RESUMO
Ontogenesis of somatostatin (SRIF) neurons and receptors was studied in fetal hypothalamic cell cultures kept in serum-free medium, and compared to the in vivo developmental pattern. Initial rise in neuronal content of SRIF occurred later in vitro than in vivo. In vitro, K(+)-induced SRIF release was only present after synaptogenesis. SRIF binding sites were measurable as early as 1 day after birth and at an equivalent time in culture, after 6 days in vitro (DIV); their affinity was in the nanomolar range. In cultured cells, binding reached a maximum at two weeks in vitro and decreased sharply thereafter as a consequence of binding site occupancy by the endogenous ligand. Indeed, pretreatment with cysteamine decreased SRIF concentration in the neuronal cultures and twice as many binding sites as in control cultures of 21 DIV were measured. Competition kinetics using unlabelled SMS 201-995 to displace [125I]SRIF revealed two distinct binding sites in the neuronal preparations (IC50 = 11 +/- 3 pM and 4.5 +/- 0.8 nM). In contrast, only the lower affinity site was present on glial cell preparations (1.7 +/- 0.4 nM). SRIF inhibited adenylate cyclase activity in glia and neurons, and the onset of SRIF coupling to the second messenger occurred earlier in vitro than in vivo. Pertussis toxin pretreatment was equally effective in neuronal and glial cell preparations to decrease SRIF binding and to inhibit adenylate cyclase activity.
