Stimulatory effect of vasoactive intestinal polypeptide on catecholamine secretion from isolated guinea pig adrenal chromaffin cells

The stimulatory effect of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion from isolated guinea pig adrenal chromaffin cell was studied. VIP (1-10 microM) induced dose-dependent CA secretion, which was slow and continued for at least 30 min. This VIP-induced CA secretion was dependent on the presence of Ca2+ in the medium, but no significant increase in Ca2+ uptake by the cells was observed during their stimulation with VIP. Studies on the intracellular free Ca2+ level ([Ca2+]i) using fura-2 showed that acetylcholine and muscarine induced a marked increase in the [Ca2+]i, but that VIP induced only a slight increase. Thus VIP may induce CA secretion by increasing the sensitivity of the secretion of CA to Ca2+.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Muscarinic stimulation of guinea pig adrenal chromaffin cells stimulates catecholamine secretion without significant increase in Ca2+ uptake

In isolated guinea pig adrenal chromaffin cells, not only nicotine, but also muscarine stimulated catecholamine (CA) secretion, the stimulation by muscarine being the greater. The secretions of CA by muscarine and nicotine were both dependent on the presence of Ca2+ in the medium, but only the latter was associated with a rapid increase in 45Ca2+ uptake. Experiments with the fluorescent Ca2+ indicator quin 2, showed that muscarine caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i). Moreover, the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both CA secretion and increase in [Ca2+]i induced by muscarine. These results indicate that in isolated guinea pig adrenal chromaffin cells, nicotine stimulated CA secretion by increasing Ca2+ uptake by the cells, whereas muscarine stimulated CA secretion by mobilizing Ca2+ from the intracellular pool.     

Spontaneous and secretagogue-induced changes in cytosolic free Ca concentration measured by microfluorimetry with fura-2 on single bovine adrenal chromaffin cells

The concentration of cytosolic Ca2+ ([Ca]in) was examined in single bovine adrenal chromaffin cells by monitoring fura-2 fluorescence with microspectrofluorimetry. To see the correlation between [Ca]in and secretion, we also measured the rates of catecholamine (CA) secretion and 45Ca efflux from populations of cells. [Ca]in was constant in the majority of single cells, but the small oscillatory changes in [Ca]in were observed in a population of cells. These spontaneous Ca oscillations, when observed, disappeared either after removal of extracellular Ca2+ or by addition of D-600 or Mn2+, but still persisted in the presence of tetrodotoxin (TTX) or after removal of extracellular Na+. In the silent cells the Ca fluctuations were often induced by Bay-K-8644. The characteristics of Bay-K-8644-induced Ca fluctuations were very similar to those of spontaneous ones. Low concentrations of nicotine (1 microM), acetylcholine (ACh; 1-2 microM), or KCl (12.5 mM) often induced oscillations riding on a steady rise in [Ca]in. These changes were rapidly suppressed by removal of either extracellular Ca2+ or Na+, or by addition of either D-600 (methoxyverapamil) or TTX. A low concentration of ACh (1 microM) or KCl (12.5 mM) also increased the rate of 45Ca efflux, but substantial secretion was not detected. On the other hand, the sustained rise in [Ca]in was evoked by 0.1 mM ACh, 20 microM nicotine, or 30 mM KCl, which was suppressed by removal of extracellular Ca2+, but was little affected by TTX. A sustained increase in 45Ca efflux upon exposure to ACh was observed, possibly reflecting the sustained rise in [Ca]in. ACh also stimulated CA secretion, which was faded out during the prolonged application. Veratridine, a Na channel activator, caused repetitive sequence of Ca transients followed by a sustained rise in [Ca]in. These results, together with the previous electrophysiological findings, suggest that: (1) the spontaneous Ca fluctuations are closely associated with occurrence of spontaneous Ca2+ and Na+ action potentials; (2) the rise in [Ca]in induced by a low concentration of nicotinic agonists of KCl is mediated by Na+ action potentials as well as gradual membrane depolarizations; (3) the oscillatory changes subsequent to a rise in [Ca]in reflect fluctuations in Ca2+ influx through the Ca2+ channels; (4) the critical [Ca]in needs to be attained before the CA secretion takes place.     

