Biomedical subjects

B G Livett

Publications and source records attributed to B G Livett.

At least 55 records · Page 3Linked to original sources

PubMed1989

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.

Adrenal Glands↗

PubMed1989

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.

Adrenal Medulla↗

PubMed1989

Developmental expression of proenkephalin A mRNA and phenylethanolamine N-methyltransferase mRNA in foetal sheep adrenal medulla.

The ontogenic expression of proenkephalin A (ProEnk A) mRNA and phenylethanolamine N-methyltransferase (PNMT) mRNA was examined in the foetal sheep adrenal medulla by the use of specific oligodeoxyribonucleotide probes. Northern blot analysis of RNA extracts from foetal adrenals demonstrated that ProEnk A mRNA was expressed as early as 60 days of gestation, a time at which the foetal adrenal is not functionally innervated. In situ hybridization on sections of foetal adrenals revealed that at 110-140 days gestation ProEnk A mRNA was expressed in chromaffin cells at the outer margin of the adrenal medulla but at earlier stages of gestation (e.g. 95 days) appeared to be expressed homogeneously throughout the whole of the adrenal medulla. In comparison, PNMT mRNA was expressed preferentially in cells at the outer margin of the adrenal medulla from the earliest stage detectable. Both PNMT mRNA and ProEnk A mRNA co-localized in cells at the outer margin of foetal adrenal of late gestations (110-140 days), a similar pattern to that seen in the adult adrenal medulla. These results indicate that, as with adult animals, in foetuses of late gestation, adrenal enkephalins are co-stored within adrenaline cells. It is likely therefore that enkephalins are co-released from the foetal adrenal with adrenaline in response to intra-uterine stress.

Adrenal Medulla↗

PubMed1989

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

The effect of phorbol esters and forskolin pretreatment 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 approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate 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 (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-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.

Adrenal Medulla↗

PubMed1988

Effect of substance P on nicotine-induced desensitization of cultured bovine adrenal chromaffin cells: possible receptor subtypes.

The neuropeptide substance P (SP) has been reassessed for its ability to modify nicotine-induced catecholamine secretion from cultured bovine, adrenal chromaffin cells. SP exhibited biphasic effects in its actions of inhibiting the nicotinic secretory response and protecting against desensitization. At low concentrations, up to 3 microM, SP partially inhibited or partially protected the nicotine response by 15-20%, and at high concentrations (30 microM), SP markedly inhibited or markedly protected the nicotinic response by 80 or 92%, respectively. The SP antagonist (D-Arg1-D-Pro2-D-Trp7,9-Leu11-SP) completely blocked both effects produced by low concentrations of SP, but not those produced by high concentrations. It is concluded that SP is more potent at protecting against desensitization than at inhibiting the nicotinic response and that SP might modulate CA release through activation of two receptor subtypes.

Adrenal Medulla↗

PubMed1988

Mammalian tachykinins modulate the nicotinic secretory response of cultured bovine adrenal chromaffin cells.

We have studied the modulatory actions of two members of the tachykinin family (neurokinin A and B) on endogenous catecholamine (CA) secretion from cultured adrenal chromaffin cells. Their ability to modulate the nicotinic response was compared to that of substance P (SP). Both neurokinin A and neurokinin B were found to have two distinct actions similar to SP, on nicotine-induced CA release: (1) an inhibitory action at low nicotine concentrations; and (2) a protective action against desensitization by high nicotine concentrations. However, on a molar basis, the efficacy of neurokinin A or B to modulate the nicotinic response (both inhibition or protection) was 30 times less than SP. We have also tested the ability of a SP antagonist (D-Arg1-D-Pro2-D-Trp7,9-Leu11-SP) to antagonize the modulatory actions of SP on the nicotinic response. The results suggest the possibility that SP's actions on the bovine adrenal chromaffin cells might be mediated through two receptor subtypes of two different affinities.

