Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.     

ATP-induced calcium mobilization in human neutrophils

Extracellular ATP at 10 microM increased the concentration of cytoplasmic free Ca2+ ( [Ca2+]i) 3-fold in human neutrophils. The [Ca2+]i was measured by fura-2 fluorescence. The effect was rapid but transient: [Ca2+]i reached a maximum within 10 s and then returned to its resting value after 2-3 min. The rise in [Ca2+]i elicited by ATP was unaffected by the removal of extracellular Ca2+, indicating that the primary source of Ca2+ is from intracellular stores. In contrast to ATP, neither ADP nor AMP, at concentrations as high as 100 microM, caused any detectable changes in [Ca2+]i. Among other nucleotide triphosphates tested, UTP was as effective as ATP in causing a transient rise in [Ca2+]i, and prevented a subsequent response to ATP. Similarly, ATP prevented a subsequent response to UTP but the second response could be obtained when the initially added ATP was removed by the addition of hexokinase. Phorbol myristate acetate, the activator of Ca2+, phospholipid-dependent protein kinase, completely inhibited the ATP-induced increases in [Ca2+]i without affecting the basal [Ca2+]i level. The results suggest that extracellular ATP stimulates human neutrophils by causing the release of calcium from intracellular storage pools by mechanisms which can be inhibited by phorbol myristate acetate.     

Effects of bradykinin on prostaglandin synthesis and cytosolic calcium in rabbit subcultured renal cortical smooth muscle cells

Smooth muscle cells were cultured from an arteriole-rich fraction of the rabbit renal cortex and characterized by their ultrastructural and immunohistochemical features, their high content in creatine kinase (60-times that of the initial preparation) and their ability to synthesize renin. Cells, studied between passages 2 and 5, produced mainly PGE2 and, to a lesser extent, PGF2 alpha. Bradykinin (BK) (0.1 nM-1 microM) induced a concentration-dependent increase in PGE2 (28-40-times basal value at 1 microM after a 5 min incubation period) and stimulated also the free cytosolic calcium concentration [( Ca2+]i) with a 2-fold maximal rise to its basal value. Both effects, inhibited by the anti-B2 receptor [Thi5.8D-Phe7] BK, were not reproduced by DesArg9 BK. A decrease in the extracellular calcium concentration and incubation in the presence of a calcium-channel blocker (lanthanum chloride) inhibited the BK-dependent rise of [Ca2+]i but not that of PGE2. Preincubation with phorbol myristate acetate increased basal and BK-induced PGE2 synthesis but prevented the effect of BK on [Ca2+]i. These results demonstrate the ability of BK to increase [Ca2+]i and PGE2 production in cultured vascular cells from the rabbit renal cortex and suggest that kinins might act on the cortical microcirculation via their direct effects on arteriolar smooth muscle cells.     

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O2-) release, membrane depolarization and an increase in cytoplasmic free Ca2+, [Ca2+]i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca2+ ionophore ionomycin was used as stimulus, an increase in [Ca2+]i clearly preceded the onset of membrane depolarization, which was followed by O2- release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutinin was used as stimulus, no demonstrable lag was seen in any of the responses. O2- release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells with a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca2+]i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O2- release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca2+]i was not affected. The dose-response curves for triggering of O2- release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca2+]i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O2- release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca2+]i, but not by augmenting an increase in [Ca2+]i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.     

Two transduction sequences are necessary for neutrophil activation by receptor agonists

The effects of 17-hydroxywortmannin (HWT), a powerful inhibitor of the respiratory burst associated with phagocytosis (Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P. H., and Payne, T. G. (1987) Exp. Cell Res. 169, 408-418), were studied in human neutrophils stimulated with chemotactic agonists or phorbol myristate acetate. At nanomolar concentrations HWT inhibited superoxide production and the release of granule contents induced by N-formyl-Met-Leu-Phe, C5a, platelet-activating factor, and leukotriene B4, but not by phorbol myristate acetate, indicating that it interferes with receptor-mediated activation of the neutrophils, without directly affecting protein kinase C (Ca2+/phospholipid-dependent enzyme), the NADPH-oxidase, or the process of granule exocytosis. Moreover, HWT did not influence agonist-induced [Ca2+]i changes, indicating that it does not interfere with the function of agonist receptors, G-proteins or the phosphatidylinositol-specific phospholipase C. By studying the effect of HWT on the respiratory burst elicited in normal and Ca2+-depleted cells by combined stimulation with N-formyl-Met-Leu-Phe and phorbol myristate acetate, evidence was obtained that two transduction sequences, both of which are G-protein-dependent, are necessary for the induction of the response by receptor agonists. One sequence is Ca2+-dependent, HWT-insensitive, and leads to activation of protein kinase C, the other is Ca2+-independent and HWT-sensitive. Ca2+ depletion, which blocks the first, and HWT, which blocks the second, can be used to show that both processes must be functional for the transduction of agonist signals into a respiratory burst response.     

Phorbol ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca2+ concentration

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.     

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.     

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase- arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.     

Calcium ions are required for the intracellular routing of insulin and its receptor.

