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+.     

Characterization of [3H]GABA release from striatal slices: evidence for a calcium-independent process via the GABA uptake system

GABA can be released by depolarization even in the absence of external Ca2+. To investigate the underlying mechanism of this phenomenon, GABA release was studied using slices prepared from rat striatum. Slices were preincubated with [3H]GABA in the presence of beta-alanine and superfused with Krebs buffer. Total tritium efflux was measured as an index of GABA release. Electrical stimulation at 2 Hz for 3 min elevated resting tritium efflux approximately two-fold. Decreasing external Ca2+ to 0.1 mM increased basal tritium efflux and reduced electrically evoked overflow, while omitting Ca2+ entirely (and adding 1 mM EGTA) increased both basal efflux and evoked overflow. Tetrodotoxin (5 microM) abolished the evoked release of tritium but did not affect the resting outflow in either normal or Ca2+-deficient conditions. In the presence of normal Ca2+, nipecotic acid (0.1-1 mM), an inhibitor of GABA transport into neurons as well as glia, enhanced both spontaneous efflux and evoked overflow of tritium. Nipecotic acid also increased spontaneous release when external Ca2+ was reduced or removed; however, under these conditions electrically evoked overflow was reduced. These results suggest that the electrically evoked release of [3H]GABA from striatal slices is of neuronal origin, but can occur in part in the absence of external Ca2+. They further suggest that this Ca2+-independent release, which may co-exist with the Ca2+-dependent release, takes place via the same carrier system utilized for high-affinity GABA uptake.     

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

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

Effect of acute hepatic encephalopathy on [3H]dopamine release from rat cerebral cortex and striatum in vitro: role of Ca2+

Hepatic encephalopathy (HE) is characterized by motor symptoms associated with disturbed functions of the dopaminergic systems, but the underlying mechanisms are not clear. A previous study from our laboratories revealed that HE, induced in rats by repeated treatment with thioacetamide, enhanced the 50 mM potassium (KCl)-stimulated release of newly loaded [3H]dopamine in both striatal and frontal cerebral cortical slices in the presence of Ca2+. In the present study we compared the effects of HE on dopamine release in striatal and frontal cerebral cortical slices and synaptosomes in the presence and absence of Ca2+. HE enhanced the KCl-stimulated [3H]dopamine release from striatal and frontal cortical synaptosomes in the presence of Ca2+ to the same extent as in slices prepared from the respective brain regions. In the absence of Ca2+ a slight reduction in dopamine release was observed in frontal cortical synaptosomes from HE rats when compared to control rats, while no effect of HE on the release was discernible in frontal cortical and striatal slices and striatal synaptosomes. We conclude that in both brain regions studied HE stimulates dopamine exocytosis triggered by Ca2+ influx without affecting the release mediated by means of plasma membrane transporters or exocytosis involving intraterminal Ca2+.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

Calcium fluxes in isolated pancreatic acini: effects of secretagogues

45Ca2+ exchange and total calcium content were measured in isolated mouse pancreatic acini. 45Ca2+ uptake could be described as the sum of a constant and a single exponential kinetic component; about 60% of total acinar calcium was exchangeable. Stimulation by bethanechol increased 45Ca2+ uptake, but the time course of uptake could be fit only by the addition of a more rapid kinetic component without any change in the total exchangeable Ca2+. 45Ca2+ washout after 1-h loading could be fit as the sum of two exponential components. Stimulation increased the rate of 45Ca2+ washout with the appearance of a third and more rapid kinetic component. There was not, however, a good correspondence between the exponential constants measured in uptake and washout protocols in unstimulated acini. Exponential constants were also affected by the concentration of calcium in the medium, further indicating the presence of nonlinearities in 45Ca2+ exchange. The dose-response relationships were similar for bethanechol stimulation of 45Ca2+ uptake and amylase release, whereas stimulation of 45Ca2+ washout reached a maximum at a higher concentration of bethanechol. Both 45Ca2+ uptake and analytical measurement of total Ca2+ showed a rapid drop in acinar Ca2+ content followed by a gradual reuptake on stimulation by bethanechol. It is concluded that the initial primary effect of secretagogues is to increase Ca2+ efflux, which is interpreted to be the result of release of sequestered calcium into the cytosol.     

