Characterization of Histamine-Induced Catecholamine Secretion from Bovine Adrenal Medullary Chromaffin Cells

Histamine activation of H1 receptors stimulates 3H release from cultured bovine adrenal chromaffin cells preloaded with [3H]noradrenaline. The initial (1-min) release induced by a high concentration of histamine was unaffected by the removal of extracellular Ca2+, whereas the more sustained response (10 min) was largely inhibited. In contrast, release induced by nicotine was dependent on extracellular Ca2+ at all times. The protein kinase inhibitor staurosporine inhibited both the initial and sustained (10-min) phases of histamine-induced release (IC50 in the region of 200 nM) but was ineffective against a direct depolarizing stimulus (56 mM K+). In contrast, the calmodulin antagonist trifluoperazine was equally effective against both stimuli. These data indicate that although a staurosporine-sensitive event (perhaps involving protein kinase C) is essential for coupling histamine receptor activation to the release processes, it is not essential for exocytosis itself. A further distinction between histamine- and depolarization-induced release was demonstrated by the differential effect of the guanine nucleotide-binding protein inhibitor pertussis toxin. Pretreatment with pertussis toxin (0.1 microgram/ml for 16 h) enhanced depolarization-induced release by approximately 1.5-fold. This pertussis toxin pretreatment was, however, approximately twofold as effective in potentiating histamine-evoked release. Thus, the characteristics of the histaminergic response are distinct from those of a depolarizing stimulus, perhaps indicating the involvement of different mechanisms in the release process.     

Natriuretic Peptides Inhibit Nicotine-Induced Whole-Cell Currents and Catecholamine Secretion in Bovine Chromaffin Cells: Evidence for the Involvement of the Atrial Natriuretic Factor R2 Receptors

Abstract: There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R_1C but not the ANF-R_1A receptor subtype. Evidence for the existence of ANF-R₂ receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R₂-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R₂ receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R₂ receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that this effect of CNP is predominantly mediated by the ANF-R₂ and not the ANF-R_1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCI-induced secretions were affected by natriuretic peptides. In the present study, we report (1) the presence of ANF-R_1C and ANF-R₂ receptor subtypes in bovine chromaffin cells, (2) the inhibition by natriuretic peptides of nicotinic whole-cell currents as well as nicotine-induced catecholamine secretion, (3) the possible mediation of these effects by the ANF-R₂ class of receptors, and (4) the specificity of this inhibition to nicotinic agonists. Because bovine chromaffin cells release ANF, BNP, and CNP together with catecholamines, all three peptides might exert negative feedback regulation of catecholamine secretion in an autocrine manner by interacting with the nondiscriminating ANF-R₂ receptor subtype.     

Expression of the Proenkephalin A Gene and [Met5]-Enkephalin Secretion Induced by Arachidonic Acid in Bovine Adrenal Medullary Chromaffin Cells: Involvement of Second Messengers

Abstract: We have previously reported that arachidonic acid (AA) increases the long-term secretion of [Met⁵]-enkephalin (ME) and the expression of proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin (BAMC) cells. To characterize the underlying signal transductional mechanisms for the AA-induced responses, the interactions of AA with several second messenger systems were studied. Long-term (24-h) treatment with AA (100 µ M ) increased both the secretion of ME and the expression of proENK mRNA. Pretreatment of BAMC cells with nimodipine (1 µ M ), but not with ω-conotoxin GVIA (1 µ M ), inhibited the secretion of ME and the expression of proENK mRNA induced by AA. Calmidazolium (1 µ M ), a calmodulin antagonist, also significantly inhibited AA-induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 µ M ), was ineffective in blocking AA-induced responses. In addition, the down-regulation of PKC by phorbol 12-myristate 13-acetate (0.1 µ M ) for 48 h did not inhibit the AA-induced responses. Forskolin (5 µ M ), an adenyl cyclase activator, alone increased the secretion of ME as well as proENK mRNA levels and, when coincubated with AA, showed an additive effect on the secretion of ME and the levels of proENK mRNA. The results suggest that the Ca²⁺/calmodulin pathway, but not the protein kinase A or PKC pathway, is partially involved in mediating the AA-induced increases of the long-term secretion of ME and the levels of proENK mRNA.     

Release of Catecholamines from Superfused Bovine Adrenal Chromaffin Cells Cultured on Microcarrier Beads

A procedure is described for the establishment of stable primary cultures of bovine chromaffin cells on microcarrier beads. The cells flatten and send out processes with varicosities over a few days and maintain their catecholamine content for 2 weeks. The beads may be incorporated into a superfusion apparatus with a chamber volume of about 150 microliters, enabling the efficient perfusion of a high density of cells. The response to the introduction of nicotine and high potassium into the perfusing medium is shown to be more rapid and more transient than hitherto described, with each secretagogue producing a different degree of preferential stimulation of noradrenaline-secreting cells.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Subcellular Distribution Studies of Diadenosine Polyphosphates—Ap4A and Ap5A—in Bovine Adrenal Medulla: Presence in Chromaffin Granules

Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 +/- 0.05 mumol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (approximately 6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 microM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 +/- 0.5 and 13 +/- 1 microM for Ap4A and Ap5A, respectively.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Met-Enkephalin [Arg6,Phe7] Immunoreactivity in Bovine Caudate and Bovine Adrenal Medulla

