Inositol 1,4,5-trisphosphate-induced Ca2+ release is regulated by cytosolic Ca2+ in intact skeletal muscle

Microinjection of inositol 1,4,5-trisphosphate (InsP ₃) into intact skeletal muscle fibers isolated from frogs (Rana temporaria) increased resting cytosolic Ca²⁺ concentration ([Ca²⁺]_i) as measured by double-barreled Ca²⁺-selective microelectrodes. In contrast, microinjection of inositol 1-phosphate, inositol 1,4-biphosphate, and inositol 1,4,5,6-tetrakisphosphate did not induce changes in [Ca²⁺]_i. Incubation in low-Ca²⁺ solution, or in the presence of L-type Ca²⁺ channel blockers did not affect InsP ₃-induced release of cytosolic Ca²⁺. Neither ruthenium red, a blocker of ryanodine receptor Ca²⁺-release channels, nor cytosolic Mg²⁺, a known inhibitor of the Ca²⁺-induced Ca²⁺-release process, modified the InsP ₃-induced release of cytosolic Ca²⁺. However, heparin, a blocker of InsP ₃ receptors, inhibited InsP ₃-induced release of cytosolic Ca²⁺. Also, pretreatment with dantrolene or azumulene, two inhibitors of cytosolic Ca²⁺ release, reduced [Ca²⁺]_i, and prevented InsP ₃ from inducing release of cytosolic Ca²⁺. Incubation in caffeine or lengthening of the muscle increased [Ca²⁺]_i and enhanced the ability of InsP ₃ to induce release of cytosolic Ca²⁺. These results indicate that InsP ₃, at physiological concentrations, induces Ca²⁺ release in intact muscle fibers, and suggest that the InsP ₃-induced Ca²⁺ release is regulated by [Ca²⁺]_i. A Ca²⁺-dependent effect of InsP ₃ on cytosolic Ca²⁺ release could be of importance under physiological or pathophysiological conditions associated with alterations in cytosolic Ca²⁺ homeostasis. Received: 15 December 1995/Received after revision and accepted: 10 May 1996     

Glucose‐induced inhibition of insulin secretion

Increase in glucose is known to elevate the concentration of cytoplasmic Ca²⁺ ([Ca²⁺]_i) in pancreatic β‐cells and stimulate insulin secretion. However, rise of glucose can also lower [Ca²⁺]_i and inhibit insulin release. In the present review, we examine the mechanisms for this inhibition and highlight its importance for the healthy β‐cell and the development of diabetes. It is possible to distinguish between 60 and 90 s of prompt inhibition and the late inhibition seen after the first‐phase peak of insulin release. The introductory inhibition is characteristic of the healthy β‐cell and mediated by sequestration of [Ca²⁺]_i in the endoplasmic reticulum. This inhibition is easily seen in studies of isolated islets but too brief to be detected in a conventional intravenous glucose tolerance test. Coupled to simultaneous rise of glucagon, the introductory suppression of insulin release is the starting point for the antiphase relation between the subsequent insulin and glucagon pulses. Another effect of the initial suppression is to increase the pool of readily releasable granules responsible for the first‐phase release of insulin. The presence of late inhibition of insulin release is an indicator of β‐cell dysfunction. Patients with type 2 diabetes often respond to intravenous bolus injection of glucose with 5–10 min of late suppression of circulating insulin.     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca²⁺] ([Ca²⁺]_i) due to Ca²⁺ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca²⁺]_i elevation during stimulation depends on: (a) the rate of Ca²⁺ release from the SR, (b) the rate of Ca²⁺ uptake by the SR, and (c) the myoplasmic Ca²⁺ buffering. We have used fluorescent Ca²⁺ indicators to measure [Ca²⁺]_i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca²⁺]_i: β-adrenergic stimulation, which potentiates the SR Ca²⁺ release; application of 2,5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca²⁺ pumps; application of caffeine, which facilitates SR Ca²⁺ release and inhibits SR Ca²⁺ uptake; early fatigue, where the rate of SR Ca²⁺ uptake is reduced; acidosis, which reduces both the myoplasmic Ca²⁺ buffering and the rate of SR Ca²⁺ uptake. Reduced tetanic [Ca²⁺]_i was observed in late fatigue, due to reduced SR Ca²⁺ release, and in alkalosis, due to increased myoplasmic Ca²⁺ buffering. Force is monotonically related to [Ca²⁺]_i, but depends also on the myofibrillar Ca²⁺ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca²⁺]_i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca²⁺ sensitivity and maximum cross-bridge force.     

