Effects of cooling on histamine-induced contractions of human umbilical artery: the role of ion channels

The effects of cooling (to 28 degrees C) on histamine (10(-9) - 3 x 10(-4) M)-induced contractions and the role of calcium (Ca(2+)), potassium (K(Ca) (2+)) and sodium (Na(+)) channel blockers in the cooling-induced responses were investigated in the endothelium-denuded human umbilical artery. Concentration-response curves to histamine were isometrically recorded at 37 and 28 degrees C (control). The same procedure was repeated at 28 degrees C in the presence of tetraethylammonium (TEA, 10(-3) M), pilsicainide (10(-6) M), ouabain (10(-6) M), caffeine (3 x 10(-4) M), verapamil (10(-6) M) and also in Ca(2+)-free medium with ethylene glycol bis-(beta-aminoethyl ether) N,N,N(1),N(1)-tetraacetic acid (EGTA). During cooling, the sensitivity, but not the maximal response, was significantly higher than 37 degrees C. Cooling to 28 degrees C after treatment with verapamil or pilsicainide decreased the sensitivity, whereas treatment with TEA and ouabain significantly increased sensitivity. Treatment with caffeine did not modify the effect of cooling. Furthermore, cooling to 28 degrees C after incubation in Ca(2+)-free solution with EGTA decreased the sensitivity to histamine. The results of this study suggest the role of Ca(2+), K(Ca) (2+) and Na(+)-ion channels in the cooling-induced changes of human umbilical arteries treated with histamine.     

The role of K+ ions on the response to carbachol of calf coronary artery and cardiac vein during cooling.

The role of K+ ions on the vasoconstrictions induced by carbachol during cooling (28 degrees C) in the endothelium of a denuded calf coronary artery and cardiac vein (noncutaneous vessel) was studied. Carbachol (10(-9) - 3 x 10(-4) M) induced concentration-dependent contractions at both 37 degrees C and 28 degrees C. The sensitivity, but not the maximal response, of carbachol (10(-9) -3 x 10(-4) M) was significantly lower at 28 degrees C than at 37 degrees C. Cooling to 28 degrees C after treatment with tetraethylammonium (TEA, 10(-3) M) or ouabain (10(-5) M), after incubation in K+-free medium increased the sensitivity to carbachol in both preparations. The results suggest a role for K+ ions in the cooling-induced changes of noncutaneous vessels.     

Interference of pentobarbital and verapamil with the reactivity of middle cerebral artery of cat exposed to experimental subarachnoid hemorrhage

1. Pentobarbital and verapamil produced concentration-dependent vasodilation in segments of middle cerebral artery of either control or exposed to experimental subarachnoid hemorrhage (SAH) cats precontracted with 5-hydroxytryptamine (5-HT, 10(-5) M) or K+ (75 mM), the potency to evoke relaxation being greater for verapamil than pentobarbital. 2. Pentobarbital (10(-4) M and 10(-3) M) or verapamil (10(-6) M) inhibited the contractions induced by 10(-5) M 5-HT or 75 mM K+ in both kinds of vessels. 3. Pentobarbital (10(-3) M) and verapamil (10(-6) M) reduced the concentration-dependent contractions induced by Ca2+ addition in control and SAH arteries previously exposed to Ca2+-free medium containing K+ or 5-HT. 4. All these vasodepressor effects of pentobarbital and verapamil were similar in control and SAH vessels, suggesting that the sensitivity of them to these agents is unaffected by SAH.     

Effects of diltiazem and other Ca2+ antagonists on guinea-pig tracheal muscle

The effects of diltiazem and 3 other Ca2+ antagonists (verapamil, nicardipine, bepridil) were studied on isolated guinea-pig tracheal preparations which were contracted with several agonists. Assessment of the contractile agonists was performed under physiological conditions as well as in Ca2+-depleted solutions. The order of potency of the contractile agonists was LTD4 greater than ACh greater than 5-HT greater than Hist greater than BaCl2 greater than TEA greater than KCl. The efficacy of the physiological agonists ACh, Hist and LTD4 was moderately depressed in Ca2+-free solutions while the responses to non-specific agonists and 5-HT were markedly reduced. Diltiazem and verapamil reduced basal tone at concentrations greater than or equal to 10(-4) M. Diltiazem displaced all agonist concentration-effect curves to the right. The four Ca2+ antagonists studied had a marked effect on non-physiological agonists as compared to that on physiological agents. Increasing Ca2+ concentration only partially reversed the inhibitory effect of diltiazem.     

