Stephen J. Bunn

Localization of angiotensin II binding sites in the bovine adrenal medulla using a labelled specific antagonist

Angiotensin II binding sites have been localized in sections of bovine adrenal glands and on living cultured bovine adrenal medullary cells using [125I]-[Sar1,Ile8]-angiotensin II and autoradiographic techniques. Binding sites were observed over both adrenaline and noradrenaline chromaffin cells. However, they were present in higher density over adrenaline cells, as determined by the distribution of phenylethanolamine N-methyltransferase mRNA by in situ hybridization histochemistry and of glyoxylic acid-induced fluorescence of noradrenaline. Binding sites were also observed in low density over nerve tracts within the bovine adrenal gland. Living cultured bovine adrenal medullary cells possessed angiotensin II binding sites. Not all cells were labelled. At least 73% of identified dispersed chromaffin cells in these cultures were labelled. Some chromaffin cells were not labelled with the ligand, and at least some non-chromaffin cells in the cultures did possess angiotensin II binding sites. The results provide direct anatomical support for the known ability of angiotensin II to elicit catecholamine secretion from perfused adrenal glands and from cultured adrenal chromaffin cells. They also suggest that some of the effects of angiotensin II on calcium fluxes and second messenger levels measured in cultured adrenal medullary cell preparations may be due to angiotensin II acting on non-chromaffin cells present in these cultures.

Effects of phorbol esters and forskolin on basal and histamine-induced accumulation of inositol phosphates in cultured bovine adrenal chromaffin cells

Abstract: The effect of phorbol esters and forskolin pretreat‐ment on basal and histamine‐induced accumulation of inositol phosphates and catecholamine release was Examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose‐dependent, Ca2+‐dependent accumulation of total inositol phosphates with an EC50 at ∼ 1 μMandjan eight‐ to 10‐fold increase at 100 μM within 30 min of jincubation. Histamine (10 μM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20‐min period. Both the inositol phosphite and catecholamine responses were completely blocked by the Hrantagonist mepyramine and were insensitive tojthe H2‐antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphatesj stimulated by histamine revealed an early and sustained rise| in inositol 1,4‐bisphosphate content but not inositol 1,4,5‐trikphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor‐promoting phorbol ester phorbol 12‐myristate 13‐acetate (PMA) resulted in a dose‐dependent, time‐dependent inhibition of histamine‐induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC5o of ∼0.5 nM), an effect not shared by the inactive phorbol ester 4‐a‐phorbol 12,13‐didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine‐induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.

The distribution of opioid binding subtypes in the bovine adrenal medulla

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1-13). Specific [3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1-13). Delta opioid sites, labelled with [3H][D-Ala2,D-Leu5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [3H][D-Ala2, NMePhe4,Gly-ol5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.

Receptor stimulated formation of inositol phosphates in cultures of bovine adrenal medullary cells : the effects of bradykinin, bombesin and neurotensin

The ability of a number of drugs and neuropeptides to stimulate phosphoinositide metabolism in cultured bovine adrenal medullary cells has been assessed. Low concentrations (10 nM) of angiotensin II, bradykinin, histamine, arginine-vasopressin, and bombesin, and high (10 microM) concentrations of oxytocin, prostaglandins E1, and E2, beta-endorphin, and neurotensin stimulated significant accumulation of [3H]inositol phosphates in adrenal medullary cells preloaded with [3H)]inositol. Bradykinin stimulated a significant response at concentration as low as 10pM, with an EC50 of approximately 0.5 nM. The response was markedly inhibited by the bradykinin B2 antagonist [Thi5,8,D-Phe7] bradykinin but not the B1 antagonist [Des-Arg9,Leu8] bradykinin. Higher concentrations of bombesin and neurotensin were required to elicit a response (10 nM and 10 microM respectively). The bombesin response was sensitive to inhibition by the bombesin antagonist [D-Arg1,D-Pro2,D-Trp7,9Leu11]-substance P. In contrast, the neurotensin response was not reduced by the NT1 antagonist [D-Trp11]-neurotensin. These results indicate there are a number of agents that can stimulate phosphatidylinositide hydrolysis in the adrenal medullary cells by acting on different classes of receptors. Such a range of diverse agonists that stimulate inositol phosphate formation will facilitate further analysis of the phosphatidylinositide breakdown in chromaffin cell function.

Co-localization of RNAs coding for phenylethanolamine N-methyltransferase and proenkephalin A in bovine and ovine adrenals.

A 29-mer oligodeoxyribonucleotide probe, complementary to the coding region of bovine phenylethanolamine N-methyltransferase (PNMT) mRNA was synthesized. Characterization of this probe by Northern blot hybridization showed that it hybridized to a single band in RNA extracted from bovine and ovine adrenal medullae. The molecular size of this hybridized band was approximately 1.0-1.2 kb which is consistent with recently reported data on the molecular weight of bovine PNMT mRNA. In situ hybridization histochemistry was carried out with this probe on bovine and ovine adrenal sections and results compared on adjacent sections with a probe against proenkephalin A (ProEnk A) mRNA synthesized previously. Both showed a similar localization to the outer margin of cells in the adrenal medulla. The results of this study provide strong evidence at the level of mRNA expression that ProEnk A mRNA is expressed preferentially in the adrenaline synthesizing cells within the adrenal medulla. Further, it demonstrates the usefulness of a synthetic oligodeoxyribonucleotide probe for the study of PNMT gene expression.

