The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured by aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68-71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca2+]c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca2+]c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca2+]c responses to 250 microM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca2+]c. Glucagon-induced elevation of [Ca2+]c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca2+]c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca2+]c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca2+]c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 microM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca2+]c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.     

The effects of calcium channel blockers on isoproterenol induced cyclic adenosine 3',5'-monophosphate generation in intact human lymphocytes

We have investigated the effects of clinically available calcium channel blockers (nifedipine, verapamil and diltiazem) on isoproterenol stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) generation in intact human lymphocytes. After preincubation of various calcium antagonists with intact lymphocytes at 37 degrees C for 15 minutes, 10 microM nifedipine or verapamil partially inhibited isoproterenol induced cyclic AMP generation in the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) while they alone had no effect on cyclic AMP level at a concentration of up to 100 microM. In contrast, 10 nM-1.0 microM nifedipine, verapamil or diltiazem potentiated cyclic AMP generation induced by isoproterenol in a dose dependent manner. Similar results were observed in the time course studies of cyclic AMP generation. These effects are somewhat similar to the effect of phenothiazine, a calmodulin inhibitor, which, at 10 microM (close to IC50), also potentiated the effects of isoproterenol. In contrast, lanthanum chloride (LaCl3), an extracellular inorganic calcium antagonist, at 1.0 mM, inhibited isoproterenol induced cyclic AMP generation. The biochemical mechanisms underlying these potentiating effects are unknown. It may be partly related to the effect of calcium channel blockers (at least for nifedipine) on preventing beta 2 adrenergic receptor desensitization. This may provide a potential mechanism for the synergistic effect between calcium channel blockers and beta 2 adrenoceptor agonists on bronchial dilatation.     

Discriminatory Effects of Forskolin and EGTA on the Indirect Cyclic AMP Responses to Histamine, Noradrenaline, 5-Hydroxytryptamine, and Glutamate in Guinea-Pig Cerebral Cortical Slices

The effects of forskolin (1 microM) and EGTA (5 mM) on indirect cyclic AMP responses in slices of guinea-pig cerebral cortex were examined. Forskolin had little effect on the direct 2-chloroadenosine-stimulated cyclic AMP response. However, it completely abolished the glutamate-induced augmentation of this response. In contrast, forskolin had very little effect on the indirect cyclic AMP responses to noradrenaline, 5-hydroxytryptamine, and histamine. Conversely, rapid removal of extracellular calcium with EGTA 2 min before addition of the indirectly acting agent markedly reduced the augmentation responses produced by these latter agonists, but had little effect on the glutamate augmentation. When EGTA was added once a steady level of cyclic AMP had been achieved with the indirect agents, it was without effect on any of the responses. Thus, calcium appears to have a role in the early, but not the later, stages of the noradrenaline, 5-hydroxytryptamine, and histamine responses. A role for protein kinase C in the glutamate augmentation response was suggested, because forskolin inhibited the augmentation of the 2-chloroadenosine response produced by phorbol esters (which mimic the actions of diacylglycerol in activating protein kinase C). We conclude that there is more than one mechanism by which the augmentation of cyclic AMP responses can occur.     

The role of calcium ion in epinephrine activation of lipolysis.

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.     

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation.

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ability of calcium to change cyclic AMP from a stimulator to an inhibitor to thymic lymphoblast proliferation

Calcium is a major regulator of thymic lymphoblast proliferation in vivo and in vitro. The proliferative activity of the lymphoblasts in thymic lymphocyte (thymocyte) populations in vitro is both constant and low in the presence of calcium concentrations between 0 and 1.0 mM, but higher concentrations increase proliferation by an endogenous cyclic AMP-mediated promotion of the initiation of DNA synthesis. Lower concentrations (10^?7 to 10^?5 M) of exogenous cyclic AMP (but not 5′-AMP) stimulate lymphoblast proliferation in a low-calcium (0.5 mM) medium, but higher concentrations do not. However, all exogenous cyclic AMP concentrations between 10^?7 and 10^?3 M (but again not 5′-AMP) block the stimulation of lymphoblast proliferation in a high-calcium (1.5 mM) medium. Exogenous cyclic AMP does not prevent calcium from “activating” lymphoblasts, but it reversibly blocks the reaction responsible for the initiation of DNA synthesis in these stimulated cells. Finally, cyclic AMP's inhibitory action, in contrast to its stimulatory action in low-calcium medium, is not specific for the cyclic nucleotide since a low, non-mitogenic concentration of cyclic GMP also prevents calcium from stimulating DNA synthesis and cell proliferation.     

