Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Calcium fluxes and contractility in isolated guinea pig atrium: effect of A23187.

The Ca2+ ionophore A23187 (10(-6) to 3 X 10(-5) M) increased the force of contraction is isolated guinea pig atria. In individual twitches, peak tension, maximum rate of tension development, time to peak tension, and total twitch duration were all increased by A23187. Tripelennamine, indomethacin, and atropine did not significantly alter the inotropic effect of A23187. Serotonin produced changes in individual twitches that differed qualitatively and quantitatively from those of A23187. Therefore, the inotropic action of A23187 is probably not mediated by release of endogenous histamine, prostaglandins, acetylcholine, or serotonin. 45Ca influx and efflux were increased by A23187. The enhanced 45Ca efflux exceeded that which would be predicted if the ionophore acted only to increase the passive Ca2+ permeability of the myocardial cell membrane. These results suggest that A23187 facilitates the entry of extracellular Ca2+ into the myocardial cell and the release of intracellular Ca2+ stores into the myoplasm. The resultant increase in intracellular Ca2+ activity could account for the positive inotropic action of A23187.     

Calcium and voltage-dependent alterations of cell volume in neuroblastoma×glioma hybrid NG108-15 cells

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.     

Role of calcium in cholinergic and adrenergic mechanisms of eccrine sweat secretion

The role of Ca2+ in eccrine sweat secretion was studied using isolated cannulated monkey palm eccrine sweat glands in vitro. Removal of Ca2+ from the incubation medium promptly abolished sweat secretion induced by methacholine or phenylephrine. In contrast, isoproterenol-induced sweat secretion lasted from 40 to 220 min in a Ca2+-free medium. The methacholine-induced maximal sweat rate was a hyperbolic function of the Ca2+ concentration in the bath and reached a plateau at 1 mM Ca2+. Higher Ca2+ concentrations rather suppressed the secretory rate. The Ca2+ ionophore A23187, but not X537A, at 3 X 10(-6) M induced copious prolonged sweat secretion after a latent period of 10 min. A23187-induced sweat secretion was not inhibited by either atropine or propranolol. D 600 (methoxyverapamil) at 10(-3) M inhibited sweat secretion induced by methacholine or by isoproterenol, although the latter lasted longer than methacholine sweating (20 vs. 5 min) in the presence of D 600. The data support the notion that Ca2+ influx into the cell plays a crucial role in cholinergic and alpha-adrenergic sweating, whereas a partial supply of Ca2+ for isoproterenol-induced sweating is derived from an intracellular store.     

Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.     

Effects of extracellular Mg2+ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions

The electrophysiological characteristics of monolayers of cultured renal epithelial A6 cells were studied under short-circuit conditions. Replacing basolateral isosmotic (260 mOsm/kg H2O) media by hyposmotic (140 mOsm/kg H2O) solutions transiently increased the transepithelial capacitance (C(T)) by 57.3+/-2.3% after 16 min. The transepithelial Na+ current (I(Na)) increased concomitantly from 4.2+/-0.7 to 26.1+/-2.6 microA/cm2 with a time course that was noticeably slower, reaching its maximum after 60 min of hypotonicity. The transepithelial conductance (G(T)) increased synchronously with I(Na). Analysis of blocker-induced noise in I(Na), using the amiloride analogue 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), showed that the hypotonic shock increased Na+ channel density (N(T)) at the apical border. The presence of 10 mM Mg2+ on both sides of the epithelium suppressed the hypotonicity-induced C(T) increase to 14.3+/-0.5%, whereas the I(Na) increase was even larger than without Mg2+. Both effects of Mg2+ were located at an extracellular, basolateral site, because apical administration was without effect, whereas the acute basolateral addition of Mg2+ at the moment of the hypotonic shock was sufficient. Interaction between Mg2+ and Ca2+ influenced the behaviour of C(T). At constant osmolality (200 mOsm/kg H2O) 10 mM Mg2+ increased I(Na), leaving C(T) unaffected, whereas 10 mM Ca2+ stimulated both I(Na) and CT. In the presence of 1 mM Mg2+, however, the Ca(2+)-induced CT increase was abolished. The failure of CT to increase during stimulation of I(Na) by Mg2+ suggests that the divalent cation activates pre-existing channels in the apical membrane. Noise analysis showed that the natriferic effects of Mg2+ were also mediated by an increase in NT. The moderate initial increase in CT in the presence of Mg2+ under hypotonic conditions, occurring in parallel with increases in GT and I(Na), reflects most likely Na+ channel insertion induced by the hypotonic treatment. However, the large, transient, Mg(2+)-sensitive increase in CT, not correlated with increases in GT and I(Na), seems to be unrelated to Na+ channel recruitment.     