The role of capacitative Ca2+ influx in the alpha 1B-adrenoceptor-mediated contraction to phenylephrine of the rat spleen.

1. The mechanism of contraction to phenylephrine in the rat spleen (mediated via alpha 1B-adrenoceptors) has been studied in functional experiments. 2. The concentration-dependent contraction of the rat spleen to cumulative additions of phenylephrine (pD2 4.8 +/- 0.1) was not significantly reduced by the selective protein kinase C (PKC) inhibitor, calphostin C (10(-6)M) or potentiated by the DAG kinase inhibitor, R59022 (10(-6) M). 3. Contraction of the rat spleen in normal Krebs solution containing Ca2+ (2.5 mM) to a single concentration of phenylephrine (3 x 10(-4) M) produced a maximal response consisting of an initial phasic component and a more slowly developing tonic component. However in Ca(2+)-free Krebs solution (containing EGTA), phenylephrine (3 x 10(-4)M) produced only a phasic contraction which was reduced to 46 +/- 3% maximum response to phenylephrine in normal Krebs solution. 4. In some tissues after the contraction to phenylephrine (3 x 10(-4) M) in Ca(2+)-free Krebs solution (containing EGTA), the phenylephrine was washed out and the tissue was allowed to recover. After 2 h, upon addition of Ca2+ (2.5 mM) to the Krebs solution (EGTA now removed) a tonic contraction developed in the tissue (97 +/- 4% maximum response to phenylephrine). 5. Cyclopiazonic acid produced a tonic contraction of the rat spleen with a maximum effect at 10(-5) M (202 +/- 8% maximum response compared with that to phenylephrine). The contraction to CPA (10(-5) M) was reduced in Ca(2+)-free Krebs solution containing EGTA (30 +/- 4% of the maximum response to phenylephrine). One hour after the end of the contraction in Ca(2+)-free Krebs solution (EGTA now removed), upon addition of Ca2+ (2.5 mM) to the Krebs solution a tonic contraction developed in the tissue (263 +/- 12% maximum response to phenylephrine).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetrodotoxin-sensitive, K+-induced relaxation of guinea-pig isolated trachealis in the presence of Ca2+-entry blocking drugs, Ca2+-free solution and after polyamine exposure.

We have previously observed a paradoxical relaxant effect of K+ on guinea-pig isolated trachealis after exposure to polyamines. The purpose of the present study was to evaluate whether the relaxation involved a reduction in the entry of extracellular Ca2+. We therefore investigated the effect of K+ in the presence of Ca2+-entry blocking drugs and in the presence of Ca2+-free solution. In the presence of nifedipine (10(-5) M), verapamil (10(-5) M) or diltiazem (10(-5) M), K+ (30 mM) induced relaxation of the trachealis muscle. The relaxation to K+ was not blocked by ouabain (10(-6) M), propranolol (10(-6) M), or indomethacin (10(-6) M). A relaxation in response to K+ was also observed in Ca2+-free solution, (with tone induced by methacholine), an effect not blocked by propranolol or ouabain. Tetraethylammonium (30 mM) (TEA), which ordinarily evokes contractile responses, induced trachealis relaxation in the presence of verapamil or nifedipine. The relaxation was unaltered by ouabain or propranolol. Tetrodotoxin (10(-6) M) (TTX) blocked 65% of the K+-induced relaxation in the presence of nifedipine and 100% of K+-induced relaxation either in a Ca2+-free solution or after polyamine exposure. TTX was without effect on TEA-induced relaxation after Ca2+-entry blocking drugs. Atropine (10(-6) M) or hexamethonium (10(-6) M) did not affect K+-induced relaxation after polyamine exposure. The concentration-response curve for K+-induced contraction in normal modified Krebs-Henseleit solution was shifted to the left by TTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitory action of melatonin on the contractile response to 5-hydroxytryptamine in various isolated vascular smooth muscles

