Comparative incorporation of proenkephalin-derived peptides, chromogranin A, and dopamine beta-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.

Synthesis of chromogranin A, dopamine beta-hydroxylase, and chromaffin vesicles.

Primary cultures of bovine adrenal medullary cells synthesize chromogranin A (CgA) and dopamine beta-hydroxylase (DBH) and incorporate them into chromaffin vesicles. The incorporation of L-[35S]methionine into CgA, DBH, and total protein was approximately linear for 8 h at methionine concentrations of 12.5, 25, and 50 microM. Newly synthesized CgA and DBH were initially incorporated into vesicles of low buoyant density that matured over 24 h into vesicles having the greater buoyant density of chromaffin vesicles. Approximately 10% of the newly synthesized CgA is released constitutively within 4 h of formation, approximately 30-40% appears to be degraded, and the remainder is incorporated into chromaffin vesicles, which can secrete CgA in response to nicotinic stimulation. Newly synthesized DBH follows a similar course. Once incorporated into chromaffin vesicles, the newly synthesized CgA and DBH appear to be stable for 2-3 days and then decline with a half-time of 3-4 days. Primary cultures of bovine adrenal medullary cells are a good model system for studying factors regulating CgA and DBH synthesis and the formation of chromaffin vesicles.

Synthesis of alpha 2-macroglobulin by bovine adrenal cortical cell cultures.

Primary cultures of bovine adrenal medullary cells synthesize and secrete a high-molecular-weight protein into the culture medium. The protein was purified from the serum-free medium of cultured cells and was identified as alpha 2-macroglobulin by gel electrophoresis, sedimentation velocity, electron microscopy, immunoprecipitation, immunodiffusion, and autoradiography. Antisera directed against the protein were prepared and used to determine the cell types that synthesize the protein. Immunohistofluorescence studies show that adrenal cortical cells present in the adrenal medullary cell cultures reacted with the antisera to the protein purified from the medium, but adrenal medullary chromaffin cells did not. Cell cultures prepared from bovine adrenal cortex also synthesize and secrete alpha 2-macroglobulin and react with the antisera.

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.

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)

Turnover and storage of newly synthesized adenine nucleotides in bovine adrenal medullary cell cultures.

The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84-88% of the labeled adenine nucleotides, ADP 11-13%, and AMP 1-3%. The turnover of 32P-adenine nucleotides and 3H-nucleotides was biphasic and virtually identical; there was an initial fast phase with a t1/2 of 3.5-4.5 h and a slow phase with a half-life varying from 7 to 17 days, depending upon the particular cell preparation. The t1/2 of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half-life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half-lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P-labeled compounds and relatively three times as much catecholamine as 3H-labeled compounds.

Relationship between the regulation of enkephalin-containing peptide and dopamine beta-hydroxylase levels in cultured adrenal chromaffin cells.

Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin-containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine beta-hydroxylase, were examined. Catecholamine-depleting drugs, such as tetrabenazine, and cyclic nucleotide-elevating drugs, including forskolin, 8-bromo-cyclic AMP, and theophylline, increase chromaffin cell enkephalin-containing peptide levels but fail to increase dopamine beta-hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin-containing peptides and dopamine beta-hydroxylase. The increased amounts of enkephalin-containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density-gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin-containing peptides, but that after treatment with catecholamine-depleting or cyclic nucleotide-related agents enkephalin-containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.

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.

Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells.

Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody-antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell.

Effects of ascorbic acid, dexamethasone, and insulin on the catecholamine and opioid peptide stores of cultured adrenal medullary chromaffin cells.

Bovine adrenal medullary chromaffin cells cultured in serum-free medium were examined for changes in their catecholamine and opioid peptide stores following exposure to dexamethasone, ascorbic acid, or insulin for 2 to 12 days. Dexamethasone failed to alter cellular catecholamine levels, measured by high performance liquid chromatography with electrochemical detection, or cellular opioid peptide content, measured by an enkephalin radioreceptor assay. Chromaffin cells cultured in medium supplemented with ascorbic acid retained high ascorbate contents for 2 to 3 days, despite the rapid loss of this vitamin from the culture medium (approximately 50% lost in 2 hr). The epinephrine and norepinephrine contents of chromaffin cell cultures supplemented with ascorbate for 7 days were increased approximately 10% compared to cultures without added ascorbic acid; ascorbate had no effect on chromaffin cell opioid peptide levels. Addition of insulin to chromaffin cell cultures produced a doubling of cellular protein and opioid peptide levels by 6 days and produced a concentration-dependent increase in the dopamine and norepinephrine contents of the cells with only a slight elevation in cell epinephrine. Chromaffin cells were also tested for the ability to resynthesize their catecholamine and opioid peptide stores following depletion as the result of secretion evoked by acetylcholine or nicotine. The cellular stores of norepinephrine and dopamine were resynthesized within 3 days following secretagogue-evoked depletion. Epinephrine stores were incompletely replenished with only 20% of the epinephrine lost via secretion recovered in 7 days. Opioid peptide levels were maximally recovered (50% of the amount secreted) within 1 day. Addition of ascorbic acid, dexamethasone, or insulin to the medium failed to enhance the recovery of catecholamine stores.

