IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.     

The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone

The proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited antigen-stimulated secretion and calcium influx in rat basophilic leukemia cells. In a glucose-free solution the inhibitory effects of CCCP were due to a decrease in the intracellular ATP concentration; however, when glucose was present there was no decrease in ATP. Instead, we found that in a glucose-containing saline solution, CCCP inhibited antigen-stimulated calcium uptake because it depolarized the plasma membrane, which in rat basophilic leukemia cells inhibits antigen-stimulated calcium uptake. In the presence of glucose, relatively low concentrations of CCCP inhibited calcium uptake while higher concentrations were required to inhibit secretion. In contrast, the initial antigen-stimulated rise in cytoplasmic calcium, measured with the fluorescent calcium indicator quin2, was not inhibited by CCCP. This suggests that the release of calcium from intracellular stores might, in some cases, be sufficient to support antigen-stimulated secretion. In the presence of CCCP the pH gradient becomes important for regulating the membrane potential across the plasma membrane. When cells were depolarized with CCCP and the external pH was increased, the membrane potential returned to resting levels and antigen-stimulated calcium uptake was restored. Inhibition of antigen-stimulated secretion by higher concentrations of CCCP could also be reversed by increasing the external pH.     

Characterization of increased K+ permeability associated with the stimulation of receptors for immunoglobulin E on rat basophilic leukemia cells.

Aggregation of immunoglobulin E-receptor complexes on the surface of rat basophilic leukemia cells stimulates an increase in plasma membrane K+ permeability that is monitored as an increase in the rate of efflux of preloaded 86Rb+. A major component of this stimulated 86Rb+ efflux appears to be due to a Ca(2+)-activated K+ channel because it is inhibited by quinidine in parallel with the inhibition of degranulation and membrane potential repolarization, it is blocked by 0.1 mM La3+, and it is dependent on external Ca2+. Depolarization of the plasma membrane by carbonyl cyanide 3-chlorophenylhydrazone inhibits stimulated Ca2+ influx and prevents antigen-induced 86Rb+ efflux, and increased external Ca2+ partially restores 86Rb+ efflux under these conditions. In addition, potentiation of antigen-stimulated Ca2+ influx by pretreatment with cholera toxin increases the initial rate of stimulated 86Rb+ efflux. Another component of antigen-stimulated K+ efflux appears to be mediated by a guanine nucleotide-binding protein because pretreatment of rat basophilic leukemia cells with pertussis toxin decreases the initial rate of antigen-stimulated 86Rb+ efflux to 40% of that for the untreated cells. Stimulated 86Rb+ efflux is also observed when ionomycin is used to increase cytoplasmic Ca2+ and to trigger membrane depolarization. The efflux stimulated by ionomycin is inhibited by quinidine but not by pertussis toxin pretreatment; thus, it appears to occur through the Ca(2+)-activated K+ efflux pathway. It is proposed that these K+ efflux pathways serve to sustain the Ca2+ influx that is necessary for receptor-mediated triggering of cellular degranulation.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells

Fura-2 fluorescence in single rat basophilic leukemia cells was monitored to study the rise in intracellular free ionized calcium ([Ca2+]i) produced by aggregation of immunoglobulin E receptors. Repetitive transient increases in [Ca2+]i were induced by antigen stimulation and were measured using digital video imaging microscopy at high time resolution. The [Ca2+]i oscillations were not dependent upon changes in the membrane potential of the cells and were observed in cells stimulated with antigen either with or without extracellular Ca2+. Transient oscillations in [Ca2+]i were also observed when calcium influx was blocked with La3+. These results suggested that during antigen stimulation of cells under normal physiological conditions, release of Ca2+ from intracellular stores makes an important contribution to the initial increase in [Ca2+]i. Oscillations in [Ca2+]i are not induced by elevating [Ca2+]i with the calcium ionophore ionomycin. Mitochondrial calcium buffering is not required for [Ca2+]i oscillations to occur. The results show that rat basophilic leukemia cells have significant stores of calcium and that release of calcium from these stores can participate in both the initial rise and the oscillations in [Ca2+]i.     

Release of calcium from intracellular stores in rat basophilic leukemia cells monitored with the fluorescent probe chlortetracycline.

Release of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortetracycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen-stimulation was not the result of secretion of granule-associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+ influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with 45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortetracycline was used to examine the rate of antigen-induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen-stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid response time.     

