a K+-rich, Ca2+-free medium, CaCl2 (0.01-10 mM) produced a concentration-dependent contraction. Zn2+ (0.01-1 mM), Cd2+ (0.01-1 mM), verapamil (0.01-100 microM) and trifluoperazine (1-100 microM) were effective antagonists of CaCl2-induced responses. 2 Zn2+ and Cd2+ in concentrations from 0.01 to 1 mM were without effect on the tone of taenia and trachea in normal Tris solution. Conversely, Zn2+ and Cd2+, in concentrations of 1 mM, caused contraction of these tissues in a K+-rich, Ca2+-free medium. Zn2+ (1 mM)-induced contractions of taenia and trachea were completely inhibited by verapamil (10 microM). 3 In taenia and trachea skinned of their plasma membranes, tension development induced by Ca2+ (10 microM or 1 microM, respectively) was unaffected by verapamil (100 microM), whereas trifluoperazine (100 microM) depressed the maximal tension produced by Ca2+. Segments of skinned preparations contracted in response to low concentrations of Zn2+ (10 microM) or Cd2+ (10 microM). 4 It is concluded that Zn2+ may suppress Ca2+-induced spasm by a direct action on the binding sites of the Ca2+ channel.     

A patch-clamp study of K(+)-channel activity in bovine isolated tracheal smooth muscle cells.

1. Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K(+)-channels in these cells were studied by the patch-clamp recording technique. 2. Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 microM. Large unitary currents of both Ca(2+)-dependent and -independent types were observed. Measured between + 20 and + 40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3. Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to much greater than + 40 mV, suggesting that both currents were carried by K(+)-channels. 4. The Ca(2+)-dependent and -independent K(+)-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100 microM) applied to the cytosolic surface of the patches. 5. Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 microM. Ca(2+)-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of endothelin on the mechanical activity and cytosolic calcium level of various types of smooth muscle.

1. Effects of porcine/human endothelin (endothelin-1), a novel vasoconstrictor peptide, on various smooth muscles were examined. 2. In rat aorta, endothelin (1 pM-30nM) induced contraction in a concentration-dependent manner. Removal of endothelium shifted the concentration-response curve to the left. When added during the sustained contraction induced by 0.1 microM noradrenaline, endothelin (1 nM) induced a relaxation that was inhibited by removing endothelium or by methylene blue. 3. In rat aorta without endothelium, endothelin (1-30 nM) increased cytosolic Ca2+ level [( Ca2+]cyt) followed by contraction. Endothelin induced less contraction than high K+ at a given [Ca2+] cyt when the concentration of endothelin was lower (1-3nm) and/or during the early phase of the contraction (less than 10 min). In contrast, endothelin induced a greater contraction than KCl after prolonged exposure to high concentrations (greater than 10 nM). 4. The increase in [Ca2+]cyt due to endothelin was strongly inhibited by 10 microM verapamil or 0.3 microM nicardipine although muscle contraction was only partially inhibited. 5.In Ca2+ -free solution, endothelin (30 nM) induced a transient increase in [Ca2+] cyt and a slow increase in muscle tension. After a prolonged incubation in Ca2+-free solution, endothelin (30 nM) still induced a slow increase in tension without changing [Ca2+]cyt. This contraction was inhibited by 1 microM sodium nitropusside or 10 microM forskolin. 6. In canine trachea and guinea-pig uterus, endothelin (30 nM) induced sustained contraction with an increase in [Ca2+]cyt. In the absence of external Ca2+, endothelin (30 nM) induced a sustained contraction in canine trachea without changing [Ca2+]cyt. In guinea-pig vas deferens, taenia caeci and ileal longitudinal muscle, endothelin induced small increases in [Ca2+]cyt and tension. 7. In permeabilized smooth muscles, endothelin (30 nM) did not change the muscle tone. 8. These results suggest that endothelin acts on the endothelium and increases the synthesis or release of endothelin-derived relaxing factor (EDRF). These results also suggest that endothelin acts directly on smooth muscle and increases [Ca2+]cyt by releasing Ca2+ from sites and increasing Ca2+ influx through the verapamil- and 1,4-dihydropyridine-sensitive pathway. Endothelin seems to decrease Ca2+ -sensitivity of contractile elements at lower concentrations and/or during the early phase of the contraction, whereas it increases Ca2+ -sensitivity at higher concentrations during the sustained phase of the contraction. Furthermore, endothelin induces a contraction that is not dependent on [Ca2+]cyt.     

Elevation of cytosolic calcium by imidazolines in mouse islets of Langerhans: implications for stimulus-response coupling of insulin release.

