Calcium and cyclic nucleotide involvement in exocrine pancreatic enzyme secretion studied with the ionophore A-23187.

The ionophore, A-23187, was an effective pancreatic secretagogue. The response to A-23187 was Ca²⁺-dependent; Mg²⁺ reduced the secretory response to the ionophore. A-23187-stimulated enzyme release was potentiated by dibutyryl cyclic AMP; in the presence of carbachol, output of pancreatic protein paralleled the response to A-23187 alone. The time-course for secretion with A-23187 was similar to that observed with carbachol. The ionophore did not affect basal cyclic AMP levels but did stimulate a rapid Ca²⁺-dependent production of pancreatic cyclic GMP which preceded the onset of the secretory response. A-23187 did not significantly alter basal or carbachol-stimulated ⁴⁵Ca efflux from isotope preloaded glands; yet in Ca²⁺-lowered media, it inhibited (reversed) the secretory response to carbachol, an effect which may have been due to an outward transport by the ionophore of cholinergic-mobilized intracellular Ca²⁺. Like carbachol, A-23187, inhibits the incorporation of amino acid into new protein, the effect being partially dependent on extracellular Ca²⁺. The data suggest that the pancreatic cholinergic receptor acts as a Ca²⁺-ionophore and that extracellular Ca²⁺ is utilized in the synthesis of cyclic GMP.     

Effect of protein kinase C activation and Ca2+ mobilization on hexose transport in Swiss 3T3 cells

Down-modulation of Ca²⁺-activated, phospholipid-dependent protein kinase (protein binase C), which was accomplished by pretreatment with phorbol-12,13-dibutyrate for 24 h, resulted in the loss of a phorbol ester-induced stimulation of hexose transport activity in Swiss 3T3 cells. In these cells, however, platelet-derived growth factor as well as Ca²⁺ ionophore A23187 were still able to induce stimulation of hexose transport activity accompanied by the elevation of intracellular free Ca²⁺ concentration. Since chelation of extracellular Ca²⁺ inhibited this stimulation, inflow of extracellular Ca²⁺ into cytoplasm seemed to be esential for the stimulatory effect of platelet-derived growth factor and A23187 on hexose transport. Epidermal growth factor and insulin also stimulated hexose transport activity regardless of the absence of protein kinase C. However, in the case of epidermal growth factor, intracellular Ca²⁺, but not extracellular Ca²⁺, was found to be necessary for the stimulation. On the other hand, insulin stimulated the hexose transport independent of both intra- and extracellular Ca²⁺.     

Requirements for different Ca2+ pools in the activation of rabbit platelets: I. Release reaction and protein phosphorylation

We examined the role of Ca²⁺, both extracellular and intracellular in origin, in the release reaction and protein phosphorylation in rabbit platelets stimulated with platelet activating factor (acetylglyceryl ether phosphorylcholine), thrombin, or ionophore A23187. In the presence of extracellular Ca²⁺, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca²⁺ transport, blocked platelet activating factor-initiated serotonin release at a half-maximal inhibitor concentration of 40 μM, compared to 350 μM for thrombin-induced release and greater than 500 μM, for A23187-induced release. Platelet activating factor-induced phosphorylation of two platelet proteins of M_r=41 000 (P7P) and 20 000 (P9P) was inhibited by TMB-8, an effect which was additive to that caused by removing extracellular Ca²⁺. TMB-8 demonstrated only minor to non-existant inhibitory effects on phosphorylation in thrombin- or A23187-stimulated platelets. In contrast to P9P phosphorylation, phosphorylation of P7P caused by platelet activating factor was more dependent on a TMB-8 sensitive step than on the availability of extracellular Ca²⁺. Experiments with buffers containing fixed concentrations of free Ca²⁺ revealed that both processes (release and phosphorylation), when stimulated by platelet activating factor and thrombin, had the same threshold requirement (1–3 μM) for extracellular free Ca²⁺. These studies provide evidence that stimulation of rabbit platelets by platelet activating factor is more dependent on a TMB-8-sensitive intracellular Ca²⁺ source than is stimulation caused by thrombin. Furthermore, our data indicate that activation of different intracellular processes involved in platelet secretion (such as P7P and P9P phosphorylation) may require Ca²⁺ from different pools.     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Participation of the microtubular-microfilamentous system on intracellular Ca transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[¹⁴C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca²⁺. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca²⁺, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca²⁺ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca²⁺. In addition, an increase in cytosolic Ca²⁺ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca²⁺ increase induced by A23187 in a Ca²⁺-free medium. On the other hand, in a Ca²⁺-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca²⁺ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca²⁺ uptake. These observations indicate that intracellular Ca²⁺ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca²⁺ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Translational suppression by Ca2+ ionophores: Reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca²⁺ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50–300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca²⁺ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca²⁺ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH₃ pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca²⁺ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca²⁺. Ionomycin produced more Ca²⁺ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca²⁺ by ionomycin were readily reversed in GH₃ cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca²⁺ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca²⁺ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca²⁺-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca²⁺ accumulation. Increased Ca²⁺ contents in response to Ca²⁺ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.Abbreviations BSA bovine serum albumin - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - PKR double-stranded RNA-regulated protein kinase - ER endoplasmic reticulum - eIF eukaryotic initiation factor     