Potentiation by Bay-K-8644 of the adrenal catecholamine secretory response to Ca re-introduction and ouabain: possible activation of Ca influx linked with Na efflux

Catecholamine (CA) secretion induced by Ca re-introduction or ouabain in the presence of Ca is markedly potentiated by Bay-K-8644 in the perfused cat adrenal. The mechanism of potentiation by this Ca agonist was investigated using perfused cat adrenal and isolated bovine chromaffin cells. The stimulatory effect of Bay-K-8644 on the response to Ca was very slight when cat adrenal was perfused with a Ca-deficient medium, in which 1 mM Mg was added or the concentration of Na was lowered. The inhibitory effect of Mg was reversed by inhibition of the Na pump with K deprivation, ouabain, or KCN. The secretion induced by ouabain during maintained depolarization at a lowered concentration (0.25 mM) of Ca, which is supposed to be due to Ca influx in exchange for Na efflux, was larger in the Bay-K-8644 treated adrenal than that in the untreated adrenal. The increase in secretion by the delayed addition of Bay-K-8644 during perfusion with a high K medium containing ouabain was larger when the concentration of Na in a high K medium was higher. When isolated chromaffin cells were stimulated with a Na-free (Tris) medium containing 0.5 mM Ca, CA secretion from and 45Ca uptake into the cells preincubated with a divalent cation-free medium were potentiated by Bay-K-8644. The stimulatory effect of Bay-K-8644 was not seen when a Ca-free treatment medium contained Mg or lacked Na, but the inhibitory effect of Mg was reversed by the addition of ouabain or KCN to the pretreatment medium. When isolated cells preloaded with 45Ca were superfused with a Ca-free medium, Ca re-introduction increased the rate of Ca efflux only under conditions in which significant increases in CA secretion and 45Ca uptake were previously observed under the static incubation system. Bay-K-8644 further increased this Ca efflux rate under these conditions. These results support the view that Ca influx linked with Na efflux is through a pathway with properties similar to those of voltage-dependent Ca channels, and suggest that this Ca pathway is activated by Bay-K-8644, which is an activator of voltage-dependent Ca channels.     

Further characterization of [3H]gamma-aminobutyric acid release from isolated neuronal growth cones: role of intracellular Ca2+ stores

We have recently shown that growth cones isolated from neonatal rat forebrain possess uptake and release mechanisms for the neurotransmitter gamma-aminobutyric acid. About half of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones is dependent on extracellular Ca2+. The remaining component of the [3H]gamma-aminobutyric acid release is unaffected by removal of extracellular Ca2+ and is resistant to blockade by the voltage-sensitive Ca2+-channel blocker methoxyverapamil. In the present series of experiments we have used caffeine to assess the possible role of intracellular stores of Ca2+ in supporting that component of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones that is independent of extracellular Ca2+. We have chosen caffeine because of its well established effect of releasing Ca2+ from smooth endoplasmic reticulum in muscle. We found that caffeine can release [3H]gamma-aminobutyric acid from isolated growth cones. This effect persists in Ca2+-free medium, in the presence of methoxyverapamil and in the absence of Na+. Furthermore, isobutylmethylxanthine could not substitute for caffeine suggesting that the caffeine effect is not due to phosphodiesterase inhibition and the subsequent rise in intracellular cyclic nucleotides. A combination of the mitochondrial poisons, Antimycin A and sodium azide had no effect on the release of [3H]gamma-aminobutyric acid induced either by caffeine or by high K+. We conclude that caffeine causes the release of Ca2+ from a non-mitochondrial store within the growth cone and that this Ca2+ store supports that component of the K+-induced release of [3H]gamma-aminobutyric acid that is independent of extracellular Ca2+.     

Ruthenium red and magnesium ion partially inhibit silver ion-induced release of calcium from sarcoplasmic reticulum of frog skeletal muscles

Effects of Ca2+-induced Ca2+ release blockers, ruthenium red (RR) and Mg2+, on Ag+-induced Ca2+ release were studied using skinned muscle fibers or fragmented heavy SR (HSR) prepared from frog muscle, and compared with those on caffeine-induced one. Exposure of the skinned fibers to 5 microM Ag+ produced a rapid and large contraction in the presence of 0.043 mM free Mg2+. When Mg2+ concentration was increased to 0.86 mM, Ag+ led to a large transient contraction, combined with a small tonic one. The transient component was completely blocked by high Mg2+ (3.64 mM), but the tonic one was not. Ca2+-ATPase activity was not stimulated by increase of Mg2+ from 0.86 to 3.64 mM. Ag+ and caffeine induced a rapid Ca2+ efflux from HSR in a dose-dependent manner. RR over a range from 1 to 10 microM dose-dependently inhibited the Ca2+ efflux induced by 10 microM Ag+. Despite increase of RR to 30 microM, however, further inhibition of the Ca2+ efflux was not produced any more (77.8 +/- 12.2% inhibition). A 10 mM caffeine-induced efflux of Ca2+ was blocked slightly by only 0.5 microM RR and almost completely by 3 microM. A slight inhibition (about 28%) of the Ca2+-ATPase activity was observed in the presence of 10 microM Ag+ in 0.5 mg SR protein/ml of medium. RR and caffeine did not affect the enzyme activity. These results indicate that frog SR could induce a rapid release of Ca2+ upon Ag+ and caffeine, suggesting that Ag+ may have two different binding sites to release Ca2+; one is on Ca2+-induced Ca2+ release channel and the other on RR-insensitive site.     

