N- and P/Q-type Ca2+ channels in adrenal chromaffin cells.

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.

Role of Cl- co-transporters in the excitation produced by GABAA receptors in juvenile bovine adrenal chromaffin cells.

GABA is the primary inhibitory neurotransmitter in the adult mammalian brain. However, in neonatal animals, activation of Cl(-)-permeable GABA receptors is excitatory and appears to depend on the expression of a Na(+)-K(+)-2Cl- cotransporter (NKCC) that elevates intracellular Cl- levels, leading to a depolarized Cl- equilibrium potential (ECl). The change from excitation to inhibition appears to involve the expression of the K+/Cl- co-transporter, KCC2, which lowers intracellular Cl- levels resulting in a hyperpolarized ECl. In this study, we show that bovine chromaffin cells from 4- to 5-mo-old animals are excited by GABA. Activation of GABAA receptors depolarizes the cells, opens voltage-dependent Ca2+ channels, elevates [Ca2+]i, and promotes the release of catecholamines. Blockade of voltage-dependent Ca2+ channels prevents the elevation of [Ca2+]i by GABA. The extrapolated anion reversal potential in these cells is approximately -28 mV, indicating a resting intracellular anion concentration of approximately 50 mM. Expression of KCC2 protein was not detected in the juvenile chromaffin cells. In contrast, clear expression of NKCC1 was observed. Blockade of NKCC1 should reduce the intracellular Cl- concentration and hyperpolarize ECl. Bumetanide, an NKCC1 blocker, reduced the elevation of [Ca2+]i by GABA. In some cells, activation of GABAA receptors inhibits responses to excitatory neurotransmitters, even though GABA itself is depolarizing. Co-activation of cholinergic and GABAA receptors in chromaffin cells produced elevations in [Ca2+]i that were comparable to those produced by cholinergic receptors alone. Our data showing the selective expression of chloride co-transporters and the resulting strongly depolarized anion reversal potential may help explain how activation of GABAA receptors causes sufficient excitation to elicit catecholamine release from chromaffin cells.

The role of dynamic palmitoylation in Ca2+ channel inactivation.

N- and P/Q-type Ca(2+) channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca(2+) channels are multisubunit proteins, consisting of a pore-forming alpha(1), and accessory beta and alpha(2)delta subunits each encoded by multiple genes and splice variants. beta subunits alter current amplitude and kinetics. The beta(2a) subunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in beta(2a). In the current manuscript, we studied steady state inactivation properties of native N- and P/Q-type Ca(2+) channels and recombinant N-type Ca(2+) channels. When bovine alpha(1B) and beta(2a) and human alpha(2)delta were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P/Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known beta subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in beta subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca(2+) channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca(2+) channels containing beta(2a) but not beta(2b) or beta(2a) subunits mutated to be palmitoylation deficient. Our results suggest that Ca(2+) channels containing beta(2a) subunits may be regulated by dynamic palmitoylation.

Evidence of elevated intracellular calcium levels in weaver homozygote mice.

1. A mutation in the G-protein-linked, inwardly rectifying K+ channel GIRK2 leads to the loss of cerebellar and dopaminergic mesencephalic neurons in weaver mice. The steps leading to cell death are not well understood but may involve constitutive influx of Na+ and Ca2+ into the neurons. 2. We found that resting [Ca2+]i was dramatically higher in cerebellar neurons from weaver mice compared to wild-type neurons. 3. High-K+ stimuli elicited much smaller changes in [Ca2+]i in weaver cerebellar neurons compared to wild-type neurons. 4. weaver cerebellar granule cells could be rescued from cell death by the GIRK2wv cationic channel blocker, QX-314. 5. QX-314 lowered resting intracellular Ca2+ levels in weaver cerebellar granule cells. 6. These results suggest that changes in resting [Ca2+]i levels and alterations in K+ channel function are most likely to contribute to the developmental abnormalities and increased cerebellar cell death observed in weaver mice.

Coexpression of cloned alpha(1B), beta(2a), and alpha(2)/delta subunits produces non-inactivating calcium currents similar to those found in bovine chromaffin cells.