Effects of manganese and other divalent cations on calcium uptake and catecholamine secretion by primary cultures of bovine adrenal medulla cells.

Primary cultures of bovine adrenal medullary chromaffin cells were used to examine the effect of replacing divalent cations in the extracellular media on secretion. When calcium was replaced by manganese, nicotine-stimulated secretion was delayed in onset for 3 to 5 minutes, but continued for approximately 60 minutes. In contrast, calcium-supported secretion began immediately on stimulation and plateaued by 10 minutes. 54Mn2+ uptake occurred on stimulation but at a lower rate than 45Ca2+ uptake. There was no delay of 54Mn2+ uptake upon stimulation and 54Mn2+ uptake was considerably prolonged compared to 45Ca2+ uptake. Replacement of calcium with strontium gave results similar to those with calcium, and, in addition, strontium was able to bring about secretion by itself in a manner similar to barium. Inhibition experiments showed that the potency for inhibiting calcium uptake was Cd2+ greater than Mn2+ greater than Ca2+ greater than Sr2+.

Inhibition of calcium uptake, sodium uptake, and catecholamine secretion by methoxyverapamil (D600) in primary cultures of adrenal medulla cells.

The calcium-entry antagonist D600 (methoxyverapamil) inhibited nicotine- and veratridine-induced 45Ca2+ uptake, 22Na+ uptake, and catecholamine secretion in primary cultures of bovine adrenal medulla cells. Inhibition of nicotine-induced effects occurred at D600 concentrations approximately 3-10-fold lower than those needed to produce similar inhibition of veratridine-induced effects. Inhibition of the veratridine-induced effects was competitive, but inhibition of the nicotine-induced effects was not competitive. These results suggest that D600, in addition to blocking "slow" Ca2+ channels and tetrodotoxin-sensitive Na+ channels also blocks nicotine transmission, possibly either by noncompetitively inhibiting the interaction of nicotine with the receptor binding site or by blockade of the receptor-associated ion conductance channel.

Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells.

The effects of the steroid glycoside digitonin on cultured bovine adrenal chromaffin cells were studied as a way to gain access to the intracellular sites of calcium action in stimulus-secretion coupling. Chromaffin cells treated with digitonin secreted catecholamines by an exocytotic mechanism when exposed to free calcium concentrations of greater than 1 microM. At free calcium concentrations of less than 100 microM, an apparently saturable release of catecholamines was observed with a half-maximal effect (EC50) of 3-4 microM. Catecholamine release at these low calcium concentrations was limited to 10-15% of the cellular content, was partially dependent on the presence of MgATP, and was inhibited by magnesium (EC50 = 6 mM). At calcium concentrations between 0.1 and 10 mM, a second, apparently saturable component of catecholamine release from digitonin-treated chromaffin cells was observed (EC50 = 0.3 mM). This release at the higher calcium concentrations was unaffected by the addition of MgATP, magnesium, antipsychotic drugs known to bind and inactivate calmodulin, or calcium channel blockers, but was temperature-dependent and was inhibited by N-ethylmaleimide (EC50 = 140 microM). Strontium and manganese can also evoke catecholamine secretion from digitonin-treated chromaffin cells. The results suggest that digitonin will be a useful preparation in elucidating the molecular mechanisms of the secretory process.

22Na+ uptake and catecholamine secretion by primary cultures of adrenal medulla cells.

