Release of pro-thyrotropin-releasing hormone connecting peptides PS4 and PS5 from perifused rat hypothalamic slices

Thyrotropin-releasing hormone prohormone contains five copies of the thyrotropin-releasing hormone progenitor sequence Gln-His-Pro-Gly, each flanked by pairs of basic amino acids and separated by intervening sequences (connecting peptides). Using a perifusion system for rat hypothalamic slices, we have studied the ionic mechanisms underlying the release of two connecting peptides originating from the thyrotropin-releasing hormone precursor: prepro-thyrotropin-releasing hormone-(160-169) (Ps4) and prepro-thyrotropin-releasing hormone-(178-199) (Ps5). Quantification of these two peptides in the effluent fluid was performed using sensitive and highly specific radioimmunoassay procedures. Reverse phase high performance liquid chromatography analysis of the effluent perifusate showed that released peptides co-eluted with synthetic Ps4 and Ps5. The secretion of Ps4 and Ps5 was stimulated by depolarizing agents such as (i) high potassium concentrations, (ii) ouabain, an Na+/K(+)-ATPase inhibitor, and (iii) veratridine, a stimulator of voltage-operated Na+ channels. The response to potassium (70 mM) was not affected by the specific Na+ channel blocker tetrodotoxin. The K+ channel blocker tetraethylammonium did not modify K(+)-evoked release of Ps4 and Ps5. These data suggest that voltage-operated Na+ channels are not involved in the stimulatory effect of high K+ on the release of Ps4 and Ps5. The lack of effect of picrotoxin, a Cl- channel blocker, on the secretion of the connecting peptides indicates that chloride ions play a minor role in the release process. In contrast, deprivation of Ca2+ in the perifusion medium suppressed K(+)-evoked release of the two peptides, indicating that voltage-operated Ca2+ channels are implicated in the release process. Taken together, the present results show that non-thyrotropin-releasing hormone peptides originating from the thyrotropin-releasing hormone precursor are secreted by mediobasal hypothalamic fragments. The release of these peptides is stimulated by depolarization through a calcium-dependent process. These data indicate that Ps4 and Ps5 may be released at the level of the median eminence into the portal circulation, suggesting that these peptides may play a role in the control of anterior pituitary cells.