Chromaffin cells express N-type calcium channels identified on the basis of their high sensitivity to block by omega-conotoxin GVIA (omega-CgTx GVIA). In contrast to neuronal N-type calcium currents that inactivate during long depolarizations and that require negative holding potentials to remove inactivation, many chromaffin cells exhibit N-type calcium channel currents that show little inactivation during maintained depolarizations and that exhibit no decrease in channel availability at depolarized holding potentials. N-type calcium channels are thought to be produced by combination of the pore-forming alpha(1B) subunit and accessory beta and alpha(2)/delta subunits. To examine the molecular composition of the non-inactivating N-type calcium channel, we cloned the alpha(1B) and accessory beta (beta(1b), beta(1c,) beta(2a), beta(2b), and beta(3a)) subunits found in bovine chromaffin cells. Expression of the subunits in either Xenopus oocytes or human embryonic kidney 293 cells produced high-threshold calcium currents that were blocked by omega-CgTx GVIA. Coexpression of bovine alpha(1B) with beta(1b), beta(1c), beta(2b), or beta(3a) produced currents that were holding potential dependent. In contrast, coexpression of bovine alpha(1B) with beta(2a) produced holding potential-independent calcium currents that closely mimicked native non-inactivating currents, suggesting that non-inactivating N-type channels consist of bovine alpha(1B), alpha(2)/delta, and beta(2a).

Differential regulation of phenylethanolamine N-methyltransferase expression in two distinct subpopulations of bovine chromaffin cells.

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.

Differences in the composition of chromaffin granules in adrenaline and noradrenaline containing cells of bovine adrenal medulla.

Several constituents of chromaffin granules were quantitatively determined in noradrenaline and adrenaline cells purified from bovine adrenal medulla. As far as secretory peptides are concerned noradrenaline granules contained slightly more secretogranin II, but much less chromogranin A than adrenaline granules. This can be explained by the dependence of the biosynthesis of chromogranin A on corticosteroids. Proteolytic processing of chromogranin A and secretogranin II was higher in noradrenaline cells which was paralleled by a higher content of the prohormone convertase PC2. Noradrenaline granules also contained a higher concentration of the vesicular monoamine transporter (vMAT2). No differences were found for dopamine beta-hydroxylase, prohormone convertase PC1, carboxypeptidase H and synaptophysin. These results indicate that the secretory cocktail of peptides released from these cells differs significantly between adrenaline and noradrenaline storing cells.

Tetraethylammonium selectively stimulates secretion from noradrenergic bovine chromaffin cells.

1. The effects of tetraethylammonium chloride (TEA) on catecholamine secretion from primary cultures of noradrenaline-rich (noradrenergic) and adrenaline-rich (adrenergic) bovine chromaffin cells were studied. TEA stimulated catecholamine secretion from both cell types but was a much more effective secretory stimulus for noradrenergic cells. 2. TEA-induced catecholamine secretion was dependent on extracellular Ca2+, was partially inhibited by nifedipine and by tetrodotoxin, and was potentiated by ouabain. Other K+ channel blocking agents including 4-aminopyridine, glibenclamide, and tolbutamide did not stimulate catecholamine secretion. 3. TEA had no effect on Ca(2+)-induced secretion from digitonin-permeabilized chromaffin cells. 4. TEA presumably evokes secretion by inhibiting K+ channels, depolarizing chromaffin cells, and activating voltage-gated Ca2+ channels in the cells. Noradrenergic cells appear to be more sensitive to K+ channel inhibition than are adrenergic cells. 5. The secretory response of the chromaffin cells to TEA increased with time in culture. 6. In addition to being a more effective secretagogue in noradrenergic cells, TEA was also more effective in stimulating catecholamine synthesis in these cells.

Histamine evokes greater increases in phosphatidylinositol metabolism and catecholamine secretion in epinephrine-containing than in norepinephrine-containing chromaffin cells.

Chromaffin cells have H1 histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol-containing phospholipids and stimulates catecholamine secretion from chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP1) and catecholamine secretion in purified cultures of norepinephrine-containing and epinephrine-containing bovine chromaffin cells. Histamine-stimulated InsP1 accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP1 accumulation in the two chromaffin cell subtypes. Histamine-stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K+, bradykinin, phorbol 12,13-dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H1 receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H1 receptors on epinephrine cells may account for the greater effects of histamine on InsP1 accumulation and catecholamine secretion in these cells.

Staurosporine activates a 60,000 M(r) protein kinase in bovine chromaffin cells that phosphorylates myelin basic protein in vitro.

