Extracellular and intracellular calcium sources mediating contractile responses of smooth muscle in bovine ovarian follicle and ovarian artery

The relative importance of extracellular and intracellular calcium sources mediating smooth muscle contraction in ovarian follicle and ovarian artery was assessed in experiments on the influence of nifedipine, D-600, amrinone, diethylstilbestrol (DES), lanthanum and/or calcium removal on contractions induced by K+ depolarization, by noradrenaline, histamine and acetylcholine. The K+-induced response was biphasic in the ovarian artery but not in the ovarian follicle. The K+-induced contraction in both preparations was greatly inhibited by nifedipine (1 microM), D-600 (10 microM) and lanthanum (2 mM). Although both phases of the responses in the ovarian artery appeared to be completely dependent on extracellular calcium, phase I was significantly more sensitive to nifedipine than phase II. Incubation in calcium-free medium for 15 min almost abolished the K+-induced contraction. Noradrenaline- and histamine-induced contractions of ovarian follicle were essentially unaffected by nifedipine (1 microM) and D-600 (10 microM) whereas the noradrenaline-induced contraction in ovarian artery was inhibited significantly by D-600 (1 and 10 microM) but not nifedipine (1 microM). In calcium-free medium containing EGTA (1 mM) the responses of ovarian follicle to noradrenaline and histamine were reduced by 26 and 22% respectively. When preparations were stimulated with noradrenaline more than one in calcium-free medium, the contraction decreased progressively compared to time-matched controls. The response was 34% of the control after 50 min in calcium-free medium containing EGTA. In the ovarian artery the response obtained (6% of control) was significantly smaller (P less than 0.05) than that in the follicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influences of sodium on the contraction induced by oxytocin in rat testicular capsule

1. The effect of tetrodotoxin (5 microM), monensin (10 microM) and the replacement of Na+ by choline (choline medium) on the contractions of the rat testicular capsule induced by oxytocin (50 and 200 nM) have been studied. 2. The sodium channel blocker tetrodotoxin did not modify the oxytocin contraction. 3. The sodium ionophore monensin produces contraction of rat testicular capsule and reduces the oxytocin-induced contraction. The monensin contraction is inhibited by amiloride (0.1 mM). 4. Replacement of Na+ by choline increases the contraction induced by oxytocin and KCl (60 mM) but inhibits that induced by noradrenaline (3 microM). 5. The increase of contraction due to oxytocin in choline medium is inhibited by amiloride (50 microM and 1 mM) and when calcium is suppressed of the incubation medium.     

Adenosine A1-receptor stimulation of inositol phospholipid hydrolysis and calcium mobilisation in DDT1 MF-2 cells.

1. The effect of adenosine receptor-stimulation on inositol phospholipid hydrolysis and calcium mobilization has been investigated in the hamster vas deferens smooth muscle cell line DDT1 MF-2. 2. Adenosine receptor stimulation increased the accumulation of total [3H]-inositol phosphates in DDT1 MF-2 cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was N6-cyclopentyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine greater than adenosine. 3. The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (KD 1.2 nM), PD 115,199 (KD 39 nM) and 8-phenyltheophylline (KD 31 nM). 4. The inositol phosphate response to 2-chloradenosine (10 microM) was not significantly altered when the extracellular Ca2+ ion concentration was reduced from 2.4 mM to 1.2 mM or 0.6 mM. Under calcium-free conditions, however, a reduced but still significant response to 2-chloroadenosine was evident (39 +/- 10% of the response in calcium-containing medium). 5. The 5-lipoxygenase inhibitor AA861 (10 and 100 microM) inhibited the inositol phosphate response to 2-chloroadenosine by 40 +/- 9% and 60 +/- 4% respectively. The cyclo-oxygenase inhibitor, indomethacin, however, was without significant effect at 1 microM. 6. 2-Chloroadenosine stimulated an increase in intracellular free Ca2+ ion concentration in fura-2 loaded DDT1 MF-2 cells in calcium-free medium containing 0.1 mM EGTA, which could be inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM). 7. These data suggest that adenosine A1-receptor stimulation results in inositol phospholipid hydrolysis and calcium mobilization from intracellular stores in DDT1 MF-2 cells.     

Mechanism of endothelin-induced contraction in guinea-pig trachea: comparison with rat aorta.