Observations on the muscarinic activation of catecholamine secretion in the chicken adrenal

Cells were isolated by collagenase digestion of chicken adrenal glands. Catecholamine secretion could be stimulated by acetylcholine, carbamylcholine, potassium or veratridine. Methacholine, muscarine and oxotremorine were also effective secretagogues whereas nicotine was not. Secretion evoked by acetylcholine was blocked by low concentrations of atropine but was relatively insensitive to hexamethonium. Atropine-sensitive secretion required both external sodium and calcium, was unaffected by tetrodotoxin, blocked by methoxy verapamil and nifedipine, and potentiated by BAY-K-8644. These data suggest that muscarinic activation of these cells facilitates tetrodotoxin insensitive depolarization, thereby opening conventional voltage-sensitive calcium channels. The mechanism by which calcium activates catecholamine secretion was investigated in cells that had been made permeable by exposure to brief intense electric fields. Catecholamine release required Mg-adenosine 5' triphosphate, was half-maximally activated by 1 microM Ca2+ and could be inhibited by high concentrations of Mg2+. At low Ca2+ concentrations, release was potentiated by 12-O-tetradecanoylphorbol 13-acetate, dioctanoylglycerol, guanosine 5'-O-(3-thiotriphosphate) and 5'-guanylylimidodiphosphate, all of which increased the apparent affinity of exocytosis for Ca2+.     

Characterization of catecholamine release from deer adrenal medullary chromaffin cells

Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca2+-channel antagonist nifedipine and approximately 20% by N-type (omega-conotoxin GVIA) or N, P/Q-type (omega-conotoxin MVIIC) antagonists. In contrast the response was unaffected by omega-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.     

Sequestration of depolarization-induced Ca2+ loads by mitochondria and Ca2+ efflux via mitochondrial Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We used fura-2 microfluorometry to investigate the role of mitochondria in regulating the increase in the cytosolic Ca2+ concentration ([Ca]in) and the mechanism(s) underlying the subsequent Ca2+ efflux from mitochondria in bovine adrenal chromaffin cells. The rate of [Ca]in decay during and following stimulation with 100 mM KCl depolarization was markedly increased when the mitochondrial Na+/Ca2+ exchanger was inhibited by clonazepam or CGP-37157(CGP). In contrast, the addition of gramicidin, which increased the cytosolic Na+ concentration, following KCl depolarization caused a secondary increase in [Ca]in. This secondary increase in [Ca]in was prevented by the addition of clonazepam or CGP, and by the removal of external Na+. The subsequent removal of clonazepam or CGP, or the delayed addition of Na+ caused a slow increase in [Ca]in. A protonophore (FCCP) applied following KCl depolarization also caused a robust, secondary increase in [Ca]in, which was insensitive to blocking by clonazepam or CGP. Neither gramicidin nor FCCP altered the [Ca]in decay when applied following stimulation with histamine or caffeine, which mobilized Ca2+ from intracellular stores. These results suggest that the large [Ca]in increase induced by Ca2+ influx, but not by intracellular Ca2+ release, is buffered by mitochondria, and that the mitochondrial Na+/Ca2+ exchanger makes a major contribution to the subsequent Ca2+ efflux from mitochondria.     