Bovine chromaffin cells contain a family of renaturable protein kinases. One of these, a 60,000 M(r) kinase (PK60) that phosphorylated myelin basic protein in vitro, was activated fourfold when cells were treated with the protein kinase inhibitor staurosporine. Because staurosporine inhibits protein kinase C, the role of this kinase in the regulation of PK60 activity was investigated. Fifty nanomolar staurosporine produced half-maximal inhibition of protein kinase C activity in chromaffin cells, whereas approximately 225 nM staurosporine was required to induce half-maximal activation of PK60. Other protein kinase C inhibitors, H-7 and K-252a, did not mimic the effect of staurosporine on PK60 activity. Chromaffin cells have three protein kinase C isoforms: alpha, epsilon, and zeta. Prolonged treatment with phorbol esters depleted the cells of protein kinase C alpha and epsilon, but not zeta. Neither activation nor depletion of protein kinase C affected the basal activity of PK60. Moreover, staurosporine activated PK60 in cells depleted of protein kinase C alpha and epsilon; thus, staurosporine appeared to activate PK60 by a mechanism that does not require these protein kinase C isoforms. Incubation of cell extracts with staurosporine in vitro did not activate PK60. Incubation of these extracts with adenosine 5'-O-(3-thiotriphosphate), however, caused a twofold activation of PK60. Although this suggests that PK60 activity is regulated by phosphorylation, the mechanism by which staurosporine activates PK60 is not known. Staurosporine has been reported to promote neurite outgrowth from chromaffin cells. The role of PK60 in mediating the effects of staurosporine on chromaffin cell function remains to be determined.

Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines.

BACKGROUND: Pheochromocytomas that are usually noradrenergic arise commonly in the adult rat adrenal medulla. The widely studied PC12 cell line, that is representative of these rat adrenal tumors, is also noradrenergic. The reasons for the absence of epinephrine production by most rat pheochromocytoma cells are unknown, and there are currently no adrenergic adrenal medullary cell lines. Pheochromocytomas are rare in mice. EXPERIMENTAL DESIGN: Tumors induced by polyoma virus in the adrenal medullas of postnatal mice were studied immunocytochemically for catecholamine biosynthetic enzymes in order to determine how their profiles of catecholamine production compared with those of rat pheochromocytomas. Clonal cell lines were established from a representative tumor and were evaluated for responsiveness to agents known to affect the development and function of normal and neoplastic rat chromaffin cells. RESULTS: Although adrenal medullary cells from normal rodents produce epinephrine before birth, polyoma-induced mouse adrenal tumor cells are immature or poorly differentiated. They synthesize norepinephrine, but not epinephrine, which during normal development is produced later than norepinephrine. They also produce relatively large quantities of dihydroxyphenylalanine, suggesting an abnormality of catecholamine biosynthesis such that tyrosine hydroxylase is not rate-limiting. Secretory granules are sparse, as demonstrated by electron microscopy or by staining for chromogranin A, and catecholamine stores are low. Further, the tumor cells appear to be phenotypically unstable, as judged from heterogeneous staining for tyrosine hydroxylase even in early passage, twice-cloned cell lines. Tumor cell morphology and catecholamine profiles appear to be unaffected or minimally affected by nerve growth factor, forskolin or dexamethasone, which are known to affect normal or neoplastic rat chromaffin cells. However, tumors formed after subcutaneous injection of cell lines into mice show up to a 10-fold increase in catecholamine stores, suggesting that the cells are subject to some forms of regulation. The cloned cell lines do not produce detectable polyoma virus, but express all three viral T antigens, including a characteristic, truncated form of large T. CONCLUSIONS: The findings suggest that the process of neoplastic transformation and/or the presence of polyoma virus T antigens results in suppression of the adrenergic phenotype in mouse adrenal chromaffin cells. T antigens might therefore be useful as tools for studying mechanisms that regulate the differentiation and maturation of chromaffin cells in normal and neoplastic states. Furthermore, although polyoma virus cannot be readily used to produce adrenergic cell lines from the mouse adrenal medulla, the lines that are produced might substitute for PC12 cells in some types of studies that require a mouse model.

Nicotinic agonists, phorbol esters, and growth factors activate two extracellular signal-regulated kinases, ERK1 and ERK2, in bovine chromaffin cells.