1. Endothelin (1 nM-0.3 microM) produced a concentration-dependent contraction of guinea-pig epithelium-containing (intact) trachea (EC50 = 30.9 nM). Endothelin was a less potent agonist than leukotriene D4 (LTD4; EC50 = 0.77 nM), but was more potent than carbachol (EC50 = 0.15 microM) or substance P (EC50 = 1.4 microM). Endothelin was a more potent contractile agent in rat endothelium-denuded aorta (EC50 = 2.1 nM) than in guinea-pig trachea. 2. Endothelin-induced contraction in guinea-pig trachea was unaffected by mepyramine (10 microM), atropine (1 microM), SK&F 104353 (10 microM), a leukotriene receptor antagonist, or SQ 29,548 (1 microM), a thromboxane receptor antagonist. The contraction produced by 0.3 microM endothelin was potentiated by cyclo-oxygenase inhibition with 5 microM indomethacin. 3. Nicardipine (0.01 or 0.1 microM) or incubation in calcium-free medium +0.1 mM EGTA for 30 min had a relatively minor or no effect on endothelin concentration-response curves in guinea-pig intact trachea, but markedly inhibited responses produced by endothelin in endothelium-denuded aorta of the rat. Increasing the EGTA concentration in calcium-free medium to 1 mM abolished endothelin-induced contraction in guinea-pig trachea. 4. In guinea-pig trachea, ryanodine (10 microM) produced a 2.1 fold shift to the right of endothelin concentration-response curves and reduced the maximum response elicited by 0.3 microM endothelin. 5. Staurosporine (0.01 microM and 0.1 microM), a protein kinase C inhibitor, was without effect on endothelin- or carbachol-induced contraction in guinea-pig trachea, but markedly inhibited the response produced by endothelin in rat aorta. 6. Endothelin (3 nM-0.3 microM) produced a concentration-dependent stimulation of phosphatidylinositol (PI) turnover in guinea-pig intact trachea, with an EC50 value of 45.9 nM. 7. Removal of the epithlium markedly potentiated endothelin-induced contraction in guinea-pig trachea, producing a 4.7 fold leftward shift in endothelin concentration-response curves and an increase in the contractile response elicited by 0.3 microM endothelin. 8. These data indicate that endothelin is a potent agonist in guinea-pig trachea whose response is markedly enhanced by removal of the airway epithelium. Endothelin-induced contraction is not mediated to a marked extent by calcium influx via dihydropyridine-sensitive calcium channels and does not involve the release of histamine, acetylcholine, leukotrienes or thromboxane. Rather, endothelin appears to produce contraction of guinea-pig trachea via a direct action which involves stimulation of PI turnover and utilization of calcium from intracellular stores and, also, calcium influx via a pathway that is not sensitive to dihydropyridine calcium channel inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thapsigargin-induced tone and capacitative calcium influx in mouse anococcygeus smooth muscle cells

The sarcoplasmic reticulum Ca-ATPase inhibitor thapsigargin (Tg; 0.4-100 nM) produced concentration-related, strong and sustained contractions of the mouse-isolated anococcygeus muscle; these contractions were dependent on extracellular calcium but were only partially reduced (by about 50%) in the presence of verapamil (10 and 100 microM). The verapamil-resistant component of the Tg-induced contraction was relaxed by the general calcium entry blockers SKF96365 (0.4-40 microM) and cadmium (50-300 microM), and by the tyrosine kinase inhibitor genistein (10-180 microM). In single smooth muscle cells loaded with Fura-2, addition of Tg (100 nM) to calcium-free medium produced a small, transient increase in fluorescence; subsequent addition of calcium (2.5 mM) produced a larger and sustained increase which was abolished on return to calcium-free conditions, but was only partially reduced by verapamil (10 microM; by about 30%). Manganese quenching of Fura-2 was enhanced in cells treated with Tg. The verapamil-resistant calcium influx was reduced by SKF96365 (20 microM) and to a lesser extent by genistein (40 microM); cadmium (200 microM) produced an initial decrease in fluorescence followed by a marked increase. These results demonstrate that, in the mouse anococcygeus, Tg can cause sustained contractions and elevations of calcium influx in the presence of verapamil; the time-course, calcium dependence and, although to a lesser extent, pharmacology of these effects generally support the proposal that excitation-contraction coupling in this tonic smooth muscle involves sustained capacitative calcium influx.     

The response of non-pregnant rat myometrium to oxytocin in Ca-free solution.

