Vasoactive intestinal polypeptide induces tyrosine hydroxylase in PC12 cells.

Physiological stress induces tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, via trans-synaptic mechanisms within the adrenal medulla. Previous studies have implicated cAMP as a second messenger capable of inducing tyrosine hydroxylase; however, it is unclear whether any receptor coupled to adenylate cyclase mediates tyrosine hydroxylase induction. Recently, vasoactive intestinal polypeptide, whose receptor is coupled to adenylate cyclase in many tissues, has been shown to meet many of the criteria for a neuromodulator within the adrenal medulla. We therefore undertook a series of studies to determine whether vasoactive intestinal polypeptide may induce tyrosine hydroxylase in PC12 cells, a cell line derived from rat adrenal medulla. Here we report that vasoactive intestinal polypeptide produces a transient, time- and concentration-dependent increase in tyrosine hydroxylase mRNA levels which is followed by a stable increase in tyrosine hydroxylase protein. The increase in tyrosine hydroxylase mRNA does not occur in a mutant PC12 cell line deficient in cAMP-dependent protein kinase activity, indicating that the effect of vasoactive intestinal polypeptide is mediated through the cAMP second messenger pathway. This is the first report demonstrating that a neuromodulator which acts on an adenylate cyclase-coupled receptor can induce tyrosine hydroxylase.     

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 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.     

Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase. Temporal correlation of acetylcholine's effect on site phosphorylation, enzyme activation, and catecholamine synthesis

Tryptic peptide fragments of tyrosine hydroxylase isolated from 32PO4-prelabeled bovine adrenal chromaffin cells are resolved into seven phosphopeptides by reverse phase-high performance liquid chromatography. All seven of the peptides are phosphorylated on serine residues. Three of these putative phosphorylation sites, peptides 3, 5, and 6, are rapidly phosphorylated (5-fold in 15 s) by both acetylcholine stimulation and potassium depolarization of the cells, and this phosphorylation is accompanied by a similarly rapid activation of the enzyme. Both phosphorylation and activation are transient and do not account for the prolonged increase in catecholamine biosynthesis produced by these stimuli. Peptides 4 and 7 show a much slower and sustained increase in phosphorylation (3-fold in 4 min) in response to acetylcholine and potassium. Phosphorylation of these peptides correlates with the sustained increase in catecholamine biosynthesis rather than enzyme activation. Peptides 1 and 2 are not stimulated by any agonist yet employed and thus show no relation to enzyme activation or catecholamine biosynthesis. Phosphorylation of all five peptides by acetylcholine or potassium is calcium-dependent. In contrast to the stimulation of phosphorylation of tyrosine hydroxylase on multiple sites, forskolin stimulates the phosphorylation of only peptide 6, and this is accompanied by a coordinated activation of tyrosine hydroxylase and increased catecholamine biosynthesis. These findings show that the phosphorylation of tyrosine hydroxylase in intact cells is more complex than predicted from in vitro results, that at least two protein kinases are involved in the secretagogue-induced phosphorylation of tyrosine hydroxylase, and that the regulation of catecholamine biosynthesis, in response to phosphorylation, appears to involve both tyrosine hydroxylase activation and other mechanisms.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), the 38- and 27-amino-acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100- to 1,000-fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP-selective type I receptor. PACAP38 elicited a sustained seven- to ninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro-NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro-NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.     

Effects of Peptides of the Secretin-Glucagon Family and Cyclic Nucleotides on Tyrosine Hydroxylase Activity in Sympathetic Nerve Endings

Previous studies have shown that certain peptides of the secretin-glucagon family stimulate tyrosine hydroxylase activity in sympathetic neurons of the superior cervical ganglion and three of its end organs, i.e., the iris, pineal gland, and submaxillary gland. To determine whether a similar regulation occurs in other sympathetic neurons, the effects of two of these peptides, secretin and vasoactive intestinal peptide, were examined in the right cardiac ventricle of the rat, a tissue innervated primarily by the middle and inferior cervical ganglia. Both peptides stimulated tyrosine hydroxylase activity, measured in situ, in this tissue. In addition, several second messenger systems were investigated as possible mediators of this peptidergic stimulation of tyrosine hydroxylase activity in autonomic end organs. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin elevated tyrosine hydroxylase activity in slices of both the right ventricle and the submaxillary gland. 8-Bromoguanosine 3',5'-cyclic monophosphate also stimulated tyrosine hydroxylase activity in both tissues, whereas nitroprusside stimulated activity only in the submaxillary slices. Furthermore, the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and/or Ro 20-1724 potentiated the stimulation by secretin, as well as the stimulations by forskolin and nitroprusside. Phorbol 12,13-dibutyrate also stimulated tyrosine hydroxylase activity in cardiac and submaxillary slices; however, no potentiation of these effects was seen following addition of either phosphodiesterase inhibitor. These data, taken together with those of previous studies, suggest a role for a cyclic nucleotide, probably adenosine 3',5'-cyclic monophosphate, in the peptidergic stimulation of tyrosine hydroxylase activity in sympathetic nerve terminals.     

