Heterogeneity of the effects of monensin on two types of contractions evoked by inhibition of Na+-K+ ATPase in the guinea-pig vas deferens

The interactions between ouabain or K+-free medium and monensin on noradrenaline (NA) release and Ca2+-evoked contractions were assessed in guinea-pig vas deferens. Ouabain (10 microM) produced large, sustained contractions and monensin (1 microM) small, transient contractions. The ouabain-evoked contraction tended to be potentiated in the presence of monensin. The marked contraction induced by ouabain plus monensin was nearly abolished by phentolamine or by treatment with 6-hydroxydopamine. Ouabain caused the release of NA from the tissue, as determined in an HPCL-ECD study. This ouabain-evoked release of NA was enhanced by the simultaneous administration of monensin. High Ca2+ (10 mM) per se, ouabain, monensin or K+-free medium did not elicit contraction of the tissue in the presence of phentolamine. However, 10 mM Ca2+ caused a large contraction 45-65 min after exposure to ouabain or K+-free medium and this contraction was suppressed by monensin. The contraction evoked by Ca2+ in the presence of ouabain was further inhibited by amiloride, an inhibitor of Na+-Ca2+ or Na+-H+ exchange transport, but not by nifedipine, a voltage-dependent Ca2+ antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of vanadate, ouabain and amiloride on the contraction of the rat testicular capsule to oxytocin

1. The modification of the contraction of the rat testicular capsule to oxytocin (OT) by vanadate (0.7, 7 and 70 microM), ouabain (0.1 mM), and amiloride (10 microM to 1 mM) have been studied. 2. OT (1 nM-6 microM) and vanadate (10 microM-3 mM) induced contraction of the rat testicular capsule in a dose-dependent way (ED50: 188 +/- 66 nM and 82.8 +/- 7.4 microM, respectively). 3. Vanadate (0.7, 7 and 70 microM) and ouabain (0.1 mM) increases the contractile effect of OT (50 and 200 nM). 4. Amiloride (10 microM-1 mM) inhibit, in a dose-dependent way, the OT-contraction. 5. Amiloride (10 microM or 50 microM) block the ouabain but not the vanadate potentiation to OT.     

Interactions between oxytocin- and calcium-modifying agents in the rat testicular capsule in vitro

We have studied the effect of drugs which affect the movement of calcium on the contractions induced by 50 and 200 nM oxytocin in the isolated testicular capsule of the rat. The ED50 for oxytocin in this preparation was 188 (+/- 66 S.E.) nM and the maximal contraction induced by oxytocin was smaller than that obtained with 10 microM of the calcium ionophore, A23187. Lanthanum (10 mM), cobalt (2 mM), EGTA (3.5 and 5 nM, 30 s exposure) and a decrease in the calcium concentration of the medium reduced the oxytocin response. The response was completely abolished after prolonged incubation with EGTA (2 mM) in a calcium-free medium. The calcium blocking agents, nifedipine and flunarizine, and the agonist, Bay K 8644, did not modify the responses to oxytocin. Verapamil, at possibly non-specific doses (10 microM), reduced the contractions while diltiazem (0.1 mM), in a prazosin (10 microM)-resistant way, and nickel (0.1 mM) increased them. Both modifiers of intracellular calcium that were used namely TMB-8 (10 microM), in a calcium-free medium, and dantrolene sodium (10 and 30 microM), with and without calcium in the medium, decreased the oxytocin response. On the whole, it seems as if both intra- and extracellular calcium were involved in the contractile effect of oxytocin, although extracellular calcium does not seem to gain access to the cell through voltage-dependent calcium channels sensitive to selective calcium entry blockers.     

Na+ influx-induced decrease of (Na+ + K+)-ATPase activity in rat brain slices: role of Ca2+.

