Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phos-phorylated 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 [ 3 H]tyrosine to [ 3 H]dihydroxyphenylalanine ([ 3 H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K + and Ba 2+ , stimulated phosphorylation of tyrosine hydroxylase and [ 3 H]DOPA production. The effects of nicotinic stimulation and elevated K + on tyrosine hydroxylase phosphorylation and [ 3 H]DOPA production were Ca 2+ -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 [ 3 H]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 Ca 2+ -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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65828/1/j.1471-4159.1986.tb13011.x.pd

Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway

1. The effects on catecholamine secretion of activation of protein kinase C and clostridial neurotoxins were examined in digitonin-permeabilized bovine adrenal chromaffin cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44281/1/10571_2004_Article_BF00711564.pd

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44286/1/10571_2004_Article_BF00711168.pd

Nicotinic Receptor Mediated Short Term Regulation Of In Situ Tyrosine Hydroxylase In Cultured Bovine Adrenal Chromaffin Cells (phosphorylation).

Nicotinic receptor stimulation of monolayer bovine adrenal chromaffin cell cultures results in catecholamine (CA) secretion and phosphorylation of a 56,000 dalton protein. Both events absolutely require extracellular calcium. Incubation of Western blots of electrophoretically transferred proteins from both one- and two-dimensional PAGE with specific tyrosine hydroxylase (TH) antibody resulted in the identification of the 56,000 dalton phosphoprotein as TH, the rate-limiting enzyme of CA biosynthesis. Studies of the nicotinic receptor mediated in situ TH phosphorylation and activation strongly indicate a role for both calcium-dependent and cAMP-dependent protein kinases. Nicotinic receptor agonists, Ba('2+) and 56 mM KCl stimulate TH phosphorylation, TH activity, and CA secretion in a calcium-dependent manner. Nicotinic receptor stimulation causes a calcium-dependent increase of in situ cAMP within the time course of nicotinic receptor stimulated TH phosphorylation and activity. Dibutyryl cAMP or forskolin (specific adenylate cyclase activator) stimulated TH phosphorylation and activity, therefore suggesting a role for cAMP-dependent protein kinase in these effects. The phorbol ester TPA also causes increased TH phosphorylation and activity, implicating a role for protein kinase C. Two-dimensional PAGE analysis of phosphoprotein showed that nicotinic agonists, Ba('2+), 56 mM KCl, and TPA caused phosphorylation of three forms of TH with different pH's, while dibutyryl cAMP and forskolin phosphorylated only two of the TH spots. The physiological significance of these observations in chromaffin cells is that nicotinic stimulation mediates a short term regulation of CA biosynthesis via the TH phosphorylation that results in increased enzyme activity.Ph.D.Biological SciencesNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127721/2/8512493.pd