The contractile response of the longitudinal muscle of non-pregnant rat myometrium to oxytocin (0.2-20 nM) consisted of a phasic and a tonic component. Ca-removal abolished the phasic component but a tonic contraction could be evoked without reduction of amplitude for 50 h. Exceptionally, the tonic contraction also disappeared gradually in Ca-free medium containing 2 mM EGTA. When oxytocin was repeatedly applied in the absence of Ca, the response became at first progressively larger before reaching a steady state. Transient addition of Ca to the medium reduced the size of the subsequent oxytocin contraction. In Ca-free medium, the tissue lost Ca slowly, but it still contained 40 mumol kg-1 after 6 h and roughly 1 mumol kg-1 wet weight after 24 h exposure. 45Ca efflux was marginally increased by oxytocin (20 nM). Caffeine (5-30 mM) produced no contraction, but slightly reduced the resting tension and strongly inhibited the oxytocin response both in the presence and in the absence of Ca. Caffeine also blocked the contraction induced by Ca added to Ca-free 40 mM K solution. However, pretreatment with caffeine (30 mM) had no effect on the following oxytocin response. A calmodulin antagonist, trifluoperazine (1-10 microM) suppressed strongly the Ca-induced contraction, but had only a weak effect on the oxytocin response in Ca-free medium. Chlorpromazine (10-100 microM) and fluphenazine (10-30 microM) had similar effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells.

1. Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin-induced changes in [Ca2+]i by dynamic video imaging. 2. Bradykinin (10 pM-10 microM)-induced an increase in [Ca2+]i over basal levels (69 +/- 2 nM; n = 353) which was concentration-dependent (log EC50 = -8.7 M) in the presence of extracellular calcium ions (2 mM). The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]- bradykinin, produced a parallel shift to the right of the bradykinin concentration-response curve (log EC50 = -7.1 M and -5.8 M in the presence of 1 microM and 10 microM antagonist respectively) yielding an apparent KD of 26 nM. 3. In the absence of extracellular calcium ions (with 0.1 mM EGTA), bradykinin (10 pM-10 microM) produced a uniform increase in [Ca2+]i from a basal level of 33 +/- 2 nM (n = 140) to approximately 180 nM in BTSM cells indicating an 'all-or-nothing' release of intracellular calcium ions. In the presence of 10 microM D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin no responses could be induced by bradykinin at concentrations below 100 nM. However, at 100 nM and 1 microM bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed. 4. In both the absence or presence of D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin, there was a concentration-dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium-rich or calcium-free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Ca deprivation and of Ca-blocking drugs on oxytocin-induced contractions of the male mouse anococcygeus

Oxytocin (4 nM)-induced contractions of the male mouse anococcygeus were rapidly and completely lost in EGTA (2 mM)-containing, Ca-free Krebs solution. Contractions were also lost, although more slowly, in Ca-free Krebs solution without EGTA; under such conditions, readdition of Ca did not by itself cause contraction, but readdition of Ca (0.1-2.5 mM) in the presence of 4 nM oxytocin resulted in a rapid contractile response. These Ca-induced responses, in the presence of oxytocin, and those to oxytocin in normal Ca-containing Krebs solution, were unaffected by nitrendipine (0.01-1 microM). Contractions to oxytocin were completely blocked by the calmodulin antagonists trifluoperazine (50 microM) and W-7 (75 microM). It is concluded that oxytocin-induced contraction of the mouse anococcygeus does not require opening of nitrendipine-sensitive Ca channels, and there is no Ca-independent component of the contractile response; the cellular mechanisms linked to the oxytocin receptor in the anococcygeus are therefore different from those in the uterus.     

Adenosine A1 receptor-mediated changes in basal and histamine-stimulated levels of intracellular calcium in primary rat astrocytes.

