Adrenomedullin (ADM) and its receptors are expressed in the adrenal cortex, where ADM is currently known to inhibit agonist-stimulated aldosterone secretion from zona glomerulosa (ZG), without affecting either basal aldosterone release or the secretory activity of zona fasciculata-reticularis (ZF/R) cells. These functional findings have been obtained using freshly dispersed adrenocortical cells, but evidence has been provided that ADM is able to enhance basal aldosterone secretion from rat capsule-ZG preparations. Hence, we investigated the effect of ADM and ADM22-52, a putative antagonist of ADM receptors, on the secretory activity of rat adrenal cell cultured in vitro for 72 h. Cultures were exposed for 3 or 24 h to 10(-7) M ADM and/or ADM22-52, in the absence or the presence of 10(-8) M ACTH, and the concentration of aldosterone and corticosterone in the culture medium was measured by radioimmune assay. ADM and/or ADM22-52 raised basal aldosterone secretion at 3 h, but not 24 h exposure, and did not affect ACTH-stimulated aldosterone production. Corticosterone secretion was not changed at 3 h. In contrast, at 24 h exposure ADM22-52 alone or with ADM decreased basal corticosterone secretion; ADM evoked a small rise in ACTH-stimulated corticosterone production, and the effect was annulled by ADM22-52. These puzzling findings are interpreted in light of the fact that i) our cultures were actually a mixture of ZG, ZF/R and medullary chromaffin cells; ii) ADM stimulates adrenomedullary cells to release catecholamines, which are able to enhance aldosterone secretion from ZG cells; and iii) the prolonged exposure to ADM may modify, under in vitro culture conditions, ZF/R cells, switching their phenotype from an ADM-unresponsive to an ADM-responsive one. Our study casts doubts on the selectivity of ADM22-52 as ADM receptor antagonist, and stresses that great caution must be used in comparing adrenal-secretion findings obtained with different in vitro techniques.