Treatment of rat brain slices with veratrine and monensin decreased (Na+ + K+)-ATPase activity in the membranes in a dose-dependent manner. The effect of monensin, like that of veratrine, was accompanied by a decrease of maximal binding sites for ouabain. The inhibitory effect of monensin on the enzyme activity was dependent on external Ca2+ at low concentrations, but not at a high concentration. The decreased enzyme activity induced by monensin was restored by subsequent incubation of the slices in a Ca(2+)-free medium containing 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), a chelator of intracellular Ca2+. The effect of monensin at a low concentration on enzyme activity was antagonized by amiloride (1 mM), bepridil (5 microM), quinacrine (30 microM) or verapamil (30 microM), but not by nifedipine (1 microM) or omega-conotoxin (1 microM). Furthermore, the inhibitory effect of monensin at a high concentration under Ca(2+)-free conditions was blocked by BAPTA-AM (30 microM) and by bepridil (100 microM) or diazepam (500 microM), inhibitors of mitochondrial Na(+)-Ca2+ exchange. Inhibitors of calmodulin, protein kinase C, phospholipase A2 and calpain did not affect the monensin-induced decrease of enzyme activity. Dithiothreitol (10 mM) blocked the effect of monensin on enzyme activity but did not affect the ionophore-induced influx of Ca2+ in the slices.     

Effects of vanadate in testicular capsule of the rat

1. The effects of sodium orthovanadate (vanadate. 10(-5) to 3 x 10(-4) M) on testicular capsule of the rat, and the modifications of these effects by the calcium chelator EGTA (2mM), the calcium entry blockers verapamil (5 X 10(-5) M), nifedipine (10(-5 M) and diltiazem (5 x 10(-5) M), the (Na+ + K+)-ATPase inhibitor ouabain, the Na+/Ca2+ exchange inhibitor amiloride, and the calmodulin antagonists trifluoperazine (10(-4) M) and W-7 (5 x 10(-5) M) have been studied. 2. Vanadate induced contraction of the rat testicular capsule in a dose-dependent way (ED50: 82.8 +/- 7.4 x 10(-6) M). 3. The contraction induced by vanadate (3 and 30 x 10(-5) M) were abolished by EGTA and not modified by verapamil, nifedipine, flunarizine or diltiazem. 4. Amiloride (1 and 5 x 10(-5) M), but not ouabain (5 x 10(-5) and 10(-4) M), inhibit in a dose-dependent way the contraction induced by two doses of vanadate (3 and 30 x 10(-5) M). 5. Trifluoperazine and W-7 significantly inhibit the contraction of testicular capsule to 3 and 30 x 10(-5) M vanadate.     

Role of sodium-calcium exchange and effects of calcium entry blockers on endothelial-mediated responses in rat isolated aorta

Acetylcholine relaxes rat aorta and increases aortic cyclic GMP levels by a mechanism (or mechanisms) dependent on the endothelium and on extracellular calcium. Therefore, the effects of representatives of different subclasses of calcium entry blockers, verapamil, nifedipine, diltiazem, and bepridil, on maximal acetylcholine (1 microM)-induced increases in cyclic GMP levels were investigated in rat isolated aorta. None of these compounds, at a concentration (3 microM) sufficient to maximally inhibit agonist-stimulated Ca2+ influx into vascular smooth muscle cells, significantly affected either the basal or the acetylcholine-stimulated tissue cyclic GMP levels. On replacing all but 20 mM Na+ by choline, a condition that might be expected to limit or even abolish Na+-Ca2+ exchange, or in the presence of amiloride (1 mM), an inhibitor of Na+-Ca2+ exchange, acetylcholine-stimulated increases in tissue cyclic GMP levels were abolished or inhibited by about 80%, respectively. In choline containing solution acetylcholine relaxant responses were abolished. The presence of amiloride, or the replacement of Na+ by choline, had no effect on increases in cyclic GMP levels evoked by sodium nitroprusside (0.3 microM), an agent that stimulates cyclic GMP formation in smooth muscle without intervention of the endothelium. Replacement of Na+ by Li+ but not the other treatments depressed basal tissue cyclic GMP levels by about 45% but did not abolish either acetylcholine- or sodium nitroprusside-induced relaxant responses. However, the time course of relaxant responses elicited by both these relaxant agonists in precontracted rat aortic rings with endothelium was altered by Li+ replacement; the half-time to relaxation to acetylcholine was increased by about 70-fold. It is concluded that calcium channels, as characterized in smooth muscle and cardiac tissue, are not involved in the stimulated liberation of an endothelial-derived relaxant factor by acetylcholine, but that an Na+-Ca2+ exchange process may be of importance.     