Combined effects of calcium and dibutyryl cyclic AMP on germinal vesicle breakdown in the mouse oocyte

Mouse oocytes were cultured in the presence of dibutyryl cyclic AMP (dbcAMP) and various agents that affect cytoplasmic calcium concentrations. Treatment that inhibited calcium uptake potentiated the inhibitory effect of dbcAMP and treatments which stimulated cellular calcium uptake overcame the effect of dbcAMP. Elevated extracellular calcium (greater than 10 mM) significantly decreased the inhibitory effect of concentrations of dbcAMP up to 150 microM when compared to control levels of calcium (1.7 mM). In addition, the calcium ionophore A23187 (greater than 1 microM) significantly overcame the effect of dbcAMP in media that contained 1.7 or 20 mM calcium. In the presence of 41 microM-dbcAMP the calcium antagonist verapamil increased (in a dose-dependent fashion) the percentage of oocytes blocked at the germinal vesicle stage, from 21% with 10 microM-verapamil to 99% with 200 microM. A similar dose-dependent, reversible potentiation of the effect of dbcAMP was found with tetracaine, which also lowers cytoplasmic calcium concentrations. These results suggest that a minimum level of cytoplasm calcium is required for the initiation of germinal vesicle breakdown and that the action of dbcAMP is mediated by its effect upon this calcium.     

Parathyroid hormone causes a transient rise in intracellular ionized calcium in vascular smooth muscle cells

The effect of parathyroid hormone on intracellular calcium concentration in vascular smooth muscle cells in culture was studied. Human PTH 1-34 (hPTH (1-34)) caused a transient rise in intracellular calcium in a dose-dependent manner at physiological concentrations. The effect of PTH was mimicked by dibutyryl cyclic AMP and inhibited by a PTH receptor antagonist. The effect of PTH was increased in parallel with extracellular calcium concentration and a sustained response was observed when extracellular calcium was 2 mM or higher. The PTH action was blocked by nisoldipin, a calcium antagonist, but not by ouabain, a Na, K-ATPase inhibitor. These data indicate that PTH increases intracellular calcium through its receptor via opening calcium channels. A possible role of this effect in the regulation of vascular tone is also discussed.     

Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells.

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.     

The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets.

A protein phosphokinase (ATP: protein phosphotransferase EC 2.7.1.37) which is stimulated by 3',5'-cyclic adenosine monophosphate (cyclic AMP) has been partially purified from both the cytoplasmic and membrane fractions of human platelets. The kinetics of both enzymes preparations are similar in respect to cyclic AMP, ATP, ADP and AMP. 5-10-minus 7 M cyclic AMP stimulated both preparations by approximately 100%. Both ADP and AMP at a concentration of 5-10-minus 5 M inhibited protein phosphokinase activity of the soluble and membrane preparation by between 50% and 70%. The response of the two enzyme preparations to calcium differed. 10 mM Ca-2+ inhibited soluble protein phosphokinase activity approximately 80% both in the presence and absence of 5-10 minus 7 M cyclic AMP whereas the same concentrations of Ca-2+ inhibited the membrane-bound enzyme by approximately 60% in the presence of 5-10-minus 7 M cyclic AMP and 40% in the absence of cyclic AMP. This observation may be of importance in understanding the mechanism of platelet aggregation.     

The role of calcium and cyclic adenosine 3',5'-monophosphate in the regulation of glycogen metabolism in phagocytozing human polymorphonuclear leukocytes.