Abstract: A radioimmunoassay specific for the COOH-terminus of Met-enkephalin [Arg⁶,Phe⁷] and a separate assay specific for the COOH-terminus of Met-enkephalin are described. Immunoreactivity by these two assays was compared in bovine caudate and bovine chromaffin granule preparation after Sephadex G75 chromatography in 50% acetic acid. When the assays were applied to the chromatography fractions of the bovine caudate extract, the majority of the immunoreactivity was found in the fractions corresponding to the heptapeptide and the pentapeptide respectively. When the chromaffin granule chromatography fractions were assayed, both of the radioimmunoassays showed that most reactivity was in several peaks in the larger molecular weight fractions. The major peak for the Met-enkephalin [Arg⁶,Phe⁷] assay had an apparent molecular weight of 2800, while with the Met-enkephalin assay the dominant peak of immunoreactivity had an apparent molecular weight of 10,000. The presence of authentic Met-enkephalin [Arg⁶,Phe⁷] in both caudate and chromaffin granule extracts was confirmed by reverse-phase chromatography of the previously sized fractions. It appears then that the processing of precursors of opioid peptides is directed, in the caudate, to the synthesis and storage of the enkephalins and of Met-enkephalin [Arg⁶,Phe⁷]; in the adrenal medulla the major products of precursor processing are a variety of polypeptides of larger sizes.     

Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.     

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.     

Histamine Affects Release and Biosynthesis of Opioid Peptides Primarily via H1-Receptors in Bovine Chromaffin Cells

Histamine is a potent secretagogue for opioid pentapeptides (Met- and Leu-enkephalin) in adrenal chromaffin cells in vitro. This effect is dependent on extracellular Ca2+ and is reduced by Ca2+ channel blockers such as Co2+, D 600, and nifedipine. Moreover, histamine also produced a profound compensatory increase in cellular peptide content after 48 h of exposure, most likely caused by a four- to fivefold increase in the mRNA levels coding for the proenkephalin A precursor. All the histamine-induced effects (acute release, changes in peptide cell content, proenkephalin A mRNA levels) are antagonized by the H1-receptor antagonist, clemastine, whereas the H2-receptor antagonists, ranitidine and cimetidine, were less effective (approximately 20% inhibition).     

Desensitization to Nicotinic Cholinergic Agonists and K+, Agents That Stimulate Catecholamine Secretion, in Isolated Adrenal Chromaffin Cells

Desensitization of catecholamine (CA) release from cultured bovine adrenal chromaffin cells was studied to characterize the phenomenon of desensitization and to attempt an elucidation of the mechanism(s) involved in this phenomenon at the level of the isolated chromaffin cell. Prior exposure of chromaffin cells to nicotinic cholinergic agonists [acetylcholine (ACh) or nicotine] caused a subsequent depression or desensitization of CA release during restimulation of the cells with the same agonists. Rates of development of and recovery from nicotinic desensitization were in the minute time range and the magnitude of nicotinic desensitization of CA release was greater at 37 degrees C than at 23 degrees C. ACh- (or nicotine)-induced desensitization was shown to be the result of two processes: (1) a Ca2+-dependent component of desensitization, possibly due to a depletion of intracellular CA stores and (2) a Ca2+-independent, depletion-independent component of desensitization. Prior exposure of cultured chromaffin cells to an elevated concentration of K+ also resulted in desensitization of K+-induced CA release in these cells. K+-induced desensitization was completely Ca2+-dependent and was shown to be the result, at least in part, of a mechanism that is independent of depletion of CA stores.     

Calcium Is Released by Exocytosis Together with Catecholamines from Bovine Adrenal Medullary Cells

We have tested the hypothesis that exocytosis is a possible export route for calcium from bovine adrenal medullary cells. After prelabelling cells in primary tissue culture with 45Ca, evoked 45Ca export and catecholamine secretion show the same time course, a similar fraction of the total pool of 45Ca and catecholamine is released, and the same concentrations of carbamylcholine or KCl are required for half-maximal triggered release. Increasing the osmolarity of the extracellular medium or treating the cells with botulinum toxin type D inhibits both evoked catecholamine secretion and 45Ca export to the same extent without inhibiting 45Ca influx. Incorporation of 45Ca into chromaffin granules is very slow, however, and incorporated 45Ca is not immediately releasable. 45Ca entering the cell during short-term stimulation is not found in the releasable pool during a second period of triggered secretion. Our data suggest that chromaffin granules are the largest pool of intracellular calcium in bovine adrenal medullary cells and that most of the calcium in chromaffin granules does not rapidly exchange with cytoplasmic Ca, but can be released directly by exocytosis. Exocytosis does not appear to play a major role in exporting Ca that enters the cell during short-term stimulation.     

Inhibition of Catecholamine Secretion from Bovine Chromaffin Cells by Adenine Nucleotides and Adenosine

ATP, ADP, and adenosine have been found to inhibit acetylcholine-stimulated secretion from isolated cells of bovine adrenal medulla (chromaffin cells). Maximal inhibition is approximately 30% under the conditions studied; half-maximal inhibition occurs at nucleotide concentration in the micromolar range. Cells must be incubated with ATP for approximately 90 s for maximal inhibition, but inhibition by adenosine occurs much faster, an observation suggesting the possibility that ATP and ADP exert their effect after being converted to adenosine. Experiments with cells preloaded with the fluorescent calcium chelator quin 2 indicate that external ATP can diminish the rise in cytosolic Ca2+ concentration that follows stimulation by acetylcholine.     

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)     

Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.