Inhibitory mechanism of monensin on high K+-induced contraction in guniea-pig urinary bladder

In this study, we examined the inhibitory mechanism of monensin on high K+-induced contraction in guinea-pig urinary bladder. The relaxant effect of monensin (0.001 - 10 microM) was more potent than those of NaCN (100 microM - 1 mM) and forskolin (3 - 10 microM). Monensin (0.1 microM), NaCN (300 microM), or forskolin (10 microM) inhibited high K+-induced contraction without decreasing [Ca2+]i level. Monensin and NaCN remarkably decreased creatine phosphate and ATP contents. Monensin and NaCN inhibited high K+-induced increases in flavoprotein fluorescence, which is involved in mitochondrial respiration. Forskolin increased cAMP content but monensin did not. Monensin increased Na+ content at 10 microM but not at 0.1 microM that induced maximum relaxation. In the alpha-toxin-permeabilized muscle, forskolin significantly inhibited the Ca2+-induced contraction, but monensin did not affect it. These results suggest that the relaxation mechanism of monensin in smooth muscle of urinary bladder may be an inhibition of oxidative metabolism.     

Vascular smooth muscle contraction induced by Na+ channel activators, veratridine and batrachotoxin.

The effects of the sodium channel activators veratridine and batrachotoxin on isolated rat aorta were investigated. Veratridine caused gradual contraction, independent of the presence of endothelium, with an EC50 of 35 microM. Batrachotoxin (1 microM) also induced contraction. Both effects were completely inhibited by the sodium channel blocker tetrodotoxin (1 microM). The veratridine (60 microM)-induced contraction was inhibited by nifedipine (0.1 microM). In the absence of extracellular Ca2+, veratridine (60 microM) did not cause contraction. Sodium nitroprusside (80 nM), acetylcholine (10 microM) and isoproterenol (1 microM) caused relaxation of rings precontracted with veratridine (60 microM). An inhibitor of endothelium-derived relaxing factor (EDRF) synthase, N omega-nitro-L-arginine methyl ester (L-NAME) (0.65 mM), enhanced the veratridine-induced contraction in rings with an intact endothelium, which suggests that EDRF was being released during the veratridine-induced contraction. These results show that the activation of sodium channels on smooth muscle cells induces a contraction that is probably mediated by Ca2+ influx through voltage-dependent Ca2+ channels.     

Mechanism of palytoxin-induced [3H]norepinephrine release from a rat pheochromocytoma cell line

Palytoxin, isolated from the zoanthid Palytoha species, is one of the most potent marine toxins. Palytoxin (1 nM-1 microM) caused a release of [3H]norepinephrine from clonal rat pheochromocytoma cells in a concentration-dependent manner. This releasing action of palytoxin was markedly inhibited or abolished by Co2+ or Ca2+ -free medium, but was not modified by tetrodotoxin. The release of [3H]norepinephrine induced by a low concentration (30 nM) of palytoxin was abolished in sodium-free medium and increased as the external Na+ concentrations were increased from 3 to 100 nM, but the release induced by a high concentration (1 microM) was unaffected by varying the concentration of external Na+ from 0 to 100 mM. The release of [3H]norepinephrine induced by both concentrations of palytoxin increased with increasing Ca2+ concentrations from 0 to 3 mM. Palytoxin caused a concentration-dependent increase in 22Na and 45Ca influxes into pheochromocytoma cells at concentrations of 0.1 nM-10 nM and 1 nM-1 microM, respectively. The palytoxin-induced 45Ca influx was markedly inhibited by Co2+, whereas the palytoxin-induced 22Na influx was not affected by tetrodotoxin. These results suggest that in pheochromocytoma cells the [3H]norepinephrine release induced by lower concentrations of palytoxin is primarily brought about by increasing tetrodotoxin-insensitive Na+ permeability across the cell membrane, whereas that induced by higher concentrations is mainly caused by a direct increase in Ca2+ influx into them.     

Veratridine activates a silent sodium channel in rat isolated aorta.

