Characteristics of the K-252a-induced increase in calcium uptake in PC12 cells.

K-252a treatment produced a 30-50% increase in the uptake of radioactive calcium by PC12 cells within 3-4 minutes. The increase in uptake was partially blocked by inhibitors of voltage-operated calcium channels, such as nifedipine, but not by inhibitors of receptor-operated calcium channels, such as nickel or suramin. Introduction of phosphatase 2A into the cells completely blocked the effect of K-252a. Long-term treatment of the cells with either K-252a or with nerve growth factor blocked the subsequent actions of either K-252a or nerve growth factor on calcium uptake, but neither altered the subsequent action of the L-channel agonist Bay K 8644 on calcium uptake. Calcium uptake was not stimulated by K-252a in PC12nnr, cells that have little or no high-affinity nerve growth factor receptors; cells expressing increased levels of high-affinity nerve growth factor receptors showed a response to K-252a comparable to that seen in parent PC12. The data suggest that the increased uptake of radioactive calcium produced by K-252a is mediated by a mechanism very similar to that serving the increased calcium uptake produced by nerve growth factor.

Signal transduction and Ca2+ uptake activated by endothelins in rat brain endothelial cells.

The activation of signal transduction pathways by endothelin-1 or endothelin-3 were investigated in rat cerebromicrovascular endothelial cells. Endothelin-1 induced a rapid increase in inositol triphosphate (IP3) formation in these cells, whereas endothelin-3 was only moderately effective at high concentrations. Both endothelins also increased uptake of 45Ca2+ in these cells. Endothelin-1-induced IP3 formation or 45Ca2+ uptake were inhibited by endothelin ETA receptor antagonist BQ-123. Ryanodine, an inhibitor of intracellular Ca2+ mobilization, selectively endothelin-1-induced 45Ca2+ uptake, whereas nickel or suramin inhibited endothelin-3-induced 45Ca2+ uptake. The results indicate that endothelin-1 elevates 45Ca2+ uptake in rat brain endothelial cells by mechanisms coupled to the mobilization of intracellular Ca2+ stores. Both endothelin-1- and endothelin-3-induced 45Ca2+ uptake were inhibited by receptor operated Ca2+ channel blocker SK&F 96365, whereas they were insensitive to dihydropyridine derivatives nifedipine and nitrendipine. The release of arachidonic acid from rat brain endothelial cells observed in response to endothelin-1 was inhibited by ryanodine or SK&F 96365, implicating participation of both intra- and extra- cellular components of Ca2+ signaling in activating endothelial secretion of vasoactive substances.

Effects of ATP and UTP in pheochromocytoma PC12 cells: evidence for the presence of three P2 receptors, only one of which subserves stimulation of norepinephrine release.

1. In pheochromocytoma PC12 cells ATP and, to a lesser extent, 2-methylthioATP stimulate phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, leading to stimulation of norepinephrine release. In contrast, although UTP also stimulates phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, there is no stimulation of norepinephrine release. 2. 2-MethylthioATP, presumably acting at P2y receptors, and UTP, presumably acting at P2u receptors, in combination elicit a phosphoinositide breakdown greater than that elicited by either alone. Intracellular levels of calcium measured with Fura-2 increase to greater levels with ATP than with UTP and are sustained, while the UTP intracellular levels of calcium rapidly return to basal values. Both ATP and UTP cause a similar influx of 45 Ca2+ presumably by stimulation of a P2 receptor directly linked to a cation channel. 3. It is proposed that PC12 cells contain two distinct G protein-coupled P2 receptors that activate phospholipase C and a P2 receptor linked to a cation channel. The P2y receptor sensitive to ATP (and to 2-methylthioATP) causes the depletion of a pool of intracellular calcium, sufficient to activate so-called "receptor-operated calcium entry". The sustained elevation of intracellular calcium after ATP treatment is proposed to result in stimulation of norepinephrine release and activation of calcium-dependent potassium channels and sodium-calcium exchange pathways. 4. The P2u receptor sensitive to UTP (and to ATP) causes only a transient elevation in levels of intracellular calcium, perhaps from a different pool, insufficient to activate so-called receptor-operated calcium entry. Further sequelae do not ensue, and the functional role of the UTP-sensitive P2u receptor is unknown.

The long-term down-regulation of dihydropyridine receptors by Bay K 8644 in PC12 cells.