The effects of melatonin on the contractile responses to 5-hydroxytryptamine (5-HT), norepinephrine (NE), angiotensin-1 (AT-1) and potassium were determined on the rabbit isolated aorta, iliac and renal arteries. Melatonin, at 10(-4) or 10(-3) M inhibited the response to 5-HT in the aorta, iliac and renal arteries. Melatonin, at 10(-3) M, had a negligible effect on the responses to NE and AT-1 in all preparations used. The potassium-induced contraction of all 3 preparations used was slightly inhibited by only the high concentration of melatonin (10(-3) M). In a Ca+-free medium with EGTA (0.1 mM), the residual response to 5-HT (10(-5) or 10(-4) M) were inhibited by melatonin (10(-4) or 10(-3) M) in all preparations used. In a Ca2+-free medium with EGTA (0.01 mM), nifedipine (10(-6) M) and 5-HT (5 X 10(-7) M), application of Ca2+ (2 mM) resulted in a tonic contraction, related to receptor operated channels, of all preparations. This Ca2+ dependent, nifedipine insensitive contraction was markedly inhibited or abolished by melatonin at 10(-5) and 10(-4) M. These results demonstrated that melatonin had a generally greater inhibitory effect on the response to 5-HT than those to NE, AT-1 and potassium in vascular smooth muscles. Also the results suggest that action of melatonin on the 5-HT induced contraction is more related to interference with Ca2+ influx through receptor operated channels than release of intracellular Ca2+ from the store site.     

Endothelin-induced contractions of tracheal smooth muscle and identification of specific endothelin binding sites in the trachea of the rat.

1. The presence of specific binding sites and the contractile activity of the novel peptide, endothelin have been investigated in rat trachea. 2. Endothelin (10(-8)-10(-5) M) induced long-lasting contraction of rat tracheal rings superfused with Krebs solution (EC50 5.4 x 10(-6) M). Contractions of the tissue to 10(-6) M endothelin were attenuated in Ca2+-free medium containing 0.1 mM EGTA but unaffected by nicardipine (10(-7) M). 3. After equilibration in Ca2+-free medium (without EGTA) a return to normal Ca2+ concentrations (2.5 mM), 30 min or 60 min following endothelin (10(-6) M), produced a sustained contraction of the tissue. 4. Specific binding sites for endothelin were identified on rat tracheal smooth muscle (KD 1.34 x 10(-10) M, maximal binding 1.2 fmol mm-2). Specific binding sites were also identified on nerve trunks. Endothelin binding was unaffected by co-incubation with nicardipine (10(-7) M) or verapamil (10(-7) M). 5. The discrepancy between the apparent KD for endothelin binding and the EC50 for endothelin-induced contraction suggests that the endothelin binding sites identified in this study may not be associated with the receptors mediating contraction. 6. These results indicate that endothelin binding sites are present on tracheal smooth muscle. The mechanism of endothelin-induced contraction, whilst being dependent on extracellular calcium, does not appear to involve binding to the dihydropyridine- or verapamil-sensitive sites on the voltage-dependent Ca2+ channel. Its long duration of action may be associated with a sustained increase in Ca2+ permeability.     

Pharmacological analysis of the effects of Bay K 8644 and organic calcium antagonists on the mouse isolated distal colon.

1. Bay K 8644 (10(-8) to 10(-6) M) induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The maximal responses were enhanced and the EC50 was lowered in the presence of tetrodotoxin (TTX; 1.5 x 10(-7) M). The responses were not affected by atropine (10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), propranolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). By contrast, the contractile responses were inhibited by Ca2+ entry blockers (verapamil, nifedipine) and abolished in Ca2+-free EGTA solution. These observations indicate that the contractile effects of Bay K 8644 are dependent on its ability to promote Ca2+ influx. 2. At 10(-4) M, Bay K 8644 provoked a slow relaxation of the preparation. Moreover, from 10(-5) M, Bay K 8644 markedly reduced the contractile responses to ACh and K+ depolarization. These inhibitory effects were comparable with those produced by nifedipine. Such data suggest that, at high concentrations, Bay K 8644 could act in part as a dihydropyridine Ca2+ channel antagonist. 3. Bay K 8644 (10(-9) M) preferentially enhanced, while nifedipine (10(-10) to 10(-8) M) as well as verapamil (3 x 10(-9) to 10(-6) M) preferentially inhibited, the tonic component of the contractile response evoked by K+ depolarizing solution. This may indicate that different populations of voltage-sensitive Ca2+ channels are involved in the biphasic response to K+ depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bay K 8644-induced potentiation of the contractile response of rabbit iliac artery to caffeine in a Ca2+-free medium