Ultrastruct pathology of phalloidin-intoxicated hepatocytes in the presence and absence of extracellular calcium

The killing of cultured hepatocytes by phalloidin can be dissociated into two phases by manipulation of the Ca2+ concentration of the medium. In the absence of extracellular Ca2+, hepatocytes are injured but not killed by phalloidin. Addition of 1.8 mM Ca2+ to the culture medium kills 60-70% of the cells by three hours. As an initial attempt to identify the mechanisms whereby Ca2+ ions irreversibly injure phalloidin-damaged hepatocytes, we have examined the ultrastructural pathology of phalloidin-intoxicated liver cells in the presence or absence of extracellular Ca2+. In the absence of extracellular Ca2+ ions, the morphologic manifestations of phalloidin intoxication reflect entirely the interaction between phalloidin, microfilaments, and the plasma membrane. In the presence of Ca2+ ions, the morphologic manifestations of the lethal effects of Ca2+ are described: the swelling of mitochondria accompanied by the accumulation of dense, amorphous precipitates; a supercontracture of microfilaments, and a loss of volume control with intracellular edema and a change in cell shape. These alterations can be attributed to the known biologic actions of Ca2+ ions on cellular structure and function. The present study allows, therefore, a preliminary identification of mechanisms by which extracellular Ca2+ ions may mediate cell death in this as well as in other similar situations.     

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.     

Release of 3H-noradrenaline from the rat vas deferens under various in vitro conditions.

The release of 3H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 X 10(-6) M) or low external Na+ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca2+ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca2+ but almost completely in absence of Ca2+. The release by high K+ (117 mM)+low Na+ (26 mM) in normal tissue was dependent on the presence of Ca2+ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca2+ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.     

Sodium ions inhibit the stimulant action of caffeine on catecholamine secretion from adrenal chromaffin cells of the guinea pig

Catecholamine secretion evoked by caffeine (40 mM) was markedly enhanced by replacing NaCl in the medium with sucrose or KCl in the absence, but not in the presence, of extracellular Ca2+ and Mg2+ in both perfused adrenal glands and isolated chromaffin cells of the guinea pig. The response to caffeine declined on repetition, but was restored completely after readmission of Ca2+. These results indicate that extracellular Na+ inhibits caffeine from stimulating catecholamine secretion, which may be mediated by a release of Ca2+ from intracellular storage sites in the adrenal chromaffin cells in the presence of extracellular Ca2+ and/or Mg2+.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

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.     

Calcium mediation of cholinergic-stimulated amylase release from mouse parotid gland.

Amylase release from mouse parotid fragments was stimulated independently by cholinergic and beta-adrenergic agents. The cholinergic agonist, carbachol, significantly increased release of amylase only in Ca2+ containing medium whereas isoproterenol-stimulated amylase release was unaffected by Ca2+ removal. The ionophore, A23187, mimicked the effect of cholinergic stimulation when Ca2+ was present in the medium. Uptake of 45Ca2+ into tissue fragments was enhanced by carbachol and A23187 but not by isoproterenol; atropine blocked the effect of carbachol. Diphenylhydantoin (DPH) and verapamil partially inhibited carbachol-stimulated amylase release and 45Ca2+ uptake, whereas diazoxide potentiated these effects; in all cases there was good parallelism between 45Ca2+ uptake and amylase release. It was concluded that the primary step in the release of amylase from mouse parotid gland in response to cholinergic agents is an increased influx of Ca2+.     

Elevation of intracellular calcium ions is essential for the H2O2-induced activation of SAPK/JNK but not for that of p38 and ERK in Chinese hamster V79 cells

The mitogen-activated protein kinases (MAPK), including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and extracellular signal-related kinase (ERK), are believed to be important biomolecules in cell proliferation, survival, and apoptosis induced by extracellular stimuli. In Chinese hamster V79 cells exposed to hydrogen peroxide (H2O2), we recently demonstrated that SAPK/JNK was activated by tyrosine kinase and intracellular Ca2+ ([Ca2+]i). In this study, we report that [Ca2+]i release from intracellular stores is important in the activation of SAPK/JNK but not p38 and ERK. H2O2-induced elevation of [Ca2+]i was observed in Ca2+-free medium. Pretreatment with thapsigargin, a Ca2+-ATPase inhibition of endoplasmic reticulum (ER), did not influence H2O2-induced elevation of [Ca2+]i in the absence of external Ca2+. An intracellular Ca2+ chelator (BAPTA-AM) inhibited H2O2-induced phosphorylation of SAPK/JNK, but an extracellular Ca2+ chelator (EDTA) or a Ca2+ entry blocker (NiCl2) did not. Activation of p38 and ERK in V79 cells exposed to H2O2 was observed in the presence of these inhibitors. These results suggest that [Ca2+]i release from intracellular stores such as mitochondria or nuclei but not ER, occurred after H2O2 treatment and Ca2+-dependent tyrosine kinase-induced activation of SAPK/JNK, although [Ca2+]i was unnecessary for the H2O2-induced activation of p38 and ERK.     

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.     

