Opiate Receptor-Mediated Inhibition of Catecholamine Release in Primary Cultures of Bovine Adrenal Chromaffin Cells

Abstract: Primary cultures of chromaffin cells from bovine adrenal medulla were used to evaluate the ability of several opiates to reduce the release of catecholamines induced by stimulation of nicotinic receptors. Etorphine, β-endorphin, Met-enkephalin[Arg⁶,Phe⁷], and the synthetic peptide [d -Ala²,Me-Phe⁴,Met(O)^s-ol]enkephalin inhibited the acetylcholine-induced release of catecholamines with an IC₃₀ varying from 10^?7 to 1 × 10^?6M. The effect was stereospecific because levorphanol (IC₃₀= 7.5 × 10^?7M) was approximately two orders of magnitude more potent than dextrorphan. Morphine (μ-receptor agonist), [d -Ala², d -Leu⁵]enkephalin (δ-receptor agonist), ethylketazocine (k -receptor agonist), and N-allylnormetazocine (σ-receptor agonist) were at least 100–1000 times less potent than etorphine. Diprenorphine (IC₅₀= 5 × 10^?7M) and naloxone (IC₅₀= 10^?6M) antagonized the effect of etorphine. High-affinity, saturable, and stereospecific binding sites for [³H]etorphine, [³H]dihydromorphine, [³H-d -Ala²,d -Leu⁵]enkephalin, [³H]ethylketazocine, and for [³H]N-allylnormetazocine, [³H]diprenorphine, and [³H]naloxone were detected in chromaffin cell membranes and in membranes obtained from adrenal medulla homogenates. However, the number of binding sites for [³H]etorphine and [³H]diprenorphine was 10–70 times higher than the number of sites measured with the other ³H ligands. The rank order of potency of these compounds for the displacement of [³H]etorphine binding correlates (r = 0.90) with the rank order of potency of the same compounds for the inhibition of acetylcholine-induced catecholamine release. These data suggest that a stereoselective opiate receptor (different from the classic μ-, δ-, k -, or σ-receptor) with high affinity for etorphine, diprenorphine, β-endorphin, and Met-enkephalin[Arg⁶,Phe⁷] modulates the function of the nicotinic receptor in adrenal chromaffin cells.     

Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells

Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of [Met5]-enkephalin immunoreactive material (ME-IRM) into the culture medium (1). Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides. The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing [Met5]-enkephalin and [Leu5]-enkephalin. Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts. Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs. Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4. The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation. Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3). Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of [Met5]-enkephalin. The content of [Met5]-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation. We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

Nicotinic receptor stimulation enhances enkephalin-like peptides processing in chromaffin cells

Acute stimulation of chromaffin cells in cultures with acetylcholine (ACh), 1,1-dimethyl-4-phenylpiperazinium (DMPP), or high potassium gave rise to a significant increase in the release of [Met5]-enkephalin immunoreactive material (ME-IRM) into the assay medium. The cellular content of ME-IRM following the actual release induced by these secretagogues remained constant suggesting the replenishment of the cellular peptides. The repletion of the peptides may occur through an enhancement of the processing rate of the proenkephalin precursor. Furthermore, the increase in secretion as well as the repletion of the cellular ME-IRM were calcium-dependent and were inhibited by the nicotinic receptor antagonist, hexamethonium, but not by atropine. These results indicate that secretion and repletion of the peptides are tightly coupled and activated by nicotinic receptor stimulation.     

