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PubMed1991

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.

Alkaloids↗
PubMed1996

Effect of intracellular acidity and ionomycin on apoptosis in HL-60 cells.

The aim was to investigate in detail the influence of intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i) on apoptosis in HL-60 human promyelocytic leukaemia cells. The pHi was controlled by changing the pH of media as well as by interfering with the pHi regulatory mechanisms with 3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazincarboxamide (HMA; an inhibitor of Na+/H+ antiport), 4-diiosothiocyanatostilbene-2,2'disulfonic acid, (DIDS; an inhibitor of Na(+)-dependent HCO3-/Cl- exchange) and nigericin (a K+ ionophore). The [Ca2+]i was increased with ionomycin, a Ca2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of 3H from 3H-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and 3H release at different pHi, it was shown that apoptosis occurred in HL-60 cells when the pHi was lowered from normal pHi of 7.4 to about 7.2-6.7 with a peak increase at pHi 6.8-6.9. Addition of 4 microM ionomycin to RPMI 1640 medium, which contained 615 microM Ca2+, elevated the apoptosis in the cells. Such an increase in apoptosis by ionomycin in HL-60 cells appeared to result from both an increase in [Ca2+]i and from a decline in pHi. The results indicate that the acidic intratumour environment may greatly affect the response of neoplastic tissues to hyperthermia, radiation and chemotherapeutic drugs which cause apoptosis.

Apoptosis↗
PubMed2003

Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca2+ ionophore on mitochondria in intact cells.

We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca(2+) ionophores: (i). ferutinin, an electrogenic naturally occurring ionophore, and (ii). the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca(2+)] ([Ca(2+)](c)), mitochondrial [Ca(2+)] ([Ca(2+)](m)) and mitochondrial membrane potential (deltapsi(m)) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca(2+)] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca(2+)](c) and [Ca(2+)](m) increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca(2+)](c) in response to ferutinin, [Ca(2+)](m) remained elevated for prolonged periods after the recovery of [Ca(2+)](c) levels despite washout of the compound. Both groups of Ca(2+) ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca(2+) and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca(2+)/2Na(+)-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.

Animals↗
PubMed2002

Effects of the ionophores valinomycin, ionomycin and gramicidin A on the element compartmentation in cultured rat hepatocytes.

The element compartmentation in cultured rat hepatocytes was studied by electron probe X-ray microanalysis of freeze-dried cryosections after exposure of the cells to the ionophores valinomycin, ionomycin or gramicidin A. The most striking effect of these ionophores is the decrease of the intracellular potassium/sodium ratio from values of approximately 10 under control conditions to values below 1 after application of the ionophores. Changes of sodium, potassium and chloride are similar in cytoplasm and nucleus. However, elemental changes are delayed or impeded in mitochondria with respect to the surrounding cytoplasm. The water portion of cytoplasm and mitochondria slightly increases. Besides that, each ionophore has specific effects on the intracellular ion distribution. As compared to gramicidin A and ionomycin, valinomycin does not change the intracellular chloride content. Ionomycin induces calcium accumulation in mitochondria. The cytotoxic effects of the studied ionophores on the intracellular element distribution are more complex than supposed from their ion selective properties in membranes.

Animals↗
PubMed1988

Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped.

A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV. DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzofurans↗
PubMed1986

Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

Aminoquinolines↗
PubMed1994

Carbachol, but not norepinephrine, NMDA, ionomycin, ouabain, or phorbol myristate acetate, increases inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices.

Ionomycin, a Ca2+ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 mM Ca2+, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 microM ionomycin was equivalent to that seen with 1 mM carbachol. Phorbol 12-myristate 13-acetate or high K+ (30 mM) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 mM Ca2+. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 mM carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-trisphosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase C beta 1 in isolated rat brain membranes.

Animals↗
PubMed1994

Central demyelination induced in vivo by the calcium ionophore ionomycin.

The effects of injecting the calcium-selective ionophore, ionomycin, into myelinated tracts in the dorsal columns of adult rat spinal cords were examined electron microscopically. In vivo, ionomycin induced a primary vesicular demyelination, together with a variable degree of axonal degeneration, in a dose-dependent manner. The results are consistent with previous demonstrations that mature oligodendrocytes are more vulnerable to alterations in levels of [Ca2+]i than other glial cells. We speculate that demyelination induced by ionomycin in vivo occurs as a result of direct activation of endogenous Ca(2+)-dependent enzymes and/or as a consequence of oligodendrocyte injury mediated via astrocytes.

