No abstract

I Secretion of catecholamines (CA) and dopamine-f-hydroxylase (DBH) activity from the retrogradely perfused cat adrenal gland was studied following ouabain infusion. Perfusion with ouabain (10-s M) for 10 min caused a gradual release of CA in the effluent which reached its peak 30 min after the ouabain pulse, and was maintained constant for at least 1 h. The effect of ouabain seemed to be irreversible. 2 Mecamylamine, while blocking the CA secretory effects of acetylcholine (ACh) perfusion, did not affect the secretion of CA evoked by ouabain. In denervated adrenal glands, ouabain-induced CA secretion was similar to that in the contralateral, innervated gland. However, physostigmine perfusion potentiated the CA secretory effects of ouabain. 3 The release of CA evoked by ouabain was accompanied by a proportional release of DBH activity. The time course of appearance of DBH activity followed the pattern of CA release. 4 The CA and DBH outputs in response to a pulse of ouabain were suppressed in the absence of calcium. Calcium reintroduction to a calcium-free perfused, ouabain-treated gland not only restored but greatly potentiated the release of CA and DBH. The amplitude of the secretory response to calcium reintroduction in ouabain-treated glands was proportional to the extracellular calcium concentration, and was antagonized by an external sodium-deficient medium. 5 These data demonstrate that ouabain releases CA from the perfused cat adrenal gland by a calcium-dependent exocytotic mechanism. The secretory effect of ouabain is not secondary to the release of ACh from cholinergic nerve terminals present in the adrenal gland, but due to a direct action on the chromaffin cell itself. In addition, the results suggest that this action is exerted through redistribution of monovalent cations secondary to the inhibition by the glycoside of the sodium pump. Such monovalent cation redistribution may cause a rise of intracellular ionized calcium levels through the activation of an internal sodium-dependent calcium influx system probably located in the chromaffin cell membrane.

SUMMARY1. Nicotine evokes the release of catecholamines from perfused cat adrenal glands in a concentration-dependent manner, the median effective concentration for nicotine being 5 /tM. Two 2 min pulses of 5 pM-nicotine, 40 min apart (S. and S,) gave net catecholamine outputs of 7-64 and 3-55 jug/8 min, respectively. The ratio S2/S1 in control glands was 0-5.2. Increasing concentrations of apomorphine (1-10 /tM) markedly inhibited catecholamine release during the second nicotine pulse (A2). At 1 /tM-apomorphine, the release during S2 was significantly reduced to 16 % of S,; with 10 ,uM-apomorphine, the secretary response was reduced further to only 3 % of S,, the ratio S2/S1 being 003.3. The presence of haloperidol, sulpiride or picobenzide (each 0 5 ,/M) during S2, completely reversed the inhibition of catecholamine release produced by apomorphine. Haloperidol itself increased the nicotinic secretary response during S2; so, while the ratio S2/S1 was 0 5 in control conditions, this ratio increased significantly to 0 95 if haloperidol (0 5 /lM) was present during S2, suggesting that the presence of this dopaminergic antagonist removed a negative feed-back mechanism that inhibits nicotine-evoked catecholamine release.4. If present during S2, dopamine (1 fSM) also markedly inhibited catecholamine release evoked by nicotine; this inhibition was again reversed by 0.5 /LM-haloperidol.5. Neither the opiate antagonist naloxone nor the a-adrenoceptor blocking agent phentolamine (at concentrations of 0-5-5 ,/M) affected the inhibition by apomorphine of the secretary response to nicotine. 6. These data strongly suggest that the cat adrenal medulla chromaffin cell membrane contains a dopaminergic receptor which modulates the catecholamine secretary process triggered by stimulation of the nicotinic cholinoceptor. The fact that dopamine is released in measurable amounts, together with adrenaline and noradrenaline, from perfused cat adrenal glands in response to nicotinic stimulation (V. Cenia, unpublished results), favours a role for this dopaminergic receptor in modulating catecholamine release from the chromaffin cell.

(10-4 M) inhibited the uptake of 86Rb into adreno-medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30-40 min) to reach its peak. NEM (10-4 M) blocked 86Rb uptake in a similar manner. 6 The results are further evidence in favour of the presence of a Na+-Ca2+ exchange system in the chromaffin cell membrane, probably involved in the control of [Ca2+]i and in the modulation of catecholamine secretion. This system is activated by increasing [Na+]j, either directly (ionophore X537A, increased [Na+]J) or indirectly (Na+ pump inhibition). However, the simple inhibition of Na+ pumping does not always lead to a catecholamine secretory response; such is the case for NEM.

Bovine isolated adrenal chromaffin cells maintained in culture at 37°C for 1–7 days become polygonal and bipolar, with typical varicosity‐like extensions. Catecholamine levels and dopamine β‐hydroxylase activity decreased after 24–48 h of culture, but recovered to normal levels 3–7 days later. Incubation of 1–7 day‐old cells in the presence of increasing concentrations of [3H]‐noradrenaline (3.91 to 125 nm) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day‐old cells took up and retained only one third of the radioactivity found in 2–7 day‐old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mm K+ was very poor in 24 h‐old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2–7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatmen. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor‐mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage‐sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.