Effects of pertussis toxin on the affinity of exocytosis for Ca2+ in bovine adrenal chromaffin cells

Effects of pertussis toxin (islet-activating protein, IAP) on the secretory function of bovine adrenal chromaffin cells in culture were studied. Treatment of chromaffin cells with IAP resulted in an increase in both basal release of catecholamine and evoked-release by either acetylcholine (ACh) or high K+. In the dose-response curve for ACh-evoked release, IAP treatment produced an increase of the maximal response without affecting the half-maximal concentration of ACh. When the cells were permeabilized with digitonin after IAP-pretreatment, Ca2(+)-dependent exocytosis was markedly increased where the affinity of exocytosis for Ca2+ was augmented. These findings suggest that IAP-sensitive GTP-binding protein (or proteins) directory controls the Ca2(+)-triggered process in the machinery of exocytosis by modulating the affinity for Ca2+ of its unknown target.     

cis-unsaturated fatty acids stimulate catecholamine secretion, tyrosine hydroxylase and protein kinase C in adrenal medullary cells

In digitonin-permeabilized bovine adrenal medullary cells, arachidonic acid and oleic acid, the cis-unsaturated fatty acids, enhanced Ca2+-induced secretion of catecholamines, whereas elaidic acid, a trans-unsaturated fatty acid and stearic acid, a saturated fatty acid, had no effect. Indomethacin, an inhibitor of cyclooxygenase and nordihydroguaiaretic acid, an inhibitor of lipoxygenase, failed to inhibit the stimulatory effect of arachidonic acid. Stimulation of catecholamine secretion by arachidonic acid was abolished by the removal of adenosine 5'-triphosphate and Mg2+ from the incubation medium. Pretreatment of the cells with phorbol 12-myristate 13-acetate, an activator of protein kinase C, enhanced Ca2+-induced catecholamine secretion. In cells pretreated with phorbol 12-myristate 13-acetate, the stimulatory effect of arachidonic acid on Ca2+-induced catecholamine secretion was greatly reduced. In digitonin-permeabilized cells, arachidonic acid and oleic acid enhanced Ca2+-induced activation of tyrosine hydroxylase in the presence of adenosine 5'-triphosphate and Mg2+, whereas elaidic acid and stearic acid did not activate the enzyme. In a soluble fraction of adrenal medullary cells, 32P incorporation to histone by protein kinase C was increased by arachidonic acid and oleic acid, but not by elaidic acid and stearic acid. These results suggest that cis-unsaturated fatty acids modulate Ca2+-induced catecholamine secretion and tyrosine hydroxylase activity by activation of protein kinase C in adrenal medullary cells.     

Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and omega-conotoxin-insensitive pathways

The fluorescent Ca2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (10-65 mM) gave rise to a very rapid increase in intracellular free Ca2+ concentration, which subsequently decayed slowly towards a "plateau". The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, -23 mV). A dihydropyridine calcium channel agonist [(+)202-791, 1 microM] raised intracellular free Ca2+ concentration further in the presence of 30 mM K+, and it enhanced the initial intracellular Ca2+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the L-type. Several dihydropyridine calcium channel antagonists [(-)202-791, nifedipine, nitrendipine; 1-5 microM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, omega-conotoxin GVIA (1-10 microM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca2+ concentration only slightly at 55 mM K+. Further, the dihydropyridine-insensitive component of the intracellular Ca2+ signal was also insensitive to omega-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation Gd3+ (40 microM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ concentration to sub-nanomolar levels, in the presence of high K+. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-dependent calcium channels, only one of which is likely to be the L-type channel. We also propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.     

Rhythmic hyperpolarizations and depolarization of sympathetic ganglion cells induced by caffeine.

Superfusion of the isolated sympathetic ganglion of the bullfrog with a caffeine-containing (1-6 mM) solution caused in many cells an initial slow hyperpolarization which was followed by a subliminal depolarization interruped by rhythmic hyperpolarizations. A hyperpolarization, similar to one of the rhythmic hyperpolarizations, could be triggered by an action potential in the presence of caffeine. The action potential itself was not markedly affected by caffeine except for its afterhyperpolarization which was prolonged. All these caffeine-induced hyperpolarizations were associated with a marked reduction of the membrane resistance, their amplitude was increased in a K+-free solution and decreased in a high-K+ solution, and their polarity was reversed at the same level at which the afterhyperpolarization was also inverted. This reversal level was not altered by omission of Na+ or C1- from the external medium. These hyperpolarizations were reversibly abolished by depletion of external Ca2+ or replacement of external Ca2+ by Mg2+. Excess of external Ca2+ caused a shortening of the interval between rhythmic hyperpolarizations. Furthermore, iontophoretic injection of EDTA into the cytoplasm markedly depressed the initial caffeine hyperpolarizatin and abolished both the rhythmic and evoked caffeine hyperpolarizations. The caffeine-induced depolarization was not affected by omission of external Cl-. It was decreased in a Na+-free medium, but completely eliminated by omission of both Na+ and Ca2+ from the external medium. Tetrodotoxin did not impair the production of the initial and the rhythmic hyperpolarizations. A strong depolarizing pulse could evoke a typical hyperpolarizing response in the presence of this compound. Dibutyryl cyclic AMP, d-tubocurarine, atropine, and phenoxybenzamine were without effect on the caffeine-induced hyperpolarizations and depolarization. It was concluded that each caffeine-induced hyperpolarization is the result of an increased K+ permeability, which is probably caused by a rise in the internal Ca2+ concentration. It was also concluded that the caffeine-induced depolarization is due to an increased membrane permeability to Ca2+ and Na+.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.     

