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.     

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.     

Temporal changes in the calcium-force relation during histamine-induced contractions of strips of the coronary artery of the pig.

1. We examined temporal changes in the relationship between cytosolic calcium concentrations ([Ca2+]i) and developed tension during histamine-induced contractions of strips of the coronary artery of the pig, by making use of simultaneous measurements of fura-2 fluorescence and force. 2. The relationship between [Ca2+]i and developed tension observed with cumulative applications of extracellular Ca2+ ([Ca2+]o), ranging from 0 mM to 10 mM, during 118 mM K(+)-depolarization was similar to that observed in chemically skinned strips of the porcine coronary artery, as noted by other investigators. [Ca2+]i at 0 mM [Ca2+]o, at 50% of maximum, and at maximum tension development were 76 nM, 424 nM, and 3050 nM, respectively. 3. Cumulative applications of histamine induced dose-dependent increases in [Ca2+]i and tension and the extent of tension for a given change in [Ca2+]i increased, i.e. greater effectiveness of [Ca2+]i-tension relationship, than seen with K(+)-depolarization. 4. When histamine 10(-5) M was applied, [Ca2+]i abruptly rose and reached the first peak within several seconds. After a slight dip at 30 s, [Ca2+]i reached a second peak at 3 min, and then gradually declined. On the other hand, tension developed rapidly reached a maximum at 4 min, then gradually declined. The relation between [Ca2+]i and tension in the early, rising phase of contraction was similar to that obtained during depolarization. At the time of maximum tension development, the relation was greater than that observed during depolarization, which persisted in the phase of declining tension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle.

Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.     

The mechanism of relaxation induced by atrial natriuretic peptide in the porcine renal artery.

1. The mechanisms underlying the relaxation of the porcine renal artery induced by atrial natriuretic peptide (ANP) were investigated, using front-surface fluorimetry with fura-2 and receptor-coupled permeabilization by alpha-toxin. 2. ANP decreased the cytosolic Ca2+ concentration ([Ca2+]i) and tension during the contraction induced by a high external K+ solution, in a concentration-dependent manner. This ANP-induced decrease in [Ca2+]i during the contraction induced by high K+ solution was composed of two phases, an initial rapid phase, followed by a maintenance phase. The initial rapid decrease in [Ca2+]i, but not the maintained decrease in [Ca2+]i, was inhibited when the tissue was treated with thapsigargin, a selective Ca2+ pump inhibitor of the sarcoplasmic reticulum. When the tissues were treated with thapsigargin and external Ca2+ was replaced by Ba2+, which cannot be transported by the Ca2+ pump, ANP did not induce a decrease in [Ba2+]i, even though the elevation of tension induced by Ba2+ was strongly inhibited. 3. In the absence of extracellular Ca2+, ANP inhibited the release of Ca2+ from the intracellular store induced by noradrenaline (NA). 4. The [Ca2+]i (abscissa scale)-tension (ordinate scale) relationship observed during the contraction induced by various concentrations of high external K+ solution was shifted downwards by the addition of 10(-8) M ANP, indicating that, at any given [Ca2+]i, the tension generated by high K+ solution was considerably inhibited by the addition of 10(-8) M ANP. The [Ca2+]i-tension curve of the contraction obtained by the cumulative application of external Ca2+ (0-3.75 mM) during depolarization with 118 mM K+ solution was shifted to the left by 3 x 10(-7) M NA. This NA-induced [Ca2+]i-tension relationship was shifted to the right by 10(-8) M ANP, indicating that the ANP-induced reduction of Ca(2+)-sensitivity operates during the contraction induced by NA. 5. In alpha-toxin-permeabilized preparations, ANP induced relaxation of tissues precontracted with a mixture of 3 x 10(-7) M Ca2+, 10(-5) M guanosine 5'-triphosphate (GTP) and 10(-6) M NA. Thus a component of ANP-induced relaxation took place by way of a reduction in the Ca2+ sensitivity of the myofilaments, independent of changes in [Ca2+]i. 6. These results indicate that ANP induces relaxation of the porcine renal artery by: (1) reducing [Ca2+]i mainly via the activation of the Ca2+ pumps located on the sarcoplasmic reticulum and sarcolemma, as well as via inhibition of agoinist-induced release of Ca2+ from the intracellular store; and (2) decreasing the Ca(2+)-sensitivity of the contractile elements.     

Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig.

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.     

Front-surface fluorometry with fura-2 and effects of nitroglycerin on cytosolic calcium concentrations and on tension in the coronary artery of the pig.

