In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme L-arginine :NO synthase (NOS) in these cells . Our results show that NO produces a large stimulation of basal CA secretion . This effect was calcium-and concentration-dependent (EC5o = 64 ± 8 IM) and was not due to nonspecific damage of the tissue by NO . N O also modulates the CA secretion evoked by nicotine in a dose-dependent manner . Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (<3 MM ; EC5o = 16 ± 3 yM), it produces a dose-dependent inhibition of the CA secretion induced by high doses of nicotine (~!30 jM ; IC50 = 52 -} 6 yM . The mechanism by which NO modu-

1 The function of y-aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2 The GABAB agonists (-)-baclofen and 3-aminopropylphosphinic acid (3-APPA) were found to induce a dose-dependent increase of basal catecholamine secretion. The ECms were 151 ± 35 1LM and 225 ± 58 tIM for baclofen and 3-APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mM of the specific GABAB antagonist CGP-35348. 3 In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose-dependent manner, the catecholamine secretion evoked by 10 jaM nicotine and 200 JiM muscimol.4 The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]J). GABAB agonists produced a dose-dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol.5 The GABAB agonists also produced a dose-dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6 The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7 The possible involvement of adenylate cyclase in the mechanism of GABAB receptor modulation of catecholamine secretion is discussed.

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The activation of GABAB receptors of adrenal chomaffin cells produces an increase of [Ca2+]i measured by fura-2 AM techniques. GABAB agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca2+ values 332 +/- 60.9 and 306 +/- 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca2+ buffer. The GABAB-induced increase of [Ca2+]i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca2+ channels as the dihydropiridine nifedipine 50 microM was able to decrease it more than 60%, while omega-conotoxin, which blocks N-type channels, did not produce any change in the GABAB-evoked Ca2+ increment. The second component was due to a release of Ca2+ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca2+]i. GABAB receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca2+ store. In a low-Ca2+ buffer after treatment with 2 microM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca2+]i, whereas 4 mM caffeine had no effect on GABAB response. This intracellular Ca2+ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABAB receptors. In fact, the specific agonists after 10 minutes incubation produced a dose-dependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 microM baclofen and 3-APPA, and it was 3.63 +/- 0.75 and 3.2 +/- 1.5 times the basal levels (7.3 +/- 0.3 pmol/10(6) cells), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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