Role of Ca2+ and Mg2+ for endothelial permeability of water and albumin in vitro

The permeability of endothelial monolayers grown on a polycarbonate filter membrane and continuously exposed to a hydrostatic pressure of 10 cm H2O was studied. The ionophores A23187 and ionomycin dose dependently (0.1-10 microM) enhanced the hydraulic conductivity of sealed endothelial cell monolayers 10 to 15-fold, at the same time the reflection coefficient of albumin dropped from 0.75 to 0.2. The effects of A23187 were dependent on extracellular Ca2+. In the absence of Ca2+, however, Mg2+ could substitute for Ca2+. Phase contrast- and scanning electron-microscopy showed that the A23187-induced effects were accompanied by gap-formation in the intercellular clefts. These gaps probably are the sites which allowed enhanced fluid exchange. Ionophore-induced effects on permeability could be modified by antagonists of calmodulin function and of arachidonate liberation and metabolism. The data suggest that alterations of endothelial Ca2+-homeostasis induce metabolic events which result in an increased permeability of an endothelial monolayer.     

Macrophages increase microparticle uptake by enterocyte-like Caco-2 cell monolayers

Caco-2 cells form an enterocyte-like monolayer that has been used to explore small intestinal microparticle uptake. They are a useful functional model for the investigation of in vivo drug delivery systems and the uptake of particulate environmental pollutants. The aim of this paper was to determine if the previously reported decrease in Caco-2 transepithelial resistance following exposure to macrophages was matched by increased microparticle uptake, especially as macrophage phagocytosis simulates removal of particles from the subepithelial compartment. Caco-2 cells were grown as a monoculture for 21 days on insert membranes. A compartmentalised model involved Caco-2 cells in the upper compartment, with THP-1-derived macrophages adhering to the base of the underlying well, the two cell populations communicating only through the shared culture medium. Caco-2 cells were also cultured in macrophage-conditioned medium and all groups were exposed apically to 2 μm latex particles for 5 or 60 min. Parameters measured were: transepithelial resistance; cytokine levels; cell dimensions and the distribution of nuclei, actin and junctional proteins. Subepithelial particle numbers, defined as those located below the insert membrane, were also counted and were significantly increased in the Caco-2/macrophage model, with over 90% associated with the macrophages. Other changes induced by the presence of macrophages included decreased transepithelial resistance levels, diffuse localisation of some junctional proteins, higher proinflammatory cytokine levels, disorganisation of cell shape and decreased cell height associated with actin reorganisation. Macrophage-conditioned medium produced a smaller transepithelial resistance decrease than the Caco-2/macrophage model and there were few other changes. In conclusion, culture of Caco-2 cells with underlying macrophages produced a lower, less organised epithelium and greater microparticle uptake.     