Mitochondrial Ca2+ flux is a critical determinant of the Ca2+ dependence of mast cell degranulation

An increase in intracellular Ca2+ ([Ca2+]i) is necessary for mast cell exocytosis, but there is controversy over the requirement for Ca2+ in the extracellular medium. Here, we demonstrate that mitochondrial function is a critical determinant of Ca2+ dependence. In the presence of extracellular Ca2+, mitochondrial metabolic inhibitors, including rotenone, antimycin A, and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced degranulation induced by immunoglobulin E (IgE) antigen or by thapsigargin, as measured by beta-hexosaminidase release. In the absence of extracellular Ca2+; however, antimycin A and FCCP, but not rotenone, enhanced, rather than reduced, degranulation to a maximum of 76% of that observed in the presence of extracellular Ca2+. This enhancement of extracellular, Ca2+-independent degranulation was concomitant with a rapid collapse of the mitochondrial transmembrane potential. Mitochondrial depolarization did not enhance degranulation induced by thapsigargin, irrespective of the presence or absence of extracellular Ca2+. IgE antigen was more effective than thapsigargin as an inducer of [Ca2+]i release, and mitochondrial depolarization augmented IgE-mediated but not thapsigargin-induced Ca2+ store release and mitochondrial Ca2+ ([Ca2+]m) release. Finally, atractyloside and bongkrekic acid [an agonist and an antagonist, respectively, of the mitochondrial permeability transition pore (mPTP)], respectively, augmented and reduced IgE-mediated Ca2+ store release, [Ca2+]m release, and/or degranulation, whereas they had no effects on thapsigargin-induced Ca2+ store release. These data suggest that the mPTP is involved in the regulation of Ca2+ signaling, thereby affecting the mode of mast cell degranulation. This finding may shed light on a new role for mitochondria in the regulation of mast cell activation.     

Concerted enhancement of calcium influx, neurotransmitter release and protein phosphorylation by a phorbol ester in cultured brain neurons

We have recently shown that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate enhances the depolarization induced, calcium dependent release of [3H]dopamine from cultured brain neurons in the rat. In the present study the effects of 12-O-tetradecanoyl-phorbol-13-acetate on the kinetic parameters of depolarization induced calcium influx and on Ca2+ dependent neurotransmitter release and protein phosphorylation were investigated. Depolarization induced neurotransmitter release from the neurons occurs in two phases: an initial, fast release and a subsequent slow release. At low extracellular Ca2+, 12-O-tetradecanoyl-phorbol-13-acetate enhanced the quantity of fast release and in addition, increased the rate constant of the slow release. These effects mimicked the effects of increasing the extracellular Ca2+. Various phorbol derivatives known to activate the Ca2+ activated phospholipid dependent protein kinase (protein kinase C) were also able to enhance the stimulated release of [3H]dopamine from the neurons. 12-O-tetradecanoyl-phorbol-13-acetate induced the incorporation of 32Pi into a protein with an apparent molecular weight of 45,000 daltons regardless of depolarization or of the presence of Ca2+. In addition, 12-O-tetradecanoyl-phorbol-13-acetate induced in unstimulated neurons, Ca2+ dependent increase in the amount of 32Pi incorporated into a 43,000 dalton protein and decrease in the amount incorporated into a 55,000 dalton protein. These changes mimicked the Ca2+ dependent changes in protein phosphorylation which occur upon stimulation of the neurons. Kinetic studies of depolarization induced Ca2+ uptake by the neurons indicated that 12-O-tetradecanoyl-phorbol-13-acetate enhanced the maximal influx of Ca2+ through the voltage sensitive Ca2+ channels by 40%. The results indicate that 12-O-tetradecanoyl-phorbol-13-acetate acts primarily on the regulation of stimulated Ca2+ entry into the cells. Consequently neurotransmitter release at submaximal extracellular [Ca2+] is enhanced.     

Effect of atropine, ouabain, antimycin A, and A23187 on "trigger Ca2+ pool" in exocrine pancreas

