Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells

Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC₅₀ of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC₅₀, 11 μM) and dehydroascorbate (EC₅₀, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote-olysis of the purified enzyme as determined by sodium do-decyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Glucocorticoids Increase Tyrosine Hydroxylase Activity in Cultured Murine Neuroblastoma

Cyclic AMP and glucocorticoids appear to have a role in regulating the activity of tyrosine hydroxylase (TH), as well as the expression of "morphological differentiation" in murine neuroblastoma. Monolayer cultures of C-1300 murine neuroblastoma (clone NBP2) were treated with the following compounds in ethanol: dexamethasone, triamcinolone acetonide, hydrocortisone, cortexolone, androstenedione, testosterone, estradiol-17 beta; or with the phosphodiesterase inhibitor Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone]. Treatment with either 200 micrograms/ml Ro20-1724 or 50 micrograms/ml dexamethasone produced significant increases in TH activity compared to alcohol controls (1.44 vs. 0.82 nmol 14CO2/mg protein/hr compared to 0.095). Triamcinolone acetonide or hydrocortisone also produced smaller, but significant, increases in TH activity compared to dexamethasone. When steroid activities were compared at 25 microM concentration and after 60 min of incubation (to maximize TH activity), triamcinolone acetonide was not as effective (62%) as dexamethasone. The relatively inactive glucocorticoid cortexolone produced a slight but significant increase, while the androgens androstenedione and testosterone and the estrogen estradiol-17 beta were without effect.     

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.     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.     

Regulation of Depolarization-Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP-Dependent Enhancement of Release from PC12 Cells

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.     

Effect of Carbachol and 56 mM-Potassium Chloride on the Cyclic AMP-Mediated Induction of Tyrosine Hydroxylase in Neuroblastoma Cells in Culture

Abstract: Tyrosine hydroxylase (TH) activity is increased two- to threefold in neuroblastoma cell line NBP² maintained in culture for 48 h in the presence of either the inhibitor of cyclic AMP-phosphodiesterase (PDE), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20- 1724), or the activator of adenylate cyclase, prostaglandin E₁ (PGE₁). Cyclic AMP levels are elevated 70–80% and 30–40% throughout the 48-h treatment with RO 20-1724 and PGE₁, respectively. Carbachol does not affect either basal TH activity or cyclic AMP levels in the cells. However, the cholinergic agonist delays the induction of TH elicited by either RO 20-1724 or PGE₁. This delay is prevented by atropine. The elevation in cyclic AMP levels elicited by either RO 20-1724 or PGE₁ is blocked for 1 h or 15 min. respectively, after treatment with carbachol. Cyclic AMP levels then begin to rise until they reach those levels observed in the presence of RO 20-1724 or PGE₁ alone by 12 h or 1 h of treatment, respectively. Time course studies demonstrate that this transient inhibition of the elevation of cyclic AMP is associated with a 48-h delay in the induction of TH elicited by either RO 20-1724 or PGE₁. In contrast, the induction elicited by 8-bromo cyclic AMP is unaffected by carbachol. A depolarizing concentration (56 mM) of KCl produces a 24-h delay in the induction of TH elicited by RO 20-1724, without affecting the concomitant elevation of cyclic AMP produced by the PDE inhibitor. Furthermore, 56 mM-KCl inhibits the induction of TH elicited by 8-bromo cyclic AMP. It thus appears that carbachol delays the induction of TH by transiently inhibiting the elevation of cyclic AMP, whereas potassium depolarization delays the induction of TH by inhibiting a process with a site of action that is distal to the elevation of cyclic AMP.     

Vasoactive Intestinal Polypeptide-Related Peptides Modulate Tyrosine Hydroxylase Gene Expression in PC12 Cells Through Multiple Adenylate Cyclase-Coupled Receptors

