Bovine Adrenal Medullary Chromaffin Cells Research Articles

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.

Multiple fluorescent ligands for dopamine receptors. II. Visualization in neural tissues

Selective dopamine receptor ligands, ( R,S)-5-(4′-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3-benzazepin-7-ol, the 4′-amino derivative of the high affinity D 1 receptor antagonist SCH 23390, the high affinity D 2 receptor antagonist N-( p-aminophenethyl)-spiperone or NAPS, and the D 2 selective agonist, 2-( N-phenethyl- N-propyl)-amino-5-hydroxytetralin or PPHT were chemically coupled to the fluorescent compounds, Bodipy, Cascade blue, coumarin, fluorescein, rhodamine, or Texas red. The utility of the 6 fluorescent moieties linked to the 3 dopamine receptor binding ligands for anatomical study of regional and cellular distribution patterns of the two dopaminergic receptor subtypes has been assessed in frozen sections of the rat striatum and compared to our previous report using the rhodamine-labeled antagonists. The regional staining for the two dopaminergic receptor binding sites supports previous work using in vitro receptor autoradiographic analyses; the D 1 receptor binding was more robust than that of D 2 receptors in the caudate nucleus. The cellular element which most frequently expressed striatal D 1 binding sites had a medium-diameter cell body. Medium-sized cells also exhibited fluorescence for the D 2 binding site, as did a much larger diameter element; potentially the cholinergic interneuron of the caudate nucleus. The pharmacological specificity for each of the different D 1 fluorescent antagonist ligands in the tissues was determined by competition with 100-fold excess of unlabeled SCH 23390 (non-specific binding), spiroperidol (binding selectivity), the stereoactive paired isomers of butaclamol, and the serotonin 5-HT 2 receptor antagonist ketanserin. The same criteria were used to assess the different D 2 fluorescent agonist and antagonist ligand derivatives. The anatomical efficacy of these novel ligands was determined using selective dichroic filters to stimulate the fluorescent moieties in the optimal excitation wavelength, and the amount of fluorescent dopamine receptor binding was photographically measured and contrasted for each of the newly synthesized fluoroprobes. Using the most pharmacologically specific and anatomically efficient of these novel fluoroprobes, we determined the localization pattern of the D 1 and D 2 dopamine receptor binding sites in tissues reported to exhibit both subtypes of the receptor. The cellular distribution of the dopamine receptor binding sites was determined concurrently using fluoroprobes in the forebrain, mesencephalon, pituitary, retina, and superior cervical ganglion of the rodent, and bovine adrenal medullary chromaffin cells were examined using the rhodamine-labeled antagonists.

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.

Role of Ca 2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells

The role of Ca 2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca 2+. 12- O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca 2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca 2+-evoked secretion. Polymyxin B at a low concentration (1 μM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca 2+. However, polymyxin B at higher concentrations (10–100 μM) greatly reduced Ca 2+-evoked catecholamine secretion. Sphingosine (10μM-1 mM), staurosporine (100nM-1 μM), and H-7 (100–500 μM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca 2+-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [ 3H]phorbol-12, 13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca 2+-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca 2+-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.

Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis.

Nicotinic receptor-mediated secretion of catecholamines from individual cultured bovine adrenal medullary chromaffin cells was measured and characterized with a voltametric microelectrode placed adjacent to the cells. Nicotine-induced secretion is associated with a large increase in chemical spikes that is temporally resolved into the apparent secretion of discrete packets of attomole quantities of easily oxidized molecules. These data are consistent with direct chemical measurement of single exocytotic events.

Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: inhibitory effect of tetanus toxin on catecholamine secretion.

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.

31P nuclear magnetic resonance study of the metabolic pools of adenosine triphosphate in cultured bovine adrenal medullary chromaffin cells.

31P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium--thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct 31P signals could be seen for ATP within the vesicular and the cytosolic pools. Comparison of the integrated areas of the beta-phosphate resonances from the two ATP pools indicated 77% of the endogenous ATP was in the vesicular pool. From this observation and the assumption that the concentration of ATP in the vesicle is 87.5 mM, the concentration of ATP calculated to be in the cytoplasmic pool was approximately 4 mM. The pH in the vesicle determined from the chemical shift of the gamma-phosphate resonance of vesicular ATP was 5.84 +/- 0.17 (n = 6), slightly higher than the intragranular pH measured in hypoxic cells (5.57 +/- 0.15, n = 8). Spin-lattice relaxation times of ATP 31P resonances in the vesicular pool were from 12 to 14 times shorter than the ATP resonances in the cytosol, corresponding to a decrease in molecular mobility due to incorporation of ATP within a catecholamine-storage complex.