Treatment of bovine chromaffin cells with nicotinic agonists, phorbol esters, and growth factors increases protein kinase activity toward microtubule-associated protein-2 and myelin basic protein (MBP) in vitro. To characterize the kinases that are activated by these agents, we separated chromaffin cell proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels into which MBP had been incorporated, allowed the proteins to renature, and then assayed MBP kinase activity by incubating the gels with [gamma-32P]ATP. Chromaffin cells contain a family of kinases that phosphorylate MBP in vitro. Two of these kinases, of M(r) 46,000 and 42,000 (PK46 and PK42), were activated by treatment of the cells with dimethylphenylpiperazinium (DMPP), phorbol 12,13-dibutyrate (PDBu), or insulin-like growth factor I (IGF-I). Activation of PK46 and PK42 by DMPP was dependent on extracellular Ca2+, whereas the effects of PDBu and IGF-I were Ca2+ independent. Down-regulation of protein kinase C by incubation of the cells with PDBu abolished the activation of PK46 and PK42 by DMPP, PDBu, and IGF-I. Staurosporine, a protein kinase C inhibitor, prevented the activation of PK46 and PK42 by DMPP and PDBu but did not block the activation of these kinases by IGF-I. Immunoblotting experiments with antiphosphotyrosine (anti-PTyr) antibodies demonstrated that agents that increased the kinase activities of PK46 and PK42 also increased the apparent PTyr content of M(r) 46,000 and 42,000 proteins. PK46 and PK42 comigrated with proteins that reacted with antibodies against extracellular signal-regulated kinases (ERKs). Thus, PK46 and PK42 appear to be the bovine homologues of ERK1 and ERK2.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters cause preferential secretion of norepinephrine from bovine chromaffin cells.

Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13-dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine-containing cells or epinephrine-containing cells. In response to 55 mM K+, catecholamine release from norepinephrine-containing cells was two times greater than that from epinephrine-containing cells. In response to phorbol 12,13-dibutyrate, secretion from norepinephrine-containing cells was 13 times greater than that from epinephrine-containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine-containing cells.

Activation of a microtubule-associated protein-2 kinase by insulin-like growth factor-I in bovine chromaffin cells.

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.

Phosphotyrosine-containing proteins in bovine chromaffin cells: effects of insulin-like growth factor I (IGF-I).

1. Antiphosphotyrosine antibodies were used to detect phosphotyrosine-containing proteins in immunoblots of bovine chromaffin cell proteins. 2. Unstimulated cells exhibited two major phosphotyrosine-containing proteins, which had Mr's of 121,000 and 70,000. Insulin-like growth factor I (IGF-I) had little effect on the phosphotyrosine content of these two proteins but greatly increased the phosphotyrosine content of three other proteins of Mr 185,000, 170,000, and 96,000. These proteins were found predominantly in the particulate fraction of cell homogenates. 3. The effects of the IGF-I were time and concentration dependent, with maximal increases in phosphorylation occurring after 1 min of treatment with 10 nM IGF-I. Na3VO4, an inhibitor of phosphotyrosine phosphatases, potentiated the effects of IGF-I. 4. Thus, the IGF-I receptor appears to function as an IGF-I-activated protein tyrosine kinase in chromaffin cells. The tyrosine kinase activity of the IGF-I receptor presumably mediates the effects of IGF-I on chromaffin cell function.

Phosphorylation of tyrosine hydroxylase in protein kinase C-deficient PC12 cells.