Incubation of human polymorphonuclear leukocytes in a glucose-free Krebs-Ringer bicarbonate buffer for 2 h resulted in glycogen depletion, decreased phosphorylase activity and increased synthase-R activity. Addition of dialyzed latex particles to starved leukocytes revealed a very rapid ingestion rate (half-maximal ingestion within 30 s). This uptake is followed by glycogenolysis associated with an immediate two-fold increase in phosphorylase a activity and a synthase-R to -D conversion within 30 s. Furthermore, in rapid time-course experiments with phagocytozing cells we found that the concentration of cyclic AMP increased by 93% within 15 s and returned to baseline values at 1 min. In a medium without added calcium and with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, phagocytosis was blocked, cyclic AMP formation decreased by 50% and phosphorylase activation was abolished, but the conversion of synthase-R to -D was preserved. Addition of calcium ions to cells suspended in a calcium-free buffer without added latex results in phosphorylase activation and glycogenolysis, but not in cyclic AMP increase or synthase-R to -D conversion. Measurements of 45Ca efflux during phagocytosis suggest an initial increase in cytosolic calcium obtained by a release of membrane-bound 45Ca. Activation of phosphorylase during phagocytosis is thus presumably due to an increase in cytosol Ca2+ and subsequent activation of phosphorylase kinase, and is independent of the simultaneous increase in concentration of cyclic AMP. Phosphorylation of synthase R to the D form does not depend on the presence of Ca2+ in the extracellular phase.     

Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Effect of intracellular cyclic AMP injection on calcium current in identifiedHelix pomatia neurons

The effect of intracellular injection of cyclic AMP (cAMP) and extracellular application of theophylline on the inward calcium current was investigated in neurons RPa3 and LPa3 ofHelix pomatia. Iontophoretic injection of cyclic AMP (current 10–35 nA, duration about 1 min) led to a decrease in amplitude of the calcium current to a new stationary level, which depended on the injection current. After the end of injection the calcium current was restored to its initial level. Current-voltage characteristic curves of the calcium current were not shifted along the voltage axis by cAMP injection, indicating that the reduction in this current was connected with a change in maximal calcium conductance. An increase in the frequency of depolarizing shifts from 0.1 to 0.5 Hz caused a decrease in the calcium current but did not affect the time course of the decrease in calcium current in response to injection of cAMP or the time course of its recovery after the end of injection. Theophylline an inhibitor of cyclic nucleotide phosphodiesterase, in a concentration of 1 mM in the external solution, lowered the amplitude of the calcium current by 50–75% of its initial value. In 40% of neurons, abolition of the action of theophylline by rinsing was incomplete, but in the rest the effect of theophylline was irreversible. It is postulated on the basis of the results that cytoplasmic compounds take part in regulation of the calcium current of molluscan neurons. The possible physiological role of this process is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 290–297, May–June, 1982.     

The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations

We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2-3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level.     

Rat pancreas adenylate cyclase VII. Effect of extracellular calcium on pancreozymin-induced cyclic AMP formation

1. 1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated.

2. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium.

3. 3. The dose-responce curve for pancreazymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium.

4. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide id obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM.

5. 5. It suffices to lower the Ca²⁺-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide.

6. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggest that a low cytoplasmic Ca²⁺-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.

Cyclic AMP calcium and the growth of mastocytoma cells

Arresting P815 mastocytoma cell growth with N6, O2'-dibutyryladenosine 3':5' cyclic monophosphate (db cAMP) and theophylline increased 45Ca2+ uptake and efflux by the cells (i.e, Ca2+ cycling) without altering cytoplasmic free Ca2+ concentrations or the amount or distribution of protein kinase C in the cells. Attempts to identify the Ca2+ channels involved using a wide variety of drugs were unsuccessful. However, the inhibitory effect of db cAMP on growth was greatly increase in medium containing low Ca2+ concentrations, confirming that interactions between Ca2+ and cyclic AMP can affect mastocytoma cell growth.