Treatment of PC12 cells with Bay K 8644 for 12 hr or more leads to an almost 80% decrease in the subsequent ability of Bay K 8644 to stimulate the uptake of radioactive calcium into the cells. This effect is a property of the S(-)isomer of Bay K 8644; pre-treatment with the R(+)isomer, now known to be a calcium channel blocker, has the opposite effect. This treatment is specific in that it does not interfere with the stimulation of calcium uptake by potassium, ATP, or nerve growth factor. Such treatment is accompanied by a 90% decrease in the ability of Bay K 8644 to stimulate the release of norepinephrine. The characteristics of the binding of [3H]isradipine to control and to treated cells indicates that the decrease in the effect of dihydropyridines is accompanied by a marked decrease in the number of dihydropyridine binding sites with no apparent change in the affinity of the remaining sites. The continued ability of depolarizing levels of potassium to stimulate calcium uptake and the induction of the protooncogene c-fos in Bay K 8644-treated cells indicates that the L-type calcium channels are still intact, but are simply unresponsive to dihydropyridine agonists.

Pardaxin-stimulated calcium uptake in PC12 cells is blocked by cadmium and is not mediated by L-type calcium channels.

Pardaxin is an excitatory neurotoxin which triggers neurotransmitter release as a result of voltage-dependent pore formation within the neuronal membrane. We have used several pharmacological manipulations of calcium influx to characterize pardaxin pore activity in PC12 cells in culture. Pardaxin stimulates the uptake of radioactive calcium into PC12 cells in a dose dependent fashion (ED50 of 0.4 microM). This stimulation is partially inhibited by nifedipine, a blocker of L-type calcium channels. Effective blockade of pardaxin stimulation was produced by the inorganic calcium channel blockers cadmium (IC50 of 10 microM) and nickel (2 mM). Homologous down regulation of L-calcium channels by the agonist Bay K-8644, inhibited the subsequent stimulation of calcium uptake by this drug, but not by pardaxin. A fluorometric analysis of pardaxin pore formation in unilamellar large liposomes indicates pardaxin pores are blocked by cadmium (10-200 microM). These data distinguish between pardaxin pores and L-type calcium channels in PC12 cells. We suggest pardaxin as a pharmacological ionophore tool to modulate neuronal calcium homeostasis and neurotransmitter release.

Intracellular calcium levels regulate the actions of nerve growth factor on calcium uptake in PC12 cells.

The uptake of divalent cations and the intracellular concentration of calcium in PC12 cells were studied by flow cytometric analysis using the calcium-sensitive dye, Fluo-3, under a variety of conditions. In particular the actions of nerve growth factor were analyzed. The data show that nerve growth factor stimulates the uptake of divalent cations and increases the intracellular calcium levels of cells attached to collagen-coated plates. The data further indicate that nerve growth factor-dependent increases in the uptake of divalent cations become less pronounced as the intracellular concentration of calcium increases. Intracellular calcium levels increase upon detachment of the cells from the plates and also with increasing cell density. Studies on the uptake of 45calcium confirmed the influence of intracellular calcium levels on nerve growth factor-stimulated calcium uptake. Thus, the effect of nerve growth factor on the uptake of divalent cations is dependent on the calcium levels in the cells, perhaps explaining why previous studies in this field have provided inconsistent results.

Nerve growth factor-stimulated calcium uptake into PC12 cells: uniqueness of the channel and evidence for phosphorylation.

Nerve growth factor stimulates the uptake of radioactive calcium into PC12 cells. This stimulation is inhibited by low concentrations of dideoxyforskolin or staurosporine, and by high concentrations of nifedipine or cadmium. On the other hand, neither dideoxyforskolin nor staurosporine inhibited the stimulation of calcium uptake caused by BK-8644 or adenosine triphosphate (ATP). Nickel inhibited only the effect of ATP on calcium uptake, and actually stimulated the effects of either BK-8644 or nerve growth factor. Down-regulation of L-calcium channels by BK-8644 blocked the subsequent stimulation of calcium uptake by this agent, but not the stimulation by nerve growth factor. Conversely, pre-treatment of the cells with nerve growth factor inhibited the subsequent stimulation of calcium uptake by nerve growth factor, but not the stimulation by BK-8644. The effects of BK-8644 and nerve growth factor on calcium uptake were additive, as were the effects of nerve growth factor and ATP. Phosphatase 2A inhibited the effect of nerve growth factor on calcium uptake, but did not influence the action of BK-8644. On the other hand, calcineurin inhibited the effect of BK-8644 on calcium uptake, but potentiated the action of nerve growth factor. Calmidazolium or fluphenazine also inhibited the effect of nerve growth factor on calcium uptake, but okadaic acid stimulated it. A comparison of the effects of these inhibitors on the actions of various calcium channel agonists shows that the channels on which the action of nerve growth factor is exerted are different than either the L-type calcium channels or the ATP-activated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor-induced increase in calcium uptake by PC12 cells.

Treatment of PC12 cells with nerve growth factor (NGF) produces a rapid and transient increase in calcium uptake into the cells. The increased uptake is maximal after 5 minutes of NGF treatment, but after 15 minutes of NGF treatment, no such increase can be observed. The effect of NGF is partially inhibited by blockers of L-type calcium channels. K-252a, an alkaloid-like kinase inhibitor that usually is found to inhibit the actions of NGF on PC12 cells, produces an increase in calcium uptake similar to, but smaller than, that seen with NGF. NGF had no effect on calcium release under these conditions.

