David C.-C. Wan

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.

Induction of phenylethanolamine N-methyltransferase mRNA expression by glucocorticoids in cultured bovine adrenal chromaffin cells

The effects of glucocorticoids on the expression of phenylethanolamine N-methyltransferase (PNMT) mRNA and proenkephalin A (ProEnk A) mRNA in cultures of bovine adrenal chromaffin cells were examined. The expression of PNMT mRNA (approx. 1.1 kilobases) was induced in the presence of glucocorticoids. This induction was of high potency with an EC50 in the range of 1-10 nM for dexamethasone, and was blocked by high concentrations of the glucocorticoid antagonist RU-38486. Cortisol, prednisolone and Reichstein substance S (11-deoxy-17-hydroxycorticosterone) were all effective in stimulating PNMT mRNA expression while cortisone, progesterone and beta-estradiol were without effect. These results indicate that the effects are mediated by specific glucocorticoid receptor activation and exhibited a strict structural requirement for the ability of glucocorticoids to induce PNMT mRNA expression. By contrast, glucocorticoids had no significant effect on the expression of ProEnk A mRNA. In summary, this study provides evidence that glucocorticoids act to regulate PNMT (but not ProEnk A) at the transcriptional level. This differential effect of glucocorticoids suggests that different mechanisms govern the expression of mRNAs required for synthesis of the co-stored secretory components, the enkephalins and adrenaline within the chromaffin cells of the adrenal medulla.

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.

Coordinate and differential regulation of proenkephalin A and PNMT mRNA expression in cultured bovine adrenal chromaffin cells : responses to cAMP elevation and phorbol esters

The expression of proenkephalin A (ProEnk A) mRNA and phenylethanolamine N-methyltransferase (PNMT) mRNA in response to cAMP analogues, forskolin and phorbol esters was examined in cultures of bovine adrenal chromaffin cells. Exposure of chromaffin cells to 1 mM dibutyryl cAMP for 24 h increased significantly the levels of ProEnk A mRNA, with no significant effect on the levels of PNMT mRNA. Cells exposed to the tumor promoting phorbol esters (phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate or 4-beta-phorbol 12,13-didecanoate) for 12 h differentially activated PNMT mRNA and ProEnk A mRNA expression. The levels of PNMT mRNA were dramatically elevated in response to low concentrations (10(-9) to 10(-8)M) of these phorbol esters, but these increases were diminished at higher concentrations (10(-7) to 10(-6) M) of the phorbol esters. These responses were synergistically potentiated by dexamethasone (1 microM), a synthetic glucocorticoid. None of these effects was seen with the biologically inactive phorbol ester, 4-alpha-phorbol 12,13-didecanoate. By contrast, the expression of ProEnk A mRNA was activated by the tumor promoting phorbol esters in a concentration-dependent manner. The results of this study demonstrate a differential stimulatory effect of second messenger mechanisms in the control of PNMT and ProEnK A mRNA expression and provide further evidence for an independent control for the enkephalin and adrenaline synthesis in these cells.

Effects of α- and β-adrenoceptor agonists and antagonists on ATP and catecholamine release and desensitization of the nicotinic response in bovine adrenal chromaffin cells

Abstract The effects of a number of α- and β-adrenoceptor agonists and antagonists on the modulation of secretion from bovine adrenal chromaffin cells were investigated. Secretion was induced by nicotine, 56 mM K + , histamine or Ba 2+ and was detected by the ATP luciferin-luciferase bioluminescence technique or by the measurement of endogenous catecholamines (CA) by HPLC coupled with electrochemical detection. ATP release from freshly isolated cells by 5μM nicotine was only weakly inhibited by adrenaline and noradrenaline and even then required high concentrations (> 500 μM), while dopamine (1 μM–1 mM) and isoproterenol (100 μM) had no effect. Clonidine (100 μM), oxymetazoline (100 μM), yohimbine (100 μM), and propranotol (5 μM) all produced inhibition of nicotine-induced ATP release with the order of potency:propranolol > oxymetazoline > clonidine = yohimbine. The inhibitory effect by propranolol could not be reversed by high concentrations of adrenaline or isoproterenol. In chromaffin cell monolayer cultures, all α 2 -adrenoceptor agents tested (clonidine, oxymetazoline and yohimbine), produced a dose-dependent, Na + -sensitive, non-competitive inhibition of nicotine-induced catecholamine release with little effect on the catecholamine release induced by K + (56 mM), histamine (10 μM) or Ba 2+ (2.2 mM). (±)Propranolol caused a similar pattern of inhibition, however, this inhibition was also observed by (+)propranolol, an isomer with little β-adrenoceptor antagonist activity. The effects of clonidine and propranolol on desensitization of nicotine-induced CA secretion were also investigated. The degree of desensitization of the nicotinic response was dependent on the concentration of nicotine to which the cells were pre-exposed. Desensitization was detected as the decrease in response to a near EC 50 concentration of nicotine (5 μM) following pre-incubation of cells to nicotine in the range of 0.3–300 μM. The desensitization had a threshold of 1 μM nicotine and was maximal at 3 μM nicotine in the pre-incubation. Both clonidine (50 μM) and (±)propranolol (5 μM) inhibited CA secretion induced by nicotine (0.3 μM–300 μM) during the pre-incubation period. However, regardless of this inhibition of secretion, neither clonidine nor propranolol had an effect on either the onset, or the rate of nicotine-evoked desensitization subsequently observed. These data suggest that inhibition of the nicotinic response and desensitization of the nicotinic response are regulated independently. These findings do not support the existence of α- or gb-adrenoceptor modulation of secretion from isolated bovine chromaffin cells.