1. In a Ca2+-free medium caffeine (10 mM) was still able to cause a phasic contraction in rabbit iliac arteries. 2. Bay K 8644 at 10(-6) but not at 10(-7) M potentiated the residual response to caffeine in a Ca2+-free medium. In a Ca2+-free medium with or without KCl (40 mM), Bay K 8644, however, caused no contraction. 3. Nifedipine (10(-6) M) did not affect the residual caffeine-response or the potentiating effect of Bay K 8644. Verapamil (10(-6) M), however, inhibited both the caffeine response and the potentiation. 4. Bay K 8644 (10(-6) M) potentiated the contractile response to Ca2+ (0.01-2.4 mM) in a Ca2+-free medium containing KCl. The potentiation was equally inhibited by nifedipine or verapamil. 5. La#+ (1 mM), EGTA (0.1 mM), or vanadate (10(-4) M) completely inhibited the Bay K 8644-induced potentiation without affecting the residual caffeine response. 6. These results suggest that the potentiating action of Bay K 8644 on the residual caffeine response in a Ca2+-deficient medium may not be related to voltage-dependent Ca2+ channels. In addition, the activity of Ca2+-ATPase in sarcolemmal membranes may be important in this potentiation.     

Transient resensitization interrupting the development of carbachol-induced desensitization in smooth muscle of guinea-pig taenia caeci: Ca2+-dependent termination of resensitization

It is important to clarify developmental mechanisms of desensitization because of their great significance in regulation of cellular responsiveness. We have found that carbachol-induced desensitization to carbachol develops in three successive phases in the presence of extracellular Ca(2+) in the smooth muscle of guinea pig taenia caeci: fast desensitization within 15 s, transient resensitization reaching a peak at 1 min and the subsequent re-development of desensitization to terminate resensitization for up to 30 min. In contrast, in the absence of extracellular Ca(2+), desensitization develops without resensitization. To further clarify the roles of Ca(2+) in the formation of the transient resensitization phase, we examined the developmental process of carbachol-induced desensitization in the absence of extracellular Ca(2+), following the induction of desensitization by a 15-s treatment with carbachol in the presence of extracellular Ca(2+). Desensitization to carbachol occurred due to pretreatment with 10(-4) M carbachol for 15 s in normal physiological solution, and continued pretreatment with carbachol in Ca(2+)-free solution containing 0.2 mM EGTA induced resensitization followed by the obscure progress of re-desensitization for up to 30 min resulting in a long-lasting phase of resensitization. These results suggest that resensitization is promptly terminated by the Ca(2+)-dependent development of subsequent desensitization for further regulation of cellular responsiveness via G(q) protein-coupled Ca(2+)-mobilizing receptors against sustained stimuli.     

Effects of verapamil on the response of the guinea-pig tracheal muscle to carbachol.

The effects of verapamil on the contraction of the guinea-pig tracheal smooth muscle induced by calcium (Ca2+) or barium (Ba2+) were investigated in three different conditions: (a) in excess K solution, (b) in the presence of carbachol, and (c) in excess K solution containing carbachol. In order to clarify the contractions, the effects of removal and readdition of the divalent cations were also investigated. In Ca2+-loaded tissues, application of carbachol in Ca-free medium produced a transient contraction, the magnitude of which decreased the longer the duration of exposure to Ca2+-free solution. In Ca2+-depleted, Ba2+-loaded tissues, application of carbachol in a Ba2+- and Ca2+-free medium produced a transient contraction the magnitude of which decreased the longer the duration of exposure to the Ba2+- and Ca2+-free solution. After exposure to Ca2+-free solution for 40 min, the sensitivity of the tissue to Ca2+ was greater in the presence of 30 microM carbachol (ED50 = 0.06 mM) than in the presence of 40 mM K+ (ED50 = 0.3 mM). The Ca2+-sensitivity in the presence of 30 microM carbachol plus K+ (40 mM) was not different from that in the presence of 30 microM carbachol alone. In Ca2+-free solution, the sensitivity of the tissue to Ba2+ in the presence of 40 mM K+ (ED50 = 1.4 mM) was not different from that observed in the presence of 30 microM carbachol (ED50 = 1.3 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Overlap in the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