1. By use of front-surface fluorometry and fura-2-loaded strips of the coronary artery of the pig, the effects of nitroglycerin (NG) on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension development were measured simultaneously. 2. Both high K+ depolarization and histamine increased [Ca2+]i and tension in a concentration-dependent manner. However, the tension development in relation to the [Ca2+]i increase ([Ca2+]i-tension relation) observed with histamine was much greater than that observed with K+ depolarization. 3. NG reduced in a concentration-dependent manner both [Ca2+]i and tension, irrespective of whether the vascular strips were in a resting state or during exposure to high K+ or to histamine stimulation. However, the extent of reduction in tension (relaxation) was greater than that expected from the reduction in [Ca2+]i based on the [Ca2+]i-tension relationship observed with K(+)-depolarization. 4. In the absence of extracellular Ca2+, NG depleted stored Ca2+ and also inhibited Ca2+ release from histamine-sensitive stores, but had no effect on the caffeine-sensitive stores. NG inhibited the caffeine-induced tension development with no change in [Ca2+]i. 5. We suggest that NG relaxes the coronary artery of the pig by reducing [Ca2+]i and also by directly controlling contractile elements through second messengers not related to changes in [Ca2+]i.     

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)     

Changes in the cytosolic Ca2+ concentration and Ca(2+)-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery

1. To investigate the underlying mechanism for the angiotensin II-induced desensitization of the contractile response during the prolonged stimulation of the vascular smooth muscle, we determined the effects of angiotensin-II on (1) cytosolic Ca2+ concentration ([Ca2+]i) and tension using fura-2-loaded medial strips of the rabbit femoral artery, (2) 45Ca2+ influx in ring preparations, and (3) Ca(2+)-sensitivity of the contractile apparatus in alpha-toxin permeabilized preparations. 2. In the presence of extracellular Ca2+, high concentrations of angiotensin-II elicited biphasic increases in [Ca2+]i and tension, which consisted of initial transient and subsequent lower and sustained phases. 3. The 45Ca2+ influx initially increased after the application of 10(-6) M angiotensin-II, and thereafter gradually decreased. At 20 min after the application, there was a discrepancy between the level of [Ca2+]i and the extent of 45Ca2+ influx. 4. The relationships between [Ca2+]i and tension suggested that the angiotensin-II-induced increase in the Ca(2+)-sensitivity of the contractile apparatus was maintained during the desensitization of smooth muscle contraction. 5. When 10(-6) M angiotensin-II was applied during the sustained phase of contraction induced by 118 mm K(+)-depolarization, at 10 min after the application, the [Ca2+]i levels were significantly lower and the tension levels were significantly higher than those prior to the application of angiotensin-II. 6. In conclusion, the decrease in [Ca2+]i, which is partially due to the inhibition of the Ca2+ influx, is mainly responsible for the desensitization evoked by high concentrations of angiotensin-II, and angiotensin-II seems to activate additional mechanisms which inhibit Ca2+ signaling during prolonged stimulation.     

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.     

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)     

Leukotriene C(4) enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway

1. An unsaturated fatty acid, leukotriene C(4) (LTC(4)), has a potent contractile effect on human airway smooth muscle, and has been implicated in the pathogenesis of human asthma. Using front-surface fluorometry with fura-PE3, the effect of LTC(4) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were investigated in porcine tracheal smooth muscle strips. 2. The application of LTC(4) induced little or no contraction despite a small and transient increase in [Ca(2+)](i). In the presence of LTC(4), however, the contractions evoked by high K(+) depolarization or a low concentration of carbachol (CCh) were markedly enhanced without inducing any changes in the [Ca(2+)](i) levels, thus indicating that LTC(4) increases the Ca(2+) responsiveness of the contractile apparatus. This LTC(4)-induced increase in Ca(2+) responsiveness could partly be reproduced in the permeabilized preparation of tracheal smooth muscle strips. 3. The LTC(4)-induced enhancement of contraction was accompanied by an increase in myosin light chain (MLC) phosphorylation and was blocked by a rho kinase inhibitor (Y-27632), but not by either a PKC inhibitor (calphostin C) or a tyrosine kinase inhibitor (genistein). 4. These results indicated that, in porcine tracheal smooth muscle, LTC(4) enhances the contraction by increasing the Ca(2+) responsiveness of the contractile apparatus in a MLC phosphorylation dependent manner, possibly through the activation of the rho-rho kinase pathway.     

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.     

The effects of a novel vasodilator, LP-805, on cytosolic Ca2+ concentrations and on tension in rabbit isolated femoral arteries.