Abstract: We investigated the receptor mechanisms by which vasoactive intestinal polypeptide (VIP) and related peptides exert their effects on tyrosine hydroxylase (TH) gene expression. VIP, secretin, and peptide histidine isoleucine (PHI) each produced increases in TH gene expression, as measured by increases in TH mRNA levels and TH activity. The concentrations at which the effects of these peptides were maximal differed for TH activity and TH mRNA. Moreover, maximal increases in TH activity were 130-140% of control, whereas maximal increases in TH mRNA were 250% of control. The concentration dependence of the increases in TH mRNA in response to the three peptides was analyzed by fitting the data to nonlinear regression models that assume either one or two components to the response. The data for secretin fit best to a model that assumes a single component to the increase in TH mRNA levels. The data derived for PHI and VIP fit best to models that assumed two components to the TH mRNA response. These data suggested that there may be more than one receptor or signal transduction mechanism involved in the response to the various peptides. We examined whether the peptides exerted their effects through common or multiple second messenger systems. The ability of maximally active concentrations of these peptides to stimulate increases in TH mRNA was not additive, indicating that the peptides work through a common receptor or signal transduction pathway. Each peptide stimulated increases in protein kinase A (PKA) activity. Secretin and VIP were ineffective in increasing TH mRNA levels in a PKA-deficient mutant PC12 cell line (A 126-1B2). Moreover, the adenylate cyclase antagonist 2′,5′-dideoxyadenosine prevented the increase in TH mRNA produced by each peptide. Thus, each peptide requires an intact cyclic AMP second messenger pathway to produce changes in TH gene expression, suggesting that the complex pattern of response to VIP and PHI revealed by concentration-response analysis was due to the actions of these peptides at multiple receptors. To evaluate this possibility, we examined the effect of several peptide receptor antagonists on the increase in TH gene expression elicited by VIP, PHI, and secretin. The secretin antagonist secretin (5–27) (20 μM) had no significant effect on VIP or PHI stimulation of TH gene expression, but reduced the effect of secretin. The VIP antagonist VIP (10–28) (20 μM) reduced the effect of VIP on increasing TH mRNA, but had no significant effect on the response of TH mRNA to secretin or PHI. Interestingly, the VIP antagonist [Ac-Tyr¹,D-Phe²]-growth hormone releasing factor [GRF(1–29)] amide (20 μM) potentiated the effect of VIP on elevating TH mRNA levels, but had no effect on secretin-stimulated TH mRNA induction. To determine whether this response was mediated through the cyclic AMP pathway, we examined the effects of the VIP antagonist [Ac-Tyr¹,D-Phe²]-GRF(1–29) amide on VIP stimulation of PKA activity. Although the antagonist had no effect alone, it enhanced stimulation of PKA activity by VIP. Taken together, these findings indicate that VIP and secretin stimulate TH mRNA through different adenylate cyclase-linked receptors and that a second VIP receptor may modulate TH induction by inhibiting VIP stimulation of PKA.     

Insulin-Like Growth Factor-I Enhances Tyrosine Hydroxylase Activation in Bovine Chromaffin Cells

Previous studies have shown that insulin-like growth factor-I (IGF-I) enhances secretagogue-stimulated Ca2+ uptake and catecholamine release in bovine chromaffin cells. This report describes the effect of IGF-I on the activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2), the major regulatory enzyme in the pathway of catecholamine biosynthesis. Tyrosine hydroxylase activity was assayed by measuring 3,4-dihydroxyphenylalanine (Dopa) accumulation in the presence of brocresine, an inhibitor of Dopa decarboxylase. Chromaffin cells cultured in serum-free medium produced approximately 40% less Dopa when stimulated by 55 mM K+ than did cells that had been cultured in the presence of serum. Incubation of cells for 3 days in serum-free medium containing 10 nM IGF-I restored high K(+)-stimulated Dopa accumulation to a level comparable to that seen in cells cultured continuously in serum-containing medium. In eight experiments, IGF-I increased high K(+)-stimulated Dopa accumulation (expressed as picomoles per minute per milligram of protein) by 96 +/- 13%. IGF-I increased the protein content of chromaffin cells by approximately 30%; consequently, its effect on tyrosine hydroxylase activity was even greater when Dopa synthesis was expressed as picomoles per minute per 10(7) cells. IGF-I also enhanced the rate of Dopa accumulation in cells stimulated by dimethylphenylpiperazinium, 8-bromo-cyclic AMP, phorbol 12,13-dibutyrate, or Ba2+. The effect of IGF-I on high K(+)-stimulated tyrosine hydroxylase activity was measurable when enzyme activity was assayed in vitro, suggesting that this effect was due to a stable modification of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

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.     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α -methyl- p -tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.