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Maintenance of Prolactin (PRL) Binding Sites in Rat Liver Cells in Suspension Culture: Effect of PRL and of Inhibitors of Various Cellular Functions^*

ALZIRA A. M. ROSA, LOUIS H. FERLAND, JEAN DJIANE, LOUISMARIE HOUDEBINE and PAUL A. KELLY

Laboratory of Molecular Endocrinology Royal Victoria Hospital, Montreal, Quebec H3A 1A1, Canada;
Laboratoire de Physiobgie de la Lactation Institute National de la Recherche Agronomique, 78350 Jouy-en-Josas, France;
Departamento de Morfologia Fisiologia da Faculdade de Ciencias Agrarias e Veterinarias de Jaboticabal 14780 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Paul A.Kelly, Laboratory of Molecular Endocrinology, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada.

Abstract

An in vitro method to study the regulation of PRL receptors has been established using adult rat liver cells cultured in a continuous suspension in L-15 medium. PRL binding averaged 28.2 ± 1.8% of the added labeled hormone per 106 cells in freshly isolated liver cells prepared from female rats treated with 17β-estradiol. When these cells were incubated at 37 C, binding rapidly declined by 50% at 10 h and 90% at 48 h. This rapid decline could be counteracted by the inclusion of ovine PRL (50 nM), which maintained initial PRL receptor levels up to 48 h of culture. Higher concentrations of PRL (2.5 nM) induced a rapid down-regulation, apparent at 2 and 10 h of culture. Cycloheximide (50 Mg/ml) induced a slight diminution of control PRL receptor levels and partially reversed the effect of 50 nM PRL. Approximately 60% of the PRL receptors were resistant to the effect of cycloheximide. On the other hand, actinomycin D (10 Mg/ml) had no effect on PRL receptor levels in control and only a very slight effect in PRL-treated cells. Dinitrophenol, which blocks metabolic oxidation, also partially reversed the effect of 50 nM PRL although it was without any significant effect on control levels. Chloroquine (100 βM) and colchicine (1 pM) failed to alter PRL binding either in the absence or presence of 50 nM PRL. Our results suggest that the existence of regulatory factors occurring in vivo, which are absent in the culture medium, could be responsible for the decline in PRL receptor levels in the control hepatocytes. PRL itself could be one of these factors. On the other hand, and in agreement with the putative actions of the drugs utilized, the mechanism of the PRL-induced maintenance of receptor levels appears to lie in part with an effect on receptor synthesis at the translational (ribsomal) level but to be independent of the internalization or of lysosomal degradation. (Endocrinology 116: 1288-1294, 1985)

Footnotes

^* This work was supported by the Medical Research Council of Canada, the National Cancer Institute, and the United States Public Health Service.

Postdoctoral Fellow of the Fundac,ao ao Amparo ao Ensino e Pesquisa do Estado de Sao Paulo, Brazil.

Received June 14, 1984.