Biochemical characterization of various populations of isolated bovine adrenal chromaffin cells

Various populations of bovine adrenal chromaffin cells were isolated first by successive digestions with collagenase (original cell preparation) followed by sedimentation through a stepwise bovine serum albumin gradient (cell layers I, II and III). At the fine structural level, the ratios between the number of adrenaline-cells and noradrenaline-cells were 1.9 in the original cell preparation and 0.9, 2.0 and 4.6 in cell layers I, II and III, respectively. The catecholamine content of each cell population was also measured by spectrofluorometry. The original cell preparation contained 20.1 and 12.2 nmol per 10⁶ cells of adrenaline and noradrenaline, respectively. Each cell layer had similar total amount of catecholamines (from 38.3 to 40 nmol per 10⁶ cells) but their adrenaline/noradrenaline content ratios varied from 0.6 in cell layer 1 to 1.3 and 3.3 in cell layers II and III, respectively. Incubation of the cells in the presence of acetylcholine (50 μM) induced a release of catecholamines which was proportional to the cell content of each amine. However, the percentage of total cell content released was much higher in cell layer I (20%) than in cell layers II (8%) and III (5%). Finally, each cell population was also analyzed for its ability to respond to a muscarinic stimulation of cyclic GMP level and to bind [³H]etorphine, a highly potent opiate agonist. Acetylcholine induced 3.15-, 2.15- and 4.21-fold increases in the levels of cyclic GMP in the original cell preparation, cell layers II and III, respectively, but not in cell layer I. Conversely, the high affinity opiate binding site for [³H]etorphine was almost exclusively confined to cell layer III (B_max of 28.4 fmol per 10⁶ cells as compared with 2.8–7.5 fmol in the other cell preparations). These results indicate that bovine adrenal chromaffin cells can be separated according to their content in adrenaline and noradrenaline and their response to nicotinic, muscarinic and opiate stimuli.     

Evidence for nuclear sites of stereospecific opiate binding in neuroblastoma cells in continuous culture.

A class of stereospecific, saturable receptors for narcotic drugs has been found in a clone of mouse neuroblastoma cells in continuous culture. Cells grown in the presence of 10(-8) M etorphine, a synthetic opiate, had an increased doubling time. The neuroblastoma cell nucleus was found to be the sole site for the high affinity binding of etorphine. It was found that these nuclear receptors (1) became saturated at a concentration of 2 X 10(-9) M etorphine, (2) had a dissociation constant of 5 X 10(-11) M etorphine, (3) were capable of binding etorphine stereospecifically at 37 degrees C but not at 4 degree C, and t4) were protein in nature as evidenced by treatment with proteolytic enzymes. More importantly, the receptor activity appeared to be chromatin-associated.     

The release of 3H-1-methyl-4-phenylpyridinium from bovine adrenal chromaffin cells is modulated by somatostatin

Besides cholinergic regulation, catecholamine secretion from adrenal chromaffin cells can be elicited and/or modulated by noncholinergic neurotransmitters and hormones. This study was undertaken to investigate the influence of somatostatin and octreotide on [3H]MPP+ secretion evoked by KCl or cholinergic agents, from bovine adrenal chromaffin cells. The release of [3H]MPP+ was markedly increased by excess KCl (50 mM), acetylcholine (50 microM-10 mM) and by the nicotinic agonists, nicotine (5-100 microM) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10-100 microM), but not by the muscarinic agonist, pilocarpine (10-100 microM). Acetylcholine-evoked release of [3H]MPP+ from these cells was mainly mediated by nicotinic receptors: a) nicotine and DMPP stimulated the release of [3H]MPP+, b) a nicotinic antagonist, hexamethonium, markedly blocked the acetylcholine-evoked response and c) pilocarpine was devoid of effect on [3H]MPP+ secretion. At all concentrations tested, somatostatin and octreotide interfered neither with [3H]MPP+ basal release nor with KCl-induced release of [3H]MPP+. However, somatostatin (0.01-0.3 microM) increased the release of [3H]MPP+ induced by a high concentration of acetylcholine (10 mM). Octreotide (1-10 microM) had no effect. These results, showing that somatostatin potentiates acetylcholine-induced [3H]MPP+ release, support the hypothesis that somatostatin may increase the release of catecholamines from adrenal medullary cells.     