Actions of somatostatin on perfused bovine adrenal glands and cultured bovine adrenal medullary cells.

The effects of somatostatin on catecholamine secretion and inositol phosphate accumulation have been studied using isolated perfused bovine adrenal glands and cultured bovine adrenal medullary cells. Somatostatin had no effect on basal adrenaline or noradrenaline secretion from either preparation. At concentrations above 1 microM, somatostatin inhibited the secretion of both catecholamines induced by 5 microM nicotine from cultured chromaffin cells. In contrast, over the concentration range 0.1 nM-10 microM, somatostatin had no effect on the secretory responses produced by 10 nM angiotensin II or 1 microM histamine. Inositol phosphate accumulation in cultured bovine adrenal medullary cells was unaffected by 0.1 nM-0.1 microM somatostatin, however at 1 and 10 microM somatostatin it was significantly increased, by 23% and 103% respectively. The effects of somatostatin (0.1 nM-10 microM) and of 50 microM muscarine on inositol phosphate accumulation were simply additive. Similarly, somatostatin at 0.1 nM and 10 nM together with 10 nM angiotensin II or 1 microM histamine produced additive inositol phosphate responses. In contrast, 1 microM somatostatin gave significantly more-than-additive (synergistic) inositol phosphate responses with angiotensin II and histamine. The results suggest that some adrenal medullary cells possess several types of receptors, and that these receptors may interact to produce non-additive responses.

Effects of opioid compounds on basal and muscarinic induced accumulation of inositol phosphates in cultured bovine chromaffin cells

The mammalian adrenal medulla expresses a variety of both opioid peptides and opioid receptors. The function of this adrenal opioid system is, however, largely unknown. We have examined the ability of a number of opioid compounds to influence basal and muscarinic stimulated accumulation of inositol phosphates in cultured bovine chromaffin cells. Muscarine produced a dose-dependent 1.5-fold increase in total inositol phosphates. This response was sensitive to atropine inhibition. The ten opioid compounds examined were chosen because between them they possess selectivity for all of the identified opioid receptor subtypes. However, none of these opioids in the concentration range 10nM-10 microM had any significant effect on either basal or muscarinic induced total inositol phosphate accumulation. We conclude that it is unlikely that opioid peptides released from either the chromaffin cells themselves or the splanchnic nerve can modulate the inositol phosphate second messenger system within the adrenal chromaffin cells.

Prostanoid responses of bovine adrenal medullary cells: lack of effect of opioids

The effects of opioid compounds on catecholamine (CA) secretion and phosphatidylinositol turnover induced by prostaglandins E1 (PGE1) and E2 (PGE2) in cultured bovine adrenal medullary cells have been studied. PGE1 induced CA secretion at 100 nM and above. PGE2 was more potent, inducing CA secretion at 1-10 nM. Both prostaglandins required extracellular calcium to induce CA release. Neither etorphine nor diprenorphine (1 nM-10 microM) affected CA secretion induced by 1 microM PGE1 or 0.1 microM PGE2. PGE1 a small increase in phosphatidylinositol turnover at 10 microM, but had no effect at lower concentrations. PGE2 was effective at 1 and 10 microM. Etorphine and diprenorphine had no effect on phosphatidylinositol turnover induced by PGE1 or PGE2. The results indicate prostaglandins can facilitate CA secretion independently of their effects on phosphatidylinositol metabolism. They also indicate that endogenous adrenal opioid peptides do not act on the opioid binding sites found on adrenal medullary cells to modify their responses to prostaglandins.

The effects of neosurugatoxin on evoked catecholamine secretion from bovine adrenal chromaffin cells.

1 The effect of neosurugatoxin (NSTX), a toxin from the Japanese ivory mollusc (Babylonia japonica), on the nicotinic response of bovine adrenal chromaffin cells was examined. 2 NSTX inhibited acetylcholine‐and nicotine‐induced catecholamine secretion from the cultured cells with an IC50 against 5 μm nicotine of 30 nm. 3 This inhibitory effect was reversible and independent of the presence of agonist. 4 NSTX had no effect on the catecholamine release from cultured cells evoked by 50 mm K+, or 1 μm histamine. 5 NSTX had no effect on the stimulation of phosphatidylinositol metabolism evoked by 100 μm muscarine. 6 These results suggest NSTX may be useful as a nicotinic receptor probe in tissues such as the adrenal and sympathetic ganglia where α‐bungarotoxin is ineffective.

Lack of effect of opioid compounds on angiotensin II responses of bovine adrenal medullary cells

Angiotensin II (10 nM) increased basal adrenaline and noradrenaline secretion from cultured bovine adrenal chromaffin cells by 2.5- to 3-fold and 4- to 6-fold, respectively, and stimulated basal accumulation of inositol phosphates more than 2-fold. Etorphine and diprenorphine in the range 10(-9) to 10(-5) M had no effect on the catecholamine secretion induced by angiotensin II, and, at 10(-8) and 10(-5) M, had no effect on angiotensin II-induced inositol phosphate accumulation. The functions of adrenal medullary opioid receptors remain to be determined.