The effects of L-, N-, P- and Q-type calcium channel antagonists and (+/-)-BayK-8644 on catecholamine release induced by pituitary adenylate cyclase-activating polypeptide (PACAP-27) were investigated in bovine cultured adrenal chromaffin cells. PACAP-27 induced the release of 4-15% of the total cellular catecholamines over 7 min, with an EC50 of 20 nM and the effect approaching maximum at 100 nM. Catecholamine release was fully dependent on the presence of extracellular calcium. The dihydropyridine nitrendipine which inhibits L-type calcium channels inhibited PACAP-27-induced secretion in a concentration dependent manner with an inhibition of 20-30% at 1 microM. In contrast, (+/-)-BayK-8644, which prolongs the opening of L-type calcium channels produced a concentration-dependent increase in PACAP-27-induced catecholamine release with 1 microM increasing release by 40-60%. Blockade of N-type calcium channels with omega-conotoxin GVIA reduced release by 5-15%. Block of P-type channels with low concentrations of omega-agatoxin IVA (< or = 30 nM) had no significant effect on release, while higher concentrations (100-300 nM) which block Q-type channels reduced release by up to 15%. omega-Conotoxin MVIIC, an antagonist of Q-type calcium channels and also of N- and P-type channels, inhibited release in a concentration-dependent manner with a near maximum effect of 30-50% produced by 300 nM. The combination of omega-conotoxin GVIA and omega-agatoxin IVA reduced release by 40-50%. Addition of omega-conotoxin MVIIC (300 nM) to the combination of omega-conotoxin GVIA (10 nM) and omega-agatoxin IVA (100 nM) did not inhibit catecholamine release more than with omega-conotoxin GVIA and omega-agatoxin IVA alone, indicating that 100 nM omega-agatoxin IVA was sufficient to block the Q-type calcium channels. When nitrendipine was used together with omega-conotoxin GVIA, omega-agatoxin IVA and omega-conotoxin MVIIC, catecholamine release induced by 20 nM or 100 nM PACAP-27 was reduced by 70-85%. Taken together these results suggest that influx of calcium through multiple different voltage-sensitive calcium channels mediate PACAP-27-induced catecholamine release from bovine chromaffin cells, and that L-, N- and Q-channels contribute to this response.

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1 The e ects of N-and L-type calcium channel antagonists and (+)-Bay K8644 on catecholamine release from chroma n cells and acetylcholine release from splanchnic nerve terminals was investigated in bovine perfused adrenal glands. 2 Adrenal glands were perfused retrogradely and preloaded with [ 3 H]-choline. Subsequent e ux of 3 Hlabelled compounds was taken as an index of acetylcholine release from the splanchnic nerve terminals. Noradrenaline and adrenaline release from the glands was measured by h.p.l.c. with electrochemical detection. 3 A maximally e ective frequency of ®eld stimulation of the adrenal nerves, 10 Hz, induced release of catecholamines and 3 H-labelled compounds. Tetrodotoxin (1 mM) abolished release of both catecholamines and 3 H-labelled compounds. A combination of mecamylamine (5 mM) and atropine (1 mM) inhibited nerve-induced catecholamine release by about 75% but did not inhibit release of 3 Hlabelled compounds. Reducing the concentration of extracellular calcium 5 fold to 0.5 mM inhibited nerve-induced catecholamine release by 80% and release of 3 H-labelled compounds by 50%. 4 (+)-Bay K8644 (1 mM), nitrendipine (1 mM), o-conotoxin-GVIA (10 nM) and the combination of nitrendipine and o-conotoxin-GVIA each had no e ect on nerve-induced release of 3 H-labelled compounds. 5 (+)-Bay K8644 (1 mM) potentiated nerve-induced catecholamine release by 75%. Nitrendipine (1 mM) reduced release by 20% but this did not reach statistical signi®cance. o-Conotoxin-GVIA (10 nM) reduced nerve-induced catecholamine release by 75%, while the combination of o-conotoxin-GVIA and nitrendipine reduced release to the same extent as o-conotoxin-GVIA alone. 6 Exogenous acetylcholine perfusion through the glands produced a concentration-dependent increase in catecholamine release. The maximally e ective concentration of acetylcholine for catecholamine release was 5300 mM, while 30 mM acetylcholine gave comparable catecholamine release to that obtained with 10 Hz ®eld stimulation. 7 (+)-Bay K8644 (1 mM), nitrendipine (1 mM) and o-conotoxin-GVIA (10 nM) each had no signi®cant e ect on catecholamine release evoked by perfusion of the gland with either a near maximally e ective concentration of acetylcholine, 100 mM, or with the lower concentration of 30 mM. 8 The results show that the o-conotoxin-GVIA-sensitive N-type voltage-sensitive calcium channels located on the chroma n cells are largely responsible for catecholamine release induced by nerve stimulation in bovine adrenal glands. In contrast, N-type calcium channels are not involved in catecholamine release induced by exogenous acetylcholine. L-type voltage sensitive calcium channels do not play a major role in nerve-induced or exogenously applied acetylcholine-induced catecholamine release. However, the L-type calcium channels do have the potential to augment powerfully nerveinduced catecholamine release. N-and L-type calcium channels do not play a major role in the presynaptic release of acetylcholine.

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.

Abstract:The effect on exocytosis of La 3ϩ , a known inhibitor of plasma membrane Ca 2ϩ -ATPases and Na ϩ / Ca 2ϩ exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La 3ϩ substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd 3ϩ Extracellular fluid is the principal source of Ca 2ϩ in neurons (Berridge, 1998). Neurotransmitter release is essentially dependent on Ca 2ϩ influx through the plasma membrane. In adrenal chromaffin cells, voltage-operated Ca 2ϩ channels (VOCCs) are responsible for the majority of the Ca 2ϩ entry that supports catecholamine release evoked by both depolarizing agonists and agonists acting at G protein-coupled receptors (Burgoyne, 1991;Artalejo et al., 1994;Lomax et al., 1997;O'Farrell and Marley, 1997Lara et al., 1998). Although mobilization of intracellular Ca 2ϩ stores modulates secretion, it fails to evoke significant levels of exocytosis in the absence of Ca 2ϩ (Kim and Westhead, 1989;Pan and Kao, 1997 Schroeder et al., 1994;Robinson et al., 1995Robinson et al., , 1996. VOCCs mediate substantial Ca 2ϩ influx in these cells and are very effective at evoking secretion. Parts of the endoplasmic reticulum come into close contact with the plasma membrane (Berridge, 1998), and some of these Ca 2ϩ stores are suitably located in chromaffin cells to influence membrane events [such as K ϩ channel activity (Kim and Kim, 1998)]. Their lack of efficacy in evoking exocytosis is likely to be because mobilization of store Ca 2ϩ fails to generate a sufficiently high subplasmalemmal [Ca 2ϩ ]. This may be because (a) the stores do not contain sufficient quantities of Ca 2ϩ , (b) the density of channels on the store membrane is too low, (c) insufficient numbers of such channels are activated at any one time, or (d) local Ca 2ϩ buffering and Ca 2ϩ reuptake and extrusion processes prevent high concentrations of Ca 2ϩ being achieved by store mobilization. This last possibility is of particular interest because the activity of the sarco/endoplasmic reticulum Ca 2ϩ -ATPases (SERCAs) and of the plasma membrane Ca 2ϩ -ATPases (PMCAs) and Na ϩ /Ca 2ϩ exchangers is acutely regulated (Verboomen et al., 1995;Matsuda et al., 1997;Guerini, 1998;Pan et al., 1998