Transsynaptic Regulation of Galanin, Neurotensin, and Substance P in the Adrenal Medulla: Combinatorial Control by Second-Messenger Signaling Pathways

The adrenomedullary content of neurotensin and substance P was examined 1, 6, and 12 days after hypoglycemic shock. The neurotensin content was increased 60-fold within 24 h and remained elevated for up to 12 days, whereas the substance P content was increased approximately sevenfold within 24 h of insulin treatment and returned to control levels by 12 days poststimulation. Because protein kinase A, protein kinase C, and calcium influx in the rat adrenal medulla are all stimulated following splanchnic nerve stimulation, the differential regulation of neurotensin and substance P biosynthesis following stimulation of these three pathways was examined in bovine chromaffin cells in vitro. Neurotensin levels were up-regulated by elevated potassium, forskolin, and phorbol ester in bovine chromaffin cells. Substance P levels were up-regulated by elevated potassium and forskolin but not by phorbol ester treatment. When chromaffin cells were treated with phorbol ester in combination with forskolin, neurotensin levels were increased in a synergistic fashion, whereas phorbol ester antagonized the forskolin-induced elevation of substance P levels. Earlier, it was reported that galanin biosynthesis, like neurotensin biosynthesis, is upregulated by depolarization, phorbol ester stimulation, and forskolin treatment in chromaffin cells in vitro. Here we report that galanin is also, like neurotensin, increased greater than 60-fold after stimulation of the rat adrenal medulla in vivo. Neuropeptide-specific combinatorial effects of stimulating the calcium, protein kinase A, and protein kinase C signaling pathways may underlie the quantitative differences between galanin and neurotensin compared with substance P up-regulation in rat adrenal medulla after splanchnic nerve stimulation in vivo.     

The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and Bay K8644 and inhibited by 8-bromo-cyclic GMP

The role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release was studied using the isolated perfused rat heart. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the protein kinase C pathway, produced a dose-dependent increase in perfusate ANP immunoreactivity. Bay k8644, a putative calcium channel activator, and forskolin, which stimulates adenylate cyclase, induced a sustained increase in ANP secretory rate. TPA in combination with either Bay k8644 or forskolin induced higher ANP secretion than the calculated additive value for each agent. 8-bromo-cyclic GMP and sodium nitroprusside, when given alone, had no effect on ANP secretion, but delayed the TPA-stimulated increase in perfusate ANP. ANP secretion appears therefore to be mediated both by the phosphoinositide and the cAMP system, whereas the cGMP pathway may be inhibitory.     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.     

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.     

Generation and initial characterization of conditionally immortalized chromaffin cells

Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.     

Inhibition of atrial natriuretic peptide secretion by forskolin in noncontracting cultured atrial myocytes

Secretory rates for immunoreactive atrial natriuretic peptide (ANP) by 7 - 8 day-old primary cultures of atrial myocytes from adult rats (with myocyte contraction inhibited by tetrodotoxin (TTX)) were (a) constant for at least two hours, and (b) significantly slowed by forskolin (1, 5, and 25 microM), dibutyryl cyclic adenosine monophosphate (1 mM), or isobutylmethylxanthine (100 microM). The substantial rates of ANP secretion which persisted in cells rendered noncontracting either by inhibiting Ca2+ influx via reduction of external [Ca2+] to less than 10(-7) M or by inhibiting sarcoplasmic reticulum Ca2+ release with 100 microM ryanodine were significantly slowed by 25 microM forskolin, but forskolin sensitivity was lost by cells exposed simultaneously to external Ca2+ concentration of less than 10(-7) M and 100 microM ryanodine. Quiescent myocytes whose ANP secretory rate was depressed by forskolin remained responsive to secretory stimulation by phorbol ester.     

Secretion of [Met]Enkephalyl-Arg6-Phe7-Related Peptides and Catecholamines from Bovine Adrenal Chromaffin Cells: Modification by Changes in Cyclic AMP and by Treatment with Reserpine

Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.     

Enhancement of Ca2+-induced catecholamine release by the phorbol ester TPA in digitonin-permeabilized cultured bovine adrenal chromaffin cells

The phorbol ester, 4 beta-phorbol 12-myristate acetate (TPA), increased the extent of catecholamine release induced by Ca2+, without affecting the basal release response in digitonin-permeabilized chromaffin cells. This finding is consistent with the hypothesis that protein kinase C has a role to play in stimulus-secretion coupling in the bovine adrenal medullary chromaffin cell.     