1. The effects of adenosine A1 receptor stimulation on basal and histamine-stimulated levels of intracellular free calcium ion concentration ([Ca2+]i) have been investigated in primary astrocyte cultures derived from neonatal rat forebrains. 2. Histamine (0.1 microM-1 mM) caused rapid, concentration-dependent increases in [Ca2+]i over basal levels in single type-2 astrocytes in the presence of extracellular calcium. A maximum mean increase of 1,468 +/- 94 nM over basal levels was recorded in 90% of type-2 cells treated with 1 mM histamine (n = 49). The percentage of type-2 cells exhibiting calcium increases in response to histamine appeared to vary in a concentration-dependent manner. However, the application of 1 mM histamine to type-1 astrocytes had less effect, eliciting a mean increase in [Ca2+]i of 805 +/- 197 nM over basal levels in only 30% of the cells observed (n = 24). 3. In the presence of extracellular calcium, the A1 receptor-selective agonist, N6-cyclopentyladenosine (CPA, 10 microM), caused a maximum mean increase in [Ca2+]i of 1,110 +/- 181 nM over basal levels in 30% of type-2 astrocytes observed (n = 53). The size of this response was concentration-dependent; however, the percentage of type-2 cells exhibiting calcium increases in response to CPA did not appear to vary in a concentration-dependent manner. A mean calcium increase of 605 +/- 89 nM over basal levels was also recorded in 23% of type-1 astrocytes treated with 10 microM CPA (n = 30). 4. In the absence of extracellular calcium, in medium containing 0.1 mM EGTA, a mean increase in [Ca2+]i of 504 +/- 67 nM over basal levels was recorded in 41% of type-2 astrocytes observed (n = 41) after stimulation with 1 microM CPA. However, in the presence of extracellular calcium, pretreatment with the A1 receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, for 5-10 min before stimulation with 1 microM CPA, completely antagonized the response in 100% of the cells observed. 5. In type-2 astrocytes, prestimulation with 10 nM CPA significantly increased the size of the calcium response produced by 0.1 microM histamine and the percentage of responding cells. Treatment with 0.1 microM histamine alone caused a mean calcium increase of 268 +/- 34 nM in 41% of the cells observed (n = 34). After treatment with 10 nM CPA, mean calcium increase of 543 +/- 97 nM was recorded in 100% of the cells observed (n = 33).(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the effects of hydralazine and nifedipine on contractions and 45Ca influx of rat aorta.

The effect of the vasodilator hydralazine has been compared with nifedipine on KCl-(K+) (60 mM) and noradrenaline-(NA) (10 microM) induced 45Ca uptake and contractile responses in rat aorta arterial strips without endothelium. Hydralazine (0.5-10 mM) was equally effective in relaxing K(+)- (IC50 = 2.2 +/- 0.17 mM) and NA- (IC50 = 3.06 +/- 0.25 mM) induced tension, the degree of relaxation depending on the dose. Nifedipine totally inhibited K(+)- (IC50 = 3.16 +/- 0.28 nM) induced contractions with lower doses than were necessary to relax (up to 54.0 +/- 4.1% with supramaximal concentrations) NA-induced contractions (IC50 = 1.48 +/- 0.12 microM). In the experiments in a calcium-free medium, nifedipine (1 microM) had no effect on the NA- (10 microM) induced contractions whereas hydralazine (1 mM) strongly inhibited them. Nifedipine did not affect the basal uptake of 45Ca but the induced uptakes were reduced to 66.3 +/- 3.2% (K+) and 65.5 +/- 4.1% (NA) of their basal values. Hydralazine did not affect the basal uptake of 45Ca nor that induced by the two vascoconstrictor agents. These results suggest that nifedipine acts on the cell membrane by blocking the movements of calcium through the voltage-dependent and receptor-operated calcium channels, whilst hydralazine has an intracellular effect.     

Vasoconstrictive responses elicited by endothelin in bovine cerebral arteries.

1. Endothelin (ET-1) induced concentration-dependent contractions, which were slowly developed in segments of bovine cerebral arteries. Furthermore, this agent produced tachyphylaxis. 2. The contractions evoked by ET-1 were markedly reduced in Ca-free medium containing 1 mM EGTA and by the Ca channel antagonist, nifedipine (1 microM), but increased by the Ca channel agonist, BAY K 8644 (10 nM). 3. The contractions caused by ET-1 were significantly reduced by the protein kinase C (PKC) inhibitor, staurosporine (1 and 10 nM). 4. These results indicate that ET-1 induced potent vasoconstrictive responses, probably mediated by PKC activation, which were mainly dependent on extracellular Ca; this Ca enters the smooth muscle cells via dihydropyridine sensitive Ca channels.     