On the usefulness of Fura-2 measurements of intrasynaptosomal calcium levels in rat cortical synaptosomes to study mechanisms of presynaptic function

Levels of [Ca²⁺]_i in rat cortex synaptosomes were measured using the Ca²⁺indicator Fura-2. Ca²⁺influx was induced by veratridine in a concentration-dependent manner (1–10 μm ). The resulting increase in [Ca²⁺]_i was inhibited by tetrodotoxin (TTX). K⁺(18 mm ) increased the [Ca²⁺]_i which was not influenced by TTX. K⁺-channel blockers such as 4-aminopyridine, α-and δ-dendrotoxin per se were ineffective. The veratridineinduced Ca²⁺influx in synaptosomes was reduced by L-type Ca²⁺-channel blockers, such as felodipine, nifedipine and PN-200-110, verapamil and diltiazem. ω-Conotoxin, an N-type Ca²⁺-channel blocker, did not inhibit the veratridine-stimulated [Ca²⁺]_i increase. Bay K 8644, an L-channel agonist, stimulated an increase of [Ca²⁺]_i in synaptosomes which was not sensitive to TTX. R-N⁶-Phenyl-isopropyl-adenosine (R-PIA) and clonidine, agonists at adenosine A₁-receptors and α₂-adrenoceptors, respectively, did not influence the veratridinestimulated [Ca²⁺]_i increase. R-PIA did not interact with Bay K 8644-stimulated [Ca²⁺]_i increase in synaptosomes. The results for all the substances used show major differences between the effects on Ca²⁺influx in synaptosomes and on the electrically evoked neurotransmitter release in slice preparations. Thus, the synaptosome preparation is not a generally applicable experimental model for the study of Ca²⁺mechanisms of presynaptic neuromodulation.     

Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles

The effect of the protein kinase C (PKC) inhibitor chelerytrine (Ch) and the PKC activator 12–0-tetradecanoyl-phorbol-13-acetate (TPA) on the cytosolic calcium concentration ([ Ca²⁺]_i) in isolated intact rabbit afferent arterioles was investigated. [Ca²⁺]_i was measured in the proximal and distal parts of the arteriole. Administration of 1 μM Ch gave rise to a peak followed by an elevated level of [Ca²⁺]_i in both these parts. Neither the peak nor the elevated level of [Ca²⁺]_i was significantly reduced by 1 μM nifedipine. The relative peak increase in [Ca²⁺]_i in response to 1 μM noradrenaline (NA) or to 10 nM angiotensin II (AII) was significantly blunted in both parts after preincubation with 1 μM Ch. Depolarization with 25 mM K⁺ increased [Ca²⁺]_i in both parts. Preincubation with Ch did not affect the increase in [Ca²⁺]_i induced by 25 mM K⁺. TPA (10 and 100 nM ) did not significantly affect the basal [Ca²⁺]_i in the afferent arteriole. The [Ca²⁺]_i response to NA or 25 mM K⁺ was not affected by TPA. We conclude that blockade of PKC increases [Ca²⁺]_i in afferent arteriolar smooth muscle by a mechanism independent of L-type voltage-sensitive calcium channels. Inhibition of PKC blunts the relative increase in [Ca²⁺]_i in response to AII and, to a lesser extent, that induced by NA. We conclude that PKC might be important in modulating the calcium changes that occur in response to these vasoconstrictors.     

Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans

Sodium azide (NaN₃), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10–15 min) application followed by removal of 3 mM NaN₃ results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3–5 mM). In addition, application of NaN₃, even at very low [Ca²⁺]_o, reversibly increases cytosolic Ca²⁺ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca²⁺, by itself, is not sufficient to trigger insulin secretion.Deceased     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Effects of 5-hydroxytryptamine (serotonin) and forskolin on intracellular free calcium in isolated and fura-2 loaded smooth-muscle cells from the anterior byssur retractor (catch) muscle ofMytilus edulis

