Stimulus-responsive and rapid formation of inositol pentakisphosphate in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with high K+ (56 mM) and nicotine (10 microM), a large and transient increase in [3H]inositol 1,3,4,5,6-pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s and then declined to the basal level at 2 min. The time course of accumulation of InsP5 was parallel to that of [3H]inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Angiotensin II (Ang II) (10 microM) rapidly accumulated InsP5, but the level was sustained for 2 min. With a slower time course and a lesser amount than InsP5, high K+, nicotine, and Ang II caused an accumulation of [3H]inositol 1,3,4,5-tetrakisphosphate and [3H]inositol hexakisphosphate. Veratridine (100 microM), maitotoxin (10 ng/ml), ATP (30 microM), platelet-derived growth factor (10 ng/ml), and endothelin (10 ng/ml) also induced the InsP5 accumulation. High K+, nicotine, veratridine, and maitotoxin induced an increase in 45Ca2+ uptake, whereas Ang II, ATP, platelet-derived growth factor, and endothelin did not cause 45Ca2+ uptake. Nifedipine, a calcium channel antagonist, inhibited the high K(+)-induced InsP5 accumulation but failed to affect the Ang II-induced InsP5 accumulation. In an EGTA-containing and Ca2(+)-depleted medium, the high K(+)-induced InsP5 accumulation was completely inhibited, whereas the InsP5 accumulation induced by Ang II was not significantly inhibited. 12-O-tetradecanoylphorbol-13-acetate inhibited partially the Ang II-induced InsP5 accumulation but failed to inhibit the high K(+)-induced accumulation. In those experiments, the changes of InsP5 accumulation were closely correlated to those of Ins(1,4,5)P3. In the chromaffin cell homogenate, [3H] Ins(1,4,5)P3 was converted eventually to [3H]InsP5 through [3H]inositol 1,3,4,6-tetrakisphosphate. Taken together, the above results suggest that InsP5 is rapidly formed by a variety of stimulants and that the formation of InsP5 may occur through two mechanisms, i.e. Ca2+ uptake-dependent and Ca2+ uptake-independent ones in cultured adrenal chromaffin cells.     

Inositol trisphosphate accumulation by high K+ stimulation in cultured adrenal chromaffin cells

Stimulation with high K+ (KCl, 56 mM) of myo-[3H]inositol-prelabelled cells increased Ca2+ uptake and [3H]inositol trisphosphate (IP3) accumulation in a concentration-dependent manner. Nifedipine, a Ca2+ channel antagonist, inhibited high K+-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Furthermore, ionomycin (1 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation. These results indicate the existence of the Ca2+ uptake-triggered mechanism of IP3 formation in cultured adrenal chromaffin cells.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium uptake-dependent and -independent mechanisms of inositol trisphosphate formation in adrenal chromaffin cells: comparative studies with high K+, carbamylcholine and angiotensin II

When [3H]inositol prelabelled cultured bovine adrenal chromaffin cells were stimulated with 56 mM KCl (high K+), 300 microM carbamylcholine (CCh) or 10 microM angiotensin II (Ang II), a rapid accumulation of [3H]IP3 was observed. At the same time, high K+ or CCh induced rapid increases in 45Ca2+ uptake, but Ang II did not induce a significant 45Ca2+ uptake. The concentration-response curve for KCl-induced [3H]IP3 accumulation coincided well with that for KCl-induced 45Ca2+ uptake into the cells. Nifedipine, a Ca2+ channel antagonist, inhibited the high K(+)-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Nifedipine at a similar concentration range also inhibited CCh-induced 45Ca2+ uptake. Although nifedipine inhibited CCh-induced [3H]IP3 accumulation, the potency was approximately 300-fold less than that for the inhibition of 45Ca2+ uptake. Nifedipine failed to affect the Ang II-induced [3H]IP3 accumulation. BAY K 8644 (2 microM), a Ca2+ channel activator, plus partially depolarizing concentration of KCl (14 mM), induced 45Ca2+ uptake and [3H]IP3 accumulation. Ionomycin (1 microM and 10 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation in a concentration-dependent manner. Pretreatment of the cells with protein kinase C activator, 100 nM 12-O-tetradecanoyl phorbol-13-acetate, for 10 min, partially inhibited CCh and Ang II-induced [3H]IP3 accumulation, but failed to inhibit the high K(+)-induced accumulation. Furthermore, the effects of high K+ and Ang II on the IP3 accumulation was additive. Ang II and CCh induced a rapid and transient increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) accumulation (5 s) followed by a slower accumulation of inositol 1,3,4-trisphosphate (1,3,4-IP3). High K+ evoked an increase in 1,3,4-IP3 accumulation but obvious accumulation of 1,4,5-IP3 could not be detected. In Ca2(+)-depleted medium, high K(+)-induced [3H]IP3 accumulation was completely abolished, whereas [3H]IP3 accumulation induced by CCh and Ang II was partially inhibited. These results demonstrate the existence of the Ca2+ uptake-triggered mechanism of IP3 accumulation represented by high K+, and also the Ca2+ uptake-independent mechanism of IP3 accumulation represented by Ang II in cultured bovine adrenal chromaffin cells. Mechanism of CCh-induced IP3 accumulation has an intermediate property between those of high K+ and Ang II.     

