Mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells.

We have studied the mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells. Na+ deprivation-induced catecholamine secretion depended on free extracellular Ca2+ concentrations and was almost parallel to 45Ca2+ influx into the cells under various experimental conditions. Furthermore, Na+ deprivation-induced 45Ca2+ influx and catecholamine secretion were actually induced by a relative Na+ concentration gradient across the plasma membrane, but not by simple omission of Na+ from the medium. These results indicate that the deprivation of Na+ from the medium changes the relative Na+ gradient across the plasma membrane and results in Ca2+ influx via a reverse mode of Na(+)-Ca(2+) exchange rather than by inducing Ca2+ entry through Ca2+ channels by eliminating the competition between extracellular Na+ and Ca2+.

Psychological stress induces heat shock protein 70 expression in rat aorta.

Psychological stress without any physical stimuli caused a rapid and marked increase in the level of heat shock protein (HSP) 70 mRNA in rat aorta, but had little effect on the other tested tissues. The maximum increase in HSP70 mRNA level in the aorta was observed at 0.5-1 hr after the stress, and then it declined. Moreover, this stress also increased the level of HSP70 protein in the aorta, but had little effect on the other tested tissues. These results indicate that exposure of rats to mild psychological stress results in the induction of HSP70, especially in the blood vessels.

Pharmacological evidence for regulation of Na(+)-Ca++ exchange by Ca++/calmodulin-dependent protein kinase in isolated bovine adrenal medullary cells.

The mechanism of the regulation of Ca++ influx via Na(+)-Ca++ exchange in response to Na+ deprivation was studied in bovine adrenal medullary cells. Protein kinase inhibitors staurosporine and (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl2,3,9,10-te trahydro-8, 11-epoxy-1H,8H, 11H-2,7b, 11a-triazadibenzo[a,g]cycloocta[c,d,e]trinden- 1-one depressed Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion in a concentration-dependent manner. However, 1 mM dibutyryl cyclic AMP and 1 microM forskolin, an activator of adenylate cyclase, had little effect on Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion. Dibutyryl cyclic GMP (1 mM) and muscarine (30 microM), which increased intracellular cyclic GMP level via stimulation of muscarinic receptors, had also little effect on the responses. Although the phorbol esters 12-O-tetradecanoyl-phorbol-13-acetate and phorbol 12,13-dibutyrate, activators of protein kinase C, enhanced Na+ deprivation-induced catecholamine secretion, these compounds failed to affect Na+ deprivation-induced 45Ca++ uptake. On the other hand, a variety of calmodulin antagonists such as calmidazolium, trifluoperazine, pimozide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion in a concentration-dependent manner. Furthermore, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zin e, which is known as an inhibitor of Ca++/calmodulin-dependent protein kinase II, also reduced Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion. Chelation of intracellular Ca++ with Quin-2 acetoxymethyl ester resulted in a decrease in Na+ deprivation-induced 45Ca++ uptake. However, these compounds that inhibited the Na+ deprivation-induced responses in the cells did not cause solely nonspecific and direct inhibition on Na(+)-Ca++ exchanger. These pharmacological observations suggest that Ca++/calmodulin-dependent protein kinase is involved in the regulation of Na(+)-Ca++ exchange in bovine adrenal medullary cells.

Role of protein kinase C in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary cells.

The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 microM) did not cause large changes in catecholamine secretion evoked by Ca2+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabilized cells with [gamma-32P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca(2+)-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 microM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 microM TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Ca(2+)-induced catecholamine secretion, and cAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA.

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.