Characterization of two different calcium entry pathways in small mesenteric arteries from rat

The effects of Ca2+ removal, nifedipine, and La3+ on contractions induced by 124 mM K+ and 10 microM noradrenaline (NA) were investigated in small mesenteric arteries from rat. Ring segments of the arteries were suspended between two steel wires in a 2.5 ml muscle bath, and the mechanical activity recorded "isometrically". The tonic components of the contractile responses to both K+ and NA were critically dependent on the presence of Ca2+ in the bath solution. Nifedipine effectively relaxed K+-contracted arteries, whereas those activated by NA were considerably less affected by the drug. Application of NA to arteries depolarized by K+ in the presence of nifedipine induced a sustained tonic contraction, which was only approximately 20% smaller than that elicited by NA in "standard" Krebs solution, implicating pharmacomechanical coupling. Unlike nifedipine, La3+ inhibited K+- and NA-induced contractions to approximately the same extent. Re-application of Ca2+ to "Ca2+-depleted" preparations exposed to K+ and/or NA induced concentration-dependent contractions. The experimental results suggested that the effects of K+ and NA on the membrane permeability to Ca2+ were additive. The Ca2+-induced contractions were more inhibited by nifedipine in K+-depolarized than in NA-exposed arteries. It is concluded that K+ and NA utilize partly different Ca2+ entry pathways to increase the myoplasmic Ca2+ concentration in rat mesenteric arteries. Whereas K+ seems to promote the influx Ca2+ by activation of CA2+ channels sensitive to the membrane potential, the nature of the receptor-operated Ca2+ entry pathway remains to be established.     

Influence of extracellular calcium and nifedipine on alpha 1- and alpha 2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca2+ and nifedipine on contractile responses to 10 microM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly alpha 2-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly alpha 1-adrenoceptors) and CMA (sensitive to both alpha 1- and alpha 2-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca2+-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K+ was more sensitive to Ca2+ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca2+, can also release Ca2+ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, alpha 1-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca2+ influx and intracellular Ca2+ release, whereas alpha 2-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca2+ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca2+-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca2+, whereas in the other types of artery, Ca2+ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca2+ influx and intracellular Ca2+ release to the contractile activation, but also in the nifedipine sensitivity of the Ca2+ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of alpha-adrenoceptor stimulated by NA and to the type of vessel studied.     

Ca2+ clearance at growth cones produced by crayfish motor axons in an explant culture

Intracellular free Ca2+ concentration ([Ca2+]i) plays an important role in the regulation of growth cone (GC) motility; however, the mechanisms responsible for clearing Ca2+ from GCs have not been examined. We studied the Ca2+-clearance mechanisms in GCs produced by crayfish tonic and phasic motor axons by measuring the decay of [Ca2+]i after a high [K+] depolarizing pulse using fura-2AM. Tonic motor axons regenerating in explant cultures develop GCs with more rapid Ca2+ clearance than GCs from phasic axons. When Na/Ca exchange was blocked by replacing external Na+ with N-methyl-d-glucamine (NMG), [Ca2+]i decay was delayed in both tonic and phasic GCs. Tonic GCs appear to have higher Na/Ca exchange activity than phasic ones since reversal of Na/Ca exchange by lowering external Na+ caused a greater increase in [Ca2+]i for tonic than phasic GCs. Application of the mitochondrial inhibitors, Antimycin A1 (1 microM) and CCCP (10 microM), demonstrated that mitochondrial Ca2+ uptake/release was more prominent in phasic than tonic GCs. When both Na/Ca exchange and mitochondria were inhibited, the plasma membrane Ca2+ ATPase was effective in extruding Ca2+ from tonic, but not phasic GCs. We conclude that Na/Ca exchange plays a prominent role in extruding large Ca2+ loads from both tonic and phasic GCs. High Na/Ca exchange activity in tonic GCs contributes to the rapid decay of [Ca2+]i in these GCs; low rates of Ca2+ extrusion plus the release of Ca2+ from mitochondria prolongs the decay of [Ca2+]i in the phasic GCs.     