Multiple effects of cocaine upon evoked secretion in bovine adrenal medullary chromaffin cells. Additional insight into the mechanism of action of cardiac glycosides.

Experiments to determine the effects of the catecholamine neuronal uptake blockers cocaine and desipramine, and of the cardiac glycoside, ouabain, upon 3H-(noradrenaline) efflux have been performed with bovine adrenal medullary chromaffin cells in tissue culture. Both cocaine and desipramine reduced 3H-noradrenaline uptake into chromaffin cells. Inhibitable uptake was 80% of total accumulation over 60 min; this degree of inhibition was produced by cocaine (30 mumol/l) or desipramine (1 mumol/l). Cocaine (30 mumol/l) had no effect upon spontaneous 3H-efflux measured over 60 min, but reduced that evoked over the same period by carbachol (EC50), veratridine (EC50) and by ouabain (100 mumol/l). Cocaine did not reduce that efflux evoked by raised levels of K+ (28 mmol/l; EC50). Desipramine (1 mumol/l), like cocaine, had no effect upon spontaneous efflux of 3H, but reduced that efflux evoked by carbachol, veratridine and ouabain. Tetrodotoxin (TTX) inhibited veratridine-evoked 3H efflux (IC50 0.2 mumol/l). The degree of inhibition caused by TTX (0.2 mumol/l) was not increased by cocaine (30 mumol/l). TTX also inhibited ouabain-evoked 3H efflux: this was reduced by 55% by a concentration of TTX (1 mumol/l) sufficient to virtually abolish veratridine-evoked efflux. Cocaine (30 mumol/l) in the presence of TTX (1 mumol/l) did not further inhibit ouabain-evoked efflux. Cocaine (30 mumol/l) did not alter 86Rb+ uptake into chromaffin cells, nor did it alter that inhibition of 86Rb+ uptake produced by ouabain (100 mumol/l) indicating that cocaine has no effect upon Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Intracellular pH in Secretion from Adrenal Medulla Chromaffin Cells

The role of intracellular pH in stimulus-secretion coupling was investigated in cultured bovine adrenal medullary chromaffin cells. NH4Cl (1-25 mM) did not affect basal catecholamine or ATP release but markedly inhibited nicotine- or high K+-induced release by up to 60%. The inhibition had a rapid onset (less than 1 min) and was maximal at about 5 mM NH4Cl. The effect of NH4Cl was largely sustained over 20 min and was reversed upon NH4Cl removal. Sodium propionate did not affect secretion but partially reversed the inhibition by NH4Cl in a concentration-dependent manner. Methylamine (10 mM) produced a similar, but slower, inhibition than NH4Cl. Monensin (1-10 microM) inhibited catecholamine secretion by 30-60%, and its effect was reduced in the presence of NH4Cl. Using the fluorescent Ca2+ probe Fura-2, we found that the increase of [Ca2+]i following stimulation was not altered by concentrations of NH4Cl which inhibited secretion maximally. Measurement of cytosolic pH (pHi) with the fluorescent probe 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) revealed an alkalinization by NH4Cl (2.5-25 mM) of 0.1-0.23 pH units and acidification by sodium propionate (10-20 mM) of 0.2-0.25 pH units, with intermediate combined effects. Monensin (1 microM) caused a cytosolic acidification of 0.26 pH units. All pHi changes were partly recovered in 15 min. Fluorescence quenching measurements using the weakly basic fluorescent probe acridine orange indicated the accumulation of the probe into acidic compartments, presumably the chromaffin granules, which was strongly reduced by both NH4Cl and monensin. From these findings we conclude that the pH of the chromaffin granule modulates secretion by affecting some step in the secretory process unrelated to the rise in [Ca2+]i.

H 1-histaminergic activation of catecholamine release by chromaffin cells

Bovine adrenal medullary chromaffin cells, prelabeled with [ 3H]norepinephrine, released a large proportion of cellular 3H-labeled catecholamines (CAs) when stimulated with nicotine, K +, histamine, γ-aminobutyric acid (GABA) and several peptidic hormones [bradykinin, angiotensin II, thyrotropin releasing hormone (TRH) and neurotensin]. The histamine-induced response was dose dependent and occurred through H 1 histaminergic receptors. Quantitatively and temporally the histamine- and nicotine-induced responses differed. Nicotine, during the first minutes, induced a large increase of [ 3H]CAs, but this response was desensitized rapidly. In contrast, histamine initially provoked a smaller release of [ 3H]CAs than nicotine but, with prolonged exposure (hours), a much greater response was found with histamine. Moreover, little desensitization was observed with histamine even during extended stimulation. External Ca 2+ was obligatory for the histamine response, and both inorganic (Co 2+ and Ni 2+) and organic (verapamil, nifedipine and D-600) Ca 2+ channel blockers significantly reduced release of [ 3H]CAs. These studies suggest that histamine as well as certain other neuroactive substances could play an important role in the physiology and biochemistry of adrenal medullary chromaffin cells.