Calcium release induced by high K+ and caffeine in cultured skeletal muscle cells of embryonic chicken

To further understand the function of excitation-contraction coupling in skeletal muscle cells developing in vitro, Ca2+ transients elicited by high-K+ depolarization in the presence and absence of extracellular Ca2+ were compared with Ca2+ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K+]O with an elevation of [Ca2+]i. Although all myotubes cultured for more than 4 days exhibited Ca2+ release independent of extracellular Ca2+, only about 50% of E9I1 and E9I2 cells maintained their response to Ca(2+)-free high-[K+]O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K+]O in the absence of extracellular Ca2+. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca2+ release from caffeine-insensitive cells induced by Ca(2+)-free high-[K+]O solution could be blocked by 100-200 microM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca2+]i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.     

Evidence that TRH stimulates secretion of TSH by two calcium-mediated mechanisms

Thyrotropin-released hormone (TRH) stimulation of thyrotropin (TSH) release from mouse thyrotropic tumor (TtT) cells is dependent on Ca2+. We demonstrate that TRH action in TtT cells does not require extracellular Ca2+ but that Ca2+ influx induced by TRH can augment TSH secretion. TRH caused a 46% increase in 45Ca2+ uptake by TtT cells in medium with 100 micro M Ca2+. The increment in 45Ca2+ uptake caused by TRH was dependent on the concentration of Ca2+ in the medium. In contrast to the effect of 50 mM K+, which also causes Ca2+ influx, TRH caused 45Ca2+ efflux and TSH release from TtT cells even when the concentration of Ca2+ in the medium was lowered below 100 micro M. TRH stimulated TSH release during perifusion in medium in which the free Ca2+ concentration was lowered to approximately 0.02 micro M, and reintroduction of Ca2+ into the medium simultaneously with TRH markedly increased TSH release. We suggest that TRH may affect Ca2+ metabolism in TtT cells by both extracellular Ca2+-independent and -dependent mechanisms and that this dual mechanism of action serves to augment further TSH secretion induced by TRH.     

Effect of Mg2+ on non-quantal acetylcholine release at the mouse neuromuscular junction

The effects of extracellular concentrations of Mg2+ on the non-quantal release of acetylcholine (ACh) from nerve terminals was studied by extra- and intracellular electrophysiological methods. Anticholinesterase-treated mouse diaphragms were used in vitro. In the presence of Ca2+, the non-quantal release was maximal in the absence of Mg2+ and was inhibited by 3 mmol/l Mg2+. The inhibitory effect of Mg2+ was antagonized by ouabain and was absent in Ca2+-free (EGTA) solutions. The non-quantal release of ACh was found to be more sensitive to inhibition by Mg2+ than the quantal one which was measured as the amplitude of miniature endplate currents.     

Nicotinic receptor-mediated intracellular calcium release in cultured bovine adrenal chromaffin cells

When cultured bovine adrenal chromaffin cells were stimulated by a nicotinic agonist, carbamylcholine (0.3 mM) or 1,1-dimethyl-4-phenylpiperazinium (50 microM), in the Ca2+-free medium containing 0.1 mM ethyleneglycoltetraacetic acid, intracellular free Ca2+ concentration ([Ca2+]i) rose from approximately 90 to 149 nM. High K+ (56 mM) and veratridine (50 microM) had no effect on the [Ca2+]i in Ca2+-free medium. The carbamylcholine-evoked rise in [Ca2+]i was blocked by hexamethonium (0.1 mM) but not by atropine (1 microM). Furthermore, the carbamylcholine-evoked rise in [Ca2+]i was inhibited by an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (10 microM) but not by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 microM). These results show the existence of intracellular Ca2+ store sites, from which Ca2+ is released upon nicotinic receptor stimulation, in cultured adrenal chromaffin cells.