Effects of extracellular calcium and calcium channel blocker on silver-induced contractures in frog skeletal muscle fibers

The mechanism by which Ag+ induces muscle contracture was elucidated by investigating the effect of external Ca2+ concentration and Ca2+ channel blocker on the maximum tension amplitude in single fibers from frog toe skeletal muscle. Five microM Ag+ induced two different types of contracture in the presence of external Ca2+ more than 0.1 mM, i.e., a phasic and a subsequent tonic contracture. The phasic contracture appeared only in fibers with intact T-tubules immersed in a solution with or without Ca2+ after a lag time of 5.7 +/- 0.9 s (N = 5). The maximum amplitude was 58% of the tetanus tension observed in the same fiber immediately before Ag+ exposure. Diltiazem at high-concentration (100 microM) inhibited the Ag+-induced phasic contracture only to a small extent (17%). The contracture was not affected by 1 microM TTX or 1 mM DAP at all. These results indicate that Na+, K+, and Ca2+ channels on the T-tubular membrane would not be attributed to the phasic tension development induced by Ag+. On the contrary, a tonic contracture did not require intact T-tubules. The amplitude and the rate of rise of the contracture depended on external Ca2+ concentrations and were inhibited by a high concentration of diltiazem. Neither 1 microM TTX nor 1 mM DAP affected them. Therefore, the tonic contracture seems to be triggered by Ca2+ which entered the muscle fiber through the surface but not T-tubular membranes.     

Tension and cyclic GMP changes in potassium depolarized rabbit colon muscle.

Potassium depolarization of rabbit colon muscle elicited a contraction consisting of 2 distinct phases, an initial rapid phasic contraction and a tonic contracture. The tonic contraction was, in contrast to the phasic contraction, dependent on the extracellular calcium for its development. There was a correlation between the tension development and the increase of the cyclic GMP level in the K+-depolarized muscle. Experimental conditions which abolished the tonic contracture, viz glucose omission and treatment with Ca2+-antagonists (verapamil, SKF 525A) also inhibited the cyclic GMP response. The changes of the cyclic GMP levels were Ca2+-dependent. K+-ions also changes the cyclic AMP content an effect which was atropine sensitive. From the experimental data obtained in this investigation we suggest that the co-variation of the tension and the cyclic GMP level in the depolarized colon muscle might depend on oscillations in a common intracellular factor, probably Ca2+.     

Influence of extracellular calcium and nifedipine on α1-and α2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca²⁺ and nifedipine on contractile responses to 10 μM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly α₂-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly α₁-adrenoceptors) and CMA (sensitive to both at,- and α₂-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca²⁺-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K⁺ was more sensitive to Ca²⁺ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca²⁺, can also release Ca²⁺ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, α₁-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca²⁺ influx and intracellular Ca²⁺ release, whereas α₂-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca²⁺ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca²⁺-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca²⁺, whereas in the other types of artery, Ca²⁺ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca²⁺ influx and intracellular Ca²⁺ release to the contractile activation, but also in the nifedipine sensitivity of the Ca²⁺ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of α-adrenoceptor stimulated by NA and to the type of vessel studied.     

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.     

Somatostatin inhibits prolactin secretion by multiple mechanisms involving a site of action distal to increased cyclic adenosine 3',5'-monophosphate and elevated cytosolic Ca2+ in rat lactotrophs

The release of prolactin (PRL) from a clonal cell-line of anterior pituitary cells (GH4C1) was inhibited by somatostatin (SRIH) in a dose-dependent manner (ED50 nM). The inhibition (20% of control levels) was detectable within 50 s and maximal within 90 s. Thyroliberin (TRH) enhancement of PRL secretion was biphasic. SRIH inhibited both phases equally. Ionomycin in combination with the phorbol ester, TPA, mimics the TRH-elicited PRL release, and SRIH partly inhibited this effect. SRIH had no effect on TRH-stimulated formation of inositol trisphosphate, and only small effects on TRH-activated adenylate cyclase. Vasoactive intestinal peptide (VIP) and forskolin stimulated cAMP formation and PRL release potently. SRIH inhibited both effects of VIP and forskolin, and there was a close correlation between the inhibition of PRL secretion and cAMP accumulation. 8-Bromo-cAMP enhanced PRL release, an effect that was also partly reduced by SRIH. The Ca2+ channel activator, BAY-K-8644 and high extracellular K+ increased PRL release, and SRIH caused a partial reduction in the release response to both secretagogues. SRIH lowered [Ca2+]i, and markedly reduced the rise in [Ca2+]i elicited by TRH, VIP and K+. SRIH did not influence the Ca2+ spikes recorded in Na+-free solution, and had no effect on the TRH-induced membrane potential changes. Our results demonstrate that SRIH may inhibit PRL release from GH4C1 cells by (1) inhibiting hormone-sensitive adenylate cyclase, (2) blocking the effect of cAMP and (3) lowering [Ca2+]i. None of these effects is, however, sufficient to explain all the effects of SRIH, suggesting that SRIH also exerts a major action at a step subsequent to cAMP accumulation and [Ca2+]i elevation. Since the GH4C1 cells possess one single class of binding sites, this implies that the same SRIH receptor is coupled to several cellular signalling systems.     

