Culture sensitization and inhibition of luteinizing hormone responsive production of cyclic AMP in luteal cells by luteinizing hormone, prostaglandin F2 alpha and [D-Trp6]-luteinizing hormone releasing hormone.

Abstract

Many cells are able to regulate their sensitivity to hormones. In order to investigate the mechanism(s) by which rat luteal cells regulate their sensitivity to LH, we have developed and characterized a cell culture model. Cultures of dispersed rat luteal cells were exposed to graded doses of bovine LH, an analogue of LH releasing hormone (LH-RH) ([D-Trp6]-LH-RH), and prostaglandin F2 alpha (PGF2 alpha) for 3 h. The media containing these hormones were then replaced with fresh hormone-free medium and the cells cultured for 24 h. In order to test the sensitivity of these cultures after 24 h, the medium was discarded and replaced by medium alone, or medium containing a standard dose of bovine LH for 1 h. The amount of cyclic AMP accumulated during this hour was used as an index of the sensitivity of the cells to LH. Control cultures became 'supersensitive' to LH with augmented production of cyclic AMP during culture but LH-receptor binding activity was not increased. During the first 2 h of culture, LH (100 ng/ml) increased accumulation of cyclic AMP by fourfold, but after 5 h of culture, stimulation of cyclic AMP accumulation by the same dose of LH was increased 32-fold, 299-fold at 13 h and 359-fold at 21 h of culture. The increase in LH-responsive accumulation of cyclic AMP with cultured was severely impaired by early exposure of cells to LH, LH-RH analogue or PGF2 alpha during the first 2 h of culture. Also, both LH-RH analogue and PGF2 alpha acutely inhibited LH-stimulated accumulation of cyclic AMP. Inhibition of culture-induced sensitization of LH responsiveness was not altered by the addition of 3-isobutyl-1-methylxanthine. Scatchard analysis of LH binding sites indicated that pretreatment of luteal cells with LH (or human chorionic gonadotrophin at an equivalent dose) reduced the number of free LH receptors when measured after 24 h of culture, but total receptor binding activity was not changed. However, a similar effect was not seen with cells treated with PGF2 alpha or LH-RH analogue. It is suggested that 'culture-induced' supersensitivity may represent either recovery of pre-isolation sensitivity or result from the loss of an endogenous factor(s) which retards the coupling of the LH receptor and adenylate cyclase. Although PGF2 alpha and LH-RH analogue have been shown to directly prevent the occupied LH receptor from activation of adenylate cyclase, the present observations have indicated that this inhibitory process was continued even when these agents were removed from the culture medium.