Effects of glucose and ouabain on transepithelial electrical resistance and cell volume in stripped and unstripped goldfish intestine

1. In goldfish intestine (perfused unstripped segments and mucosal strips) the serosal addition of ouabain (10(-4) M) resulted in a vanishment of the transepithelial potential difference and in a continuous increase in transepithelial resistance. 2. Incubation of mucosal strips with ouabain resulted in an increase in sodium content which was greater than the decrease in potassium content. The resulting increase in cation content was accompanied by an increase in chloride content and an increase in water content. 3. Histological examination showed that exposure to ouabain resulted in a swelling of the epithelial layer as compared to the control situation. 4. The ouabain induced resistance increase is greater in the presence of glucose, 3-OMG or fructose than in the presence of mannitol. Phlorizin (10(-4) M) inhibits the extra resistance increase induced by mucosal glucose but is without effect on the fructose induced extra resistance increase. The initial velocity and the magnitude of the glucose induced extra resistance increase depends on the glucose concentration. 5. The results suggest that in goldfish intestine ouabain induces cellular swelling with a concomitant collapse of the lateral intercellular spaces, which is the cause of the increased transepithelial resistance. The additional changes in resistance induced by sugars suggest that the cell membrane is more permeable to glucose, 3-OMG and fructose than to mannitol. The resulting changes in osmotically active material within the epithelial cell influence the cross-sectional area and consequently the conductivity of the paracellular shunt pathway. The hypothesis that these sugars do not induce a resistance change in the absence of ouabain is discussed.     

Properties of the larval neuromuscular junction in Drosophila melanogaster.

The anatomy and physiology of the Drosophila larval neuromuscular junction were studied. 2. The dependence of muscle resting potentials on [K+]o and [Na+]o follows the Goldman-Hodgkin-Katz equation (PNa/PK=0-23). Chloride ions distribute passively across the membrane. 3. The mean specific membrane resistance of muscle fibres is 4-3 X 10(3) omega cm2, and the mean specific membrane capacitance is 7-1 muF/cm2. The muscle fibre is virtually isopotential. 4.Transmitter release is quantal. Both the miniature excitatory junctional potential and the evoked release follow the Poisson distribution. 5. Transmitter release depends on approximately the fourth power of [Ca2+]o. If Sr2+ replaces Ca2+, it depends on approximately the fourth power of [Sr2+]o. Mg2+ reduces transmitter release without altering the fourth power dependence on [Ca2+]o.     

Adenosine relaxation of isolated vascular smooth muscle.

Adenosine relaxed hog carotid media strips contracted with norepinephrine (NE) and potassium (K+). Adenosine (3 X 10(-6)M) was more effective in relaxing the NE contractures than those produced by K+. In both cases, adenosine's efficacy decreased with increasing concentrations of the stimulating agent. A high adenosine concentration (1 X 10(-3)M) was necessary to elicit relaxation of completely depolarized (124 mM K+) media strips and equimolar concentrations of aminophylline caused greater relaxation than did adenosine. Adenosine inhibited the Ca2+ dose-response curves of strips stimulated with 20 mM and 30 mM K+ and its effect was dependent on the Ca2+ concentration. Neither 1 X 10(-6)M nor 1 X 10(-4)M adenosine produced any change in the cAMP content of vascular strips. Only at high concentrations did adenosine increase the cAMP content of vascular strips, but the increase was signficantly more than that observed with the same dose of aminophylline. The present results are consistent with the possibility that adenosine relaxes vascular smooth muscle by directly altering Ca2+ permeability and/or membrane potential; they do not support a role for cAMP in the adenosine-induced relaxation of vascular smooth muscle.     

Excessive intracellular Ca2+ inhibits glutamate-induced Na(+)-K+ pump activation in rat hippocampal neurons.