To investigate the existence of silent Na+ channels, isolated rat aorta was treated with veratridine (0.1 mM) and the resulting Ca2+ uptake was determined. After 30-min incubation the total tissue uptake of Ca2+ and Ca2+ uptake increased from 2.325 +/- 0.017 to 2.614 +/- 0.080 nmol/mg wet weight (ww) and from 162.6 +/- 9.7 to 218.1 +/- 13.0 pmol/mg ww, respectively. The veratridine-induced Ca2+ uptake was blocked by tetrodotoxin (1 microM; to 17 +/- 5%) but not altered by amiloride (10 microM-1 mM). Activation of Na+/Ca2+ exchange by Na+ removal increased Ca2+ uptake from 74.2 +/- 4.5 to 97.3 +/- 5.3 pmol/mg ww, which was suppressed by amiloride (10 microM-1 mM). Nifedipine (10 nM) and verapamil (0.1 microM) at concentrations at which depolarization-induced Ca2+ uptake was diminished did not attenuate veratridine-induced Ca2+ uptake. Phenytoin at 0.1 mM reduced the Ca2+ uptake induced by veratridine or by depolarization. R 56865 (0.1 microM) and R 59494 (1 microM), novel anti-ischemic compounds inhibiting slowly inactivating Na+ channels, suppressed the veratridine-induced but not the depolarization-induced Ca2+ uptake. Guanidinium uptake was increased by veratridine (0.1 mM) from 371.2 +/- 7.2 to 574.8 +/- 45.9 pmol/mg ww. These results suggest that the rat aorta possesses a Na+ channel which is electrically silent under normal conditions but could be activated by veratridine.     

The inhibition of agonist- or depolarisation-evoked formation of inositol phosphate by excitatory amino acids in rat cerebral cortex is due to the neurotoxic action of this class of neurotransmitter and is mediated by sodium influx.

Previous work has shown that excitatory amino acids inhibit agonist or depolarisation evoked formation of inositol phosphate in brain. In this paper, possible mechanisms by which this may be occurring have been investigated. The inhibition of carbachol-stimulated formation of inositol phosphate by kainic acid (KA) was abolished if the tissue was incubated in a sodium-free medium. The sodium channel activator, veratridine (10 microM) and the sodium ionophore, monensin (3 microM), also inhibited the response of inositol phosphate to carbachol; tetrodotoxin (300 nM) reversed the effect of veratridine but not monensin or KA. Incubation with cadmium (0.3 mM) or removal of extracellular calcium did not alter the effects of KA, monensin or veratridine. The effects of KA were significantly reduced with the Na+/K(+)-ATPase inhibitor, ouabain (10-100 microM). Inhibition by KA was still observed in tissue that had been prestimulated with KA and then washed to remove the agonist. Incorporation of [3H]inositol into inositol lipids was significantly reduced by KA, in the absence or presence of carbachol. It is suggested that the inhibition of the turnover of stimulated phosphoinositide, by excitatory amino acids, is related to the neurotoxic actions of these transmitters and is mediated by Na+ influx, with a consequent activation of Na+/K(+)-ATPase, depletion of cellular ATP and reduction in synthesis of inositol lipid.     

The vasorelaxant effect of evocarpine in isolated aortic strips: mode of action

The effect of evocarpine (EVO), a quinolone alkaloid isolated from Evodiae fructus, on Ca2+-blocking activity has been examined. In the isolated rat thoracic aorta evocarpine significantly inhibited the contraction induced by 60 mM K+ with an IC50 of 9.8 microM, and that induced by external Ca2+ in the depolarized muscle in concentrations of 10-100 microM. The relaxant effect of evocarpine and verapamil was antagonized by Bay K8644. The increase of 45Ca2+-influx induced by 60 mM K+ was significantly inhibited by 100 microM evocarpine. In the isolated rabbit thoracic aorta 100 microM evocarpine had no effect on the norepinephrine-induced contraction in normal medium or on the phasic contraction in Ca2+-free medium or on the transient relaxation induced by activation of the Na+ pump. The content of cyclic AMP or cyclic GMP was unchanged. These results suggest that evocarpine inhibits Ca2+ influx through voltage-dependent calcium channels.     