Animals↗
PubMed1988

Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate.

Gel-filtered human platelets that had been pre-labelled with [32P]Pi were stimulated with thrombin, ionomycin or the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The effect of the hexacationic aminoglycoside antibiotic, neomycin, on platelet physiological responses, such as aggregation and secretion, as well as changes in phosphoinositide metabolism was studied. Neomycin strongly inhibited thrombin-induced aggregation and secretion whereas the antibiotic had no effect on ionomycin- or TPA-induced platelet functions. The thrombin-induced enhancement of inositol phospholipid metabolism was strongly inhibited by the presence of neomycin whereas the TPA- or ionomycin-induced increase in inositol [32P]polyphospholipids remained unaffected. The inhibitory effect of some other aminoglycoside antibiotics was compared to that of neomycin and the data demonstrate that the inhibition of platelet secretion and phosphatidic acid production was dependent on the cationic charge of the antibiotic. It is suggested that neomycin inhibits signal transduction in platelets at a level prior to the inositol-phospholipid-specific phosphodiesterase.

Blood Platelets↗
PubMed1992

Intracellular pH on protein kinase C and ionomycin potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP production in rat pinealocytes.

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.

Ammonium Chloride↗
PubMed1983

The action of the ionophore ionomycin in guinea-pig intestinal smooth muscle.

The ionophore ionomycin produced concentration-dependent (5 X 10(-9) to 5 X 10(-6) M) contractions in guinea-pig ileal longitudinal smooth muscle. Responses were dependent on extracellular Ca2+, consistent with the known role of this Ca2+ source in supporting excitation-contraction coupling to a variety of stimulants in this tissue. Responses were insensitive to atropine (10(-6) M) but were dependent upon extracellular Na+ and were completely blocked by low concentrations of the Ca2+-channel antagonists nicardipine, YC-93 (5 X 10(-7) M), and D-600 (5 X 10(-6) M). The behaviour of ionomycin is very similar to that shown by A 23187 in this tissue. Ionomycin, like A 23187, can apparently activate D-600 sensitive Ca2+ channels in the guinea-pig intestinal smooth muscle rather than simply translocating Ca2+EXT.

Animals↗
PubMed1994

Effects of CGRP, forskolin, PMA, and ionomycin on pHi dependence of Na-H exchange in UMR-106 cells.

We examined the effects of calcitonin gene-related peptide (CGRP), forskolin, phorbol 12-myristate 13-acetate (PMA), and ionomycin on the intracellular pH (pHi) dependence of Na-H exchange in UMR-106 cells. In the nominal absence of CO2-HCO3-, each agent increased pHi, measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). From the rate of pHi recovery (dpHi/dt) from an acid load, and intracellular buffering power, we computed the pHi dependence of the total acid-extruding flux (JTotal). All four agents increased JTotal. From dpHi/dt data obtained in the presence of ethylisopropyl amiloride (EIPA, a blocker of Na-H exchange), we determined the EIPA-resistant component of JTotal (JEIPA/R). We estimated the Na-H exchange flux (JNa-H) as the difference JTotal-JEIPA/R-CGRP, forskolin, and PMA produced similar increases in the slope of the JNa-H vs. pHi-relationship. The net effect of these agents, as well as ionomycin, was to increase JNa-H over a broad pHi range. Ionomycin alkaline shifted the JEIPA/R vs. pHi relationship; the other agents had no effect. Our results indicate that CGRP increased JTotal by stimulating Na-H exchange, with little effect on EIPA-resistant processes. A signaling pathway involving only adenosine 3',5'-cyclic monophosphate, only protein kinase C, or only Ca2+ cannot account for the effects of CGRP on both pHi and pHi dependence of JNa-H. Thus, CGRP probably affects UMR-106 pHi physiology via more than one pathway.

Animals↗
PubMed1995

Effects of Ca2+ ionophore ionomycin on insulin-stimulated and basal glucose transport in muscle.