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.

Histamine activates proenkephalin A mRNA but not phenylethanolamine N-methyltransferase mRNA expression in cultured bovine adrenal chromaffin cells

The effects of histamine on the regulation of proenkephalin A (ProEnk A) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression were examined in cultures of bovine adrenal chromaffin cells. Prolonged incubation with histamine resulted in a concentration-dependent increase in the levels of ProEnk A mRNA with little effect on the levels of PNMT mRNA. The activation of ProEnK A mRNA by histamine followed a slow time course, reaching 2-3 fold basal levels after 48 h incubation. This activation was antagonized by the H1-antagonist mepyramine but not by the H2-antagonist cimetidine indicating involvement of H1-histamine receptors. The histamine-induced activation of ProEnK mRNA was blocked by the RNA synthesis inhibitor actinomycin D, suggesting that the novo synthesis of ProEnkA mRNA is a requirement for activation. In the presence of the calcium channel blocker D600, the histamine-induced increase in ProEnk A mRNA was greatly reduced, though not abolished. Prolonged incubation with histamine also caused a substantial release of catecholamines and opioid peptides from these cells. These results suggest that the synthesis and release of opioid peptides is controlled by histamine via H1-receptors. The differential effects of histamine on ProEnk A mRNA and PNMT mRNA expression suggest that different regulatory mechanisms are called upon to regulate the synthesis of opioid peptides and adrenaline in response to stimulation of the chromaffin cells.

Synthesis of a specific oligodeoxyribonucleotide probe and its use for studying proenkephalin A gene expression

A 44-mer, complementary to the coding region of bovine adrenal medullary proenkephalin A mRNA (ProEnkA mRNA) has been synthesized as a specific probe for this mRNA. Northern-blot hybridization analysis identified a single band in an RNA extract from bovine, ovine and porcine adrenal medullae. The molecular size of these hybridized bands (1400-1600 nucleotides) was in excellent agreement with that reported previously using cloned cDNA probes. In situ hybridization on bovine and ovine adrenal sections revealed that ProEnkA mRNA was localized selectively in cells at the outer margin of the medulla, a region rich in adrenaline-containing cells. This study both confirms and extends previous findings with cloned cDNA probes on the presence of high concentrations of ProEnkA mRNA in cells at the periphery of the adrenal medulla. In addition, it demonstrates the usefulness of synthetic oligodeoxyribonucleotides as specific and sensitive probes for the study of proenkephalin A gene expression.

Coordinate and differential regulation of proenkephalin A and PNMT mRNA expression in cultured bovine adrenal chromaffin cells: responses to secretory stimuli.

The expression of proenkephalin A (ProEnk A) mRNA and phenylethanolamine N-methyltransferase (PNMT) mRNA in response to nicotine and to a number of secretagogues was examined in cultured bovine adrenal chromaffin cells. Prolonged incubation with nicotine (10 microM) resulted in a 2-fold increase in ProEnk A mRNA but had no significant effect on the level of PNMT mRNA. Similarly, prolonged stimulation with high K+ (56 mM) induced a time-dependent elevation in the level of ProEnk A mRNA reaching 4-fold basal level after 24 h incubation. By contrast, the level of PNMT mRNA was not changed by treatment with high K+. The increase in the level of ProEnk A mRNA by high K+ was abolished by the presence of 10 microM D600, a calcium channel blocker. Unlike the effects of high K+, treatment of the cells with the sodium channel activator veratridine significantly elevated the levels of both ProEnk A and PNMT mRNA. This increase in ProEnk A and PNMT mRNA levels was however less affected by D600. Stimulation of the cells with Ba2+ (1.1 mM) also stimulated the levels of ProEnk A and PNMT mRNA and this action required the presence of extracellular Ca2+. This was in contrast to the effect of Ba2+ in stimulating catecholamine secretion, which was inhibited by Ca2+ and enhanced in Ca2(+)-free buffer. The results of the present study indicate that membrane depolarization and entry of extracellular Ca2+ play an important role on the regulation of ProEnk A and PNMT mRNAs, in addition to their well-known actions on hormone secretion. Furthermore, these results suggest that the expression of ProEnk A mRNA and PNMT mRNA are under independent regulation in response to secretory stimulation.

Vasoactive intestinal peptide stimulates proenkephalin A mRNA expression in bovine adrenal chromaffin cells.

The effects of vasoactive intestinal peptide (VIP) and substance P (SP) on the amount of proenkephalin A (ProEnk A) mRNA in cultures of bovine adrenal chromaffin cells were examined. Exposure of chromaffin cells to 5 microM VIP for 24 h produced a significant elevation in ProEnk A mRNA. The stimulatory effect of VIP could be abolished by the presence of the calcium channel blocker D600 or actinomycin D but was not affected by the nicotinic antagonist hexamethonium. The results suggest that VIP may induce transcription of ProEnk A mRNA by a Ca2+-dependent, non-cholinergic mechanism. By contrast, SP (5 microM) had no effect on the amount of ProEnk A mRNA. Since VIP is found in nerve terminals and the ganglion cells within the adrenal medulla, this peptide could be an endogenous regulator of adrenal enkephalin gene expression.