Mechanism of inhibition of rat caudal artery contraction by amiloride

Amiloride (N-amidino-3,5-diamino-6-chloropyarzine carboxamide), a potassium sparing diuretic, decreases blood pressure and inhibits vascular smooth muscle contractility in rats. The purpose of these experiments was to investigate the mechanisms by which amiloride inhibits contraction of rat caudal artery. Caudal artery rings were mounted in a muscle chamber that was continuously flushed with circulated bathing medium and the contractions were recorded through a force transducer. The results demonstrate that amiloride competitively inhibited the norepinephrine-stimulated contraction of arterial rings (pA2 = 5.2). This suggests that amiloride and norepinephrine (NE) interact at the same site. The concentration required to produce half-maximum inhibition (IC50) of contraction produced by 10(-6) M norepinephrine was 48 +/- 6 microM. Amiloride (100 microM) shifted the concentration-response curves of norepinephrine and methoxamine to the right without significantly affecting the maximum contraction produced by these agonists. The EC50 value and the threshold concentration of both norepinephrine and methoxamine were increased by about 10-fold in the presence of 100 microM amiloride. Amiloride-induced inhibition of norepinephrine-stimulated contraction was reversible following removal of the drug. Amiloride-induced inhibition of vascular smooth muscle contraction was not blocked by ouabain or monensin. Amiloride at concentrations up to 100 microM did not inhibit K+ stimulated contractions. From these results we conclude that amiloride reversibly inhibits vascular smooth muscle contraction by competing with alpha-adrenoceptor agonists for binding to alpha 1-adrenoceptors.     

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)     

Practical usage concentrations of monensin have non-specific actions other than as a sodium ionophore in rat parotid acinar cells.

Monensin is used as a sodium ionophore to examine the effect of Na+ on cellular function in a variety of cell types. In the present study, we investigated the effects of different concentrations of monensin on the signal transduction system in exocrine parotid acinar cells. Monensin increased cytosolic free Na+ concentration, measured by the Na+ indicator sodium-binding benzofuran isophthalate in a concentration-dependent manner (0.01 to 100 microM). Likewise, monensin concentration-dependently increased amylase release and intracellular Ca2+ concentration in the presence and the absence of extracellular Ca2+. Low concentrations (0.01 to 1 microM) of monensin did not release Ca2+ from non-mitochondrial intracellular pools in permeabilized cells with saponin but high concentrations (10 and 100 microM) of monensin which are of practical usage did. Monensin itself did not change the cyclic AMP accumulation, whereas high concentrations (10 and 100 microM) but not low concentrations (0.01 to 1 microM) of monensin inhibited cyclic AMP accumulation elevated by isoproterenol in the presence and absence of extracellular Na+. These results indicate that high concentrations of monensin, which are practically used, have nonspecific actions in rat parotid acinar cells, and lower concentrations of monensin are recommended for use as a sodium ionophore.     

Atropine-resistant relaxation induced by high K+ in iris dilator muscle of the rat and pig.

1. The effects of high K+ ion concentration on the isometric tension in dilator muscle strips of the rat and porcine iris were examined. A high K+ solution, prepared by the replacement of Na+ in the medium with equimolar K+, was applied in the presence of 1 microM phentolamine, 1 microM propranolol and 1 or 10 microM atropine. High K+ (greater than 20 mM) induced a biphasic response; an initial phasic contraction followed by relaxation rather than tonic contraction. 2. An additional application of a Ca2+ antagonist, 1 microM nifedipine or nicardipine, almost completely blocked the K(+)-induced initial contraction and enhanced the following relaxation. The effect of K+ under these conditions was concentration-dependent in the range 20 to 80 mM. The maximum amplitude of the atropine-resistant relaxation induced by high K+ corresponds to 50-75% of that produced by acetylcholine in the absence of atropine. A similar K(+)-induced relaxation was observed in the porcine iris dilator. 3. The atropine-resistant relaxation in the rat iris dilator was not affected by pretreatment with 10 microM ouabain. The relaxation induced by 40 or 80 mM K+ in the porcine dilator was slightly enhanced or not affected, respectively, in the presence of 1 microM ouabain. Application of 10 microM ouabain per se induced relaxation in the porcine iris dilator. 4. The low Na+ ion concentration present in high K+ solutions was not responsible for the K(+)-induced relaxation since the complete replacement of Na in the medium with Tris did not affect significantly the relaxation produced by high K(+)-containing solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Astroglial cell death induced by excessive influx of sodium ions