Effects of 5-hydroxytryptamine (5-HT) and forskolin on intracellular free calcium concentration ([Ca²⁺]_i) were studied in suspensions of fura-2 loaded smooth-muscle cells from the anterior byssus retractor catch muscle ofMytilus edulis. The successive addition of 5 mM carbachol (CCh) and 100 mM KCl to the suspension evoked a transient elevation of [Ca²⁺]_i from the resting value of 124±2.7 nM (mean ± SE,n=18) to 300–400 nM, which was associated with contraction. The change in [Ca²⁺]_i induced CCh was concentration-dependent with the EC₅₀ of 10^–5 M. The resting [Ca²⁺]_i was unaffected by 10 M 5-HT. The change in [Ca²⁺]_i induced by 5 mM CCh was suppressed by 5-HT from 167±14.0 (n=11) to 124±14.9 (n=8) nM whereas that induced by 100 mM KCl was enhanced from 321±31.9 to 405±17.6 nM (n=8). 5-HT applied during the decaying phase of the CCh response caused a rapid decline in [Ca²⁺]_i. In both the responses to CCh and KCl, the falling phase was accelerated by 5-HT. 10 M forskolin, a potent activator of adenylate cyclase, mimicked the effects of 5-HT as did a membrane-permeant cyclic AMP analogue, 8-parachlorophenylthio cyclic AMP (cpt-cAMP). Application of 100 M cpt-cAMP partially suppressed the Ca²⁺ _i response to CCh and enhanced that to KCl.d-Tubocurarine (500 M) added during the decaying phase of the response induced by 100 M CCh, caused a rapid decline in [Ca²⁺]_i similar to that caused by both 5-HT and forskolin. In essentially Ca²⁺-free sea water, or in the presence of 10 M D600 in seawater containing 4 mM, Ca²⁺, the response to CCh was partially suppressed, whereas that to KCl was completely abolished, demonstrating a CCh-induced release of intracellularly stored Ca²⁺. The remaining component of the response to CCh, in either Ca²⁺-free sea water or in the presence of D600, was abolished by both 5-HT and forskolin. The results suggest that 5-HT has multiple effects on [Ca²⁺]_i in the ABRM, and implicate cyclic AMP in this effect, and that one of the mechanisms underlying these responses is the inhibition of an agonist-induced release of stored Ca²⁺. In addition, that Ca²⁺ _i is at, or close to resting values during the catch state.     

Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca²⁺ concentration [Ca²⁺]_i through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca²⁺]_i oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin–Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca²⁺]_i increases in renal epithelia. Spontaneous [Ca²⁺]_i increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 ± 6.7% (n = 23) of the cells showed spontaneous [Ca²⁺]_i increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca²⁺]_i oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca²⁺]_i oscillatory activity. The association between spontaneous [Ca²⁺]_i increases and nucleotide signalling was further tested in 132–1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca²⁺]_i increases. Transfection with either hP2Y₆ or hP2Y₂ receptors revealed a striking degree of oscillations. Similar spontaneous [Ca²⁺]_i increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y₂ knock out mice (0.050 ± 0.020 events per second, n = 8) compared to the wild type (0.147 ± 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca²⁺]_i oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function. C. S. Geyti and E. Odgaard contributed equally to the publication.     

Nitric oxide‐induced signalling in rat lacrimal acinar cells

The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5‐diaminofluorescein (DAF‐2). We initiated investigations by adding NO from an external source by means of the NO‐donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5′‐phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura‐2 loaded lacrimal acinar cells, resulted in a cGMP‐dependent protein kinase‐mediated release of Ca²⁺ from intracellular stores, leading to a rise in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). The Mn²⁺ quenching studies revealed that the Ca²⁺ release was not accompanied by Ca²⁺ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by β‐adrenergic stimulation and not by a rise in [Ca²⁺]_i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca²⁺ release from intracellular stores via G kinase activation. However, the changes in [Ca²⁺]_i are relatively small, suggesting that this pathway plays a modulatory role in Ca²⁺ signalling, thus not by itself causing fast transient increases in [Ca²⁺]_i. In addition, we suggest that endogenously produced NO activated by β‐adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.     

Integrin stimulation induces calcium signalling in rat cardiomyocytes by a NO-dependent mechanism

The myocardial stretch-induced increase in intracellular [Ca²⁺] ([Ca²⁺]_i) is considered to be caused by integrin stimulation. Myocardial stretch is also associated with increased nitric oxide (NO) formation. We hypothesised that NO is implicated in calcium signalling following integrin stimulation. Integrins of neonatal rat cardiomyocytes were stimulated with a pentapeptide containing the Arg-Gly-Asp (RGD) sequence. [Ca²⁺]_i was measured with Fura2, [NO]_i was measured with DAF2 and phosphorylation of focal adhesion kinase (FAK) was monitored with immunofluorescence techniques. Integrin stimulation increased both [NO]_i and [Ca²⁺]_i, the latter response being inhibited by ryanodine receptor-2 (RyR2) blockers and by N^G-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, but resistant to GdCl₃, diltiazem and wortmannin. Integrin-induced intracellular Ca²⁺ release thus appears to be independent of the influx of extracellular Ca²⁺ and phosphatidylinositol-3 kinase activity. In addition, integrin stimulation induced phosphorylation of FAK. Our results provide evidence for an integrin-induced Ca²⁺ release from RyR2 which is mediated by NO formation, probably via FAK-induced NOS activation.     