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.     

Kinetics of the Ca(2+)-activated K+ channel in rat hippocampal neurons

The kinetics of the large-conductance Ca(2+)-activated K+ channel (235 pS in symmetrical 150 mM K+) were examined in the inside-out mode of the patch clamp technique. The open probability of the channel increased when [Ca2+]i, [Sr2+]i, or [Ba2+]i was increased. The [Ca2+]i-response relation was fitted with a Hill coefficient of 2 and half-maximum concentrations of 185, 80, 14.5, and 5.5 microM at -40, -20, +20, and +40 mV, respectively. The channel was blocked by TEA or Ba2+. The open-time histogram showed a single exponential component and the closed-time histogram showed at least two exponential components at various [Ca2+]i. Increasing [Ca2+]i decreased the time constant of the slow component of the closed-time histogram. Cell-attached patch recording revealed activation of the large-conductance Ca(2+)-activated K+ channel (BK channel) during the action potential. The deactivation time course was consistent with the fast after-hyperpolarization. A minimum model of the channel, close(2)-close(1)-open, where the transition from close(2) to close(1) requires the binding of 2 Ca2+, reconstructed quick activation of the channel if [Ca2+]i of 40 microM was assumed.     

Cytosolic calcium levels in vascular smooth muscle

Increase in cytosolic Ca2+ level ([Ca2+] cyt) is prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+] cyt and muscle tension give direct information for the Ca2(+)-regulation of smooth muscle. A fluorescent Ca2+ indicator, fura-2, is used for this purpose. Comparison between [Ca2+] cyt and muscle tension in vascular smooth muscle indicates that, although high K+ and receptor-agonists such as norepinephrine and prostaglandin F2 alpha induce sustained contraction by the sustained increase in [Ca2+] cyt, greater contraction is produced by receptor-agonists than high K+ at a given [Ca2+]cyt. Phorbol ester show similar effects as receptor-agonists, and it potentiates a high K(+)-induced contraction with little effect on [Ca2+]cyt. These results suggest that the contraction of smooth muscle is due to the increase in [Ca2+]cyt. Furthermore, receptor-agonists stimulate phosphatidylinositol turnover and generates diacyl glycerol which activates protein kinase C and may consequently increase the Ca2+ sensitivity of contractile elements. The [Ca2+]cyt -dependent portion of these contractions is inhibited by Ca2+ channel blockers such as verapamil by the decrease in [Ca2+]cyt. By contrast, increase in cyclic AMP by isoproterenol and forskolin inhibits smooth muscle contraction by the decrease in [Ca2+]cyt also by the decrease in the Ca2+ sensitivity of contractile elements. Increase in the cyclic GMP level by sodium nitroprusside show effects quite similar to those of cyclic AMP. Thus, contractility of vascular smooth muscle seems to be regulated by [Ca2+]cyt and also by Ca2+ sensitivity of the contractile elements. Furthermore, at least part of the receptor-mediated changes may be due to activation of protein kinase C.     

Effects of nifedipine on potassium-induced contraction and noradrenaline release in cerebral and extracranial arteries from rabbit