We have previously shown that elimination of buffer Ca2+ markedly reduced maximum 5-HT-induced contractions. We have now investigated the effect of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists on 5-HT- and K+-induced contractions in rat aorta to explore the possibility of a relationship between blockade of L-type Ca2+ channels and 5-HT2 receptor antagonism. Sodium nitroprusside, felodipine, nifedipine, diltiazem, cinnarizine, verapamil, ritanserin, cyproheptadine, ketanserin and mianserin inhibited 5-HT-induced contractions of rat aorta with mean IC50 values (concentration (M) resulting in 50% inhibition) of 2.2 x 10(-11), 6.6 x 10(-11), 1.5 x 10(-9), 1.7 x 10(-9), 3.2 x 10(-7), 5.4 x 10(-7), 9.7 x 10(-10), 1.9 x 10(-8), 5.0 x 10(-7) and 6.4 x 10(-7), respectively. The same compounds antagonized K+-induced rat aortic contractions with the rank order of potency (mean IC50, M): felodipine (7.0 x 10(-11)) > nifedipine (4.8 x 10(-9)) > sodium nitroprusside (4.1 x 10(-8)) > verapamil (5.5 x 10(-8)) > cyproheptadine (6.2 x 10(-8)) > diltiazem (4.1 x 10(-7)) > cinnarizine (1.3 x 10(-6)) > ritanserin (1.8 x 10(-6)) > ketanserin (9.0 x 10(-6)) > mianserin (2.0 x 10(-5)). These data are indicative of a highly significant correlation (r=0.81, P=0.03) between potency against 5-HT-induced contraction and that against contractile response to K+ depolarization, and suggest overlap of the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.     

The role of diacylglycerol and activation of protein kinase C in alpha 1A-adrenoceptor-mediated contraction to noradrenaline of rat isolated epididymal vas deferens.

1. The mechanism of contraction to noradrenaline (pEC50 5.6 +/- 0.1) in the rat epididymal vas deferens (mediated via alpha 1A-adrenoceptors) has been studied in functional experiments. 2. Contractions to noradrenaline at 10(-6) M were potentiated by the diacylglycerol (DAG) kinase inhibitor R 59022 (3 x 10(-7) M) from 49 +/- 4% to 63 +/- 3% maximum response and the time taken from initiation of contraction to the maximum response was reduced from 16 +/- 2 s to 9 +/- 1 s. The same contractions were not significantly potentiated by the DAG lipase inhibitor, U-57,908, 10(-5) M (51 +/- 2% control and 53 +/- 4% in the presence of U-57,908) nor was the time taken from initiation of contraction to the maximum response significantly altered (17 +/- 1 s control and 16 +/- 1 s in the presence of U-57,908). 3. Concentration-dependent contractions to noradrenaline (NA) were reduced by staurosporine (10(-7) M) and the selective protein kinase C inhibitor, calphostin C (10(-6) M) from 68 +/- 2% (NA, 3 x 10(-6) M) to 28 +/- 2% and 20 +/- 2% respectively and from 94 +/- 2% (NA, 3 x 10(-5) M) to 50 +/- 2% and 44 +/- 2% respectively. Contractions to K+ (40 +/- 2% maximum response to NA) were also significantly reduced by staurosporine (10(-7) M) (35 +/- 2%) but not by calphostin C (43 +/- 3%). 4. The phorbol ester, phorbol-12,13-dibutyrate (PDBu), produced a phasic, concentration-dependent contraction (10(-7) M - 10(-4) M) which was 41 +/- 2% of the maximum response to NA at 10(-4) M PDBu. The contraction to PDBu (10(-5) M) was reduced by calphostin C (10(-6) M) from 33 +/- 5% to 4 +/- 1% maximum response to NA. 5. Non-cumulative contractions to NA (10(-8) M - 10(-4) M) were abolished in Ca(2+)-free Krebs solution containing EGTA (1 mM) and were reduced in the presence of nifedipine (10(-6)M) in normal Krebs solution by 91 +/- 2% at 10(-4)M NA. The contraction to PDBu (10(-5)M, 33 +/- 5% maximum response to NA) was also abolished in Ca(2+)-free Krebs solution containing EGTA (1 mM) or by the presence of nifedipine (10(-6)M) in normal Krebs solution. 6. When NA (10(-4)M) was added to vasa deferentia in Ca(2+)-free Krebs solution containing EGTA (1 mM), following its wash out (and with EGTA later removed from the Krebs solution), readdition of Ca2+ (2.5 mM) to the Krebs solution produced no response. Cyclopiazonic acid (10(-5)M), which can deplete Ca2+ from intracellular stores, also produced no contraction. Therefore influx of extracellular Ca2+ is not a consequence of depletion of intracellular Ca2+ stores (capacitative Ca2+ influx). 7. Pre-incubation of tissues for 30 min with either cyclopiazonic acid (10(-5)M) or ryanodine (10(-4)M), which can both deplete intracellular Ca2+ stores, did not reduce the contractions to NA (3 x 10(-6)M). Pre-incubation of vasa deferentia with cyclopiazonic acid (1 or 3 min, when any rise in [Ca2+]i produced by cyclopiazonic acid might still exist) did not potentiate the contraction to PDBu (10(-5)M). Thus mobilization of intracellular Ca2+ may not be required for the activation of protein kinase C involved in these contractions. 8. In conclusion, the contraction of the rat epididymal vas deferens to NA mediated by alpha 1A-adrenoceptors appears to depend upon activation of protein kinase C by diacylglycerol, resulting in the influx of extracellular Ca2+ through voltage-gated Ca2+ channels. There was no evidence for a role of inositol trisphosphate in the contraction to noradrenaline in this tissue.     