Ca2+ oscillations in pancreatic β-cells exposed to leucine and arginine

Dual-wavelength microfluorometry with the fura-2 indicator was employed for continuous recordings of cytoplasmic Ca²⁺ (Ca²⁺) in individual pancreatic β-cells isolated from ob/ob-mice. When added to a medium containing 3 mmol l^-1 glucose, both 10 mmol r¹ leucine and 20 mmol l“¹ arginine induced rises in Ca_i²⁺ with periodic fluctuations. In the case of leucine, this increase was preceded by initial lowering followed by high-amplitude oscillations with a periodicity of 2–0 min. In a glucose-free medium arginine had no effect, and leucine was unable to induce more than a single peak of Ca_i²⁺ increase. When present at a concentration of 1 mmol l^-1, leucine sometimes induced a couple of high-amplitude oscillations at 3 mmol l^-1 glucose but lowered Ca_i²⁺ permanently in a glucose-free medium. It is likely that the high-amplitude oscillations of Ca_i²⁺ are related to the electrical activity of the β-cells. Provided that some glucose was present, leucine initiated a similar type of Ca_i²⁺ response as obtained during glucose-induced insulin release. The observed leucine effect is therefore compatible with a role of glycolysis in generating high-amplitude Ca_i²⁺ oscillations and pulsatile insulin release.     

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal

We correlated dynamic changes in free cytosolic [Ca²⁺] ([Ca²⁺]_i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca²⁺]_i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca²⁺ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses. The presynaptic Ca²⁺ transient was graded by varying the amplitude of extracellular stimulating pulses. The probability of the release event, P, estimated for each stimulation strength, reached a maximum (P=1) when the Ca²⁺ signal became maximal and remained at this level at higher stimulation strength, despite the subsequent decrease in the amplitude of the Ca²⁺ transient. A gradual, linear increase in stimulation amplitude (V_stim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca²⁺ signals and corresponding averaged amplitudes of eIPSCs. Analysis of the eIPSC demonstrated that the decrease in both the mean eIPSC amplitude and the mean quantal content of release resulted from a reduction in the probability of multivesicular release, i.e. in the disappearance of failures and in the decrease of individual eIPSC amplitude. The Ca²⁺ signals of similar amplitude resulted in both random and determinate (non-random) neurotransmitter release. We conclude that depolarisation-induced elevation of [Ca²⁺]_i within the terminal is necessary but not sufficient for activation of vesicular release of neurotransmitter.     

The role of cytosolic Ca2+ in the secretion of NaCl in isolated in vitro perfused rectal gland tubules of Squalus acanthias

 In many exocrine glands cytosolic Ca²⁺ ([Ca²⁺]_i) plays a pivotal role in stimulation-secretion coupling. In the rectal gland of the dogfish Squalus acanthias this appears not to be the case and it is believed that secretion is mainly controlled by the Cl^– conductance of the luminal membrane. We have examined this question in a study of isolated in vitro perfused rectal gland tubules (RGT). Three types of measurements were performed: (1) measurements of [Ca²⁺]_i by the fura-2 technique; (2) measurements of transepithelial electrical parameters, i.e. transepithelial voltage (V _te), transepithelial resistance (R _te), the equivalent short-circuit current (I _sc) and the voltage across the basolateral membrane (V _bl), and (3) whole-cell patch-clamp measurements of cellular voltage (V _m), conductance (G _m) and membrane capacitance (C _m). The data indicates that carbachol (CCH) increases [Ca²⁺]_i by increasing store release and Ca²⁺ influx. Other agonists, producing cytosolic cAMP, also increased [Ca²⁺] by enhancing Ca²⁺ influx. CCH hyperpolarized these cells and enhanced G _m significantly. The effect of CCH on V _te and I _sc was most marked under control conditions and disappeared in RGT otherwise stimulated by agonists that lead to cAMP production. It is concluded that [Ca²⁺]_i plays a major role in the stimulation of NaCl secretion in RGT by enhancing the basolateral K⁺ conductance. cAMP-producing agonists enhance [Ca²⁺]_i by increased Ca²⁺ influx. CCH releases Ca²⁺ from respective stores. CCH, unlike the cAMP-producing agonists, only increases basolateral K⁺ conductance. It modulates secretion especially under conditions in which the cAMP pathway is not fully activated. Received: 25 November 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca²⁺]_i to a stable plateau of 10–20 nmol/l above resting [Ca²⁺]_i was observed. Lower osmolalities resulted in a triphasic increase of [Ca²⁺]_i to a peak value. In a first phase after the volume change, lasting 20–40 s, [Ca²⁺]_i increased slowly by about 30 nmol/l. Thereafter [Ca²⁺]_i increased more rapidly within 20–30 s to a peak value. This peak was 189±45 nmol/l (190 mosmol/l, n=9) and 243±41 nmol/l (160 mosmol/l, n=20) above resting [Ca²⁺]_i. The peak was then followed by a decline of [Ca²⁺]_i over the next 2–3 min to a stable plateau value of 28±6 (n=6) and 32±11 nmol/l (n=11) above resting [Ca²⁺]_i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca²⁺-free bath conditions the peak value for the cell-swelling-induced [Ca²⁺]_i transient was reached significantly later (60–100 s, compared to 40–60 s under control conditions). The peak values under Ca²⁺-free conditions were not significantly lower. This indicates that the [Ca²⁺]_i peak was mostly of intracellular origin. No [Ca²⁺]_i plateau phase was observed under Ca²⁺-free bath conditions. With the use of the fura-2-Mn ²⁺ quenching technique an increased Ca²⁺ influx induced by hypotonic cell swelling was shown (160 mosmol/l; n=4). This influx started immediately after or simultaneously with the cell swelling and preceded the [Ca²⁺]_i peak for more than 50 s.This study was supported by DFG grant Gr 480/10.     