We have studied the role of the cytosolic-free calcium concentration ([Ca2+]i) on the early and later internalization steps of insulin and its receptor. As before, we find that the rate of 125I-insulin internalization in HL60 cells remains normal when [Ca2+]i is lowered 10 times below normal resting level by the use of an intracellular Ca2+ chelator. By contrast, the subsequent intracellular steps, i.e. insulin receptor recycling and insulin degradation, are inhibited in calcium-depleted cells. Under low [Ca2+]i conditions, the association of 125I-insulin with late endosomes and lysosomes is also reduced. This suggests that calcium ions are required for fusion processes occurring at the endosomal or postendosomal stage of internalization. Thus, by regulating insulin receptor recycling and by controlling insulin degradation, Ca2+ ions play a key role in the regulation of insulin action.     

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (<2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t_1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 μM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.     

Extracellular ATP itself elicits superoxide generation in guinea pig peritoneal macrophages.

Extracellular ATP itself elicited the generation of superoxide (O2-) in guinea pig peritoneal macrophages associated with an increase in cytosolic calcium ([Ca2+]i). The ATP-induced O2- generation was completely inhibited by pretreatment with pertussis toxin (PT) accompanied by the suppression of [Ca2+]i mobilization. Pre-exposure to a small amount of phorbol myristate acetate (PMA) primed the ATP-induced generation of O2- without a change of [Ca2+]i. The results suggest that ATP-induced O2- generation is mediated by [Ca2+]i mobilization and by PT-sensitive G protein.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

Defective signal transduction in CD4-CD8- T cells of lpr mice

Mice homozygous for the lpr gene develop a lymphoproliferative disorder due to expansion of a subset of CD4-CD8- T cells. Triggering of the T-cell receptor in these lpr T cells does not lead to translocation of protein kinase C or phosphorylation of CD3, interleukin-2 production, or proliferation, whereas a combination of phorbol ester and calcium ionophore does. Stimulation with concanavalin A or anti-CD3 induces phosphoinositide hydrolysis. The rise in inositol bisphosphate, inositol triphosphate, and inositol tetrakisphosphate, identified by HPLC, is similar in +/+ and lpr T cells. The concentration of cytoplasmic free calcium ([Ca2+]i), however, under basal and stimulated conditions is significantly lower in lpr T cells. The lower basal [Ca2+]i may explain why induction of proliferation with phorbol ester and calcium ionophore requires a higher concentration of ionophore in these cells than in normal T cells. The lower [Ca2+]i obtained on stimulation may contribute to the activation defect of CD4-CD8- lpr T cells.     

Intracellular Ca(2+) regulates the cellular iron uptake in K562 cells

Fluorescence quenching was used to study the kinetics of the transferrin receptor (TfR)-mediated iron uptake in the calcein-loaded K562 cells. It was found that elevation of intracellular free Ca(2+) ([Ca(2+)](i)) by thapsigargin (TG) speeds up the initial rate of iron uptake and increases the overall capacity of the cells in taking up iron. Depletion of intracellular Ca(2+) or complete chelation of extracellular Ca(2+) results in complete inhibition of the iron uptake in cells. To gain insight into molecular mechanism, IANBD-labeled transferrin (Tf) and microscopic fluorescence imaging were used to observe the endocytosis and recycling of the Tf-TfR complex in single live cells. The study showed that the preincubation of cells with TG or phorbol myristate acetate (PMA), the direct activator of protein kinase C (PKC), accelerated the endocytosis and recycling of the complex in a dose-dependent manner. W-7, the calmodulin antagonist, and GF109203X, a selected cell-permeant inhibitor of PKC, can reverse the acceleration. Analysis of actin polymerization in controlled, [Ca(2+)](i)-elevated and W-7-treated cells revealed that the actin polymerization is enhanced as [Ca(2+)](i) is raised, but reduced by W-7. The results suggest that the regulation of actin polymerization by intracellular Ca(2+) may play a central role in Ca(2+)-dependent iron uptake.     

Calcium concentration and amylase secretion in guinea pig pancreatic acini: interactions between carbachol, cholecystokinin octapeptide and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on amylase secretion and cytoplasmic free calcium concentration ([Ca2+]i) were investigated in dispersed guinea pig pancreatic acini. Carbachol evoked dose-dependent increases in amylase secretion and [Ca2+]i with half-maximal responses at 2.5 and 5 microM, respectively. Carbachol-induced calcium transients could be blocked by atropine. In the presence of a maximal effective dose of carbachol, cholecystokinin octapeptide caused no further increase in [Ca2+]i, suggesting that both agonists act on the same pool of trigger calcium. TPA (10(-9)-10(-6) M) stimulated amylase secretion with no change in [Ca2+]i. Maximum amylase secretion occurred at 0.5 microM TPA. Preincubation of acini in the presence of TPA resulted in a time- and dose-dependent inhibition (IC50 = 30 nM) of the carbachol-induced rise in [Ca2+]i, the maximal effect being observed within 3 min. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate was ineffective in inhibiting the carbachol-stimulated rise in [Ca2+]i. These findings suggest that, in addition to stimulating amylase secretion, probably through protein kinase C, TPA may also exert a negative feedback control over secretagogue-induced calcium transients.