1. LP-805, 8-tert-butyl-6,7-dihydropyrrolo-[3,2-e]-5-methylpyrazolo- [1,5a]-pyrimidine-3-carbonitrile, is a newly synthesized potent vasodilator. To investigate the cellular mechanisms of vasorelaxation induced by LP-805, we simultaneously determined the effects of LP-805 on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension of smooth muscle of rabbit femoral arterial strips, with or without the endothelium, using front-surface fluorometry and fura-2. 2. In the absence of the endothelium, LP-805, in a concentration-dependent manner, decreased [Ca2+]i and tension during the contraction induced by K(+)-depolarization, at relatively low concentrations ([K+]o < or = 30 mM). The decreases in [Ca2+]i and tension were fully antagonized by treatment with 2 x 10(-6) M glibenclamide. The [Ca2+]i-tension relationship in the LP-805-induced relaxation was similar to that of K(+)-depolarization-induced contractions. 3. LP-805, in a concentration-dependent manner (IC50 for inhibition of tension; 1.7 x 10(-6) M), decreased both [Ca2+]i and tension during the steady-state of contractions induced by 1 x 10(-7) M noradrenaline (NA) in the strips without the endothelium. Glibenclamide completely inhibited these reductions of [Ca2+]i and tension. At the steady-state of relaxation induced by LP-805 during NA-induced contraction, [Ca2+]i-tension relation was shifted to the left of that obtained with high K(+)-induced contraction. 4. NA induced transient increases in [Ca2+]i and tension in the absence of extracellular Ca2+. LP-805 (up to 3 x 10(-6) M) had no effect on these intracellular Ca2+ mobilisation and tension development induced by NA. 5. In strips with an intact endothelium, LP-805 decreased both [Ca2+]i and tension during contraction induced by 1 x 10(-7) M NA. The concentration-response curve for inhibition of [Ca2+]i and tension obtained in the presence of the endothelium was shifted to the left from that obtained in the absence of endothelium. IC50 for the inhibition of tension obtained in the strips with the endothelium was 4.0 x 10(-7) M. Treatment with 1 x 10(-4) M NG-nitro-L-arginine (L-NOARG) attenuated reductions of both [Ca2+]i and tension induced by LP-805 and the concentration-response curve shifted to the right and overlapped that obtained in the absence of the endothelium. Treatment with glibenclamide almost fully overcame the reduction of [Ca2+]i induced by LP-805, while the reversion of tension was 50% at most.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some effects of nipradilol, a beta-antagonist possessing a nitroxy group, on smooth muscle of the pig coronary artery.

1. The effects of nipradilol, a beta-adrenoceptor antagonist which possesses a nitroxy group, on cytosolic Ca2+ concentration ([Ca2+]i), and on tension development were simultaneously measured by front-surface fluorometry and fura-2-loaded strips in the proximal portion of pig coronary arteries. 2. Nipradilol reduced in a concentration-dependent manner both the [Ca2+]i and tension, irrespective of whether the strips were unstimulated or exposed to either high K+ or histamine containing solutions. However, both in the case of contractions induced by high K+-depolarization and histamine stimulation, for a given [Ca2+]i elevation the tension which developed in the presence of nipradilol was smaller than that generated in its absence, so that the [Ca2+]i-tension curves during the contraction were shifted to the right. 3. In the absence of extracellular Ca2+, the [Ca2+]i elevation due to the release of Ca2+ from histamine-sensitive store was inhibited by nipradilol. Nipradilol had no effect on the [Ca2+]i elevation due to the release of Ca2+ from caffeine-sensitive stores; however, it did inhibit the caffeine-induced increase in tension. A derivative of nipradilol, which lacked a nitroxy molecule (Nip(-N)), had no effect on the [Ca2+]i and tension elevated by histamine or caffeine in the absence of extracellular Ca2+. 4. The beta-adrenoceptor agonist, isoprenaline, reduced [Ca2+]i tension when applied to steady state contractions induced by high K+, or at the peak level of tension to histamine. The reduction of [Ca2+]i and tension induced by isoprenaline was inhibited by Nip(-N) in a concentration-dependent manner and nipradilol inhibited the isoprenaline-induced relaxation with bell-shaped concentration-response curves. At lower concentrations, nipradilol acted as a beta-blocker, the IC50- value being smaller than that of Nip(-N), and at higher concentrations, it acted as a nitrovasodilator. 5. Thus, it is suggested that, at lower concentrations, nipradilol, an antianginal drug, acts as a beta-adrenoceptor antagonist. At higher concentrations, it relaxes the proximal portion of the coronary artery by directly reducing [Ca2+]i and the Ca2+-sensitivity of the myofilaments, apparently due to the presence of the nitroxy molecule.     

Mechanisms controlling caffeine-induced relaxation of coronary artery of the pig.