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca²⁺ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca²⁺ ions, although the origin of Ca²⁺ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca²⁺ ions whereas those by the other three chemicals utilized intracellular Ca²⁺ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).     

Secretion of granule enzymes from alveolar macrophages : Regulation by intracellular Ca-buffering capacity

Rabbit alveolar macrophages exposed to the ionophore A23187 in the presence of extracellular Ca²⁺ take up about 12 nmoles of Ca²⁺/1 × 10⁶ cells. This uptake induces a slight, but significant, extracellular release of granule enzymes, β-glucuronidase and lysozyme, but not of the cytoplasmic marker, lactate dehydrogenase. If either EGTA is added to the medium or Mg²⁺ replaces Ca²⁺ no stimulation of secretion is observed. If the energy supply is decreased by treating the macrophages with mitochondrial inhibitors such as oligomycin, cyanide, or rotenone and antimycin, Ca²⁺-dependent secretion is potentiated several fold. Selective release of granule enzymes from macrophages exposed to A23187 and Ca²⁺ is also stimulated by cytochalasin B (CB).     

Regulation of endothelial cell gap formation and barrier dysfunction: Role of myosin light chain phosphorylation

Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (<15 sec) increase in [Ca²⁺]_i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8-fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca²⁺ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4-fold increase). Down-regulation or inhibition of PKC activity attenuated thrombin-induced MLC phosphorylation (～40% inhibition) and both thrombin- and PMA-induced albumin clearance (～50% inhibition). Agents which augmented [cAMP]_i partially blocked thrombin-induced MLC phosphorylation (～50%) and completely inhibited both thrombin- and PMA-induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML-7 or KT 5926) or Ca²⁺/calmodulin antagonism (with either trifluoperazine or W-7) attenuated thrombin-induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK-dependent and independent mechanisms and are significantly modulated by both PKC and cAMP-dependent protein kinase A activities. © 1995 Wiley-Liss, Inc.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Pancreatic acinar cells: use of Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling

The Ca⁺⁺ ionophore A23187 had no effect on the release of amylase by mouse pancreas fragments in the absence of Ca⁺⁺ but when Ca⁺⁺ was re-added to the medium amylase release was observed in a pattern which mimicked that produced by normal stimulants. Uptake of ⁴⁵Ca⁺⁺ by pancreatic fragments was increased by A23187. Tetracaine and dinitrophenol at concentrations which block cholinergic stimulated enzyme release blocked ionophore induced release whereas atropine did not. None of the inhibitors studied affected the ionophore induced Ca⁺⁺ uptake.     

Effects of Ca ionophore A23187 and Ca deficiency on pancreatic phospholipids and amylase release in vitro

The Ca²⁺ ionophore A23187 elicits a transient increase in pancreatic amylase release in vitro, and this is accompanied by a transient decrease in phosphatidyl inositol concentration. Effects of ionophore A23187 and carbachol on amylase release and phosphatidylinositol breakdown are dependent on medium Ca²⁺. These results suggest that major secretagogue-induced, pancreatic phospholipid changes follow, rather than precede, changes in Ca²⁺ in the pancreas.     