Calcium influx and protein kinase C activation involved in uterine vasoconstriction in guinea pigs

The mechanical responses of circular segments of uterine arteries to different combinations of vasoactive agents and putative inhibitors of calcium fluxes were examined using a sensitive in vitro method. Exposure to high potassium (127 mM) or to noradrenaline (NA; 0.1 mM) resulted in a rapid, initial increase in tension of the vascular preparation to reach a sustained level of contraction (approximatively 12 mN) which lasted for at least 15 min. The protein kinase C activator, 4-beta-phorbol-12,13-esther-dibutyrate (10 microM), induced a slowly developing but sustained contractile response with a maximum (PDBumax) of only 4 mN. Addition of the calcium ionophore, A23187, to PDBu-contracted vessels did not increase tension, while the maximum tension evoked by A23187 per se (3 microM) was 5 mN. Ionomycin had only a small contractile effect on the uterine artery. The contraction evoked by depolarization (potassium) or alpha 1-adrenoceptor stimulation (NA) was decreased in nominally calcium-free medium containing EDTA (0-100 microM) or EGTA, while uterine arteries loaded intracellularly with the calcium chelator, quin-2, responded to the vasoconstrictors almost as well as the control preparations. Blockade of calcium influx with Cd2+ (greater than 0.01 mM), nifedipine (greater than 3 microM), verapamil (greater than 1 microM) and TMB-8 (greater than 10 microM) reduced the tension evoked by potassium somewhat more than it reduced the contractions induced by NA, while the opposite was seen in the presence of Ni2+ (greater than 0.1 mM). Inhibition of calmodulin-dependent enzymes by W7 (greater than 10 microM) reduced the maximum tension evoked by potassium and NA to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of calcium entry blockers on contractions evoked by endothelin-1, [Ala3,11]endothelin-1 and [Ala1,15]endothelin-1 in rat isolated aorta.

1. The aim of the present study was to examine the contractile responses evoked by the recently characterized vasoactive peptide endothelin-1 (ET-1) and by two of its structural analogues, [Ala3,11]ET-1 and [Ala1,15]ET-1 in endothelium-denuded rat isolated aorta, and also to assess the extent of dependence of these responses on extracellular calcium entry. 2. ET-1 (0.3 to 10 nM), [Ala3,11]ET-1 (2.25 to 225 nM) and [Ala1,15]ET-1 (0.04 to 1.36 microM) evoked concentration-dependent contractions in normal, calcium-containing medium with the order of potency: ET-1 greater than [Ala3,11]ET-1 greater than [Ala1,15]ET-1. 3. Preincubation of tissues for 60 min with diltiazem (1 microM) induced a significant 3 fold rightward shift of concentration-effect curves to ET-1 without affecting maximal responses elicited by 10 nM of this peptide, whereas the same treatment failed to modify concentration-effect curves to [Ala3,11]ET-1. 4. Preincubation of tissues for 60 min with nifedipine (0.1 or 1 microM) markedly inhibited contractions elicited by either ET-1 (10 nM) or [Ala1,15]ET-1 (0.41 microM). Furthermore, when added cumulatively to tissues maximally contracted by ET-1 (10 nM), nifedipine (3 nM to 1 microM) induced concentration-dependent relaxations with an IC50 value of 21.8 +/- 5.9 nM. Maximal relaxation to nifedipine, 1 microM, amounted to 56.9 +/- 11.5%. 5. Submaximal concentrations of ET-1 (3 nM), [Ala3,11]ET-1 (75 nM) and [Ala1,15]ET-1 (0.41 microM), gave about 85% of maximal contractions elicited by noradrenaline (1 microM) in normal, calcium-containing medium. These contractile responses were all reduced by about 70% in calcium-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of some inhibitors of arachidonic acid metabolism in rat uterus in vitro

The effect of four eicosanoid synthesis inhibitors (ESIs): mepacrine (6 x 10(-6) to 10(-4) M), nordihydroguaiaretic acid (NDHGA, 10(-6) to 10(-5) M), indomethacin (2 x 10(-6) and 2 x 10(-5) M) and imidazole (10(-5) and 10(-4) M), were studied on contractions induced by cumulative doses (4 and 20 mU/ml) of oxytocin (OT) on the uterus of rats both in natural estrus and ovariectomized. ESIs were also assayed on contractions induced by carbachol (10(-4) M) and KCl (60 mM) in rat uterus under natural estrus, and OT (10 mU/ml)-induced contractions in rat uterus incubated in calcium-free EDTA treated medium. Mepacrine, NDHGA and indomethacin, but not imidazol, inhibited in a dose-dependent way contractions induced by OT in the uterus. The effect was higher in ovariectomized than in natural estrus rats. Mepacrine and NDHGA, but not indomethacin or imidazole, inhibited contractions induced by carbachol and relaxed tonic contractions to KCl (60 mM). Mepacrine, NDHG and indomethacin also inhibited tonic contractions by OT in calcium-free EDTA treated medium. Our results suggest that mepacrine, NDHGA and indomethacin, independently of inhibition of eicosanoids synthesis, reduce the entry of extracellular calcium and/or the release of intracellular calcium.     