Acute stimulation of ganglionic tyrosine hydroxylase activity by secretin,VIP and PHI

We have examined the ability of a number of neuropeptides to increase tyrosine hydroxylase (TH) activity in the superior cervical ganglion in vitro. Secretin and vasoactive intestinal peptide (VIP) both increased TH activity, whereas angiotensin II, bombesin, bradykinin, cholecystokinin octapeptide, insulin, luteinizing hormone-releasing hormone, [D-Ala², Met³]enkephalinamide, motilin, neurotensin, somatostatin, and substance P produced no effects. Secretin and VIP increased TH activity with an EC₅₀ of 5 nM and 0.5 μM, respectively. The effects of these peptides were not altered by prior decentralization of the ganglia, by addition of hexamethonium (3 mM) and atropine (6 μM), or by lowering the concentration of calcium in the medium to 0.1 mM. Addition of carbachol (3 μM) potentiated the effects of both secretin and VIP on TH activity. Several gastrointestinal peptides with structural similarities to secretin and VIP were examined for their ability to increase TH activity. Glucagon, gastric inhibitory peptide and human pancreatic tumor growth hormone-releasing factor produced no effect at a concentration of 10 μM, while PHI increased enzyme activity.     

The influence of localized chemical modifications of the basic pancreatic trypsin inhibitor on static and dynamic aspects of the molecular conformation in solution.

Porcine vasoactive intestinal peptide stimulated adenosine 3':5'-monophosphate (cyclic AMP) production in rat intestinal epithelial cells. The stimulation was dependent on time and temperature and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Under optimal conditions (at 15 degrees C, with 0.2 mM 3-isobutyl-1-methylaxanthine, at a cell concentration up to 18 microgram DNA/ml), the cyclic AMP production produced by vasoactive intestinal peptide was constant for 10 min and stopped after 15 min incubation, at either low (1 nM) or high (30 nM) concentration of the peptide. This plateau effect was demonstrated not to be due to an inactivation of vasoactive intestinal peptide in the medium nor to an alteration of receptors for the peptide. Cyclic AMP production was sensitive to a concentration as low as 0.1 nM vasoactive intestinal peptide. Maximal stimulation of cyclic AMP levels by vasoactive intestinal peptide was observed with 30 nM vasoactive intestinal peptide and represented an 11-fold increased above basal. The dorse-response curve was monophasic with a Km of 2.3 x 10(-9) M. No cooperative effects were detected by Hill analysis. The positive non-linear relationship observed between stimulation of cyclic AMP production and occupancy of binding site was not time-dependent as indicated by experiments performed after 15, 45 and 120 min incubation. Maximal and half-maximal responses were obtained at about 70% and 7% occupation of binding sites, respectively. Chicken vasoactive intestinal peptide and porcine secretin were agonists of porcine vasoactive intestinal peptide with a 6-times and a 120-times lower potency, respectively. Among secretin analogs that were found to have low affinity for vasoactive intestinal peptide binding sites, [4-alanine, 5-valine]secretin, that resembles vasoactive intestinal peptide at the first seven amino acids at the N-terminal end, was a partial agonist of vasoactive peptide at the first seven amino acids at the N-terminal end, was a partial agonist of vasoactive intestinal peptide and others failed to stimulate cyclic AMP production. Glucagon (10microM), gastric inhibitory peptide (0.1 microM), substance, P, neurotensin, octapeptide of cholecystokinin, bovine pancreatic polypeptide, human gastrin I with leucine at residue 15, Leu-enkephalinand somatostatin (1 microM) did not alter cyclicAMP levels. Non-peptide mediators such as dopamine, serotonin, acetylcholine and histamine, tested at 10 microM, were also ineffective. Prostaglandins E2, E1 and isoproterenol, tested at 10 microM, induced an increase of cyclic AMP levels above basal but were 9.5, 13.7 and 17.5 times less efficient than vasoactive intestinal peptide, respectively. Thus vasoactive intestinal peptide is a unique stimulus of cyclic AMP production in rat intestinal epithelial cells.     

Synergistic Interaction of Muscarinic and Opioid Receptors with Gs-Linked Neurotransmitter Receptors to Stimulate Adenylyl Cyclase Activity of Rat Olfactory Bulb

We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with /-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal a0denylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect ¹²⁵ICRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5′-O-(3′-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarinic and opioid effects. Thus, the synergistic interaction appears to result from opioid- and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors.     