Na(+) influx has been implicated to play an important role in the mechanisms of neuronal cell damage under ischemia as well as in neurodegenerative disorders. Thus far, however, the effects of Na(+) influx on astrocytic damage have not been studied extensively. In the present study, we have examined the effects of Na(+) influx induced by veratridine (Na(+) channel opener), monensin (Na(+) ionophore), and glutamate (co-transportation with Na(+)) on rat cultured astroglial damage. Cells were incubated with bicarbonate buffer with 25 mM glucose containing either 100 microM veratridine, 10 microM monensin, or 1 mM glutamate with or without 1 mM ouabain for 20 h. Cellular damage was evaluated quantitatively by lactate dehydrogenase (LDH) release or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction. Veratridine, monensin, or glutamate alone did not induce significant astroglial damage. Veratridine and monensin co-incubated with ouabain, which inhibits active extrusion of Na(+) by Na(+),K(+)-ATPase, thereby enhances intracellular Na(+) accumulation, caused significant cell death (P<0. 001, approximately 50% cell damage), whereas glutamate did not. Na(+)-free solution substituted by choline (impermeable cation) attenuated cell damage induced by veratridine and monensin markedly, while Li(+) substitution (permeable cation) rather exacerbated. Nifedipine (100 microM), a blocker of L-type Ca(2+) channel, reduced veratridine-induced glial damage by 50%. Neither bepridil nor benzamil, a blocker of Na(+)-Ca(2+) exchanger, had any protection. Cyclosporin A (1 or 10 microM), an inhibitor of mitochondrial permeability transition or 10 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (zVAD-fmk), which inhibits a broad range of caspases, did not show protective effects.     

Involvement of sodium/calcium exchange in the diclofenac-induced spasmolytic effect on rat uterus

• 1.1. The effect of diclofenac (10–100 μM) on rat uterus contraction and its modification by ouabain (0.1 mM), amiloride (0.1 and 1 mM), ouabain (0.1 mM) plus amiloride (1 mM) and the replacement of sodium by choline have been assayed.
• 2.2. Diclofenac produces dose-dependent relaxation of vanadate (0.3 mM)-induced contraction (EC₅₀, 17.3 ± 1.8 μM). This effect is significantly reduced in choline medium (EC₅₀, 49.1 ± 4.5 μM) and by ouabain in sodium-medium (EC₅₀, 52 ± 7 μM).
• 3.3. Amiloride displaces, in a dose-dependent way, the diclofenac-induced relaxant effect. However, ouabain plus amiloride did not produce a sinergic effect.
• 4.4. Our results suggest that diclofenac produces relaxation of vanadate-induced contraction by activation of Na⁺/Ca²⁺-exchange.

     

Differential regulation of norepinephrine- and prostaglandin F2alpha-induced contraction by extracellular Na+ in rat aorta

The purpose of this study was to test whether extracellular Na+ differentially regulates agonist-induced contraction in vascular smooth muscle. Exposure of rat aorta to 20 nM extracellular Na+ by substitution of 123 mM Na+ with N-methyl-D-glucamine or choline, inhibited norepinephrine-induced contraction to a greater magnitude than contraction to prostaglandin F2alpha. In the absence of extracellular Ca2+ and in 20 mM Na+ solution containing 123 mM N-methyl-D-glucamine, the norepinephrine and prostaglandin F2alpha contraction remained unaltered. In contrast, in the absence of extracellular Ca2+ and in 20 mM Na+ solution containing 123 mM choline, the norepinephrine and prostaglandin F2alpha contraction were decreased and increased, respectively. Contraction to the phorbol ester, phorbol dibutyrate, was inhibited in 20 mM extracellular Na+ solution containing N-methyl-D-glucamine. Removal of extracellular Ca2+ inhibited the phorbol dibutyrate contraction, and 20 mM extracellular Na+ solution containing N-methyl-D-glucamine did not inhibit the phorbol dibutyrate contraction elicited in the absence of extracellular Ca2+. Complete replacement of extracellular Na+ with choline, and concomitant treatment with nifedipine to reduce the elevated basal tone after Na+ replacement, also resulted in greater inhibition of norepinephrine- as compared with prostaglandin F2alpha-induced contraction. Ethylisopropylamiloride, a Na+/H+ exchange inhibitor, did not alter norepinephrine contraction, as determined in the presence of nifedipine to reduce the elevated basal tone due to ethylisopropylamiloride. Acidification, which may result from decreased Na+/H+ exchange, inhibited the prostaglandin F2alpha-induced contraction to a greater magnitude than contraction to norepinephrine. These results demonstrate that extracellular Na+ selectively regulates agonist-induced contraction. The study further suggests that the selectivity may be related to an extracellular Na+-dependent process that is activated by protein kinase C, such as Na+/Ca2+ exchange, and is unrelated to the release of intracellular Ca2+ and Na+/H+ exchange.     

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)