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Physiological Concentrations of L-Leucine Control the Release of Prolactin from Cultured Rat Pituitary Cells^*

MICHAEL CROSS and AMEAE M. WALKER

Division of Biomedical Sciences, University of California Riverside, California 92521

Abstract

In an attempt to further define the optimum experimental conditions for the in vitro study of PRL endocrinology, the effect of extracellular leucine concentration on PRL secretion was assessed. Rat pituitary cells, maintained in short term monolayer culture, were washed free of serum and placed in Minimum Essential Medium (MEM; 0.39 mM leucine) supplemented with nonessential amino acids and 0.1% gelatin. After the establishment of a constant rate of basal PRL release, the MEM was replaced with leucine-free MEM (supplemented as above), and the rate of PRL release was followed. PRL release from the cells in the leucine-free medium fell within 15 rnin to a rate 30% of that seen in the controls. Return to the control rate was achieved by the addition of L-leucine only. The Disomer, isoleucine, valine, proline, leucyl-leucine, and leucineagarose were ineffective. In the short term, the rate of PRL synthesis was found to be independent of extracellular leucine and the inhibition of release seen upon removal of leucine, and the subsequent recovery upon replacement of leucine was also observed when this experiment was conducted in the presence of 50 µg/ml cycloheximide. The L-leucine recovery was found to be dose dependent, with an increase in leucine concentration from 20 to 40 µg/liter causing a tripling of the PRL release rate. The addition of 10^–7 M TRH showed that leucine can be limiting in the response to this stimulant and allowed a response to as little as 10 mg/liter leucine to be discerned. From these experiments, it is concluded that physiological concentrations of leucine specifically and profoundly affect the release of PRL by a mechanism independent of protein synthesis. This leucine requirement for release has not been previously reported. (Endocrinology 114: 80, 1984)

Footnotes

^* This work was supported in part by NIH Grants AM-28534 and RR-05816. Part of this work was conducted in the laboratory of Dr. Marilyn Farquhar and has been presented in preliminary form [J Cell Biol 83:434a, 1979 (Abstract)].

Recipient of a UCR Student Minigrant.

To whom requests for reprints should be addressed.

Received April 11, 1983.