An endogenous polypeptide of rat brain has been identified that is capable of displacing 1,4-benzodiazepines and the esters of the 3-carboxylic acid derivatives of beta-carbolines from their specific synaptic binding sites. This polypeptide was termed diazepam-binding inhibitor (DBI). Previous studies have shown that DBI injected intraventricularly in rodents elicits "proconflict" responses and antagonizes the "anticonflict" action of benzodiazepines. An antiserum to this peptide, directed toward an immunodeterminant near its amino terminus, makes it possible to detect, measure, and study the neuronal location of this peptide in rat brain. In the rat cerebral cortex, DBI immunoreactivity is located in neurons that are not GABAergic (GABA, gamma-aminobutyric acid); in the cerebellum and hippocampus, however, it might be present also in GABAergic neurons.
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
The development of mesencephalic catecholaminergic neurons in the embryonic and fetal mouse was analysed in tissues fixed with 5% acrolein using polyclonal rabbit antibodies against tyrosine hydroxylase (TH), the first enzyme in catecholamine synthesis. The first TH positive cells were identified as early as day 8.5-9 of gestation and some expressed TH while apparently still migrating from the proliferative layer. The number of catecholamine cells increased dramatically by embryonic day 9.5-10; at gestation days 10.5-11 numerous TH positive cells bearing many neurites were localized in the ventral part of the mesencephalon but they were not yet separated into two different groups (A9 and A10). After 13 days of gestation two separate catecholaminergic groups could be visualized, although many TH positive cells with long neurites (putative dopaminergic neurons) could still be seen at the edges of the ventricle, and appeared to be moving towards the ventral mesencephalon. On the basis of these results the possibility that catecholamine cells that are produced early during the development of the midbrain may have neurotrophic and/or morphogenetic roles is discussed.
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