Contractions of rat uterine smooth muscle induced by acetylcholine and angiotensin II in Ca2+-free medium

The effects of acetylcholine (ACh, 10(-4)M) and angiotensin II (Ang II, 10(-6) M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. Both ACh and Ang II produced transient contractions, the amplitude of which can be taken as a measurement of the amount of Ca2+ present in a drug-sensitive Ca2+ store. The degree of filling of this store depended on the external Ca2+ concentration, and on the presence of contractile responses during the Ca2+ loading period. The existence of two pathways (either direct or transcytoplasmic) is suggested for Ca2+ uptake into the internal Ca2+ store. The rate of filling of the Ca2+ store in 2.1 mM-Ca2+-containing solution was faster (time to half-maximal response, t 1/2 = 29 +/- 2.2 s, n = 4) than the rate of depletion in Ca2+-free solution (t 1/2 = 3 +/- 0.3 min, n = 3). The gradual depletion of this store was much slower at 18 degrees C than at 35 degrees C, and in the presence of vanadate which is known to inhibit Ca2+-ATPases. Methoxyverapamil (D600, 10(-6)-10(-5) M) had no appreciable effect on the direct Ca2+ uptake or on the release of Ca2+ from the store by ACh and Ang II. Mn2+ (10(-3) M) completely inhibited the direct pathway to the internal Ca2+ store and also reduced the release of Ca2+. ACh and Ang II induced repetitive depolarizations close to zero potential which did not parallel the transient contractions as a function of the time of perfusion in Ca2+-free solution. Applications of 2 mM EGTA, 135 mM K+ or Ca2+ antagonists which suppressed or reduced the drug-induced depolarizations did not affect appreciably the drug-induced contractions. These results suggest that myometrial cells have an intracellular Ca2+ store sensitive to different stimulus substances. This store is not affected by depolarization of the plasma membrane and is certainly different from that described in voltage-clamp experiments.     

The mechanisms of alpha(2)-adrenoceptor agonist-induced contraction in longitudinal muscle of the porcine uterus

The aim of the present study was to clarify the cellular mechanisms underlying the alpha(2)-adrenoceptor-mediated contraction of porcine myometrium (nonvascular smooth muscle). Acetylcholine (3 nM-1 microM), clonidine (1 nM-10 microM) and 5-bromo-N-[2-imidazolin-2-yl]-6-quinoxalinamine (UK14304) (1 nM-10 microM) in Krebs solution caused a concentration-dependent contraction in the longitudinal muscles of the porcine uterus with similar EC(50) values and maximum responses. A lowered external Ca(2+) concentration and verapamil (10 nM-10 microM) decreased the contractile response to clonidine and UK14304 more markedly than the response to acetylcholine. However, in Kumagai solution, neither clonidine nor UK14304 caused contractile responses, but acetylcholine remained effective. The effects of alpha(2)-adrenoceptor agonists on intracellular Ca(2+) concentration ([Ca(2+)](i)) and smooth muscle force were measured simultaneously using fura-PE3-loaded muscle preparations. Clonidine and UK14304 caused increases in [Ca(2+)](i) and force of the longitudinal muscle. The increases in [Ca(2+)](i) and muscle force were markedly inhibited by verapamil and in Ca(2+)-free solution (EGTA, 1 mM). In the absence of external Ca(2+), clonidine caused only a small increase in [Ca(2+)](i) in Ca(2+)-loaded preparations compared with those increases caused by carbachol, histamine, and oxytocin. Ca(2+) (2.5 mM) caused increases in [Ca(2+)](i) and force of the longitudinal muscles in a Ca(2+)-free high K(+) solution. Clonidine concentration dependently potentiated the Ca(2+)-induced contraction without significantly changing the increase in [Ca(2+)](i), and this potentiation was inhibited by yohimbine. These results suggested that clonidine increases the Ca(2+) sensitivity of the contractile elements through activation of alpha(2)-adrenoceptors. During the development of the contractile response to clonidine (1 microM, 0-5 min), tissue cyclic AMP levels did not change significantly. In vitro treatment with pertussis toxin (1 microg/ml for 2 h) significantly decreased the contraction induced by clonidine without affecting the responses to carbachol and high K(+). The present results indicate that in porcine myometrium, alpha(2)-adrenoceptor stimulation caused contraction of the longitudinal muscles by mechanisms largely dependent on the influx of extracellular Ca(2+), probably through voltage-dependent Ca(2+) channels (VDCCs), and that the potentiation of the Ca(2+) sensitivity of the contractile elements is another mechanism of the contractile responses. These actions involve a pertussis-toxin-sensitive G protein (probably G(i) type) in the signal transduction pathway.     