45Ca2+ fluxes have been analyzed in dispersed acinar cells prepared from rat pancreas. Sudden addition of carbamylcholine (CCh) to 45Ca2+-preloaded acinar cells at quasi-steady state for 45Ca2+ resulted in a quick 45Ca2+ release followed by a slower 45Ca2+ reuptake with net accumulation of 45Ca2+. Subsequent sudden addition of atropine caused a further transient increase in cellular 45Ca2+ followed by a slow decrease to a steady-state value. 45Ca2+ release could not be evoked a second time by pancreozymin when prestimulated with CCh. However, if CCh stimulation was abolished by an interposed step of atropine, restimulation by cholecystokinin-pancreozymin was possible. Addition of A23187 or antimycin A to cells induced a fast decrease in cellular 45Ca2+. This effect was not additive to the CCh effect. In ouabain-pretreated cells, the CCh-induced sudden loss of cellular 45Ca2+ was blocked by 60%. The following slow reuptake of 45Ca2+ was blocked completely. Subsequent addition of atropine caused a fast uptake of cellular 45Ca2+ with no secondary decline. The data are consistent with the following model: acetylcholine releases Ca2+ from a cellular "trigger pool" into the cytosol located in or near the cell membrane. Then Ca2+ is extruded from the cell via Ca2+ pumps partly by a Na+-dependent Ca2+ transport system (quick phase of 45Ca2+ release). Subsequently, due to increased Ca2+ permeability of the plasma membrane as induced by acetylcholine, Ca2+ influx occurs and Ca2+ is taken up from the cytosol into intracellular Ca2+ pools (slow 45Ca2+ reuptake phase). Atropine causes refilling of the trigger Ca2+ pool and return of the increased Ca2+ permeability of the plasma membrane back to the unstimulated state.     

N-methyl-D-aspartate, kainate and quisqualate release endogenous adenosine from rat cortical slices

N-Methyl-D-aspartate, kainate, and quisqualate released endogenous adenosine from superfused slices of rat parietal cortex. N-Methyl-D-aspartate-evoked adenosine release was blocked by D,L-2-amino-5-phosphono-valeric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), indicating that it was receptor-mediated, although it did not show the expected potentiation in the absence of Mg2+. In contrast, N-methyl-D-aspartate-evoked release of [3H]noradrenaline from the same slices was markedly potentiated in Mg2(+)-free medium. Therefore, the lack of Mg2+ modulation of N-methyl-D-aspartate-evoked adenosine release was not due to depolarization-induced alleviation of the Mg2+ block in the slices. Kainate-evoked adenosine release was diminished by the non-specific excitatory amino acid antagonist, gamma-D-glutamyl-glycine, and kainate- and quisqualate-evoked adenosine release was diminished by 6,7-dinitroquinoxaline-2,3-dione, indicating that these agonists release adenosine by acting at non-N-methyl-D-aspartate receptors. Tetrodotoxin decreased N-methyl-D-aspartate- and kainate-evoked adenosine release by 40% and 19% respectively, indicating that release was mediated in part by propagated action potentials in the slices. Total release of adenosine by N-methyl-D-aspartate, kainate or quisqualate was not diminished in the absence of Ca2+. A second exposure to kainate following restoration of Ca2+ to slices previously depolarized in the absence of Ca2+ resulted in an amount of adenosine release equal to an initial release by slices in the presence of Ca2+, a result suggesting the presence of separate Ca2(+)-dependent and Ca2(+)-independent pools of adenosine. The present experiments demonstrate that activation of all three major subtypes of excitatory amino acid receptors in the cortex releases adenosine, possibly from separate Ca2(+)-dependent and -independent pools. Adenosine released from the cortex following excitatory amino acid stimulation may, by acting at inhibitory P1 purinoceptors, diminish excitatory neurotransmission and protect against excitotoxicity.     

Histidine Containing Peptides and Polypeptides as Nucleic Acid Vectors

Nucleic acid transfer in mammalian cells is drastically improved with devices which increase their delivery in the cytosol upon endocytosis. In this chapter, we describe the effect on plasmid DNA (pDNA) and oligonucleotide (ODN) transfer, of an histidine-rich peptide (H5WYG), histidylated oligolysine (HoK), and histidylated polylysine (HpK) designed on the basis of the membrane destabilization capacity of poly-L-histidine at a pH close to that of the endosomes. We report that H5WYG, which permeabilizes the cell membrane at pH 6.4, favors the transfection mediated by lactosylated polylysine/pDNA complexes and, by lowering the pH of extracellular medium, allows the loading of the cytosol and the cell nucleus with ODN. We show that HoK forms small cationic spherical particles of 35 nm with ODN and HpK rod or toroid cationic particles of 100 nm with pDNA. PEGylation stabilizes these particles at physiological salt concentration. We also show that (i) HoK/ODN complexes yield a more than 20-fold increase of the biological activity of antisense ODN towards the inhibition of transient as well as constitutive gene expression and (ii) HpK/pDNA complexes yield a transfection efficiency of 3–4.5 order of magnitude higher than do polylysine/pDNA complexes. We also provide evidence that the effect of these polyhistidylated molecules is mediated by imidazole protonation in endosomes. Overall our data show that polyhistidylated molecules constitute interesting devices for an efficient cytosolic delivery of nucleic acids, and that ionic complexes between histidylated polylysine and a pDNA are attractive for developing a nonviral gene delivery system.