Effects of epinephrine and norepinephrine on glucose release from chinook salmon (Oncorhynchus tshawytscha) liver incubated in vitro

The effects of two catecholamines, epinephrine (EP) and norepinephrine (NE), on carbohydrate metabolism were studied by incubating chinook salmon liver in vitro. Basal release of glucose over the course of a 5-h incubation was 7.93 +/- 1.70 mumol/g dry weight. Both EP and NE (2 X 10(-7) M) stimulated glucose release rapidly during the first hour. After 5 h, EP and NE significantly increased glucose release over basal levels to 43.55 +/- 9.01 and 32.75 +/- 6.17 mumol/g dry weight, respectively. Epinephrine- and NE-stimulated glucose release was dose dependent, with a minimum effective dose of 10(-9) M. ED50 for both agents was approximately 2 X 10(-7) M; maximal stimulation occurred at 10(-5) M. No difference in potency between the two catecholamines was found. The effects of adrenergic agonists and antagonists were also studied. Alpha-agonists, methoxamine and phenylephrine, had no effect on glucose release. Isoproterenol, a beta-agonist, stimulated glucose release in a manner similar to EP. The beta-antagonist, propranolol, inhibited both catecholamine- and isoproterenol-stimulated glucose release. Alpha-antagonists (phentolamine, prazosin, and yohimbine) had no effect on either catecholamine- or isoproterenol-stimulated glucose release. Epinephrine and NE stimulate glycogen phosphorylase activity; propranolol inhibits catecholamine-stimulated phosphorylase activity. These results indicate that catecholamines stimulate glucose mobilization in salmon liver by promoting glycogenolysis mediated through beta-adrenergic receptors.     

Effects of bombesin and gastrin releasing peptide on catecholamine secretion from rat adrenal gland, in vitro

The effects of bombesin and gastrin releasing peptide (GRP) on the release of catecholamine were investigated by using isolated rat adrenal gland. Bombesin and GRP stimulated an epinephrine (E) release with dose-dependency. A half maximal effect of bombesin was observed at 1.2 X 10(-9) M, and a maximal release of E occurred at 1 X 10(-6) M of bombesin. The stimulatory effect of GRP on the E release was very similar to that of bombesin. Although both these peptides also stimulated a norepinephrine (NE) release, a significant effect was detected at concentrations of bombesin and GRP above 1 X 10(-7) M. Nicotine and pilocarpine stimulated both E and NE releases dose dependently, but the effect of pilocarpine on E and NE release was 1/100 or less potent than that of nicotine. Bombesin-induced catecholamine releases were not inhibited by hexamethonium or atropine that fully impeded the stimulatory effects of nicotine or pilocarpine. In addition, bombesin had additive effects on the nicotine- or pilocarpine-induced E and NE releases. These data strongly suggest that bombesin or GRP plays a physiological role as one of the important regulators in catecholamine secretion in the adrenal gland.     

Nicotinic Stimulation of [3H]Acetylcholine Release from Mouse Cerebral Cortical Synaptosomes

The effects of nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) on the release of newly synthesized [3H]acetylcholine in mouse cerebral cortical synaptosomes were examined. Nicotine and DMPP produced increases in [3H]acetylcholine release, over the level of spontaneous release, of 24% and 30%, respectively, of a maximum depolarization-induced release produced by 50 mM potassium. The maximum effect was achieved at a concentration of 1 X 10(-4) M for both agents. The time course of release indicated a slow onset of action, reaching a maximum effect at 15 min of incubation. Both nicotine and DMPP also produced a slightly greater release of total tritium, measured in the absence of cholinesterase inhibition, than of [3H]acetylcholine. The release induced by nicotine was completely antagonized by hexamethonium and was largely (58%) calcium-dependent. Nicotine also produced an increase in [3H]choline accumulation into synaptosomes. These results indicate that the nicotinic agonists nicotine and DMPP can produce a moderate enhancement of acetylcholine release by a receptor-mediated action on cholinergic nerve terminals in the central nervous system.     