Physiologic increases in extracellular sodium salt enhance coronary vasoconstriction and Ca2+ entry

High dietary sodium salt has been suggested to increase the risk of coronary vasospasm and coronary artery disease. However, whether high-sodium salt directly affects the mechanisms of coronary artery contraction is unclear. This study investigated whether physiologic and supraphysiologic increases in extracellular concentrations of sodium chloride ([NaCl]e) enhance the Ca2+ handling mechanisms of coronary smooth muscle contraction. Isometric contraction and 45Ca2+ influx were measured in endothelium-denuded porcine coronary artery strips incubated in Krebs solution (2.5 mM Ca2+) containing increasing [NaCl]e (120, 121, 123, 126, 130, 140, and 150 mM). Increasing [NaCl]e for 30 min did not increase the resting coronary tone or 45Ca2+ influx. 5-Hydroxytryptamine (5-HT) caused concentration-dependent increases in contraction and 45Ca2+ influx. Preincubation of coronary strips in increasing [NaCl]e for 30 min did not change the median effective dose of 5-HT. However, the magnitude of the 5-HT contraction and 45Ca2+ influx was significantly increased at 121-126 mM [NaCl]e. Preincubation with 2,4-dichlorobenzamil (10-5 M), inhibitor of the Na+/Ca2+ exchanger, or KB-R7943 (10-5 M), selective inhibitor of the reverse mode of the Na+/Ca2+ exchanger, abolished the increases in 5-HT contraction and 45Ca2+ influx at 121-126 mM [NaCl]e. Preincubation in Krebs solution containing 120 mM NaCl plus 1-6 mM LiCl or N-methyl-d-glucamine did not increase 5-HT contraction or 45Ca2+ influx. Higher [NaCl]e (140-150 mM) increased 5-HT-induced 45Ca2+ influx but inhibited 5-HT contraction. 5-HT (10-5 M)- and caffeine (25 mM)-induced contraction in Ca2+-free (2 mM EGTA) solution, a measure of Ca2+ release from the intracellular stores, was not affected by small increases in [NaCl]e (121-126 mM) but was inhibited at higher [NaCl]e (130-150 mM). Thus increases in [NaCl]e within the physiologic range enhance coronary smooth muscle contraction to 5-HT by a mechanism possibly involving Ca2+ entry via the reverse mode of the Na+/Ca2+ exchanger, but not Ca2+ release from the intracellular stores. The reduction of coronary contraction with supraphysiologic [NaCl]e in both Ca2+-containing and Ca2+-free Krebs could be related to excessive increases in ionic strength and may mask significant coronary vasoconstrictor effects of physiologic increases in [NaCl]e.     

Relaxant effects of xanthines, a beta 2-receptor agonist and Ca2+ antagonists in guinea-pig tracheal preparations contracted by potassium or carbachol

Potassium (124 mM K+ Krebs) produced a biphasic contractile response in the guinea-pig isolated trachea. An initial phasic contraction was followed by a larger and sustained contraction. Repeated potassium-induced contractions in spontaneously contracted guinea-pig tracheas were not reproducible. However, reproducible K+ responses were obtained in the presence of indomethacin (10(-6) M) that almost abolished the spontaneous tone. This suggested that endogenous cyclooxygenase products were variably released by K+ and interfered with its contractile effects. Both phases of K+-induced contractions were inhibited in Ca2+-free/EGTA Krebs. In contrast, about 80% of the contractile response to carbachol persisted in this medium. Tracheas contracted by potassium (indomethacin present) were completely relaxed by theophylline and enprofylline but only partly relaxed by terbutaline. All bronchodilators completely relaxed carbachol-contracted preparations. Each bronchodilator was 2-3 times less potent to relax K+- than carbachol-induced contractions. In sharp contrast, two Ca2+ antagonists, verapamil and nimodipine, preferentially relaxed K+-induced contractions. The results obtained with Ca2+ antagonists, which are poorly effective in asthma, compared to the established antiasthma drugs, xanthines and beta 2-receptor agonists, may indicate that depolarization-induced mechanisms contribute little to bronchoconstriction in asthma.     