1. We studied the effects of caffeine on coronary artery smooth muscle of the pig by measuring changes in isometric tension, cytosolic free Ca(2+) concentration ( [Ca2+]i) and transmembrane potential. 2. In the absence of tone, caffeine induced a concentration-dependent transient contraction of coronary artery strips, followed by sustained relaxation. Simultaneously with the relaxation, caffeine, 25 mM, hyperpolarized the smooth muscle cells by 7.7 +/- 0.9 mV. 3. Caffeine caused a concentration-dependent relaxation of strips precontracted with 10(-5)M acetylcholine (ACH). A supramaximal relaxing concentration of 25 mM caffeine produced an additional transient increase in [Ca2+]i on the Ca2+ plateau of ACh tonic contraction, which was followed by a decrease in [Ca2+]i to a level slightly below the basal concentration. This relaxation was accompanied by a hyperpolarization of 7.3 +/- 0.9 mV. 4. KCI 120 mM (high K+) contracted the strips with a concomitant depolarization of 38.6 +/- 1.6 mV and sustained increase in [Ca2+]i. Caffeine caused a concentration-dependent relaxation of high K+-induced contraction. Caffeine, 25 mM, decreased the Ca2+ plateau to a level that remained above the basal concentration of Ca2+ but did not change the membrane potential. 5. When strips were placed in a Ca(2+)-free medium with EGTA 2mM, and, in addition, ACh was applied successively three times, both intracellular and extracellular mobilizable Ca2+ pools were depleted. In these conditions, phorbol 12,13 dibutyrate (PDBu) 10(-7) M and prostaglandin F 2 alpha (PGF 2 alpha) 10(-5) M contracted the strips. Caffeine (25 mM) inhibited these contractions with no change in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of extracellular and intracellular sources of Ca2+ in sarafotoxin S6b-induced contraction of strips of the rat aorta.

1. The effect of sarafotoxin S6b (sarafotoxin), a vasoconstrictor peptide, on cytosolic Ca2+ concentration ([Ca2+]i) and force in rat aortic strips loaded with fura-2 was determined by front-surface fluorometry. The objective was to elucidate the role of extracellular and intracellular Ca2+ in the mechanism of action of this peptide. 2. In the presence of extracellular 1.25 mM Ca2+, sarafotoxin induced a biphasic response consisting of an initial rapid increase in [Ca2+]i followed by a secondary sustained increase. Tension developed slowly but was sustained during the application of sarafotoxin. Diltiazem (10 nM-0.1 mM) partially inhibited both the increases in [Ca2+]i and tension. 3. In the presence of extracellular Ca2+, the force developed in relation to the increase in [Ca2+]i ([Ca2+]i-force relationship) observed with sarafotoxin was much greater than that observed upon K+ depolarization. In the presence of diltiazem the sarafotoxin-induced [Ca2+]i-force relationship was shifted even further to the left. 4. In the absence of extracellular Ca2+, sarafotoxin induced a transient increase in [Ca2+]i and a sustained contraction. Extending the incubation time in Ca(2+)-free physiological solution, resulted in smaller responses. However, after 60 min in Ca(2+)-free solution, sarafotoxin induced a sustained contraction but no change in [Ca2+]i. This residual contraction was inhibited by H-7, which is known to inhibit protein kinase C. 5. After treatment with caffeine to reduce intracellular stored Ca2+, sarafotoxin could still elicit increases in [Ca2+]i and in tension, showing that the caffeine-sensitive intracellular Ca2+ store partially overlaps with the sarafotoxin-sensitive store.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of okadaic acid on cytosolic calcium concentrations and on contractions of the porcine coronary artery.

1. We investigated the effects of okadaic acid (OA), a phosphatase inhibitor derived from a 38-carbon fatty acid and isolated from the black sponge, genus Halichondria, on cytosolic Ca2+ concentration ([Ca2+]i) and tension developed in porcine coronary arterial strips loaded with fura-2. 2. Both in the presence (1.25 mM) and absence of extracellular Ca2+, OA (over 10(-6) M) induced a concentration-dependent, slow and progressive increase in tension. Calcium removal had no effect on the maximum level of tension, time between application of the drug and the onset of tension, or the time required to reach the maximum tension. However, there was a slight concentration-dependent increase in [Ca2+]i, only in the presence of extracellular Ca2+. 3. At a lower concentration that did not cause contraction or increase [Ca2+]i, OA (10(-6) M) inhibited tension development but not the Ca2+ transient on readmission of Ca2+ in 118 mM K+-depolarizing solution. OA inhibited the maximum levels of the developed tension, without affecting the KD value (598 +/- 204 nM for control vs 678 +/- 464 nM after OA treatment) or the Hill coefficient (1.78 +/- 0.10 for control vs 1.98 +/- 0.47 for OA treatment). 4. It is concluded that high concentrations of OA induce a contraction independent of extracellular Ca2+ and without any changes in [Ca2+]i. Lower concentrations of OA inhibit the Ca2+-dependent contractions. The lack of effect on KD values suggests that the [Ca2+]i-sensitivity of the contractile apparatus is not affected by this inhibition of contraction.     

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)