Vasoactive intestinal polypeptide and acetylcholine coexist with neuropeptide Y,dopamine-β-hydroxylase,tyrosine hydroxylase,substance P or calcitonin gene-related peptide in neuronal subpopulations in cranial parasympathetic ganglia of rat

Summary Immunohistochemistry has been used to demonstrate that neuropeptide Y, dopamine--hydroxylase, calcitonin gene-related peptide or substance P are colocalized with vasoactive intestinal polypeptide and choline acetyltransferase in subpopulations of neurons in cranial parasympathetic ganglia of rat. These comprise the ciliary, sphenopalatine, otic, glossopharyngeal-vagal and internal carotid ganglia. In the ciliary and glossopharyngeal-vagal ganglia tyrosine hydroxylase is also found in such neurons. The findings emphasize that the combined localization of dopamine--hydroxylase and neuropeptide Y or the presence of tyrosine hydroxylase is not exclusively a marker for peripheral adrenergic neurons. Further, the co-localization of calcitonin gene-related peptide and substance P is not a decisive indication that a neuron is sensory in nature. It is discussed whether the presence of the enzymes and peptides other than vasoactive intestinal polypeptide is a remnant of a different expresion during ontogenesis or indicates target-specific functions in the adult.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the presence of cholinergic muscarinic receptors negatively linked to adenylate cyclase in the iris-ciliary body

Adenylate cyclase activity can be stimulated in the rabbit iris-ciliary body directly by forskolin or through receptor-mediated mechanisms by vasoactive intestinal peptide (VIP) and the β-adrenoreceptor agonists isoproterenol and salbutamol. Increases in the level of c-AMP observed following application of forskolin, isoproterenol and VIP are decreased by carbachol in a dose-dependent manner. The carbachol response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophyline suggesting the involvement of a Gi-protein. Carbachol attenuation of elevated c-AMP levels can be inhibited by the muscarinic antagonist atropine but not by the specific muscarinic receptor antagonist pirenzepine. This is in contrast to carbachol stimulation of inositol phosphate accumulation, where both atropine and pirenzepine inhibit the muscarinic response. Thus there exist two distinct muscarinic receptors in the iris-ciliary body, one linked to adenylate cyclase and the other to the hydrolysis of phosphoinositides.     

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad,Bufo marinus

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.     

Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.     

The distribution of putative neurotransmitters in the nervous system of the dipteran Chironomus tentans insect larva: An immunohistochemical study using antisera to 5-hydroxytryptamine,tyrosine hydroxylase,methionine-enkephalin,proctolin and bombesin

Using the indirect immunofluorescence technique, the localization and distribution of transmitters, transmitter-related enzymes and neuropeptides was studied in the larvae of the dipteran species Chironomus tentans. Immunoreactivity could be seen for 5-hydroxytryptamine, tyrosine hydroxylase (the rate-limiting enzyme in the catecholamine synthesis), and the neuropeptides methionine-enkephalin (met-enk), proctolin and bombesin. The immunoreactivity was confined both to cell bodies as well as to nerve fibers within ganglia and along the alimentary canal. Furthermore, tyrosine hydroxylase immunoreactivity could also be seen in epithelial cells locally distributed along a short, middle part of the alimentary tract. These latter cells were regarded as endocrine-like cells. No immunoreactivity could be found with certainty for the enzyme phenylethanolamine-N-methyltransferase (PNMT) nor for the peptides vasoactive intestinal polypeptide (VIP), dynorphin, substance P, somatostatin, thyrotropin releasing hormone (TRH), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), neurotensin, galanin and cholecystokinin (CCK).     

Nitric oxide synthase in guinea pig sympathetic ganglia: Correlation with tyrosine hydroxylase and neuropeptides

Nitric oxide synthase (NOS) has previously been reported in a small population of postganglionic sympathetic neurons in the guinea pig. The present study of paravertebral ganglia and the inferior mesenteric ganglion aimed to classify these neurons according to their content of neuropeptides (calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide) and the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, by means of immunohistochemical and histochemical double-labelling techniques. NOS-containing neurons belonged to the non-catecholaminergic population of postganglionic neurons, and partial coexistence was found with neuropeptide Y and vasoactive intestinal peptide immunoreactivities but not with calcitonin gene-related peptide. However, most of the NOS-containing neurons contained none of the neuropeptides, thus representing a hitherto unrecognized population of postganglionic neurons. The findings show that NOS is localized to small but neurochemically highly specific populations of postganglionic neurons, which most likely reflects an association with target- and function-specific pathways.     

Vasoactive Intestinal Peptide Stimulates Catecholamine Biosynthesis in Isolated Adrenal Chromaffin Cells: Evidence for a Cyclic AMP-Dependent Phosphorylation and Activation of Tyrosine Hydroxylase

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.     

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]P_i 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 μM veratridine producing a 5-fold increase in ³²P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 μM), 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.     

Muscarinic Receptors in Chromaffin Cell Cultures Mediate Enhanced Phospholipid Labeling but Not Catecholamine Secretion

Abstract: The addition of either carbachol or muscarinic agonists to cultured bovine adrenal chromaffin cells results in a selective stimulation of phosphatidate (PhA) and phosphatidylinositol (PhI) labeling from ³²Pⁱ and [³H]glycerol that can be inhibited by the inclusion of atropine, but not d -tubocurarine. In contrast, increased catecholamine secretion is observed on the addition of carbachol or nicotinic agonists and is inhibited by d -tubocurarine but not by atropine. Added calcium is essential for catecholamine secretion but not for stimulated phospholipid labeling. Chelation of endogenous Ca²⁺ with EGTA does, however, inhibit the stimulated phospholipid labeling. These results suggest that stimulated phospholipid labeling in the bovine chromaffin cell and catecholamine secretion are separate and distinct processes.