Nerve growth factor and K-252a increase catecholamine release from PC12 cells.

PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. The cells contain catecholamines and secrete them in response to depolarizing stimuli and cholinergic agonists. Treatment of the cells with nerve growth factor produces a number of very rapid changes, including the structural rearrangement of the cell membrane, the generation of a number of different second messengers, and the phosphorylation of several proteins. The present studies show that nerve growth factor treatment increases the release of dopamine and norepinephrine from the cells within a few minutes and does so independently of its effect on their metabolism. The experiments indicate that this effect on nerve growth factor is dependent on the presence of extracellular calcium and can be blocked by calcium channel antagonists. K-252a, an alkaloid-like material, usually found to inhibit the actions of nerve growth factor on PC12 cells, also increases the release of catecholamines under these conditions.

Nicardipine versus nifedipine: multicentre controlled trial in essential hypertension.

Ninety-five hypertensive outpatients of both sexes, aged 23 to 65 years with diastolic blood pressures above 105 but below 120 mmHg (greater than 14.0 but less than 16.0 kPa), after one week on a placebo were randomly assigned either to nicardipine plus a placebo (40 mg/day - 48 patients) or nifedipine sustained-release plus a placebo (20 mg/day - 47 patients) for an additional six weeks. The study groups were homogeneous and comparable. After the run-in period the average blood pressure was 181 +/- 17/116 +/- 9 mmHg (24.1 +/- 2.3/15.5 +/- 1.2 kPa) in the nicardipine and 177 +/- 22/116 +/- 9 mmHg (23.6 +/- 2.9/15.5 +/- 1.2 kPa) in the nifedipine group (p greater than 0.10). In the acute oral test (nicardipine 40 mg to all the subjects; blood pressure measured at 30 min intervals during two hours) almost identical hypotensive effects within and between groups were observed (mean arterial pressure decrease of 11%, after 120 min; p less than 0.05). At the end of this trial blood pressure decreased further to 152 +/- 12/94 +/- 11 mgHg (20.3 +/- 1.6/12.5 +/- 1.5 kPa) (mean decrease of 20%; p less than 0.01) on nicardipine and to 145 +/- 12/94 +/- 11 mmHg (19.3 +/- 1.6/12.5 +/- 1.5 kPa) (mean decrease of 20%; p less than 0.01) on nifedipine. There were no significant changes in pulse rate. The observed between-group differences were trivial (p greater than 0.10). The laboratory data did not alter appreciably during this study. Three patients on nicardipine and four on nifedipine reported headache, palpitations and flushing: one patient on nicardipine and two on nifedipine were as a result excluded from the trial. It was concluded that nicardipine and nifedipine sustained-release were comparably effective and well-tolerated drugs suitable as the first-line agents for the management of mild to moderate hypertension.

Alteration in the uptake and storage of cardiac noradrenaline in spontaneously hypertensive rats.

Uptake and storage of noradrenaline administered to Spontaneously hypertensive rate (SHR) (14) was significantly reduced compared with normotensive Wister rats (NR). Initial uptake of noradrenaline at 10 minutes was only 19 per cent less in SHR than in NR. However, 60 minutes following the administration of noradrenaline, its concentration in the heart of SHR was 60 per cent less than that in NR. Noradrenaline administered to NR disappeared from the heart in 10 hours, and that from SHR in 5 hours only.

Control of adenylate cyclase from secretory vesicle membranes by beta-adrenergic agents and nerve growth factor.

Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of purified secretory vesicle membranes from the adrenal medulla is inhibited by I-isoproterenol and I-epinephrine, as well as by nerve growth factor (NGF). The effect of these agents was found to be dose-dependent and, in the case of the catecholamines, saturable. NGF was active at concentrations as low as 10(-8) M. Oxidized NGF was only minimally active, and insulin was completely inactive. Neither dopamine nor phenylephrine had activity. Inhibition of cyclase by either isoproterenol or epinephrine was blocked by I-propranolol, a specific beta-antagonist, but propranolol by itself had no effect on adenylate cyclase activity. The data indicate that the secretory vesicle membrane has beta-adrenergic receptors coupled to the adenylate cyclase. Propranolol was also found to block the NGF-induced inhibition of cyclase. We conclude that the granule membrane has beta-adrenergic receptors as well as NGF-reactive sites, and that the two may be functionally linked.

The effect of nerve growth factor on cycle AMP levels in superior cerival ganglia of the rat.

Nerve growth factor (NGF) produces a several-fold increase in the cyclic AMP concentration in rat superior cervical ganglia in organ culture within 5 min. An increase can be seen with as little as 40 ng/ml of NGF. Oxidized NGF is without effect. The increase in the cAMP concentration produced by NGF is prevented by the addition of antiserum to NGF.