Mechanisms involved in the vasodilator effect of curine in rat resistance arteries

The vasodilator effect of curine was investigated in the rat small mesenteric arteries. In either endothelium-intact or endothelium-denuded mesenteric arteries, curine induced a concentration-dependent relaxation in rings pre-contracted with noradrenline (10 microM; IC 50 = 4.8 +/- 1.3 microM and 4.8 +/- 1.5 microM, respectively) and KCl (80 mM; IC 50 = 6.0 +/- 1.3 microM and 13.0 +/- 5.6 microM, respectively). Curine also inhibited (IC 50 = 4.6 +/- 0.9 microM) the concentration-response curves induced by noradrenaline. Contractions dependent on calcium-influx elicited by KCl (80 mM) and noradrenaline (10 microM) were inhibited by curine (10 microM). Finally, contractions induced by noradrenaline (10 microM), in calcium-free medium, were strongly inhibited by curine (10 microM). The above results suggest that the inhibition of influx of calcium ions through voltage-operated calcium channels and non-selective channels, and mobilization of intracellular calcium stores sensitive to noradrenaline are involved in the vasodilator effect of curine.     

Caffeine acting on pregnant rat myometrium: analysis of its relaxant action and its failure to release Ca2+ from intracellular stores.

1. The effect of caffeine on mechanical activity was studied in pregnant rat myometrium. 2. In muscle cells with intact plasmalemmae, caffeine (0.1-50 mM) produced no contraction whatever the experimental conditions. 3. Caffeine (0.1-10 mM) inhibited, in a concentration-dependent manner, contractions induced by electrical stimulation, potassium-rich (60 mM K+) solution, sodium-free solution or oxytocin (22.5 nM). 4. In Ca2(+)-free solution, various substances (oxytocin, sodium orthovanadate and prostaglandin E2) evoked sustained contractions that were suppressed by caffeine (5-10 mM). When caffeine (greater than 5 mM) was applied during Ca2(+)-loading of the tissue (2.1 mM Ca2+, 5 min) in the presence of a K(+)-rich solution, the subsequent transient contraction induced by a short application (10s) of oxytocin (22.5 nM) in Ca-free solution was reduced (63 +/- 3.5% reduction for 20 mM caffeine, n = 4). 5. In saponin-skinned strips, application of caffeine (5-10 mM) during loading of the Ca2(+)-store increased the subsequent contraction induced by myo-inositol 1,4,5 trisphosphate (IP3, 10 microM). Caffeine (10-30 mM) decreased calcium-activated contractions in skinned fibres lacking a functional internal Ca-store. This effect was reduced by the cyclic AMP-dependent protein kinase inhibitor Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp (8 microM). 6. In conclusion, it is suggested that the inability of caffeine to cause spasm of rat myometrium is due to the absence of a caffeine-sensitive calcium-release channel in the sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex effects of Gillichthys urotensin II on rat aortic strips.

The aim of this study was to determine whether the fish neuropeptide, Gillichthys urotensin II (GUII), possesses significant biological activity on rat aortic strips. On intact strips, pre-contracted by noradrenaline (100 nM), low concentrations (0.1-0.5 nM) of GUII produced relaxations, while higher concentrations (1-10 nM) caused further contraction. On strips rubbed to remove endothelial cells, relaxations were absent but contractile responses to higher concentrations of GUII remained. GUII (0.02-10 nM) produced dose-related contractions of quiescent, intact aortic strips. These contractions consisted of two components, tonic and phasic, and were potentiated in rubbed strips and in the presence of the antioxidant drug hydroquinone (10 microM). Mepacrine (40 microM) and p-bromophenacyl bromide (50 microM) completely abolished contractions to GUII, but indomethacin (10 microM) and nordihydro-guaiaretic acid (10 microM) were without effect. The phasic, but not the tonic, component of the contractile response was inhibited by nitrendipine (200 nM), and was absent in bathing medium from which Ca2+ had been omitted. Addition of EGTA (2 mM) to Ca2+-free bathing medium abolished the residual tonic component. GUII-induced contractions were completely abolished by the calmodulin antagonists trifluoperazine (50 microM) and W-7 (30 microM). It is concluded that GUII, previously considered devoid of significant activity on mammalian tissues, produces potent endothelium-dependent relaxations and endothelium-independent contractions of rat aorta, and possible mechanisms underlying each response are discussed.     