The present study was designed to evaluate the effects of the calcium antagonist nifedipine on potassium-evoked contractions and release of noradrenaline from sympathetic nerves in rabbit basilar and facial arteries. Contractions were measured isometrically in a small volume organ bath. While noradrenaline (NA) produced strong contractions in facial arteries, the majority of the basilar arteries responded only to the highest concentrations of NA employed (greater than 10 microM) with weak contraction. Prazosin (1 microM) and phentolamine (1-10 microM) effectively antagonized the responses to NA in both types of vessel. In contrast, contractions evoked by potassium (K+, 124 mM) were only slightly reduced by the alpha-adrenoceptor blocking agents, indicating that the participation of endogenous NA in maintaining the contractile response to K+ is either small or negligible in the vessel types studied. Nifedipine concentration-dependently inhibited K+-induced contractions in basilar and facial arteries, the former being significantly more affected as evidenced by the maximum inhibitions (approximately 80% compared to approximately 60%) and IC50 values (approximately 10 nM vs. approximately 30 nM). A combination of nifedipine (0.3 microM) and prazosin (1 microM) or phentolamine (1 microM) further suppressed the K+-evoked contractile response in facial arteries, but failed to do so in basilar arteries, when compared with the effect of nifedipine alone. The depressant effect of the alpha-adrenoceptor blockers was, however, still obtainable after reserpine treatment of the facial artery in vitro. Fluorescence histochemical demonstration of noradrenaline revealed a dense network of adrenergic nerve fibres in the walls of the basilar and facial artery. The vessels were also shown to accumulate 3H-NA and release it upon depolarization with K+. The uptake and subsequent release of 3H-NA were significantly reduced by desipramine (10 microM). Nifedipine (0.3-3.0 microM) failed to alter the K+-evoked 3H-NA efflux from sympathetic nerves in neither of the two vessel types. It may be concluded that nifedipine effectively inhibits K+-evoked contractions in isolated basilar and facial arteries from rabbit without interfering with nerve-mediated NA release. Possible explanations for this selective effect of nifedipine on muscle contraction are discussed.     

3H]acetylcholine release and the change in cytosolic free calcium level induced by high K+ and ouabain in rat brain synaptosomes

High K+ (50 mM) increased both [3H]acetylcholine ([3H]ACh) release and cytosolic free calcium level ([Ca2+]i) in rat brain synaptosomes in the presence of extracellular Ca2+. Ouabain (5 x 10(-8) to 5 x 10(-4) M) also caused a dose-dependent increase in [3H]ACh release, but not in [Ca2+]i, in the absence of Ca2+. The effects of high K+ and ouabain on [3H]ACh and/or [Ca2+]i, were inhibited by the intracellular Ca2+ antagonist TMB-8 (10(-4) M). These results suggest that unlike high K+, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca2+]i, but sensitive to TMB-8.     

The relationship between acetylcholine-evoked Ca2+-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.     

Characteristics of contractile response of isolated portal veins from chronic portal hypertensive rats under altered levels of external K+, Ca2+, and norepinephrine concentrations: a comparison with normal Wistar rats

We studied contractile properties of portal veins isolated from chronic portal hypertensive rats (PHR) resulting from liver cirrhosis, a model obtained by repeated subcutaneous injections of CCl4 (2 mg/kg) twice weekly for over 45 weeks. Portal venous pressure in vivo was significantly higher in PHR (167.0 +/- 38.7 mmH2O) than in the control normal Wistar rats (NWR) (102.0 +/- 25.5 mmH2O). A pair of portal veins from PHR and NWR were mounted longitudinally in an organ bath and perfused with Tyrode's solution with different K+, Ca2+, and norepinephrine concentrations. The isometric tension was measured by a strain-gauge. Under control conditions, spontaneous phasic contractile force, corrected by cross-sectional area, was greater, and the frequency was lower in PHR than in NWR preparations. The averaged peak contractile force measured at different [K]o (5.4-86.4 mM) was also greater in PHR than in NWR. Force of the tonic contraction measured at different [Ca]o (0.45-5.4 mM), under conditions of 86.4 mM [K]o was significantly larger in PHR than in NWR preparations. However, the Ca2+ sensitivity of both preparations was the same. D-600 (greater than or equal to 0.1 microM) inhibited the tonic contraction in both preparations with an identical sensitivity to the drug. In the presence of norepinephrine (10 microM), the Ca2+ sensitivity of the tonic contraction increased both in PHR and NWR preparations. The increase was more pronounced in PHR and was completely reversed in the presence of the alpha 1-adrenoceptor blocker, prazosin (0.1 microM). The alpha 1-adrenoceptor sensitivity to norepinephrine was not altered in PHR preparations. The rate of Ca2+ release and uptake of intracellular Ca2+ seemed identical in both preparations. Thus, in the absence of norepinephrine, the phasic and tonic contractile forces of portal veins from PHR are larger than that of NWR, probably due to increased membrane Ca2+ permeability. The PHR preparations have a higher affinity for external Ca2+ in the presence of norepinephrine, an additional factor contributing to elevation of portal blood pressure in the presence of chronic liver cirrhosis.     