1. The effects of increased intracellular Ca2+ concentration ([Ca2+]i) on Na(+)-K+ pump activity in CA1 pyramidal neurons of rat hippocampal slices were investigated. The postglutamate hyperpolarization (PGH), which follows glutamate (GLU)-induced depolarization (GD), was used as an index of Na(+)-K+ pump activity, as was a ratio of PGH area to the preceding GD area (PGH ratio). 2. Perfusion of slices with saline containing Ca2+ ionophore (A23187, 10 microM) inhibited the PGH without producing apparent signs of cell deterioration. A 60-100% (85 +/- 15%, mean +/- SD) reduction in the PGH ratio occurred after 20-50 min of A23187 superfusion in 12 of 18 neurons tested. Complete abolition of the PGH occurred in 8 of these 12 cells exposed to A23187 for 30-120 min. 3. Application of A23187 in Ca(2+)-free/high-Mg2+ solution did not abolish the PGH, although small (less than 50%; 37 +/- 10%) reductions in the PGH ratio were observed after perfusion of 50 min or longer in five neurons tested. 4. Intracellular injection of the Ca2+ chelator bis-(o-amino-phenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 300-400 mM) blocked inhibition of the PGH by A23187. After 50 min of perfusion with Ca2+ ionophore, no reduction of the PGH ratio was observed in five neurons tested. 5. Rundown of the PGH without apparent change in membrane properties was observed in three neurons that were stable for greater than 2-3 h, allowing repetitive GLU applications. 6. Block of the PGH produced by a Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitor (strophanthidin) prolonged the duration of GDs because of a delay in repolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ removal effects on bullfrog gastric mucosa in the presence of drugs

Bullfrog gastric mucosa can secrete acid in vitro for more than 24 hr. In Ca2+-deficient media, the acid secretion measured by pH stat method gradually decreased to cease about 200 min after the start of immersion in the media. When A23187 or quinidine, which are known to mobilize Ca2+ from the intracellular pools, was present on the submucosal side, a prolongation of the duration of acid secretion in the absence of Ca2+ was found. Dibutyl c-AMP also induced a similar prolongation. Histamine or theophylline not only prolonged the duration of acid secretion but also transiently increased the acid secretory rate over the control level. Based upon the known fact that there are different intracellular Ca2+ compartments, i.e, one related to the process of acid secretion and the other to the maintenance of junctional complexes, the present results of drug effects were discussed especially in regard to their Ca2+ mobilizing effects. Cessation of acid secretion in a K+- and Ca2+-deficient solution occurred much more quickly than that in a Ca2+-deficient or a K+-deficient medium.     

Polymorphonuclear leucocytes as a model of Ca++ and Mg++-dependent cellular activation. Effect of calcium entry blockers

Flunarizine inhibited FMLP- and A23187-induced aggregation, enzyme release and O2- generation from human PMN as a function of its concentration. A23187-dependent PMN aggregation was also studied in media devoid of Ca++ or Mg++. Flunarizine (2.4 X 10(-5)M) significantly affected not only Ca++-supported but also Mg++-sustained PMN aggregation. The inhibiting effect of the drug was reversed by increasing the level of Ca++ (1.2 mM) or Mg++ (2 mM). Nifedipine, another Ca++-entry blocker, was shown to inhibit enzyme release and O2- generation induced by FMLP and A23187 as a function of its concentration, but only slightly affected PMN aggregation at very high concentration (10(-4)M). A role for flunarizine as a specific Mg++-entry blocker is suggested.     

Extracellular calcium is not an absolute requirement for tumoricidal activation of RAW-264 macrophage-like cell line

The purpose of these studies was to establish whether extracellular calcium (Cao2+) plays a role in the process of activation of RAW-264 macrophages for tumor cell killing. We found that these cells were capable of developing a significant level of cytolytic activity under treatment with lymphokine (LK) and lipopolysaccharide (LPS), in the absence of Cao2+ and that responses developed in Ca2+-free media were only 6-18% lower in comparison with the responses developed in the presence of Cao2+. The determination of 45calcium uptake in RAW-264 cells treated with LK and LPS showed that the rate of 45calcium uptake has displayed no increase during either the course of activation or in activated, highly cytolytic cells. Finally, three calcium channel blockers examined here: verapamil, diltiazem and flunarizine, with concentrations ranging from 1 X 10(-7) M - 2.5 X 10(-5) M, showed no inhibitory effect on the process of activation. Nifedipine, another calcium channel blocker, inhibited the development of cytolytic activity with concentrations ranging from 1 X 10(-6) M - 2.5 X 10(-5) M. It could be argued, however, that this inhibition was nonspecific, since this agent was 13 times more potent with regard to the calcium ionophore A23187-induced release of beta-glucuronidase, the function which is entirely dependent on Cao2+. Taken together, these results suggest that Cao2+ is not an absolute requirement for the process of tumoricidal activation of RAW-264 macrophages but it may play some supportive role in this process.     