In order to study the role of protein kinase C in the regulation of tyrosine hydroxylase phosphorylation in PC12 cells, the effects of various agonists on diacylglycerol accumulation in PC12 cells were measured and the ability of these agonists to increase the phosphorylation tyrosine hydroxylase in protein kinase C-deficient cells was evaluated. Bradykinin (10 microM) and elevated extracellular K+ (55 mM) increased the accumulation of [3H]diacylglycerol in PC12 cells that had been prelabeled with [3H]arachidonic acid, and so might be expected to activate protein kinase C in these cells; in contrast, nerve growth factor did not increase diacylglycerol accumulation in PC12 cells. Protein kinase C-deficient PC12 cells were prepared by incubating the cells for 24 h with 1 microM phorbol dibutyrate. This treatment resulted in the loss of approximately 90% of the protein kinase C activity in the cells. Control and protein kinase C-deficient cells were incubated with 32Pi for 90 min and then stimulated with various agonists. 32P-labeled tyrosine hydroxylase was isolated from the cells by polyacrylamide gel electrophoresis and subjected to tryptic hydrolysis. 32P-containing phosphopeptides were separated by two-dimensional thin-layer electrophoresis and chromatography, visualized by autoradiography, and quantitated by scintillation counting Treatment of control cells with phorbol dibutyrate increased the incorporation of 32P into one tryptic phosphopeptide (referred to as T3) in tyrosine hydroxylase. Phorbol dibutyrate did not increase the phosphorylation of this peptide in protein kinase C-deficient cells. Bradykinin or 55 mM K+ increased the incorporation of 32P into four tyrosine hydroxylase phosphopeptides, including peptide T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of elongation factor 2 in the rat superior cervical ganglion.

Elongation factor 2 (EF-2) and its associated kinase, Ca2+/calmodulin-dependent protein kinase III, have recently been identified as a major protein phosphorylation system in mammalian tissues. We have measured the phosphorylation of EF-2 in 32P-labeled superior cervical ganglia. Phosphorylation of EF-2 was increased by preganglionic stimulation or by treatment of the ganglion with dimethylphenylpiperazinium or veratridine. Increases in EF-2 phosphorylation presumably reflect the activation of Ca2+/calmodulin-dependent protein kinase III by nicotinic stimulation and depolarization. The phosphorylation of EF-2 may help to coordinate neuronal protein synthesis with neuronal activity.

Vasopressin stimulates the phosphorylation of an 83,000 Mr protein in the superior cervical ganglion.

1. 32P-Labeled proteins from the superior cervical ganglion of the rat were separated by two-dimensional gel electrophoresis and visualized by autoradiography. 2. The most heavily labeled phosphoprotein in the ganglion had a relative molecular weight of 83,000 and a pI of 4.5. Phosphorylation of this protein was increased by phorbol 12,13-dibutyrate, an activator of the Ca2+/phospholipid-dependent protein kinase, protein kinase C. This protein appears to be similar or identical to a specific protein kinase C substrate that has been described in other tissues (Blackshear, P. J., et al., J. Biol. Chem. 261:1459-1469, 1986). 3. Phosphorylation of this protein was also increased by treatment of the ganglion with phospholipase C (Bacillus cereus) but was not increased by 8-bromo-cyclic AMP or by nicotinic agonists. Vasopressin increased the hydrolysis of inositol-containing phospholipids in the ganglion and also increased the labeling of the 83,000 Mr protein. Thus, vasopressin appears to activate protein kinase C in the ganglion. 4. Muscarine, which also increased phospholipid metabolism in the ganglion, did not increase the phosphorylation of the 83,000 Mr protein. Muscarine and vasopressin stimulate phospholipid metabolism in different structures within the ganglion (Horwitz, J., et al., J. Pharmacol. Exp. Ther. 237:312-317, 1986). Muscarine may increase phospholipid metabolism in structures that do not contain significant amounts of the 83,000 Mr protein.

Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases.

Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.

Nicotinic and muscarinic agonists, phorbol esters, and agents which raise cyclic AMP levels phosphorylate distinct groups of proteins in the superior cervical ganglion.

The phosphorylation of proteins in the superior cervical ganglion of the rat was investigated. Ganglia were incubated with 32Pi, and the 32P-labeled proteins in the ganglion were separated by two-dimensional electrophoresis and visualized by autoradiography. Approximately 40 distinct phosphoproteins could be visualized by these methods. The most heavily labeled ganglionic protein was an acidic protein with an Mr of approximately 83,000. Tyrosine hydroxylase was identified as a doublet of two closely-migrating radioactive spots. Treatment of intact ganglia with depolarizing agents, nicotinic and muscarinic agonists, phorbol esters, and agents that increase the content of cyclic adenosine 3':5'-monophosphate in the ganglion stimulated the incorporation of 32Pi into distinct but overlapping groups of phosphoproteins. All of these agents increased the phosphorylation of tyrosine hydroxylase. In contrast, only phorbol esters and muscarinic agonists increased the phosphorylation of the 83,000 ganglionic phosphoprotein. Our data are consistent with the idea that the various classes of agonists may activate distinct protein kinases in the ganglion.