1. Microfluorimetry techniques with fura-2 were used to characterize the effects of efaroxan (200 microM), phenotolamine (200-500 microM) and idazoxan (200-500 microM) on the intracellular free Ca2+ concentration ([Ca2+]i) in mouse isolated islets of Langerhans. 2. The imidazoline receptor agonists efaroxan and phentolamine consistently elevated cytosolic Ca2+ by mechanisms that were dependent upon Ca2+ influx across the plasma membrane; there was no rise in [Ca2+]i when Ca2+ was removed from outside of the islets and diazoxide (100-250 microM) attenuated the responses. 3. Modulation of cytosolic [Ca2+]i by efaroxan and phentolamine was augmented by glucose (5-10 mM) which both potentiated the magnitude of the response and reduced the onset time of imidazoline-induced rises in [Ca2+]i. 4. Efaroxan- and phentolamine-evoked increases in [Ca2+]i were unaffected by overnight pretreatment of islets with the imidazolines. Idazoxan failed to increase [Ca2+]i under any experimental condition tested. 5. The putative endogenous ligand of imidazoline receptors, agmatine (1 microM-1 mM), blocked KATP channels in isolated patches of beta-cell membrane, but effects upon [Ca2+]i could not be further investigated since agmatine disrupts fura-2 fluorescence. 6. In conclusion, the present study shows that imidazolines will evoke rises in [Ca2+]i in intact islets, and this provides an explanation to account for the previously described effects of imidazolines on KATP channels, the cell membrane potential and insulin secretion in pancreatic beta-cells.     

Effects of diltiazem on calcium concentrations in the cytosol and on force of contractions in porcine coronary arterial strips.

1. Using front-surface fluorometry with fura-2-loaded porcine coronary arterial strips, we simultaneously measured effects of a Ca2+ antagonist, diltiazem, on cytosolic Ca2+ concentrations [( Ca2+]i) and on tension development. 2. In the presence of extracellular Ca2+ (1.25 mM), histamine concentration-dependently induced abrupt (the first component) and then sustained (the second component) elevations of [Ca2+]i. In the absence of extracellular Ca2+, histamine induced transient elevations of [Ca2+]i, and the time course was similar to that of the first component observed in the presence of extracellular Ca2+. Histamine caused a greater contraction for a given change in [Ca2+]i than did potassium, at [Ca2+]i over 300 nM. 3. Diltiazem, 10(-8)M to 10(-5)M, concentration-dependently inhibited the second component of [Ca2+]i elevation and tension development induced by histamine (10(-5) M). Only at higher concentrations (over 10(-5) M) did diltiazem inhibit the first component of increases in [Ca2+]i and tension development induced by histamine, both in the presence and absence of extracellular Ca2+. 4. Diltiazem (10(-6) M) inhibited increases in [Ca2+]i and tension development induced by cumulative applications of extracellular Ca2+ during K(+)-depolarization. The curve of [Ca2+]i against tension of these Ca2(+)-induced contractions obtained in diltiazem-treated strips overlapped with that obtained in untreated strips. This suggests that diltiazem has no direct effects on contractile elements. 5. In contrast, the histamine-induced Ca2(+)-tension curve (second component) was shifted in parallel to the left by diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolving mechanisms of action of alverine citrate on phasic smooth muscles

BACKGROUND AND PURPOSE: We have investigated the mechanisms underlying the paradoxical ability of the antispasmodic, alverine, to enhance spontaneous activity in smooth muscles while suppressing evoked activity. EXPERIMENTAL APPROACH: The effects of alverine on spontaneous and induced contractile activity were examined in preliminary experiments with various smooth muscles. More detailed effects were also investigated by recording membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and tension from single-bundle detrusor smooth muscle (DSM) of the guinea-pig urinary bladder. KEY RESULTS: Alverine (10 microM) increased the frequency and amplitude of spontaneous action potentials, transient increases in [Ca2+]i and associated contractions. Alverine also decreased action potential rate of decay, suggesting inhibition of L-type Ca channel inactivation. Charybdotoxin (50 nM) but neither cyclopiazonic acid (10 microM) nor Bay K 8644 (10 microM) attenuated alverine-induced enhancement of spontaneous contractions. Alverine suppressed contractions produced by high K (40 mM) or ACh (10 microM), without affecting electrical responses and with little suppression of increases in [Ca2+]i. This feature was very similar to that of the effects of the Rho kinase inhibitor Y-27632 (10 microM). CONCLUSIONS AND IMPLICATIONS: Alverine may increase Ca influx during action potentials due to inhibition of the inactivation of L-type Ca channels, but may also suppress evoked activity by inhibiting the sensitivity of contractile proteins to Ca2+. The proportional contribution of Ca-dependent and Ca-independent contractions in DSM may differ between spontaneous and evoked activity, necessitating further investigations into the interactions between these pathways for assessing the therapeutic potential of alverine to treat DSM dysfunction.