Intracellular uptake and α-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells

Summary Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca²⁺ and Mg²⁺ (t1/2=1 min) and much slower in the presence of Ca²⁺ or Mg²⁺ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca²⁺ and Mg²⁺, and by equivalent interaction of ionophore with membranes of whole and sonicated cells.A23187 (10 m) increased amylase release 200% in the presence of extracellular Ca²⁺ and Mg²⁺. In the absence of Ca²⁺ (but in the presence of Mg²⁺) A23187 did not increase amylase release. A23187 (10 m) also produced Ca²⁺-dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 m) increased amylase release 140% without increasing LDH release or permeability to trypan blue.     

Implication of Ca2+-Dependent Protein Tyrosine Phosphorylation in Carbachol-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract: The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca²⁺. PLD activity was assessed by measuring the formation of [³H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65–70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44^MAPK, and 42^MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca²⁺-free buffer, CCh-induced [³H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca²⁺ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca²⁺. Extracellular Ca²⁺ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca²⁺-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca²⁺-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca²⁺ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)₃, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca²⁺- and calmodulin-dependent and that HNMPA-(AM)₃-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.     

Influence of calcium on phosphorylation of a synaptosomal protein

Synaptosomal proteins isolated from rat cerebral cortex were phosphorylated endogeneously in the presence of [γ-³²P]ATP. The phosphorylated proteins were found to be membrane bound by differential and density gradient centrifugation. In contrast to the phosphorylation of all synaptosomal proteins, phosphorylation of one protein (C), 41 000–43 000 daltons, was inhibited by Mg²⁺ and stimulated by Ca²⁺. In addition, the ionophores X537A and A23187, as well as papaverine, selectively enhanced phosphorylation of protein C without affecting phosphorylation of the other proteins. Cyclic AMP did not influence the phosphorylation of protein C but markedly affected the phosphorylation of other synaptosomal proteins. It appears that the phosphorylation of protein C is stimulated by agents which trigger the release of neurotransmitters (Ca²⁺, X537A, A23187 and papaverine), and is inhibited by Mg²⁺, which inhibits release. It is proposed that the phosphorylation of protein C is related to membranal events underlying the release of neurotransmitters.     

红细胞在钙离子和离子载体A23187作用下的流变特性研究

用新激光衍射法研究了钙离子及离子载体A23187对红细胞流变特性的影响.用不同浓度的钙离子及离子载体A23187分别处理红细胞后,测量其取向指数和小变形指数.结果表明离子载体A23187较细胞外钙离子浓度对红细胞流变特性的影响更大.而且,最大取向指数和最大小变形指数随着钙离子及离子载体A23187浓度的增加而降低.离子载体A23187浓度增加导致红细胞变形能力明显降低.     

Phospholipid-sensitive calcium-dependent protein kinase and its endogenous substrate proteins in rat pancreatic acinar cells

Phospholipid-sensitive Ca²⁺-dependent protein kinase and its endogenous substrate proteins were examined in acinar cells from rat pancreas. The enzyme was clearly demonstrable by DEAE-cellulose chromatography of acinar cell extract. At least four endogenous substrate proteins (M_r = 38K, 30K, 22K and 15K) for this Ca²⁺-activated kinase were found in the acinar cell extract. These substrate proteins were maximally phosphorylated in the combined presence of Ca²⁺ and phosphatidylserine. Calmodulin was partially effective as a cofactor for phosphorylation of the 38K substrate protein, but ineffective for the other three. A slight Ca²⁺/phospholipid-dependent phosphorylation of 38K and 30K proteins, but not of 22K and 15K proteins was seen in extract of isolated pancreatic islets. The K_a for Ca²⁺ for phosphorylation of the endogenous acinar cell proteins was decreased more than ten-fold in the combined presence of phosphatidylserine and unsaturated diacylglycerol. The presence of this Ca²⁺/phospholipid-dependent protein kinase/ protein phosphorylation system provides a potential mechanism of action for Ca²⁺ as a regulator of exocrine pancreatic function.     

The peripheral Ca2+ pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.