Vasopressin responses in electrically coupled A7r5 cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca²⁺ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca²⁺]_i rise that was about 5 times higher than the amplitude of the spontaneous Ca²⁺ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca²⁺ spiking, which persisted after [Ca²⁺]_i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca²⁺ waves within one cell propagated much more slowly than spontaneous [Ca²⁺]_i rises. We conclude that agonist-induced [Ca²⁺]_i increases in an electrically coupled cell monolayer can be asynchronous.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats

Aim: The aim of this study was to determine the effects of motilin on [Ca²⁺]_i regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs). Methods: Antral cells were isolated and cultured from neonatal rats, and then the [Ca²⁺]_i in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope. Results: We show that motilin dose-dependently increased [Ca²⁺]_i concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10⁻⁵ mol L⁻¹) or the calcium chelator Egtazic (EGTA, 0.1 mmol L⁻¹) significantly suppressed motilin (10⁻⁶ mol L⁻¹) induced [Ca²⁺]_i increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10⁻⁵ mol L⁻¹), guanosine triphosphate regulatory protein antagonist NEM (10⁻⁵ mol L⁻¹), phospholipase C (PLC) inhibitor compound 48/80 (1.2 μg mL⁻¹) and ryanodine at high concentration (10⁻⁵ mol L⁻¹), the motilin-induced [Ca²⁺]_i increase was only partially blocked. The protein kinase C inhibitor d -sphingosine (10⁻⁶ mol L⁻¹), however, did not show any inhibitory effect on motilin-induced [Ca²⁺]_i elevation. Conclusions: Our study suggests that motilin-stimulated [Ca²⁺]_i elevation in ASMCs is probably due to sustained extracellular Ca²⁺ influx and Ca²⁺ release from Ca²⁺ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca²⁺]_i release is accompanied with guanosine triphosphate-binding protein-coupled receptor–PLC–inositol tris-phosphate signalling cascades.     

Spatial distribution of intracellular,free Ca2+ in isolated rat parotid acini

The spatial distribution of intracellular, free Ca²⁺ ([Ca²⁺]_i) in rat parotid acini was measured by imaging fura-2 fluorescence from individual acinar cells by means of a digital imaging microscope. Upon cholinergic stimulation in a Krebs-Ringer bicarbonate buffer at (37° C), [Ca²⁺]_i increased synchronously at both the basolateral and luminal membranes as well as in all cells of the secretory endpiece, reaching peak [Ca²⁺]_i levels 1 s after stimulation. Atropine addition caused a rapid down-regulation of [Ca²⁺]_i, which, however, never reached prestimulatory levels. When acini were stimulated in a medium containing 5 nM Ca²⁺, the Ca²⁺ mobilization arising from internal pools caused an increase in [Ca²⁺]_i predominantly near the basolateral area, where the endoplasmic reticulum is located, and standing Ca²⁺ gradients were observed for up to 10 s. A mathematical model is developed to simulate the time courses of the Ca²⁺ profiles through the cytoplasm using estimated values of the Ca²⁺ diffusion coefficients and the cytosolic Ca²⁺ buffering capacity. It is concluded that under physiological conditions, the Ca²⁺ release from the endoplasmic reticulum is responsible for the activation of the basolaterally located K⁺ channels. Furthermore, Ca²⁺ influx from the interstitium is responsible for much of the rise in [Ca²⁺]_i near the luminal membranes, where the Cl^– channels are supposed to be located.