There is evidence that an increase in sarcoplasmic Ca2+ stimulates glucose transport in muscle. Recent studies have provided the apparently conflicting finding that a sustained increase in cytosolic Ca2+ has little effect on basal glucose transport but inhibits insulin-stimulated transport. This study was done to try to explain this discrepancy. Continuous exposure of rat epitrochlearis and soleus muscles to the Ca2+ ionophore ionomycin (2 microM) had no effect on basal 2-deoxyglucose (2-DG) transport but blunted, by approximately 40%, stimulation of 2-DG transport by insulin. Decreasing Ca2+ in the medium to a very low level prevented this inhibition. Ionomycin induced a small increase in adenosine 3',5'-cyclic monophosphate (cAMP); however, studies with the protein kinase A (PKA) inhibitor HA-1004 provided evidence that activation of PKA by cAMP does not mediate the inhibition of glucose transport. When muscles were allowed to recover in the absence of ionomycin for 15 min, basal 2-DG transport was significantly increased. Our results agree with previous studies showing that a sustained influx of Ca2+ into the cytoplasm can inhibit insulin-stimulated glucose transport. They further show that stimulation of glucose transport by Ca2+ is also inhibited. A recovery period that allows this inhibition to wear off unmasks the stimulation of glucose transport by an increase in sarcoplasmic Ca2+.

Animals↗
PubMed1996

Presynaptic inhibition of calcium-dependent and -independent release elicited with ionomycin, gadolinium, and alpha-latrotoxin in the hippocampus.

1. Presynaptic inhibition of synaptic transmission in the hippocampus was investigated by comparing the effects of several agonists on miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs). 2. The Ca2+ ionophore ionomycin increased the frequency of mEPSCs and mIPSCs but did not affect their amplitude. Ionomycin-induced release required extracellular Ca2+ and was prevented by pretreatment with botulinum neurotoxin serotype F, like evoked synaptic transmission. Unlike evoked transmission, however, this increase did not involve activation of voltage-dependent Ca2+ channels because it was insensitive to Cd2+. 3. Both the lanthanide gadolinium and alpha-latrotoxin produced increases in the frequency of mEPSCs and mIPSCs, but their actions were independent of extracellular Ca2+. 4. Adenosine, the gamma-aminobutyric acid-B (GABAB) receptor agonist baclofen, and a mu-opioid receptor agonist strongly reduced the frequency of synaptic currents triggered by all three secretagogues. 5. We conclude that activation of these presynaptic receptors can reduce high frequencies of vesicular glutamate and GABA release by directly impairing transmitter exocytosis. Presynaptic inhibition of gadolinium- and alpha-latrotoxin-induced release indicates that this impairment occurs without changes in intraterminal Ca2+ homeostasis and when vesicle fusion is rendered Ca2+ independent, respectively. 6. The inhibition of ionomycin-induced release provides additional evidence for a direct, neurotransmitter receptor-mediated modulation of the proteins underlying vesicular docking or fusion as an important component of presynaptic inhibition of evoked synaptic transmission.

Adenosine↗
PubMed1982

Some characteristics of histamine secretion from mast cells treated with ionomycin.

The ionophorous antibiotic ionomycin released histamine from rat peritoneal mast cells in both the presence and absence of added calcium ions. The response under the latter conditions was potentiated by brief pretreatment of the cells with chelating agents. The interaction between the ionophore and exogenous calcium was complex. Supramaximal concentrations of calcium potentiated the release induced by low levels of ionomycin but markedly inhibited the secretion evoked by larger amounts of the compound. Dispersed mesenteric mast cells of the rat and guinea pig also responded to ionomycin but were less reactive than the peritoneal cells.

Animals↗
PubMed1988

Calcitonin gene-related peptide and calcitonin secretion from a human medullary thyroid carcinoma cell line: effects of ionomycin, phorbol ester and forskolin.

Calcitonin gene-related peptide (CGRP) and calcitonin are secreted together from medullary thyroid carcinoma (MTC) cells. Interactions of cytosolic free calcium concentration (Cai2+) and the protein kinase C and A pathways on the secretion of immunoreactive CGRP and calcitonin have been investigated in a human MTC cell line. Ionomycin (10 mumol/l) raised the concentration of Cai2+, concomitant with a transient stimulation of the secretion of CGRP and calcitonin. 12-O-tetradecanoylphorbol-13-acetate (TPA; 16 nmol/l) did not affect the concentration of Cai2+, but caused a gradual rise of the secretion of CGRP and calcitonin. Combined addition of 10 mumol ionomycin/l and 16 nmol TPA/l resulted in additive stimulation of CGRP and calcitonin secretory responses. Forskolin (10 mumol/l) alone did not change the concentration of Cai2+, marginally enhanced (P greater than 0.1) the release of CGRP and calcitonin and increased by 23-fold the cellular levels of cyclic AMP (cAMP). Ionomycin and TPA did not change cellular cAMP. Forskolin synergistically enhanced (P less than 0.01) the ionomycin-induced early phase as well as the TPA-induced late phase of the CGRP and calcitonin secretory responses. In conclusion, increased concentrations of Cai2+ together with protein kinase C and A activation mediate the secretion of CGRP and calcitonin in MTC cells.

