The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca21 ionophore ionomycin. PMA also enhanced secretion induced by Ba2 . In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion -70% at 0.5 jIM Ca2+ and 30% at 10 jiM Ca2+. PMA enhanced the maximal response to Ca2+ =25% and decreased the Ca2+ concentration required for half-maximal secretion :30 %. The effects of PMA on chromaffin cells were associated with a 2-to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.The exocytotic release of catecholamines from bovine adrenal chromaffin cells is normally triggered by the influx of extracellular Ca2l and the rise in cytosolic Ca" concentration that occur on stimulation of nicotinic receptors (1-4). Membrane depolarization in the presence of Ca2+ or the substitution of Ba2+ for Ca2+ also stimulates exocytosis. Although the mechanism of exocytosis was first described using biochemical techniques in the adrenal medulla, the underlying mechanisms triggered by Ca2+ are not understood.One reaction that could be involved in exocytosis is protein phosphorylation. Previous studies concerning the role of protein phosphorylation in secretion from adrenal chromaffin cells revealed that two proteins, one of 56-60 kDa and one of 100 kDa, were phosphorylated when secretion was stimulated by secretagogues (5, 6). The 56-60-kDa protein was identified as tyrosine hydroxylase (7), the ratelimiting enzyme in catecholamine biosynthesis, and is probably not' directly involved in exocytosis. The 100-kDa protein has not been identified but its phosphorylation is not well correlated with secretion.Protein kinase C is Ca2+-dependent and requires acidic phospholipids for activity (8,9). Diglyceride in the presence of phosphatidylserine increases the Ca2+ sensitivity of the enzyme to micromolar or perhaps submicromolar concentrations (10). The ester phorbol 12-myristate 13-acetate (PMA) can substitute for diglyceride in vitro (11) and similarly increases the Ca2+ sensitivity of the enzyme. Most importantly, PMA activates protein kinase C and protein phosphorylation in intact platelets and enhances serotonin secretion (11,12).In the present study, we have investigated the effects of PMA on catecholamine secretion and protein phosphorylation in bovine adrenal chromaffin cells in monolayer culture. We have used both intact chromaffin cells and cells treated with a low concentration...

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.

The levels of serotonin (5-HT), 5 hydroxyindoleacetic acid (5-HIAA), dopamine (DA), homovanillic acid (HVA), norepinephrine (NE), and tyrosine hydroxylase (TH) activity were measured in the focus (spiking) and nonfocus (nonspiking) regions of the temporal neocortex of 20 patients with intractable complex partial seizures. The levels of 5-HT, DA, 5-HIAA, and HVA were higher in the focus when compared to the nonfocus. Values for NE and TH activity were not different when focus and nonfocus were compared. The ratios of metabolite to precursor for 5-HT and DA were not significantly different between the focus and the nonfocus, suggesting that the changes observed were the result of a modification in the synthesis and release of these amines. Such changes in the epileptic focus could be caused by altered transsynaptic regulatory processes, which occur as a result of neuronal loss, gliosis, or neuronal sprouting.

Calcium is the most important physiological regulator of PTH secretion. Peak PTH secretion occurs at an intracellular calcium concentration of about 200 nM, regardless of the extracellular calcium concentration. We suggest, therefore, that intracellular calcium concentration is a regulator of PTH secretion that maintains calcium homeostasis. Other factors may be responsible for modulation of the intracellular calcium concentration, ultimately modulating PTH secretion. The "paradoxical" nature of the dependence of PTH secretion on the calcium concentration may be explained by considering PTH secretion to be unusual in a quantitative, rather than a qualitative, fashion. A possible mechanism for the control of PTH secretion by intracellular calcium, which involves calcium-activated potassium channels, is proposed. The parathyroid cell plasma membrane contains several sensors or channels by means of which the cell senses extracellular calcium. It is not clear whether these entities are coupled to each other or whether they function independently. Guanine nucleotide regulatory proteins are transducers of extracellular signals, including calcium. Several other second messengers that influence PTH secretion have also been described, but possible interactions between these messengers have not yet been determined.