Differential inhibitory effects of nitroglycerin on contractile responses to the alpha-adrenoceptor agonists, methoxamine and clonidine, in rabbit aorta

The vasoinhibitory action of nitroglycerin was examined on contractile responses to methoxamine and clonidine in isolated rabbit aorta. Nitroglycerin at 10(-5) M, but not 10(-6)-10(-8) M, shifted the concentration response curve for methoxamine to the right. Nitroglycerin (10(-8)-10(-5) M), however, noncompetitively inhibited responses to clonidine in a concentration dependent manner. Nitroglycerin (10(-5) M) had no effect on responses to potassium (10-70 mM), but slightly inhibited responses to Ca2+ (0.1-5 mM) in a Ca2+-free medium containing potassium. Nifedipine (10(-6) and 10(-5) M), however, almost abolished responses to both potassium and Ca2+ but had no effect on responses to either methoxamine or clonidine. Agonist-antagonist interactions using prazosin and yohimbine revealed that responses to both methoxamine and clonidine were due to activation of alpha 1-adrenoceptors. Results with phenoxybenzamine suggested that the aorta has more receptor reserve for methoxamine than for clonidine. Furthermore, in tissues pretreated with phenoxybenzamine, nitroglycerin (10(-5) M) inhibited the maximal contractile response to methoxamine (3 x 10(-4) M). The maximal response to clonidine in tissues pretreated with phenoxybenzamine was not affected by nitroglycerin (10(-8) M). Nitroglycerin (10(-9)-10(-4) M) had greater inhibitory effect on residual responses to clonidine (10(-5) M) than that to methoxamine (10(-5) M) in a Ca2+-free medium containing EGTA. The contractile responses to Ca2+ (2 mM) in a Ca2+-free medium containing EGTA, nifedipine, and either methoxamine (5 x 10(-7) M) or clonidine (3 x 10(-7) M) were inhibited by nitroglycerin (10(-9) - 10(-5) M). The effect of nitroglycerin was greater on responses in the presence of clonidine than methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of barium-induced contraction in the vascular smooth muscle of rabbit aorta.

In a solution containing 1.5 mM Ca2+, cumulative application of 0.3-10.0 mM Ba2+ induced a concentration-dependent contraction of the rabbit aorta. This contraction was reduced by the Ca2+ channel inhibitors, verapamil (10(-6) M), nifedipine (10(-7) M) and lanthanum (2.0 mM), and was potentiated by the Ca2+ channel facilitator, Bay K8644 (10(-7) M). In a Ca2+-free solution containing EGTA (1.0 mM), cumulative application of Ba2+ still induced a concentration-dependent contraction, the maximum contractile tension of which was comparable to that in the presence of 1.5 mM Ca2+. The Ba2+-induced contraction which was not dependent on the external Ca2+ was also inhibited by verapamil, nifedipine and lanthanum and was potentiated by Bay K8644. A high concentration (65.4 mM) of K+ potentiated this Ba2+-induced contraction whereas noradrenaline (10(-6) M) did not have such an effect. In order to deplete the releasable Ca2+ store in the cell, the muscle strip was treated with noradrenaline (10(-6) M) and/or caffeine (20.0 mM) in a Ca2+-free solution. In such a Ca2+-depleted muscle, Ba2+ still induced a contraction of a similar magnitude to that without such treatment. Further, the second application of Ba2+ in a Ca2+-free solution induced a similar contraction to that induced by the first application of Ba2+. These results suggest that Ba2+ depolarizes the cell membrane and opens the voltage-dependent Ca2+ channels resulting in a Ca2+ influx in the presence of Ca2+. In the absence of external Ca2+, Ba2+ may enter the cell through the voltage-dependent Ca2+ channels and induce contraction without mobilizing the Ca2+ store which is sensitive to noradrenaline and caffeine.