Stimulatory Action of γ-Aminobutyric Acid on Catecholamine Secretion from Bovine Adrenal Chromaffin Cells Measured by a Real-Time Monitoring System

The present study was designed to evaluate the role of gamma-aminobutyric acid (GABA) in the secretory function of cultured chromaffin cells using the method of real-time monitoring. GABA evoked the secretion of catecholamines (CA) from adrenal chromaffin cells in a dose-dependent manner. Bicuculline 10(-5) M inhibited the stimulatory action of GABA. Diazepam 5 X 10(-6) and 2.5 X 10(-5) M facilitated the secretory response evoked by 7 X 10(-5) M GABA by 22% and 96%, respectively, which was antagonized by Ro 15-1788. This finding suggests that GABA-benzodiazepine receptor coupling can function in the secretion of CA from the adrenal chromaffin cells in a manner similar to that observed in the brain. GABA-evoked release of CA was reduced by 1 microM nifedipine to 16% of control, suggesting the involvement of voltage-sensitive Ca2+ channels in the mechanisms of the CA-releasing action of GABA in these cells. From these findings, the involvement of GABAergic mechanisms in the regulation of adrenal medullary function can be proposed.     

Differential Release of Enkephalin and Enkephalin-Containing Peptides from Perfused Cat Adrenal Glands

We have compared the enkephalin-like material derived from proenkephalin released from perfused cat adrenal glands stimulated with pilocarpine (5 X 10(-4)M) and nicotine (5 X 10(-6) M). In addition, two doses of acetylcholine (10(-5) and 10(-4) M) and 50 mM K+ were tested. Free Met-enkephalin immunoreactivity and total Met-enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin-containing fragments, were coreleased with catecholamines. Free Met-enkephalin immunoreactivity represented 13% of total immunoreactivity for nicotinic stimulation, 46% for pilocarpine, 33% for 10(-5) M acetylcholine, 22% for 10(-4) M acetylcholine, and 16% for 50 mM K+. Analysis of the perfusate by gel filtration showed that 80% of the total Met-enkephalin immunoreactivity whose release was induced by pilocarpine was eluted in fractions corresponding to fragments of low molecular weight, whereas these fractions accounted only for 10% of the total Met-enkephalin immunoreactivity whose release was induced by nicotine. HPLC analysis of low-molecular-weight peptide fractions revealed that Met-enkephalin, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-Arg-Phe represented 69% of total Met-enkephalin immunoreactivity whose release was induced by pilocarpine. These results indicate that selective activation of muscarinic receptors is followed by release of low-molecular-weight material, whereas nicotine application also yielded high-molecular-weight peptides. Furthermore, increasing the acetylcholine concentration from 10(-5) to 10(-4) M and using 50 mM K+ increased proportionally the high-molecular-weight peptide secretion. Results are discussed in relation to the existence of a heterogeneous population of granules either in the same cell or in different cells, containing proenkephalin-derived peptides. (ABSTRACT TRUNCATED AT 250 WORDS)     

Opiate-prostaglandin interactions in the regulation of insulin secretion from rat islets of Langerhans in vitro

The inadequate insulin secretory response to glucose stimulation in non-insulin dependent diabetes has been attributed to many factors including high PGE2 levels blunting the secretory response, and to the existence of inhibitory opiate activity in vivo. The purpose of the present work was to see if there was a connection between these two independent theories. Radioimmunoassayable PGE2 in islets of Langerhans was found to be proportional to islet number and protein content and was typically 4 to 5pg/micrograms islet protein. Indomethacin (2.8 X 10(-5) M), sodium salicylate (1.25 X 10(-3) M) and chlorpropamide (7.2 X 10(-5) M) all lowered islet PGE2 levels and stimulated insulin release in vitro. Dynorphin (1-13), stimulated insulin release at a concentration of 6 X 10(-9) M, while lowering islet PGE2. Conversely, at a higher concentration, (6 X 10(-7) M), dynorphin had no stimulatory effect on insulin secretion and did not lower PGE2 levels in islets or in the incubation media. The stimulatory effects of dynorphin and sodium salicylate on insulin secretion were blocked by exogenous PGE2 (10(-5) M). PGE2 at a lower concentration (10(-9) M) did not exert any inhibitory effect on dynorphin- or sodium salicylate-induced insulin release. This concentration of exogenous PGE2 stimulated insulin release in the presence of 6mM glucose. Results from these experiments suggest that since an opioid peptide can lower endogenous PGE2 production in islets and since the stimulatory effects of the opioid peptide are reversed by exogenous PGE2 there may be interactions between these two modulators of insulin secretion.     