Differential regulation of natriuretic peptide biosynthesis in bovine adrenal chromaffin cells

Chromaffin cells synthesize and secrete two forms of natriuretic peptides which are also found in the heart and in the central nervous system. While atrial tissue predominantly contains atrial natriuretic factor (ANF), brain tissue appears to produce relatively larger amounts of brain natriuretic peptide (BNP) also identified as aldosterone secretion inhibitory factor (ASIF), suggesting tissue-specific differential regulation of these two peptides. This report compares the modulation of the biosynthesis and secretion of ASIF with that of ANF using cultured chromaffin cells as a model system. Cholinergic nicotinic activation and KCl depolarization induce a 5-fold increase of the corelease of ASIF and pro-ASIF in cell culture medium concomitantly with a 3-fold stimulation of ANF and pro-ANF cosecretion. While the combined treatment with phorbol ester and forskolin produces a 2-fold increase in total ANF level, it induces a synergistic 20-fold elevation of total ASIF level. These results indicate that chromaffin cell secretagogues induce the cosecretion of both the precursor and mature forms of ASIF and ANF. The preferential stimulation of ASIF production is revealed by the combined treatment rendering the ASIF to ANF proportion similar to that in brain.     

Regulation of chromaffin cell secretion and protein kinase C activity by chronic phorbol ester treatment

Bovine adrenal chromaffin cells were exposed to phorbol esters to determine the effects of reduced levels of protein kinase C on secretion of hormones. Treatment with active phorbol esters such as 4 beta-phorbol 12, 13-didecanoate (PDD) reduced levels of protein kinase C activity with a maximal 80-90% reduction in activity after 16-24 h treatment (greater than or equal to 500 nM PDD). Treatment with PDD also inhibited catecholamine secretion from chromaffin cells evoked by nicotine, barium, and scorpion venom (50-70%, t1/2 approximately 6 h) and by veratridine (80%, t1/2 less than 15 min). Secretion induced by these agents in phorbol ester-treated cells returned to that of untreated cells by 3-4 days despite no recovery of protein kinase C activity. Potassium-evoked secretion was not inhibited by phorbol ester treatment. Catecholamine secretion from digitonin-permeabilized cells was more sensitive to calcium between 1 and 24 h, but not greater than or equal to 48 h, after addition of phorbol ester. The results suggest that phorbol esters inhibit secretion by activation of protein kinase C resulting in inhibition of ion channels or receptors but not of the secretory machinery itself; hence, protein kinase C may usually machinery itself; hence, protein kinase C may usually attenuate secretory responses in the adrenal chromaffin cell.     

Differential Regulation of Phenylethanolamine N-Methyltransferase Expression in Two Distinct Subpopulations of Bovine Chromaffin Cells

Abstract: 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 ～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.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels ∼2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10⁻⁷ M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.     

Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester

Rat brain neuropeptide Y precursor (prepro-NPY) cDNA clones were isolated and sequenced in order to study regulation of the prepro-NPY gene. Rat prepro-NPY (98 amino acid residues) contains a 36-residue NPY sequence, followed by a proteolysis/amidation site Gly-Lys-Arg, followed by a 30-residue COOH-terminal sequence. The strong evolutionary conservation of rat and human sequences of NPY (100%) and COOH-terminal peptide (93%) suggests that both peptides have important biological functions. In the rat central nervous system, prepro-NPY mRNA (800 bases) is most abundant in the striatum and cortex and moderately abundant in the hippocampus, hypothalamus, and spinal cord. The rat adrenal, spleen, heart, and lung have significant levels of prepro-NPY mRNA. Regulation of the prepro-NPY mRNA abundance was studied in several rodent neural cell lines. PC12 rat pheochromocytoma and N18TG-2 mouse neuroblastoma cells possess low basal levels of prepro-NPY mRNA, while NG108-15 hybrid cells possess high levels. Treatment of PC12 cells with a glucocorticoid such as dexamethasone or elevation of cAMP by forskolin increased the prepro-NPY mRNA level 2-3-fold or 3-10-fold, respectively. In N18TG-2 cells dexamethasone and forskolin synergistically increased prepro-NPY mRNA 7-fold. Treatment of PC12 cells with the protein kinase C activator phorbol 12-myristate 13-acetate alone elevated prepro-NPY mRNA marginally, but the phorbol ester plus forskolin elicited 20-70-fold increases, which were further enhanced to over 200-fold by dexamethasone and the calcium ionophore A23187. These results indicate that NPY gene expression can be positively regulated by synergistic actions of glucocorticoids, cAMP elevation, and protein kinase C activation.     

The phorbol ester TPA enhances A23187--but not carbachol- and high K+-induced catecholamine secretion from cultured bovine adrenal chromaffin cells

The effect of the phorbol ester TPA on catecholamine secretion was studied in cultured bovine adrenal chromaffin cells. The pretreatment of chromaffin cells with TPA caused the enhancement of catecholamine secretion induced by the calcium ionophore, A23187. By contrast, neither carbachol- nor high K+-induced secretion was changed by TPA pretreatment. These results support the concept that protein kinase C plays an important role as a factor transducing the Ca2+ signal to the exocytotic process of catecholamine secretion in bovine adrenal chromaffin cells.     

Long-Term Activation of Protein Kinase C by Nicotine in Bovine Adrenal Chromaffin Cells

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells

We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen-activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin-induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells.