Histochemical and biochemical studies demonstrate that -aminobutyric acid (GABA), glutamic acid de- Chromaffin cells of adrenal medulla specialize in the production, storage, and secretion of catecholamines (CAs). These processes are modulated by nicotinic receptors that are located in chromaffin cell membranes and are innervated by splanchnic cholinergic axons. However, recent evidence has challenged this simplistic view on the neuronal modulation of adrenal medullary function. This evidence includes a documentation of the presence in adrenal medulla of opioid peptides (1-3), substance P (4), somatostatin (5), vasoactive intestinal peptide (6), and histamine (7). Though we know that receptors for opioids (8) and substance P (9) interact with cholinergic receptors, our understanding of the molecular mechanisms operative in the modulation of medullary function is far from being complete. The present report shows that primary cultures of chromaffin cells prepared from bovine adrenal can synthesize, store, release, and inactivate y aminobutyric acid (GABA); moreover, GABA receptors appear to modulate the secretion of CA mediated by acetylcholine (AcCho). The GABA receptors of medulla include a recognition site for benzodiazepines and function in a manner reminiscent of brain GABAergic receptors. MATERIALS AND METHODSCell Preparation. Primary cultures of chromaffin cells were prepared from bovine adrenal glands, obtained at a local slaughterhouse (Treuth and Son, Catonsville, MD), according to Kilpatrick et al. (10) with minor modifications (8). These cultures were maintained with a Dulbecco's modified Eagle medium (DME medium, GIBCO), 5 mM Hepes (pH 7.4), 10% fetal calf serum (GIBCO), 100,000 units of penicillin per liter, 10 mg of streptomycin (GIBCO) per liter, 40 mg of gentamycin (Shering) per liter, 50,000 units of mycostatin (Squibb) per liter, and 5 ,.M fluorodeoxyuridine (Sigma).CA Release. The chromaffin cells were placed in 24-well plastic dishes (Costar). Each well contained -2.5 x 105 cells in 1 ml of DME medium and was kept for 5-7 days at 370C in 5% C02/95% air. The cells were tightly attached to the plastic after 5-7 days in culture. At this time the dishes were placed in a 370C water bath, the DME medium was aspirated, and the cells were washed twice with Locke solution (8) and then were incubated at 370C for 10 min in 500 u1l of Locke solution with or without the drug to be studied. Epinephrine and norepinephrine released in the medium or contained in the cells was measured with an HPLC coupled to electrochemical detection (8).Uptake

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Summary1. The concentrations of adrenaline and noradrenaline in the adrenal vein and the adrenal gland of the cat were studied in response to different stimuli leading to increased catecholamine (CA) secretion. 2. Haemorrhage and hypoglycaemia, but not acute exposure to cold or intravenous administration of cocaine, induced considerable increases in total catecholamine secretion.3. The ratio of the concentration of adrenaline to noradrenaline in adrenal vein plasma during the control period was higher than the ratio in the adrenal gland itself. 4. Haemorrhage increased noradrenaline secretion considerably more than adrenaline secretion so that the ratio of the concentration of adrenaline to noradrenaline in adrenal vein plasma was significantly lower than in the adrenal gland itself. 5. Hypoglycaemia induced by insulin increased catecholamine secretion, with the adrenaline to noradrenaline ratio significantly higher than in the adrenal gland itself. 6. Hypothermia resulted in a fall of the initial high ratio of adrenaline to noradrenaline, to a value similar to that in the adrenal gland. 7. Neither cocaine nor changes in adrenal plasma flow affected the adrenaline to noradrenaline ratio in adrenal vein blood. 8. It is concluded that preferential release from the adrenal gland of either adrenaline or noradrenaline is possible in vivo in response to different stimuli.

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