Isolation of Gamma-Amino Butyric Acid from Pig Hypothalami and Demonstration of its Prolactin Release-Inhibiting (PIF) Activityin VivoandinVitro*

Abstract

A non-retarded fraction with prolactin-release inhibiting factor (PIF) activity obtained by chromatography of a concentrate of porcine hypothalami on carboxymethyl-cellulose was chromatographically distinct from catecholamines. This fraction was purified further by 6 steps involving chromatography on Sephadex G-25, countercurrent distribution, free-flow electrophoresis and chromatography on triethylaminoethyl cellulose. The PIF-active substance was isolated and identified as GABA by: amino acid analyses using Na and L based buffers for resolution of biological fluids, TLC of underivatized material and phenylthiocarbamyl derivatives and mass spectroscopy. Natural and synthetic GABA inhibited prolactin, but not LH [luteinizing hormone] release in vitro from isolated rat pituitary halves at doses as low as 0.1 .mu.g/ml. Inhibition was proportional to the dose; natural and synthetic GABA possessed identical PIF activity. Synthetic GABA also decreased prolactin release in monolayer cultures of rat pituitary cells and inhibited TRH[thyrotropin-releasing hormone]-stimulated prolactin release. The inhibition of prolactin release in vitro by GABA could not be blocked by perphenazine, which inhibits PIF activity of catecholamines. GABA also suppressed prolactin release in vivo, although large doses were needed. Either rapid i.v. injection or infusion of GABA in doses of 1-100 mg in rats significantly decreased serum prolactin levels, which were previously elevated by pretreatment with monoiodotyrosine perphenazine, chlorpromazine, haloperidol or sulpiride. .beta.-hydroxy GABA significantly depressed prolactin release, but .beta.-(p-chlorophenyl)-GABA (Lioresal, CIBA) and 4 other analogs of GABA were not effective in vivo and/or in vitro. GABA can apparently inhibit prolactin release by a direct action on the pituitary gland, but whether this effect is physiologically meaningful still remains to be determined.