Reserpine Increases Chromaffin Cell Enkephalin Stores Without a Concomitant Decrease in Other Proenkephalin-Derived Peptides

Reserpine increases the levels of enkephalins in adrenal medullary chromaffin cells; however, the origin of the newly apparent pentapeptides has been the subject of debate, because no increase in the levels of proenkephalin mRNA has been observed. The present study was performed for determining if the reserpine-induced increase in context of enkephalins was derived from processing of preexisting fragments of proenkephalin. Bovine chromaffin cell enkephalins and larger enkephalin-containing peptides were separated by reversed-phase HPLC and identified by approximate molecular weight, elution with peptide standards, and enkephalin sequences contained. Treatment of the cells with reserpine increased the levels of enkephalins and of enkephalin-containing peptides of up to approximately 3 kilo-daltons without reducing the levels of larger enkephalin-containing peptides. Similar results were obtained with another catecholamine-depleting drug, tetrabenazine. In contrast, treatment of chromaffin cells with theophylline or forskolin increased the levels of both enkephalins and enkephalin-containing peptides of all sizes. The results suggest that new synthesis of proenkephalin is required for the effects of reserpine, although proenkephalin processing is also altered by this drug.     

Insulin-like growth factor I enhances proenkephalin synthesis and dopamine beta-hydroxylase activity in adrenal chromaffin cells

Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin-derived enkephalin-containing peptides and the activity of dopamine beta-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine beta-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was approximately 1 nM, with maximal effects observed at 10-30 nM. In contrast, insulin was 1000-fold less potent. Pretreatment of chromaffin cells with IGF-I increased the rate of [35S]proenkephalin synthesis 4-fold compared to untreated cells. Total protein synthesis increased only 1.5-fold under these conditions. These results suggest that IGF-I may be a normal regulator of chromaffin cell function.     

Comparative Incorporation of Proenkephalin-Derived Peptides, Chromogranin A, and Dopamine β-Hydroxylase into Chromaffin Vesicles

The incorporation of enkephalin-containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse-labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met-enkephalin sequences in gradient fractions determined. 35S-ECPs were incorporated into particles suggestive of immature vesicles within 1-2 h after radiolabeling. Vesicle maturation, measured by co-equilibration of 35S-ECPs and total ECPs in the gradients, was complete within 9-12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S-labeled dopamine beta-hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Summary Bovine chromaffin cells maintained in culture for eight days were loaded with [³H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K⁺. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus.The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K⁺ produced 91±2% (n = 5) depletion in the [³H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells. Under the same conditions of stimulation, the distribution of the clathrin fluorescence was unaltered suggesting either that K⁺induced stimulation of the chromaffin cells does not change the cellular distribution of clathrin, or that the changes in the distribution of clathrin are of such low magnitude that they escape detection by fluorescence microscopy.     

Processing of Proenkephalin in Adrenal Chromaffin Cells

The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.     

Regulation of Proenkephalin Synthesis in Adrenal Medullary Chromaffin Cells

The synthesis of proenkephalin was assessed in primary cultures of bovine adrenal medullary chromaffin cells by incubation of the cells with [35S]methionine, digestion of proenkephalin-derived peptides with trypsin and carboxy-peptidase B, and quantitation of radioactivity incorporated into Met-enkephalin following reversed-phase HPLC. Nicotine, histamine, and vasoactive intestinal peptide each enhanced the rate of proenkephalin synthesis approximately 10-fold when examined between 16 and 32 h after the drug or hormone addition. Inclusion of nifedipine (1 microM) partially blocked the stimulatory effect of nicotine, but not that of vasoactive intestinal peptide or histamine, or proenkephalin synthesis. Theophylline, tetrabenazine, and angiotensin II also increased the rate of proenkephalin synthesis (three- to eight-fold). These increases in the apparent rate of proenkephalin synthesis were not attributable to altered [35S]methionine specific radioactivity or rates of turnover and did not reflect similar increases in total protein synthesis. The half-life for turnover of Met-enkephalin sequences was 3-4 days in the cultured chromaffin cell. These studies directly show that proenkephalin synthesis is the primary regulatory step in control of chromaffin cell opioid peptide content.     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.     

The in situ kinetics of dopamine beta-hydroxylase in bovine adrenomedullary chromaffin cells. Intravesicular compartmentation reduces apparent affinity for the cofactor ascorbate

The Km of dopamine beta-hydroxylase for its cofactor, ascorbic acid, was determined in situ in primary cultures of bovine adrenomedullary chromaffin cells and in isolated chromaffin vesicles. A range of intravesicular ascorbate concentrations in chromaffin cell cultures (1.1-31.2 mM) was achieved by varying the number and concentration of ascorbate additions to the culture media. The rate of octopamine synthesis from tyramine displayed a Michaelis-Menten relationship with respect to ascorbate concentration and an apparent Km of dopamine beta-hydroxylase for ascorbate of 15.0 +/- 2.0 mM was determined. In isolated chromaffin vesicles, with an initial intravesicular ascorbate concentration of approximately 10 mM, ascorbate consumption during beta-hydroxylation occurred as a first order process. This indicated that dopamine beta-hydroxylase was not saturated at this initial ascorbate concentration. When isolated chromaffin vesicles were prepared with different intravesicular ascorbate concentrations, the rate of octopamine synthesis displayed a Michaelis-Menten relationship with respect to ascorbate with an apparent Km of 17.0 +/- 5.0 mM. Ascorbate consumption also occurred as a first order process in ascorbate-loaded chromaffin-vesicle ghosts which had initial ascorbate concentrations of approximately 30 mM but which were depleted of other small molecules such as catecholamines. These results indicate that the in situ Km of dopamine beta-hydroxylase for ascorbate (approximately 15 mM) is 25-fold higher than it is for the purified or partially purified enzyme assayed under optimal conditions in vitro (0.6 mM). The factor(s) which decreases the enzyme affinity for ascorbate, relative to in vitro, resides in the chromaffin vesicle interior and is also retained in chromaffin-vesicle ghosts. The mechanism of this effect remains to be determined. The Km value determined in these experiments is close to the estimated intravesicular ascorbate concentration of bovine chromaffin granules in vivo (4), suggesting that the availability of ascorbate could become a factor in regulating the rate of dopamine beta-hydroxylation.     

Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.     

Regulation of Cyclic AMP Levels by Calcium in Bovine Adrenal Medullary Cells

Both nicotine and histamine have been reported to increase cyclic AMP levels in chromaffin cells by Ca(2+)-dependent mechanisms. The present study investigated whether Ca2+ was an adequate and sufficient signal for increasing cyclic AMP in cultured bovine adrenal medullary cells. Depolarization with 50 mM K+ caused a two- to three-fold increase in cellular cyclic AMP levels over 5 min, with no change in extracellular cyclic AMP. This response was abolished by omission of extracellular Ca2+ and by 100 microM methoxyverapamil, and was unaffected by 1 microM tetrodotoxin and by 1 mM isobutylmethylxanthine. Veratridine (40 microM) also increased cellular cyclic AMP levels by two- to fourfold. This response was abolished by either methoxyverapamil or tetrodotoxin. The Ca2+ ionophore A23187 (10-50 microM) had little or no effect on cellular cyclic AMP levels. When the concentration of K+ used to depolarize the cells was reduced to 12-15 mM, the catecholamine release was similar to that induced by 50 microM A23187, and the cyclic AMP response was almost abolished. The results suggest that Ca2+ entry into chromaffin cells is a sufficient stimulus for increasing cellular cyclic AMP production. The possible involvement of a Ca2+/calmodulin-dependent isozyme of adenylate cyclase is discussed.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.     

Regulation of rat adrenal medullary enkephalins by glucocorticoids

Opioid peptides and their precursors of the proenkephalin family are found in the chromaffin cells of the rat adrenal medulla in low quantities. However, if the gland is denervated, there is a 10 to 20-fold increase in enkephalin-containing (EC) peptides consisting mostly of the precursor proenkephalin. The denervation-induced rise in medullary EC peptides is blocked by hypophysectomy, and partially reinstated by corticosterone, dexamethasone or ACTH treatment. In the intact rat, intermediate doses of corticosterone or dexamethasone reduce the denervation-induced increase in EC peptides, while a high dose of dexamethasone restores this response. These results indicate that glucocorticoids exert a permissive effect in vivo on the denervation-induced stimulation of EC peptide biosynthesis.     

Vasoactive intestinal peptide and substance P increase levels of enkephalin-containing peptides in adrenal chromaffin cells

The neuropeptides substance P and vasoactive intestinal peptide (VIP), reported to exist in the splanchnic nerve terminals innervating the adrenal medulla, elevate the levels of enkephalin-containing peptides (ECPs) in cultured bovine adrenal medullary chromaffin cells. Cellular ECP stores were increased over 48 hr by 72 and 46 percent, respectively, following incubation with 5 microM VIP or 10 microM substance P, maximally effective concentrations. The results suggest that VIP and substance P may be trans-synaptic modulators of chromaffin cell ECP stores.     

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.     

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two-Dimensional Electrophoresis and Identification by Lectin Binding

The proteins of highly purified chromaffin-granule membranes were separated by one- or two-dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organelles. Two-dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin-binding bands are discussed: neither cytochrome b561 nor the F1-like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin-A binding proteins, as well as dopamine beta-hydroxylase, are present in the chromaffin granule lysate.     

Metorphamide Levels in Chromaffin Cells Increase After Treatment with Reserpine

Exposure of bovine chromaffin cells in primary culture to 0.01-1 microM reserpine caused a dose- and time-dependent increase in intracellular levels of the amidated enkephalin peptide metorphamide. Maximal levels (approximately 800% of control) were obtained at 0.1 microM reserpine and increased levels were apparent by 16 h of treatment. Metorphamide increases were at least fivefold more than that of either Met- or Leu-enkephalin, suggesting that reserpine stimulates both enkephalin processing and amidation in the secretory vesicle. Treatment with elevated potassium, which increases enkephalin levels by stimulating production of preproenkephalin messenger RNA, elicited an increase in metorphamide levels equivalent to, but not greater than, the increase in Met-enkephalin pentapeptide. The ratio of Met-enkephalin to metorphamide in untreated chromaffin cells is approximately 140:1, whereas the final Met-enkephalin: metorphamide ratio in reserpinized chromaffin cells is approximately 30:1, similar to the Met-enkephalin:metorphamide ratio in enkephalinergic neurons of the CNS.     

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD-2) cloned from the C-1300 tumor. 8Br-cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta-hydroxylase activity. In contrast, catechol O-methyltransferase, monoamine oxidase and aromatic-l -amino-acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta-hydroxylase were similar with respect to dose response relationships and with respect to time course of onset. Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20-1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta-hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD-2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta-hydroxylase activity. The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta-hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta-hydroxylase in adrenergic cells.