In vivo interactions between prejunctional alpha 2- and beta 2-adrenoceptors at the level of the adrenal medulla

The combined effect of a beta 2-antagonist and an alpha 2-agonist on the release of adrenal catecholamines was studied in the anaesthetized and vagotomized dog. The electrical stimulation of the splanchnic nerve (5-V pulses of 2 ms duration for 3 min at a frequency of 3 Hz) produced a significant rise in adrenal catecholamine release in the adrenal vein. Intravenous injection of a beta 2-antagonist significantly reduced this response and a subsequent injection of an alpha 2-agonist further reduced the release of catecholamines. However, if the alpha 2-agonist is injected first, the release is not different compared with the control stimulation, and the subsequent injection of the beta 2-antagonist also did not modify the release in response to electrical stimulation. These results suggest that the blockade of presynaptic beta 2-receptors reduces the release of adrenal catecholamines without interfering with the activation of the alpha 2-adrenoceptors. In contrast, the pretreatment with the alpha 2-agonist, which does not modify the release of catecholamine at 3 Hz, seems to interfere with the inhibitory effect of the beta 2-antagonist.     

Role of prostaglandins in calcium-induced corticosteroid secretion by isolated frog interrenal gland

The role of prostaglandins (PGs) in calcium-induced corticosteroid secretion by frog adrenal (interrenal) gland has been examined in vitro using a perifusion technique. Increasing concentrations of CaCl2 (4-10 mM) stimulated in a dose-dependent manner aldosterone, PGE2 and 6-keto-PGF1 alpha production, whereas TXB2 was not affected. The kinetics of the adrenal response to CaCl2 indicated that the increase in PG output always preceded that of steroid. Administration of cobalt (4 mM), a calcium-channel inhibitor, blocked the calcium-induced stimulation of PGs and corticosteroids. Infusion of indomethacin (5 X 10(-6) M), a specific cyclooxygenase inhibitor, significantly decreased the basal production of PGs and steroids, and prevented the stimulatory effect of CaCl2 (6 mM). Infusion of the calcium ionophore A 23187 (10(-6) M), for 20 min, induced a marked stimulation of PG and steroid production. Taken together, these data support the notion that biosynthesis of prostaglandins is associated with calcium-induced corticosteroid secretion in frog adrenal cells.     

Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.     

McN-A-343, a specific agonist of M1-muscarinic receptors, exerts antinicotinic and antimuscarinic effects in the rat adrenal medulla

Pirenzepine, McN-A-343 and oxotremorine were used to determine the subtypes of muscarinic receptors involved in the secretion of catecholamines from the isolated perfused adrenal gland of the rat. In the presence of 0.1 microM pirenzepine, the concentration-secretion curve for muscarine was shifted in parallel to the right by almost one log unit. With 0.5 microM the shift was over two log units. The apparent dissociation constant for pirenzepine was about 1.12 X 10(-8) M. Perfusion with McN-A-343 (1-30 microM) did not evoke the secretion of catecholamines. A further increase to very high concentrations (100-1000 microM) caused only a modest secretion (about 50 ng/5 min with 300 microM as compared to the same amount of secretion obtained with 1 microM muscarine). Secretion evoked by nicotine was significantly reduced (30%) by 3 microM McN-A-343, and the inhibition increased (90%) with higher concentrations (100 microM). McN-A-343 also produced concentration-dependent inhibition of catecholamine secretion evoked by muscarine. A significant effect was observed at 30 microM and reached a maximum level at 300 microM. Oxotremorine, like McN-A-343 was a partial agonist on the muscarinic receptors; but unlike McN-A-343, did not block the stimulatory effects of nicotine. Although the pirenzepine data suggest that M1 receptors are responsible for the secretion of catecholamines in the rat adrenal medulla, this conclusion is not supported by the results obtained with the M1-receptor agonist, McN-A-343, which proved to be an effective blocker of muscarinic as well as nicotinic receptors.     

Diadenosine Tetraphosphate Is Co-Released with ATP and Catecholamines from Bovine Adrenal Medulla

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1-2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.     

Effects of stimulation by carbachol on the metabolism of the bovine adrenal medulla

1. A method is described that has made it possible to achieve a great decrease in the catecholamine and adenine nucleotide contents of the perfused bovine adrenal gland by the infusion of carbachol. 2. Although the catecholamines secreted were recovered in the perfusion medium, no evidence was obtained that the nucleotides are secreted by the gland. 3. It is concluded that the secretion of catecholamines is accompanied by extensive chemical alteration of the adenine nucleotides of the chromaffin granules. 4. The secretory response and the spontaneous release of catecholamines depends on the presence of Ca²⁺ in the perfusing Tyrode solution. 5. Anoxia does not have a significant effect on the carbachol-induced secretion of catecholamines. 6. Strips of bovine adrenal medullary tissue perfused with oxygenated Tyrode solution show an increased oxygen consumption when carbachol is added.     

Adenosine activating A(2A)-receptors coupled to adenylate cyclase/cyclic AMP pathway downregulates nicotinic autoreceptor function at the rat myenteric nerve terminals

In addition to the somatodendritic region, myenteric motoneuron terminals are endowed with nicotinic autoreceptors. We aimed at investigating the effect of nicotinic receptor (nAChR) activation on [3H]-acetylcholine ([3H]-ACh) release from longitudinal muscle-myenteric plexus of the rat ileum and to evaluate whether this could be modulated by adenosine, an endogenous neuromodulator typically operating changes in intracellular cyclic AMP. The nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1-30 microM, 3 min) increased [3H]-ACh release in a concentration-dependent manner. DMPP (30 microM)-induced [3H]-ACh outflow was attenuated by hexamethonium (0.1-1 mM), tubocurarine (1-5 microM), or by removing external Ca2+ (plus EGTA, 1 mM). In contrast to veratridine (0.2-10 microM)-induced [3H]-ACh release, the DMPP (30 microM)-induced outflow was resistant to tetrodotoxin (1 microM) and cadmium (0.5 mM). Pretreatment with adenosine deaminase (0.5 U/mL) or with the adenosine A(2A)-receptor antagonist, ZM 241385 (50 nM), enhanced nAChR-induced transmitter release. Activation of A(2A) receptors with CGS 21680C (3 nM) reduced the DMPP-induced release of [3H]-ACh. CGS 21680C (3 nM) inhibition was prevented by MDL 12,330A (10 microM, an adenylate cyclase inhibitor) and by H-89 (10 microM, an inhibitor of protein kinase A), but was potentiated by rolipram (300 microM, a phosphodiesterase inhibitor). DMPP-induced transmitter release was decreased by 8-bromo-cyclic AMP (1 mM, a protein kinase A activator), rolipram (300 microM), and forskolin (3 microM, an activator of adenylate cyclase). Both MDL 12,330A (10 microM) and H-89 (10 microM) facilitated DMPP-induced release of [3H]-ACh. The results indicate that nAChR-induced [3H]-ACh release is triggered by the influx of Ca2+, independent of voltage-sensitive calcium channels, presumably directly through nAChRs located on myenteric axon terminals. It was also shown that endogenous adenosine, activating A(2A) receptors coupled to the adenylate cyclase/cyclic AMP transducing system, is tonically downregulating this nAChR-mediated control of [3H]-ACh release.