Calcium permeability changes and neurotransmitter release in cultured rat brain neurons. I. Effects of stimulation on calcium fluxes

The permeability of neuronal membranes to Ca2+ is of great importance for neurotransmitter release. The temporal characteristics of Ca2+ fluxes in intact brain neurons have not been completely defined. In the present study 45Ca2+ was used to examine the kinetics of Ca2+ influx and efflux from unstimulated and depolarized rat brain neurons in culture. Under steady-state conditions three cellular exchangeable Ca2+ pools were identified in unstimulated cells: 1) a rapidly exchanging pool (t1/2 = 7 s) which represented about 10% of the total cellular Ca2+ and was unaffected by the presence of Co2+, verapamil, or tetrodotoxin; 2) a slowly exchanging pool (t1/2 = 360 s) which represented 42% of the total cellular Ca2+ and was inhibited by Co2+, but not by verapamil or tetrodotoxin; 3) a very slowly exchanging pool (t1/2 = 96 min) which represented 48% of the total cell Ca2+ was observed only in the prolonged efflux experiments. The rate of exchange of 45Ca2+ in the unstimulated cells was dependent on the extracellular Ca2+ concentration (half-saturation at 70 microM). Depolarization of the neurons with elevated K+ causes a rapid and sustained 45Ca2+ uptake. The cellular Ca2+ content increased from 56 nmol/mg protein in unstimulated cells to 81 nmol/mg protein during 5 min of depolarization. The kinetics of the net 45Ca2+ uptake by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s-1 (t1/2 = 72 s) into a single additional compartment. The other cellular Ca2+ pools were apparently unaffected by stimulation. The stimulated 45Ca2+ uptake was inhibited by Co2+ and by the Ca2+ channel blocker verapamil but not by the Na+ channel blocker tetrodotoxin. Ca2+ uptake into this compartment was dependent on the extracellular Ca2+ concentration (half-saturation at 0.80 mM Ca2+). Predepolarization of the cells with high K+ for 10-60 s prior to the addition of the radioactive calcium did not alter the rate of 45Ca2+ incorporation into the stimulated cells. It is concluded that the rapidly exchanging, the slowly exchanging, and the depolarization-induced Ca2+ pools observed in intact brain neurons are physically as well as kinetically distinct from each other. In addition, the depolarization-induced component observed in stimulated cells represents movement of the Ca2+ ions through a single class of voltage-sensitive Ca2+ channels. These Ca2+ channels are inhibited by Co2+ ions and by verapamil and are not inactivated during depolarization of the brain neurons.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Cyclosporin A inhibits degranulation of rat basophilic leukemia cells and human basophils. Inhibition of mediator release without affecting PI hydrolysis or Ca2+ fluxes.

Cyclosporin A (CSA) inhibits IgE receptor-mediated exocytosis from rat basophilic leukemia (RBL) cells and human peripheral blood basophils in a dose-dependent manner over the therapeutic range of CSA concentrations achieved in vivo. Half-maximal inhibition was observed at 0.2 micrograms/ml CSA. The effect of CSA on several biochemical parameters involved in receptor-mediated activation of RBL cells was examined. Maximum inhibition of secretion occurred when CSA was added 5 min before activation, and inhibition was nearly maximum when the drug was added 2 min before the cells were triggered. The same results were observed when RBL cells were stimulated with A23187, a calcium ionophore. These results suggest a mechanism other than inhibition of protein synthesis is involved. Inhibition by CSA of release by either secretagogue persisted, even if CSA was removed from the buffer before the cells were triggered. No inhibition was observed of either receptor-mediated phosphatidylinositol hydrolysis, 45Ca2+ uptake, or the rise in the intracellular concentration of free Ca2+ under the same conditions that produced greater than 80% inhibition of serotonin release. These results demonstrate that the early events in signal transduction are not affected, and suggest that the intracellular target for CSA participates in a later stage of exocytosis. Furthermore, the data suggest that CSA suppresses cells other than T lymphocytes and predict that patients on CSA therapy may have altered response to allergens.     