The uptake of 22Na+ and secretion of catecholamines by primary cultures of adrenal medulla cells under the influence of a variety of agonists and antagonists were determined. Veratridine, batrachotoxin, scorpion venom, and nicotine caused a parallel increase in 22Na+ uptake and Ca2+-dependent catecholamine secretion. Ba2+, depolarizing concentrations of K+, and the Ca2+ ionophore Ionomycin stimulated secretion of catecholamines but did not increase the uptake of 22Na+. Tetrodotoxin inhibited both 22Na+ uptake and catecholamine secretion evoked by veratridine, batrachotoxin, and scorpion venom, but had no effect on 22Na+ uptake and catecholamine secretion caused by nicotine. On the other hand, histrionicotoxin, which blocks the acetylcholine receptor-linked ion conductance channel, blocked nicotine-stimulated 22Na+ uptake and catecholamine secretion, but only partially inhibited veratridine-stimulated catecholamine secretion and had no effect on veratridine-stimulated 22Na+ uptake. The combination of veratridine plus tetrodotoxin, which has been shown to prevent nicotine-stimulated secretion of catecholamines by adrenal medulla cells, also prevented nicotine-stimulated 22Na+ uptake by the primary cultures. These studies demonstrate the presence of tetrodotoxin-sensitive Na+ channels in adrenal medulla cells which are functionally linked to Ca2+-dependent catecholamine secretion. However, These channels are not utilized for Na+ entry upon activation of nicotinic receptors; in this case Na+ entry occurs through the receptor-associated ion conductance channel.

Inhibition of 45Ca2+ uptake and catecholamine secretion by phenothiazines and pimozide in adrenal medulla cells cultures.

The inhibition by several phenothiazine drugs and pimozide of the uptake of 45Ca2+ and secretion of catecholamines by cultured adrenal medulla cells stimulated with nicotine, veratridine, 50 mM K+, ionomycin and Ba2+ was studied. The inhibition of 45Ca2+ uptake, except for ionomycin, closely parallelled the inhibition of catecholamine secretion. The nicotine-and veratridine-stimulated effects were several fold more sensitive to inhibition by the drugs than were those stimulated by 50mM K+, ionomycin and Ba2+; the ionomycin-stimulated effects were least sensitive to inhibition. These studies indicate that the drugs have multiple effects on stimulus-secretion coupling in adrenal medulla cells. It is suggested that inhibition of the veratridine- and nicotine-stimulated events is due to membrane perturbations caused by the drugs, that inhibition of the 50mM K+- and Ba2+-stimulated events is due to alterations in the voltage sensitive membrane Ca2+ channel, and that inhibition of secretion elicited by ionomycin may be due to inhibition of Ca2+-calmodulin reactions or to more profound non specific membrane effects.

Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells.

The uptake of 45Ca2+ and secretion of catecholamines by primary cultures of adrenal medulla cells were studied. Nicotine, veratridine, potassium, and Ionomycin stimulate both the accumulation of 45Ca2+ and the secretion of catecholamines. Nicotinic antagonists block 45Ca2+ uptake induced by nicotine, tetrodotoxin blocks 45Ca2+ uptake induced by veratridine, and D600 or secretion induced by Ionomycin. The EC50 for nicotine is 3 microM for catecholamine secretion and 10 microM for 45Ca2+ uptake, while the EC50s for veratridine-stimulated uptake and secretion are approximately the same (75 microM). Kinetic studies show that the uptake of Ca2+ is rapid and appears to precede the secretion of catecholamines, and that the rate of uptake declines rapidly. The 50 mM-K+ show saturation kinetics with respect to external calcium concentrations at about 2 mM. On the other hand, the uptake of 45Ca2+ stimulated by nicotine does not become saturated at external calcium concentrations of 10 mM although the secretion of catecholamines reaches a maximum at external calcium concentrations of 2 mM. The data suggest that depolarizing agents such as veratridine and 50 mM-K+ stimulate 45Ca2+ entry through voltage-sensitive calcium channels, while nicotinic agonists stimulate calcium entry through the acetylcholine receptor ion channels as well as through voltage-sensitive calcium channels.

The adrenal medulla of the diabetic mouse (C57BL/KsJ, db/db): biochemical and morphological changes.

1. Anatomic and biochemical indices of adrenal medullary function were studied in mice (Mus musculus) with hereditary mellitus (C57BL/KsJ, db/db). 2. In the diabetic mice increases in medullary catecholamine content and in the activities of tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase were accompanied by increases in adrenal weight and size. 3. Morphometric study of adrenals from diabetic mice showed that the medulla was increased in size but had a lower cell density indicating that medullary hypertrophy as well as hyperplasia were probably responsible for this size increase. 4. These observations are consistent with the occurrence of chronic adrenal medullary stimulation in the diabetic syndrome of these mice.