Influence of acid-base changes on carbachol- and potassium-induced contractions of taenia coli of the rabbit

The influence of acid-base changes (variations of extracellular pH within the range 6.2-8.2 and parallel changes in (HCO3-) and PCO2 at constant external pH) on contractions induced by carbachol and K+ was studied on preparations from taenia coli of the rabbit. Extracellular acidosis increased the concentration of carbachol necessary to evoke a given muscle tension and reduced the maximum response. During a carbachol-induced contraction acidosis reduced and alkalosis enhanced muscle tension. Changes in extracellular pH within the range 6.2-8.2 did not affect the initial, rapid phase of the contraction induced by 145 mmol/l of K+. However, with 40-80 mmol/l of K+ acidosis reduced the phasic response. The tonic contraction following the initial phasic response in 145 mmol/l K+ was enhanced by extracellular acidosis and decreased by alkalosis, whereas at a K+-concentration of 70 mmol/l the amplitude of the tonic contraction was greater at pH 7.4 than at pH 6.2. Propranolol 10(-6) mmol/l did not affect the influence of extracellular pH upon the K+-contracture. An increase of both PCO2 and (HCO-3) at constant extracellular pH did not alter the response of the muscle to carbachol. The tonic contraction during K+-stimulation was diminished by a parallel increase in PCO2 and (HCO-3) and at 47 mmol/l (HCO-3) it was almost abolished. A rise in PCO2 from 5 to 14 kPa without changes in (HCO-3), which caused extracellular pH to drop from 7.4 to 6.3, increased the tonic contraction in the same way as a fall in extracellular pH alone. These results seem to indicate that extracellular acidosis within the pathophysiological range (1) decreases the sensitivity of the polarized smooth muscle membrane to cholinergic stimulation, (2) reduces: the rate of release of stored Ca++, the transmembrane flow of Ca++ into the muscle cell, and the rate of Ca++ inactivation or extrusion during K+-contraction. The reduction of the tonic contracture with increasing PCO2 and (HCO-3) at constant extracellular pH is most likely attributable to the (HCO-3) ion.     

The inhibitory effect of CR-1409 on amylase secretion and intracellular Ca2+ mobilization in rat pancreatic acini in vitro

The inhibitory effects of CR-1409, a new glutaramic acid derivative developed as a cholecystokinin (CCK) receptor antagonist, on caerulein-stimulated amylase secretion and on intracellular Ca2+ ([Ca2+]i) mobilization were studied in isolated rat pancreatic acini. Pancreatic acini were prepared by collagenase digestion method and loaded with 1 microM fura-2/AM for measurement of the intracellular free Ca2+ concentration. Amylase release was examined by a perifusion method. Stimulation with 10(-10) M caerulein, 10(-5) M carbachol, or 10(-8) M gastrin-releasing peptide (GRP) led to biphasic amylase release and increase in [Ca2+]i. CR-1409 at 1 and 5 microM inhibited, by 50 and 84%, respectively, the amylase secretion and increase in [Ca2+]i induced by 10(-10) M caerulein, and 25 microM CR-1409 completely inhibited both amylase secretion and increase in [Ca2+]i induced by caerulein. However, 25 microM CR-1409 did not inhibit unstimulated secretion of amylase or the secretions induced by carbachol and GRP, which are also mediated by changes in intracellular Ca2+. We conclude that CR-1409 acts as a specific inhibitor of the CCK receptor in the pancreas, and is useful in studies on the involvement of the release and action of CCK in vitro.     

The effects of lanthanum and thulium on the mechanical responses of rat vas deferens.

1. The contractile responses of rat vas deferens to noradrenaline and K+ are composed of phasic and tonic components both of which are dependent upon the concentration of extracellular Ca2+. 2. Lanthanum, La3+, and thulium ions, Tm3+, inhibited the noradrenaline and K+ induced responses, complete inhibition being obtained at approximately 10(-3) M-Ln3+. 3. La3+ and Tm3+ were equally effective in inhibiting noradrenaline and K+ responses. The phasic and tonic components of the noradrenaline response were equally sensitive to lanthanide cations, Ln3+, but the phasic component of the K+ response was more sensitive than the tonic component. 4. 170Tm binding did not show any saturable component over the concentration range in which inhibition of the pharmacological response was obtained. 5. It is suggested that the actions of Ln3+ in the rat vas deferens are mediated through some kind of membrane stabilization rather than via a specific Ca2+ binding site concerned with excitation-contraction coupling, the mechanism previously postulated for the Ln3+ action in guinea-pig ileal longitudinal muscle.     

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.