STRUCTURAL REQUIREMENTS FOR STIMULATION BY THYROTROPIN-RELEASING-HORMONE OF ALPHA-MSH RELEASE FROM RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) PITUITARY FRAGMENTS IN-VITRO

Juvenile rainbow trout (Oncorhynchus mykiss) pituitary glands were isolated and the neurointermediate lobes (NILs) were removed from the partes distalis. NILs were surgically fragmented, pooled, and superfused in vitro with culture media, then treated with 3-min pulses of native thyrotropin-releasing hormone (TRH) or TRH analog; 10-min fractions were collected and stored (-20 degrees C) for subsequent analysis of alpha-melanocyte-stimulating hormone (alpha-MSH) content by specific radioimmunoassay. After 1 - 2 h of superfusion, alpha-MSH release from the NIL remained relatively constant; alpha-MSH-like immuno-reactivity was not detected in eluate from the partes distalis in a series of parallel experiments. Native TRH stimulated acute releases of alpha-MSH from the NILs with a minimum effective dose of 10(-9) M and an estimated ED(50) of 1.73 x 10(-9) M on the basis of increasing dose - response experiments; decreasing dose- response data provide an estimated minimum effective dose and ED(50) of 10(-9) and 1.57 x 10(-9) M, respectively. No up- or down-regulatory effect was observed when NIL fragments were treated with repeated large (10(-6) M) doses of TRH. By comparison, increasing pulse concentrations of pGlu-3-Me-His-Pro-NH2 (MeTRH) stimulated alpha-MSH release with a minimum effective dose of 10(-10) M and an estimated ED(50) of 1.56 x 10(-9) M. Substitution of the histidine residue with phenylalanine decreased the stimulatory actions of TRH so that the minimum effective dose was 10(-6) M. Substitution at either the amino terminus ([Glu(1)]TRH and [1-Me-(S)-dihydroorotyl(1)]TRH) or carboxy terminus (pGlu-His and TRH-GISI) resulted in near complete loss of bioactivity. To the best of our knowledge, this is the first investigation of the structural requirements for TRH biological activity in the teleost pituitary. Our data indicate that the structural criteria for TRH stimulation of a-MSH from the teleost pituitary are highly conservative; modification of TRH beyond the methylation of the histidine residue results in massive loss of biological activity. These data are consistent with our previous demonstrations of highly conservative structural requirements for TRH-receptor recognition in the trout pituitary and hypothalamus.