Robert L. Perlman

Glucocorticoids Increase Catecholamine Synthesis and Storage in PC 12 Pheochromocytoma Cell Cultures

Abstract: Glucocorticoids, cholera toxin and high plating density all increase the activity of tyrosine 3‐monooxygenase (TH) in cultured PC12 pheochromocytoma cells. Glucocorticoids increase enzyme activity in cells treated with cholera toxin and in cells grown at high plating density. Glucocorticoids also increase the content of stored catecholamines in the cells. In cells cultured under routine conditions, glucocorticoids primarily increase the stores of dopamine. The addition of ascorbate to the culture medium increases the storage of norepinephrine in both steroid‐treated and untreated cells. Incubation of the cells in media containing 56 nM K+ causes the release of the same percentage of stored dopamine from steroid‐treated as from untreated cells. Steroid‐treated cells contain more dopamine than do untreated cells and therefore, in response to high K+, the steroid‐treated cells secrete more dopamine than do untreated cells. We conclude that the activity of tyrosine 3‐monooxygenase in PC12 cells can be regulated by several distinct mechanisms; that glucocorticoids cause a coordinate increase in TH activity and in catecholamine storage; that steroids increase the storage of catecholamines in a releasable pool; and that the steroid‐induced increase in catecholamine storage may result in increased secretion of catecholamines from steroid‐treated cells.

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

SummaryNormal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.

Measurement of inositol phospholipid metabolism in PC12 pheochromocytoma cells

Publisher Summary PC12 cells are a clonal cell line derived from a rat pheochromocytoma. These cells are widely used as models for both adrenal chromaffin cells and sympathetic neurons. When PC 12 cells are grown in the absence of nerve growth factor (NGF), they appear as small, round, undifferentiated cells. In the presence of NGF, the cells stop dividing, enlarge, and extend long neuronal-like processes. NGF also increases the number of muscarinic cholinergic receptors on PC12 cells. Muscarinic agonists stimulate the metabolism of inositol-containing phospholipids in PC12 cells. The ability to grow large amounts of a homogeneous population of cells, to manipulate the properties of the cells by changing the extracellular environment, and to stimulate phospholipid metabolism with specific agonists provides valuable opportunities for studying neuronal phospholipid metabolism in PC12 cells. The chapter describes the growth PC12 cells.

Purification of adrenal medullary chromaffin cells by density gradient centrifugation

Abstract A method has been developed for the isolation of chromaffin cells from guinea pig adrenal glands. Crude suspensions of adrenal cells are prepared by the digestion of adrenal glands with collagenase. Chromaffin cells are then purified from these crude suspensions by isopycnic centrifugation through a 5–25% (w/v) gradient of metrizamide. More than 90% of the cells in the preparation are viable chromaffin cells, as judged by trypan blue exclusion and fluorescence histochemistry. A more convenient method for monitoring the purity of chromaffin cell preparations, using neutral red staining, is also described. Chromaffin cells are the primary neutral red staining cell in the adrenal gland, and more than 90% of the purified chromaffin cells stain with this dye. Purified chromaffin cells contain 400 ± 50 nmol of epinephrine/mg protein (133 ± 16 nmol/10 6 cells), and secrete epinephrine in response to acetylcholine. At a concentration of 100 μM, acetylcholine causes a 10- to 20-fold increase in the secretion of epinephrine from the cells. The method described in this paper is a useful procedure for the preparation of pure, functional chromaffin cells.

Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System

Abstract: The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomie tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.

Catecholamine release from the adrenal medulla

Chromaffin cells in the adrenal medulla are specialized for the synthesis, storage, and secretion of catecholamines. These cells are innervated by preganglionic sympathetic neurons in the splanchnic nerves, and, because of their unique blood supply, are exposed to unusually high concentrations of glucocorticoids in the venous drainage from the adrenal cortex. Splanchnic nerve stimulation appears to be the most important determinant of adrenomedullary function. Chromaffin cells synthesize catecholamines from tyrosine. Splanchnic nerve stimulation leads to an increase in the activity of several of the catecholamine biosynthetic enzymes, and to an increase in the rate of catecholamine biosynthesis. Glucocorticoids cause the induction of the enzyme noradrenaline N-methyltransferase, and so are particularly important for the synthesis of epinephrine. Catecholamines are stored, together with ATP, Ca2+, and protein, in secretory vesicles known as chromaffin granules. Splanchnic nerve stimulation is the physiological stimulus for catecholamine secretion. Stimulation of the splanchnic nerves results in the release of ACh from nerve endings in the adrenal medulla. ACh causes an increase in the permeability of the chromaffin cells to Ca2+, and thereby leads to the entry of Ca2+ into the cells. Ca2+ then causes the secretion of catecholamines and of other chromaffin granule constituents from the chromaffin cells by exocytosis. The biochemical mechanisms of exocytosis, and the mechanism by which Ca2+ stimulates this process, are still unknown.

Phorbol 12,13-Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat

Abstract: Phorbol 12, 13‐dibutyrate (PDBu) increased the production of 3,4‐dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half‐maximal at a concentration of approximately 0.1 μM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+‐free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8‐Br‐cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 μM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion.

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [32P]Pi and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of 32P into tyrosine hydroxylase, with 50 microM veratridine producing a 5-fold increase in 32P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 microM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca2+. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of 32P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.

CATECHOLAMINE SECRETION BY HAMSTER ADRENAL CELLS

Abstract— Suspensions of isolated adrenal cells were prepared by digesting hamster adrenal glands with collagenase, and the secretion of catecholamine from these cells was studied. Acetylcholine (ACh) produces a dose‐dependent increase in catecholamine secretion; half‐maximal secretion is produced by 3 μm‐ACh, and maximal secretion by 100 μm‐ACh. The cholinergic receptor in these cells appears to be nicotinic, since catecholamine secretion is stimulated by the nicotinic agonists nicotine and dimeth‐ylphenylpiperaziniurn, but not by the muscarinic agonists pilocarpine or oxotremorine. ACh‐induced catecholamine secretion is inhibited by hexamethonium, tubocurarine, and atropine, but is not inhibited by α‐bungarotoxin. ACh‐induced catecholamine secretion is dependent upon the presence of extracellular Ca2+, and appears to occur by exocytosis, since the release of catecholamine is accompanied by the release of dopamine β‐monooxygenase, but not of lactate dehydrogenase. These biochemical studies complement the morphological evidence for exocytosis in hamster adrenal glands, and indicate that catecholamine secretion from hamster chromaffin cells is similar to that from chromaffin cells of other species.

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.