Calcium antagonists inhibit positive chronotropic responses to alpha 1-adrenoceptor activation in rat isolated atria

Positive chronotropic responses of rat isolated atria to phenylephrine were reduced by propranolol (0.3 microM) and the residual response was further depressed by the selective alpha 1-adrenoceptor antagonist prazosin (1 nM) but not yohimbine (10 nM), confirming that a component of the response to phenylephrine was due to activation of alpha 1-adrenoceptors. When beta-adrenoceptors were blocked by propranolol, the positive chronotropic response to phenylephrine was enhanced by increasing the calcium concentration and by the calcium channel activator Bay K 8644 (0.1 microM), whereas the response was decreased by lowering the calcium concentration and by the calcium antagonists verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM). In the presence of prazosin, when phenylephrine acts only on beta-adrenoceptors, calcium antagonists had no effect on the response. In rat isolated aortic strips in a calcium-free, high K+ (40 mM) solution, verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM) shifted the calcium-induced contraction curves to the right, but prazosin (10 nM) had no effect, indicating that it is not a calcium antagonist. The calcium antagonists in the concentrations stated above had no effect on phenylephrine-induced contractions of rat aortic strips in normal Krebs-Henseleit solution, indicating that they did not block alpha 1-adrenoceptors in these concentrations. Taken together, these data suggest that the positive chronotropic effect of phenylephrine resulting from activation of alpha 1-adrenoceptors involves an increased influx of calcium through channels that are sensitive to organic calcium antagonists.     

Study of the mechanism of the relaxant action of (+)-glaucine in rat vas deferens.

1. Effects of the aporphinoid alkaloid, (+)-glaucine, on rat vas deferens were investigated. 2. (+)-Glaucine (2-18 microM) competitively inhibited contractions induced by noradrenaline and methoxamine with a pA2 value of about 6. 3. (+)-Glaucine (2 and 18 microM) did not change the accumulation of tritium during incubation of the vas deferens with [3H]-noradrenaline. 4. (+)-Glaucine (0.3 nM-0.1 mM) inhibited specific [3H]-prazosin binding to membranes from rat vas deferens with a pKi value of 6.63, which is close to the pA2 value obtained against noradrenaline and methoxamine in functional studies. 5. In electrically-stimulated rat vas deferens, (+)-glaucine (0.3-10 microM) enhanced twitch contractions and competitively antagonized the inhibitory effect of clonidine with a pA2 value of 5.91. 6. In tissues incubated in depolarizing calcium-free high-potassium medium, (+)-glaucine (30-80 microM) inhibited Ca(2+)-induced contractions with depression of the maximal response at higher doses and with a pD'2 value of 3.65. Furthermore, (+)-glaucine (50 microM) did not modify basal 45Ca uptake but strongly inhibited the influx of 45Ca induced by K+. 7. These results suggest that (+)-glaucine has non-selective alpha 1- and alpha 2-adrenoceptor blocking properties. At higher doses, (+)-glaucine shows calcium antagonist activity which may be responsible, at least in part, for the inhibition of the contractions induced by Ca2+ in calcium-free high-potassium medium.     

Effect of calcium and calmodulin antagonists on contractile responses of the human uterine artery.

1. The dependence on extracellular calcium of contractile responses of intramyometrial arteries (0.5-2 mm diameter), as well as the effects of various types of calcium antagonists on these responses, were studied. Contractions were induced by K-depolarization (K) and noradrenaline (NA). 2. Whereas the K response was completely abolished in a calcium-free medium containing 2 mM LaCl3, the NA response was substantially maintained. 3. Nimodipine strongly inhibited the K response but had a relatively weak effect on the NA response; the IC50 values for the K and NA responses being 2 nM and 6 microM, respectively. Corresponding values for verapamil were about 0.7 and 10 microM. 4. Calmodulin antagonists, particularly trifluoperazine and flunarizine, caused a greater inhibition of the NA than of the K response. 5. These results indicate that besides the extracellular calcium which appears to be the major source of activator calcium, there is an intracellular pool of calcium which can be utilized to activate, albeit to a limited extent, drug-induced contractile responses.