Maitotoxin, a Ca2+ channel activator candidate

Effects of maitotoxin, the most potent marine toxin, were studied using a rat pheochromocytoma cell line, PC12h. A low concentration (10(-8) g/ml) of maitotoxin induced a profound increase in CA2+ influx into PC12h cells and the Ca2+-dependent release of [3H]norepinephrine from them. The effects of maitotoxin were not affected by treatment with tetrodotoxin (10(-6) M) and were observed even in the absence of external Na+. Furthermore, these effects were markedly inhibited or abolished by treatment with verapamil (30-300 microM), Mn2+ (5 mM), or tetracaine (1 mM). These results suggest that maitotoxin activates the voltage-dependent calcium channels of PC12h cells.     

Evidence that a pertussis-toxin-sensitive substrate is involved in the stimulation by epidermal growth factor and vasopressin of plasma-membrane Ca2+ inflow in hepatocytes.

1. In hepatocytes, epidermal growth factor (EFG) (a) increased the rate of 45Ca2+ exchange in cells incubated at 1.3 mM extracellular Ca2+, (b) increased the activity of glycogen phosphorylase a and the intracellular free Ca2+ concentration (measured with quin2) in a process dependent on the concentration of extracellular Ca2+, and (c) enhanced the increase in glycogen phosphorylase activity which follows the addition of Ca2+ to cells previously incubated in the absence of Ca2+. It is concluded that EGF stimulates plasma-membrane Ca2+ inflow. 2. The effects of the combination of EGF and vasopressin on the rate of 45Ca2+ exchange and on the rate of increase in glycogen phosphorylase activity were the same as those of vasopressin alone. 3. The amount of 45Ca2+ released by EGF from internal stores was about 30% of that released by vasopressin. No detectable increase in [3H]inositol mono-, bis- or tris-phosphate was observed after the addition of EGF to cells labelled with myo-[3H]inositol. 4. In hepatocytes isolated from rats treated with pertussis toxin, the effects of EGF and vasopressin on phosphorylase activity (measured at 1.3 mM-Ca2+) and on the rate of Ca2+ inflow (measured with quin2) were markedly decreased compared with those in normal cells. 5. Treatment with pertussis toxin did not impair the ability of vasopressin to release Ca2+ from internal stores, but decreased vasopressin-stimulated [3H]inositol polyphosphate formation by 50%. 6. It is concluded that the mechanism(s) by which vasopressin and EGF stimulate plasma-membrane Ca2+-inflow transporters in hepatocytes involves a GTP-binding regulatory protein sensitive to pertussis toxin, and does not require an increase in the concentration of inositol trisphosphate comparable with that which induces the release of Ca2+ from the endoplasmic reticulum.     

Adrenergic regulation of glucose metabolism in rat heart. A calcium-dependent mechanism mediated by both alpha- and beta-adrenergic receptors

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.     

Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor

5'-(N-Ethyl)carboxamidoadenosine (NECA), an analog of adenosine, transiently stimulated a rat tumor mast cell (RBL-2H3 cells) to cause a release of inositol phosphates and an increase in levels of Ca2+ in the cytosol. It failed, however, to stimulate a sustained uptake of 45Ca2+ or secretion. The effects of other agents that act on P1- or P2-purinergic receptors suggested that NECA and other adenosine agonists acted via a novel subtype of adenosine membrane receptor. Although the order of potency of agonists was characteristic of A2-adenosine receptors, there was no indication of the involvement of adenylate cyclase, and antagonists such as isobutylmethylxanthine, 8-phenyltheophylline, and 8-p-sulfophenyltheophylline inhibited the responses to either NECA or antigen. The fact that stimulation of inositol phospholipid hydrolysis by NECA in washed, permeabilized RBL-2H3 cells was blocked by pertussis toxin as well as by cholera toxin suggested instead that the NECA-sensitive receptor activated phospholipase C via a G-protein. In contrast to NECA, antigen stimulation resulted in a pertussis toxin-resistant, sustained hydrolysis of inositol phospholipids, increases in free intracellular Ca2+, accelerated influx of 45Ca2+, and secretion from RBL-2H3 cells. In combination with NECA, all responses to antigen were markedly enhanced, and the enhancement was selectively blocked by pertussis toxin. The ability of antigen, but not NECA, to provoke secretion may be dependent primarily on the sustained activation of a cholera toxin-sensitive Ca2+ influx pathway that serves to amplify stimulatory signals for secretion. These studies also suggested that phospholipase C could be activated through different G-proteins via different receptors within the same cell.     

Maitotoxin-Evoked γ-Aminobutyric Acid Release Is Due Not Only to the Opening of Calcium Channels

The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of gamma-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 microM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.     

Vasoactive intestinal polypeptide and muscarine mobilize intracellular Ca2+ through breakdown of phosphoinositides to induce catecholamine secretion. Role of IP3 in exocytosis

We have recently shown that vasoactive intestinal polypeptide (VIP) is as potent as acetylcholine in inducing the secretion of catecholamines from the rat adrenal medulla. In the present study we have investigated the molecular mechanism involved in the exocytotic secretion of catecholamines by VIP and the effects of VIP on Ca45 uptake and phosphoinositide breakdown and compared them with those of the classical cholinergic agonists. We now show that omission of Ca2+ from the perfusion medium had almost no effect on VIP-induced secretion; however, addition of 1 mM EGTA to calcium-free medium abolished the secretion. Stimulation with VIP did not result in a net increase in Ca45 uptake and it was not modified by a protein kinase C activator, phorbol ester. All these effects of VIP were comparable to those of muscarine. VIP (0.3 to 10 microM) and muscarine (30 to 100 microM) produced time-and concentration-dependent increase (up to 700%) in the production of [3H]inositol phosphates. The production of [3H]inositol phosphates by VIP and muscarine occurred in calcium-free and EGTA medium. The effect of VIP on [3H]IP, [3H]IP2, and [3H]IP3 production was reduced by (1 to 30 microM) VIP antagonist (an analogue of growth hormone-releasing factor, Ac-Tyr1hGRF) and 1 to 20 microM naloxone. Although nicotine produced a brisk secretory response, there was no change in [3H]inositol phosphates. We conclude that inositol 1,4,5-trisphosphate generated upon activation of VIP and muscarine receptors is linked to exocytotic secretion of adrenal medullary hormones through release of internal Ca2+ ions.     

Lowering of the extracellular Na+ concentration enhances high-K+-induced formation of inositol phosphates in the guinea-pig ileum.