Effect of thioloxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) on 45Ca2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca2+ net uptake was enhanced by tolbutamide (100 micrograms/ml), glibenclamide (0.5 micrograms/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca2+ net uptake in the presence of (5.6-16.7 mM) glucose. It is suggested that 45Ca2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

Study of the transit of an integral membrane protein from secretory granules through the plasma membrane of secreting rat basophilic leukemia cells using a specific monoclonal antibody

The monoclonal antibody 5G10 reacted specifically with an 80-kD integral membrane protein in rat basophilic leukemia (RBL) cells. Immunofluorescence microscopy studies of RBL cells, fixed and permeabilized, revealed that the 80-kD protein was located in the membrane of cytoplasmic vesicles. The vesicles were identified as secretory granules by their content in immunoreactive serotonin. Expression of the 5G10 antigen on the surface of unstimulated RBL cells was low. However, RBL cells stimulated to secrete with anti-dinitrophenyl IgE followed by dinitrophenyl-bovine serum albumin or with the Ca2+ ionophore A-23187 displayed an increased expression of the antigen on their surface. Surface exposure of the 5G10 antigen was maximal at 5 min after stimulation of secretion. Removal of dinitrophenyl-bovine serum albumin from the incubation medium resulted in internalization of 50% of the antigen within 10 min.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Cholera toxin increases the rate of antigen-stimulated calcium influx in rat basophilic leukemia cells

Cholera toxin pretreatment has been found to cause a 3-fold increase in the initial rate of antigen-stimulated secretion of serotonin from rat basophilic leukemia (RBL) cells. Under similar conditions, cholera toxin enhances the antigen-stimulated rise in cytoplasmic free ionized calcium levels and causes a 2-3-fold increase in the rate of antigen-stimulated influx of 45Ca. In intact RBL cells cholera toxin pretreatment potentiates the antigen-stimulated production of inositol phosphates, but in permeabilized cells, with strongly buffered free calcium levels, no effect of cholera toxin pretreatment on the antigen-stimulated activation of cellular phospholipase activities is observed. In addition, pretreatment of cells with tetradecanoylphorbol acetate inhibits antigen-stimulated production of inositol phosphates by greater than 95%, while the stimulated influx of 45Ca remains unaffected. These data indicate that the antigen-stimulated influx of calcium into RBL cells can be dissociated from the production of inositol phosphates in these cells. The observed effects of cholera toxin on exocytosis and Ca2+ influx in RBL cells are not due to the elevation of cellular cyclic AMP levels since a variety of agents capable of elevating cellular cyclic AMP levels do not mimic these effects. Together, these data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the pathway responsible for the antigen-stimulated influx of calcium into RBL cells.     

Mercuric chloride inhibition of vasopressin release from the isolated neurointermediate lobe of the rat pituitary

The effects of HgCl2 and ouabain on vasopressin release and Ca2+ uptake and distribution was examined in the neurointermediate lobe of the rat pituitary. HgCl2 (0.5 mM) inhibited vasopressin release by approx. 90% in both basal and potassium depolarized states. With 0.1 mM HgCl2 vasopressin release was inhibited by 50% in the depolarized state, but release was not effected in basal state. On the other hand, ouabain (0.5 mM) caused a 3-fold stimulation of vasopressin release in the depolarized state. Both HgCl2 (0.5 mM) and ouabain (0.5 mM) increased net 45Ca+2 uptake by about 80% in groups of neurointermediate lobes. Following 45Ca+2 uptake, HgCl2 (0.5 mM), which is absorbed by the neurointermediate lobe, produced an increase in cytosolic 45Ca+2 content and a decrease in mitochondrial 45Ca+2 content compared to control. In comparison, ouabain (0.5 mM), which does not penetrate the neurointermediate lobe, gave no change in cytosolic 45Ca+2, but an increase in mitochondrial 45Ca+2. These results suggest that HgCl2 inhibits vasopressin release from the neurointermediate lobe of the rat pituitary at a point distal to Ca+2 uptake by the gland.     

The role of calcium in the secretion of surfactant by rat alveolar type II cells

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.     

Activation of rat basophilic leukemia cells. Temporal identification of the signal calcium influx mediated by the receptor-operated channel pathway

Antigen-induced 45calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45Ca2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE-receptor occupancy at low receptor occupancies, and is sensitive to the anti-allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin-2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45Ca2+ uptake curve. These qualities suggest that the early peak at 2-3 min, rather than any later 45Ca2+ uptake, comprises the initial signalling Ca2+ pool. Maximal apparent inhibition by cromolyn for 45Ca2+ uptake was about 65% and required a 10-15-min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5-6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn-binding protein [Corcia, A. et al. (1986) EM BO J. 5, 849-854]. This, as well as the effects of cromolyn, support the assumption that cromolyn-binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.