Catecholamine depletion and accumulation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) in adrenal medullary chromaffin cells

Cultures of bovine adrenal medullary chromaffin cells converted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to 1-methyl-4-phenylpyridinium (MPP(+)) by a pargyline-sensitive mechanism. Both MPTP and MPP(+) accumulated in these cells. Upon subcellular fractionation, distribution of MPP(+) paralleled that of catecholamines, suggesting storage in chromaffin vesicles. In contrast, MPTP was found in both soluble and particulate fractions, but not in chromaffin vesicles. MPTP produced a rapid depletion of 20-25% of chromaffin cell catecholamines, an action blocked by the nicotinic antagonist d-tubocurarine but not by the monoamine oxidase inhibitor pargyline. This depletion was not mimicked by MPP(+). Addition of > OR = 50 microM MPTP to chromaffin cell cultures caused additional, progressive catecholamine loss from 1 to 6 days of drug exposure that was mimicked by MPP(+) and not blocked by pargyline or d-tubocurarine. Cultures of bovine adrenal cortical cells also converted MPTP to MPP(+) and accumulated both of these drugs, but at a slower rate and to a lesser extent than chromaffin cells. Although chromaffin cells form MPP(+) from MPTP and store MPP(+), these studies suggest that catecholamine depletion in these cells results from the actions of MPTP and MPP(+) which are not stored in chromaffin vesicles. MPTP evokes catecholamine secretion from chromaffin cells via nicotinic acetylcholine receptors. MPTP and free MPP(+) are also toxic to the cells, probably through blockade of metabolic processes. These findings suggest that cells with a high capacity for MPP(+) uptake but limited vesicular storage capacity, i.e. sustained high cytoplasmic drug levels, would be susceptible to the toxic actions of this drug.

Alpha 2-adrenoceptor blockade prevents cardiac glycoside-evoked neurotransmitter release from sympathetic nerves in dog saphenous vein.

1 The effect of alpha-adrenoceptor antagonists upon neurotransmitter release evoked by cardiac glycosides from sympathetic nerve terminals has been investigated in dog saphenous vein. 2 In rings of saphenous vein preloaded with [3H]-noradrenaline, acetylstrophanthidin (ACS) caused a concentration-dependent efflux of 3H (EC50 ca. 4.4 microM) that was attenuated by phentolamine and yohimbine but not by prazosin. 3 In helical strips of saphenous vein superfused with ACS at EC50 the efflux of 3H-compounds in general, and of [3H]-noradrenaline in particular, occurred after a short delay and increased with time to a maximum reached at 75 min. Phentolamine and phenoxybenzamine, but not prazosin reduced the efflux of [3H]-noradrenaline and of total 3H-compounds throughout the time-course of the ACS-evoked effect. 4 In helical strips of saphenous vein the glycoside ouabain also caused an increase in [3H]-noradrenaline and in total 3H-efflux that was attenuated by phentolamine. 5 By contrast with the above, in bovine adrenal medullary chromaffin cells, which appear to have no functional alpha-adrenoceptors, ACS caused a small, but significant increase in 3H-efflux which was not prevented by phentolamine. 6 Phentolamine, at concentrations that attenuate markedly the ouabain- or ACS-evoked increase in 3H-efflux from dog saphenous vein, did not cause significant inhibition of cocaine-sensitive [3H]-noradrenaline uptake nor did it reduce the extent of the 3H-efflux evoked either by tyramine or by reduced extracellular Na+. These findings imply that phentolamine does not affect ACS-evoked neurotransmitter release by an action on the catecholamine uptake mechanism. 7. It is concluded that the cardiac glycoside-evoked increase in neurotransmitter release from noradrenergic nerve terminals ofdog saphenous vein is modulated by a mechanism that involves an alpha2- adrenoceptor.

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.

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 μM VIP or 10 μM substance P, maximally effective concentrations. The results suggest that VIP and substance P may be trans-synaptic modulators of chromaffin cell ECP stores.

Metabolic pools of ATP in cultured bovine adrenal medullary chromaffin cells.

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells.

Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.