1. Formation of inositol phosphates (InsPs) was measured in cross-chopped slices or dispersed cells, isolated by collagenase treatment, of guinea-pig ileum longitudinal smooth muscle pre-labelled with [3H]inositol. 2. Elevation of the extracellular K+ concentration by equimolar replacement of Na+ induced accumulation of InsPs in the dispersed cells and in the tissue slices. These effects were blocked by neither tetrodotoxin (1 microM) nor atropine (10 microM), and were approximately additive with carbachol-induced accumulation. 3. In the tissue slices, the response to K+ was partially inhibited by nifedipine (10 microM) and by CdCl2 (0.3 mM), but the carbachol-induced response was not altered. 4. Accumulation of InsPs induced by KCl-excess solution (high-K+ solution without Na+ replacement) was suppressed strongly by nifedipine and completely by CdCl2. The response to KCl excess was approx. 40% of that to high K+ with Na+ replacement. 5. Low-NaCl solution (replacement of NaCl with equimolar sucrose) also produced InsPs, and this was not blocked by either nifedipine (10 microM) or CdCl2 (0.3 mM). 6. The formation of InsPs by a maximally effective concentration of carbachol (1 mM) in the presence of KCl excess or low NaCl was greater than the additive effect of the two stimuli on their own. Enhancement of the carbachol-induced response by KCl excess disappeared in the presence of CdCl2 (0.3 mM). 7. These data suggest that formation of InsPs induced by high-K+ solution with equimolar replacement of Na+ consists of two components, i.e. high-K+-induced inositol-phospholipid hydrolysis by Ca2+ entry through voltage-sensitive channels, and low-Na+-induced formation of InsPs, insensitive to Ca2+ antagonists, but that both of them do not contribute significantly to the activation of phospholipase C by muscarinic stimuli.     

The actions of diazepam and diphenylhydantoin on fast and slow Ca2+ uptake processes in guinea pig cerebral cortex synaptosomes

The activities of diazepam and diphenylhydantoin as inhibitors of the fast and slow phases of 45Ca2+ uptake in response to K+ depolarization and of [3H]nitrendipine binding were examined in guinea pig cerebral cortex synaptosomes. The slow phase of 45Ca2+ uptake was abolished in Na+-free media (choline substitution) and was more sensitive to inhibition by 3,4-dichlorobenzamil and represents a Na+-dependent Ca2+ uptake process. The fast component of uptake represents activation of voltage-dependent Ca2+ channels. Diazepam (to 300 microM) was selectively active against the fast component of 45Ca2+ uptake. The benzodiazepines Ro 11-3624 and Ro 11-3128 were similarly selective with a modest stereoselectivity against the fast component of 45Ca2+ uptake. Diphenylhydantoin (100 and 200 microM) blocked nonselectively both fast and slow phases of Ca2+ uptake. Diazepam (60 microM) and diphenylhydantoin (200 microM) blocked [3H]nitrendipine binding in a competitive manner. Diazepam and diphenylhydantoin probably exert at least part of their anticonvulsant activity by inhibition of voltage-dependent Ca2+ channels.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.

Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.     

Maitotoxin-Induced Release of γ-[3H]Aminobutyric Acid from Cultures of Striatal Neurons

The potent marine toxin, maitotoxin, induced the release of gamma-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10(-6) M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10(-4) M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10(-6) M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Effects of ATP on Phosphatidylinositol-Phospholipase C and Inositol 1-Phosphate Accumulation in Rat Brain Synaptosomes

Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1-phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [1-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (approximately 20 microM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.     

Separate Binding and Functional Sites for ω co-Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.     

Ca2+ channel activating function of maitotoxin, the most potent marine toxin known, in clonal rat pheochromocytoma cells

Actions of maitotoxin, the most potent marine toxin known obtained from toxic dinoflagellate, Gambier-discus toxicus, were studied using clonal rat pheochromocytoma cells (PC12), rat liver mitochondria and liposomes. Maitotoxin induced a profound release of norepinephrine and dopamine from PC12 cells and the molar ratio of norepinephrine to dopamine was almost the same as that stored in the cells. This releasing action was apparent at a concentration of 5 X 10(-10) g/ml or more, the releasing rate increased with an increase in the concentration of applied maitotoxin and attained maximum at about 10(-6) g/ml. The [3H]norepinephrine release induced by maitotoxin was abolished in the absence of external Ca2+ and increased with increasing concentration of external Ca2+ up to 10 mM. The release gradually decreased as the external Na+ concentrations were reduced from 130 to 20 mM, but maitotoxin is still able to induce a profound release in the absence of external Na+. The releasing action of maitotoxin was markedly suppressed by various Ca2+ channel blockers, such as Mn2+, verapamil, and nicardipine, and by a local anesthetic, tetracaine. The inhibitory actions of Ca2+ channel blockers were antagonized by external Ca2+ and became less obvious in the higher Ca2+ concentration range. Maitotoxin did not exhibit any ionophoretic activities on rat mitochondrial and liposomal membranes. These results suggest that maitotoxin has the ability to activate voltage-dependent Ca2+ channels of PC12 cells.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.