Adrenal Medulla↗

PubMed1988

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

The effects of a number of alpha- and beta-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 Ba2+ 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 5 microM nicotine was only weakly inhibited by adrenaline and noradrenaline and even then required high concentrations (greater than 500 microM), while dopamine (1 microM-1 mM) and isoproterenol (100 microM) had no effect. Clonidine (100 microM), oxymetazoline (100 microM), yohimbine (100 microM), and propranolol (5 microM) all produced inhibition of nicotine-induced ATP release with the order of potency:propranolol greater than oxymetazoline greater than clonidine = yohimbine. The inhibitory effect by propranolol could not be reversed by high concentrations of adrenaline or isoproterenol. In chromaffin cell monolayer cultures, all alpha 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 microM) or Ba2+ (2.2 mM). (+/-)Propranolol caused a similar pattern of inhibition, however, this inhibition was also observed by (+)propranolol, an isomer with little beta-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 EC50 concentration of nicotine (5 microM) following pre-incubation of cells to nicotine in the range of 0.3-300 microM. The desensitization had a threshold of 1 microM nicotine and was maximal at 3 microM nicotine in the pre-incubation. Both clonidine (50 microM) and (+/-)propranolol (5 microM) inhibited CA secretion induced by nicotine (0.3 microM-300 microM) 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1988

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.

Adrenal Medulla↗

PubMed1988

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.

Adrenal Medulla↗

PubMed1988

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.

Adrenal Medulla↗

PubMed1988

Characterization of a dipeptidyl aminopeptidase from bovine adrenal medulla.

A dipeptidyl aminopeptidase was partially purified from a supernatant fraction of bovine adrenal medulla by gel filtration and anion-exchange chromatography. From gel filtration, the apparent molecular weight of the enzyme was 68,100 and its pH optimum was 9.5. Its Km for hydrolysis of the synthetic substrate arginylarginine-beta-naphthylamide was 5.5 X 10(-6) M. The enzyme was inhibited by metal ion chelating agents and thiol blocking agents, suggesting the requirement for both a metal ion and an active cysteine residue for its activity. Several peptides were cleaved by the dipeptidyl aminopeptidase involving the sequential removal of dipeptides from the N-terminus. Biologically active peptides, such as leucine-enkephalin, methionine-enkephalin, and angiotensin II, were hydrolyzed by the dipeptidyl aminopeptidase although opioid peptides with a length greater than five amino acid residues were not susceptible to hydrolysis. Other peptides with a blocked N-terminus (neurotensin, bombesin) or a proline residue adjacent to a potential cleavage site (substance P) were not hydrolyzed. The ability of this dipeptidyl aminopeptidase to degrade certain neuropeptides suggests that it could be involved in neuropeptide degradation.

Adrenal Medulla↗

PubMed1988

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 microM 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 microM histamine. 5. NSTX had no effect on the stimulation of phosphatidylinositol metabolism evoked by 100 microM muscarine. 6. These results suggest NSTX may be useful as a nicotinic receptor probe in tissues such as the adrenal and sympathetic ganglia where alpha-bungarotoxin is ineffective.

Adrenal Glands↗

PubMed1987

Effects of opioid compounds on desensitization of the nicotinic response of isolated bovine adrenal chromaffin cells.

Opioid compounds have been assessed for their ability to modify desensitization of nicotine-induced catecholamine secretion from cultured, bovine, adrenal chromaffin cells. Dynorphin-1-13 and metorphamide produced protection against desensitization of the nicotinic response at concentrations between 1 and 20 microM while etorphine and morphine only produced this effect at 100 microM. The opioid antagonists, naloxone and diprenorphine, at 100 microM mimicked the weak ability of the opioid agonists to protect against nicotinic desensitization. All opioid compounds tested were considerably more potent at inhibiting nicotine-induced catecholamine secretion from the cells than at protecting against desensitization of this response. It is concluded that adrenal opioid peptides probably do not act on adrenal opioid binding sites characterised from ligand binding studies to prevent the nicotinic response from desensitizing. They are unlikely, therefore, to be involved in such a mechanism to maintain catecholamine secretion during stress.

Adrenal Medulla↗

PubMed1987

Differences between the mechanisms of adrenaline and noradrenaline secretion from isolated, bovine, adrenal chromaffin cells.

Cultured, bovine, adrenal medullary chromaffin cells have been used to study the secretion of adrenaline (A) and noradrenaline (NA) in response to two consecutive stimulation periods. Cells were first exposed to nicotine, then rapidly washed before being exposed to elevated K+ levels. Both A and NA cell types secreted their catecholamine in response to nicotine; however, NA cells secreted 3 times more of their catecholamine stores as did A cells. The NA cells were desensitized to stimulation by K+ if they had previously been exposed to nicotine. A cells, on the other hand, were up to twice as responsive to K+ stimulation after having been stimulated with nicotine, in spite of substantial depletion of their catecholamine stores. This sensitization of A cells to K+ was neither due to carryover of nicotine from the first stimulation period, nor to accumulation of calcium in the cells during the nicotinic stimulation. The results suggest that stimulus-secretion coupling or the exocytotic machinery in A and NA chromaffin cells is different. These differences may contribute to the ratio A:NA secreted from the adrenal medulla during stress.