Epinephrine and norepinephrine syntheses are regulated by a glucocorticoid receptor-mediated mechanism in the bovine adrenal medulla

The bovine adrenal medulla was investigated regarding the presence of glucocorticoid binding protein and the increases in ornithine decarboxylase (ODC) activity and epinephrine and norepinephrine by dexamethasone. Scatchard analysis of specific cytosol [ 3H] dexamethasone-binding study indicated a single class of high affinity (kd, 35 ± 5 nM) and limited binding sites (150 ± 26 fmoles/mg protein). Competition studies of various steroids indicated a high affinity for dexamethasone and hydrocortisone. Sedimentation in sucrose density gradients revealed a 7.3 S binding peak in the cytosol. Dexamethasone caused an increase in ornithine decarboxylase (ODC) activity within 1 to 2 hours after which the norepinephrine and epinephrine contents increased 16 hours after the peak of ODC activity in a dose dependent manner of dexamethasone in bovine adrenal medullary chromaffin cells in primary monolayer culture. These data suggest that the bovine adrenal medulla is a target organ of glucocorticoid hormone and that norepinephrine and epinephrine syntheses are regulated by a glucocorticoid receptor-mediated mechanism.

Regulation of opioid peptide synthesis and processing in adrenal chromaffin cells by catecholamines and cyclic adenosine 3':5'- monophosphate

Primary cultures of bovine adrenal medullary chromaffin cells were used to study the regulation of opioid peptide (OP) synthesis. Chromaffin cells continuously exposed to tetrabenazine, a drug that depletes cellular catecholamine stores, increase their OP contents between 32 hr and 6 days of treatment. At no time following tetrabenazine addition were increases in opiate receptor-inactive enkephalin-containing peptides (IECPs) observed. Because IECPs may serve as precursors to OPs, these results suggest increased processing of OP precursors following treatment with catecholamine-depleting drugs in addition to an increased rate of OP precursor synthesis. The increases in cellular OP levels induced by tetrabenazine were approximately proportional to the depletion in cellular catecholamines produced by this drug. Also, the effects of tetrabenazine on chromaffin cell OP and IECP contents were mimicked by inhibitors of catecholamine biosynthesis and other agents that decreased catecholamine stores, but not by supplementing the culture medium with catecholamines or catecholamine receptor agonists. Addition of 8-bromo-cAMP or forskolin, an activator of adenylate cyclase, to chromaffin cell cultures increased both OP and IECP stores. Inhibitors of cyclic nucleotide phosphodiesterase also increase chromaffin cell OP and IECP contents, although it is unclear whether these increases result from increased cyclic nucleotide levels. Hence, both alterations in some intracellular catecholamine pool and elevations of cAMP levels may trigger increases in the synthesis and processing of OPs and IECPs in the adrenal medullary chromaffin cell.

The relationship between arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cells.

Increased arachidonic acid release occurred during activation of catecholamine secretion from cultured bovine adrenal medullary chromaffin cells. The nicotinic agonist 1,1-dimethyl-4- phenylpiperazinium (DMPP) caused an increased release of preincubated [3H]arachidonic acid over a time course which corresponded to the stimulation of catecholamine secretion. Like catecholamine secretion, the DMPP-induced [3H]arachidonic acid release was calcium-dependent and was blocked by the nicotinic antagonist mecamylamine. Depolarization by elevated K+, which induced catecholamine secretion, also stimulated arachidonic acid release. Because arachidonic acid release from cells probably results from phospholipase A2 activity, our findings indicate that phospholipase A2 may be activated in chromaffin cells during secretion.

Flux of catecholamines through chromaffin vesicles in cultured bovine adrenal medullary cells.

Primary cultures of bovine adrenal medullary chromaffin cells were pulse-labeled with [3H]dopamine or [3H]norepinephrine and examined for radioactive and total catecholamine contents by high performance liquid chromatography after additional incubations of 15 min to 10 days. [3H]Dopamine was rapidly taken up by chromaffin vesicles in situ and converted to norepinephrine with a half-time of approximately 6 h. [3H] Norepinephrine taken up by the cells was metabolized in three phases. 1) During its brief transit through the cytoplasm, 20 to 35% of this amine was converted to [3H]epinephrine. 2) Following vesicular accumulation, 65 to 70% of the remaining [3H]norepinephrine was methylated to form [3H]epinephrine with a half-time of approximately 30 h, corresponding to the rate of vesicular catecholamine loss from reserpine-treated cells. 3) The residual [3H]norepinephrine decreased with a half-time of 5 days, probably representing loss from norepinephrine-storing cells. [3H]Epinephrine formed endogenously had a half-life in the cultures of approximately 15 days. These data suggest that leakage of norepinephrine from chromaffin vesicles into the cytoplasm limits the rate of dopamine conversion to epinephrine in the adrenal medulla. The kinetic data indicate that approximately 18% of the endogenous norepinephrine and 73% of the endogenous dopamine are present in epinephrine cells.