The primary structure of frog neurotensin (f NT) has recently been determined and it has been shown that f NT is a potent stimulator of α-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of f NT on the electrical activity of cultured frog melanotropes by using the patch-clamp technique and we have determined the pharmacological profile of the receptors mediating the effect of f NT. In the cell-attached configuration, f NT (10–7 M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, f NT (10–7 M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10–10 to 10–6 M) of f NT or the C-terminal hexapeptide NT(8–13) caused a dose-dependent increase in the frequency of action potentials with EC 50 of 2 × 10–8 and 5 × 10–9 M, respectively. The stimulatory effect of f NT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp 11]NT(8–13) > Boc-[D-Trp 11]NT(8–13) > Boc-[Lys 8,9, Nal 11]NT(8–13) > Boc-[Ψ11,12]NT(8–13). In contrast, the cyclic pseudopeptide analogs of NT(8–13), Lys-Lys-Pro-D-Trp-Ile-Leu and Lys-Lys-Pro-D-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10–5 M) stimulated the spike discharge but did not block the response to f NT. In contrast, SR 142948A (10–5 M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of f NT. The specific nts2 receptor ligand levocabastine (10–6 M) had no effect on the basal electrical activity and the response of melanotropes to f NT. In cells which were dialyzed with guanosine-5′-O-(3-thiotriphosphate) (10–4 M), f NT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5′-O-(2-thiodiphosphate) (10–4 M) completely blocked the effect of f NT. Pretreatment of cells with cholera toxin (1 µg/ml) or pertussis toxin (0.2 µg/ml) did not affect the electrical response to f NT. Intracellular application of the G o/i/s protein antagonist GPAnt-1 (3 × 10–5 M) had no effect on the f NT-evoked stimulation. In contrast, dialysis of melanotropes with the G q/11 protein antagonist GPAnt-2A (3 × 10–5 M) abrogated the response to f NT. The present data demonstrate that f NT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by f NT can be accounted for by activation of a G q/11-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.