Activation of Ca2+-dependent Cl- and K+ conductances in rat and mouse parotid acinar cells

Isolated acinar cells from rat and mouse parotid glands were studied with patch-clamp whole-cell current recordings. Acetylcholine (ACh) stimulation caused a transient inward current at a membrane potential of -70 mV, and a sustained outward current at a membrane potential of 0 mV, in quasi physiological Na+, K+ ion gradients, except the zero-Cl- ion gradient condition across the membrane. The reversal potential obtained from the ACh-evoked steady current was about -75 mV, in this ionic condition. When major Cl- ions of both the pipette and the bath solution were replaced, either by glutamate or by sulphate, only a large outward current was observed, at a membrane potential of -60 mV, in the presence of ACh. The addition of Ca2+-ionophore A23187 caused responses similar to those evoked by ACh. The reversal potential of A23187-induced current was close to the K+ equilibrium potential of -90 mV, in a Cl- -free condition. When K+-free NaCl solution was used in the pipette and the bath, A23187 caused only a large inward current, at a membrane potential of -60 mV. The reversal potential of A23187-evoked current was about -15 mV, in a symmetrical K+-free, NaCl condition. These results suggest that the ACh and A23187 activate Cl- as well as K+ conducting pathways via an increase in [Ca2+]i in the parotid acinar cells. The A23187-evoked large K+ current could not be explained solely by a rise in open probability of the channels.     

Evidence for two calcium-dependent steps and a sodium-dependent step in the mechanism of cell killing by calcium ions in the presence of ionophore A23187

Elevated intracellular Ca2+ appears to play an important role in the mechanism of cell killing in certain pathologic states such as ischemia. The authors have examined aspects of the biochemical mechanism of cell killing by elevated intracellular Ca2+ using as a model system cultured fibroblasts treated with ionophore A23187 in Ca2+-containing medium. Evidence has been obtained for two Ca2+-mediated steps and a Na+-mediated step in the cell killing process. The first Ca2+-mediated step occurs in low extracellular Ca2+ concentrations (1-100 microM) and exhibits a variety of characteristics in common with the arachidonic acid release response stimulated under the same conditions. These results are consistent with the arachidonic acid release response constituting or closely monitoring the initial injury process. The second Ca2+-mediated process is achieved at near physiologic extracellular Ca2+ concentrations in the absence of A23187. Killing of cells injured by the two Ca2+-dependent steps requires extracellular Na+ ions at half or more the physiologic concentration.     

Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin-3-dependent macrophage-like cell line.

An interleukin 3 (IL-3)-dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)-prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca(2+)-dependent and alkaline pH-optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5-10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane-associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL-3-dependent M phi-like cells.     

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.     

Histamine and human heart

Heart fragments obtained from human right atrium contain 1.5 +/- 0.2 micrograms of histamine per gram of wet tissue. Human heart spontaneously synthesizes significant amounts of PGI2, PGF2 alpha, PGE2 and TxA2. The Ca2+ ionophore A 23187 (0.5-3 micrograms/ml) dose-dependently induces histamine release and prostanoid production. Histamine (0.5-1 micrograms/kg/min) infusion in 10 normal donors produced an increase in heart rate and a significant depression of the ST segment. Selective H1 receptor stimulation in patients undergoing cardiac catheterization resulted in a decrease of the mean aortic pressure and of coronary vascular resistance.