Calcitonin↗
PubMed1992

Phorbol esters inhibit ionomycin-induced hydrolysis of phosphoinositides and phosphatidylcholine in bovine corneal epithelial cells.

The effects of phorbol esters on phospholipase C (PLC) activity towards phosphoinositides and phosphatidylcholine (PC) in bovine corneal epithelial cells were examined. The cells were labeled with 32Pi, myo[3H]inositol or methyl[14C]choline, and PLC stimulated by incubation of the cells with Ca2+ ionophore, ionomycin. The PLC activity was assessed by monitoring the loss of radioactivity from the labeled phospholipids or the accumulation of their radioactive metabolites. The data from this study can be summarized as follows: Addition of 20 microM ionomycin to the prelabeled cells resulted in a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and somewhat slower hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) with concomitant several-fold increase in phosphatidic acid (PA). The effects of the ionophore were time- and dose-dependent. Incubation of the cells with phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused increased radioactivity in PC and PA, whereas the radioactivity in PI and PIP2 remained unchanged. The effects of PDBu were inhibited by staurosporine and H-7, and inactive derivatives of phorbol esters failed to exert any effect on phospholipid metabolism. Pretreatment of the corneal epithelial cells with PDBu or PMA abolished the ionomycin-induced hydrolysis of phosphoinositides and PC. The data suggest that activation of protein kinase C by phorbol esters in corneal epithelial cells results in inhibition of PLC activity towards phosphoinositides and PC through a mechanism probably involving phosphorylation of the enzyme.

Animals↗
PubMed1985

Dual actions of phorbol esters on cytosolic free Ca2+ concentrations and reconstitution with ionomycin of acute thyrotropin-releasing hormone responses.

We have used phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), to study the actions of protein kinase C (a TPA receptor) on cytosolic free Ca2+ concentrations [( Ca2+]i) and hormone secretion in rat pituitary cells (GH cells), and to elucidate the role of diacylglycerol (a protein kinase C activator) in thyrotropin-releasing hormone (TRH) action. TPA had a dual action on [Ca2+]i, inducing a stimulatory phase from 300 (basal) to 420 nM, which was interrupted in 30-60 s by an inhibitory phase which transiently lowered [Ca2+]i to 240 nM and rose in 3-10 min to yield the stimulatory phase. TPA-mediated changes in [Ca2+]i were induced by other phorbol esters and mezerein but not by phorbol or activators of kinases different from protein kinase C. Both phases of TPA action on [Ca2+]i were abolished by 5-min pretreatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (1.33 mM) or Ca2+ channel antagonists (verapamil or nifedipine). TPA also enhanced the rate of sustained hormone secretion without inducing a burst of hormone release (unlike TRH). Also, stimulation of secretion by TPA was not inhibited by Ca2+ channel antagonists and was resistant (10%) to EGTA. Simultaneous addition of TPA with the ionophore ionomycin (100 nM) reconstituted a TRH-like spike, nadir and plateau of [Ca2+]i. Ionomycin generated the spike in [Ca2+]i by releasing TRH-sensitive Ca2+ stores, while TPA induced the nadir (inhibitory phase), and a nifedipine/verapamil-sensitive plateau of [Ca2+]i (stimulatory phase). Concurrent (but not separate) addition of ionomycin and TPA also reconstituted a TRH-like burst of hormone secretion. These and previous results indicate that activation of protein kinase C by TPA or diacylglycerol (which is elevated by TRH) and a simultaneous spike in [Ca2+]i are required for burst secretion. Diacylglycerol may also mediate the TRH-induced nadir and plateau of [Ca2+]i; the latter process contributes to Ca2+-dependent stimulation of steady secretion by TRH.

Animals↗