Adrenal Medulla↗

PubMed1987

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.

Adrenal Medulla↗

PubMed1987

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)

Acetylcholine↗

PubMed1987

Sensory fibres modulate histamine-induced catecholamine secretion from the rat adrenal medulla and sympathetic nerves.

1. We have studied the mechanism of catecholamine secretion induced by histamine from the adrenal medulla and sympathetic noradrenergic neurones in the rat, and the role of capsaicin-sensitive sensory nerves in this secretion. 2. Histamine at a dose of 1 mg/kg induced adrenaline and noradrenaline secretion by a non-neurogenic mechanism. In contrast, at a dose of 3 mg/kg it induced adrenaline and noradrenaline secretion by both non-neurogenic and neurogenic mechanisms. 3. The adrenaline released in response to histamine at 3 mg/kg was exclusively of adrenal origin whereas the noradrenaline released was of non-adrenal origin (most probably noradrenergic sympathetic nerves). As with its action on the adrenal, histamine induced noradrenaline secretion from these extra-adrenal tissues by both neurogenic and non-neurogenic mechanisms. 4. When adrenaline secretion from the adrenal gland was impaired by adrenal denervation and/or adrenalectomy, the plasma noradrenaline secretion was increased. This is most probably due to compensation from the rest of the sympathetic nervous system. This compensatory increase in noradrenaline was abolished by hexamethonium, which indicates that it was mediated by a cholinergic mechanism. 5. Pre-treatment of rats as neonates subcutaneously with capsaicin (a selective neurotoxin for certain sensory nerves) at a dose of 50 mg/kg, had no effect on the non-neurogenic secretion of catecholamine induced by histamine. In contrast, capsaicin pre-treatment abolished the neurogenic catecholamine secretion in response to histamine as well as the neurogenic compensatory increase in plasma noradrenaline levels that occurred when adrenaline secretion by the adrenal gland was impaired. 6. In the present study, by using histamine as a form of stress, we have been able to provide evidence not only (a) to confirm a role for capsaicin-sensitive sensory fibres in modulating neurogenic adrenaline secretion from the adrenal gland, but also (b) to suggest a similar role for these fibres in modulating neurogenic noradrenaline release from sympathetic noradrenergic nerves in response to histamine and in response to impairment of adrenaline secretion by the adrenal gland.

Adrenal Medulla↗

PubMed1986

Metorphamide, a novel endogenous adrenal opioid peptide, inhibits nicotine-induced secretion from bovine adrenal chromaffin cells.

Opioid peptides are found in high concentrations in the adrenal medulla. Recently, a novel opioid octapeptide, metorphamide, possessing an amidated C-terminal, was characterized and also found to be present in adrenal tissue. We have studied the ability of this novel peptide to modify nicotine-induced secretion from isolated bovine adrenal chromaffin cells. Exocytosis was monitored by measuring adenosine triphosphate (ATP) release on-line by the luciferin-luciferase bioluminescence method, or by measuring endogenous catecholamine release by high-performance liquid chromatography (HPLC) with electrochemical detection. Metorphamide inhibited 5 microM nicotine-induced ATP release from fresh chromaffin cells by almost 50% at 5 microM. Metorphamide at concentrations less than 1 microM had no effect on 5 microM nicotine-induced adrenaline and noradrenaline release from cultured cells, but at higher concentrations inhibited their release equally, with an IC50 of approximately 10 microM. By contrast, Met5-enkephalin inhibited the release of both catecholamines equally with an IC50 of greater than 1 mM, making metorphamide greater than 100-fold more potent than Met5-enkephalin in this system. Naloxone (10 microM) and diprenorphine (1 microM) failed to antagonise the inhibitory action of metorphamide on nicotine-induced catecholamine release. Metorphamide inhibited the nicotinic response in a non-competitive manner, and failed to affect either adrenaline or noradrenaline release induced by elevated potassium ion concentrations. The results suggest metorphamide acts on naloxone- and diprenorphine-resistant receptors to inhibit chromaffin cell nicotinic secretion and that the novel amidated C-terminal of the peptide is important for this action.

Adenosine Triphosphate↗