Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release

The present study was designed to investigate the effect of the opioid agon ist FK 33-824 on basal and hCG-induced progesterone (P-4), cAMP and cGMP se cretion and on the phosphoinositide-specific phospholipase C signalling sys tem in separated porcine small (SLCs) and large luteal cells (LLCs). Unit g ravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P-4 and cyclic nucleotide release, S LCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% ai r, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-814 and hC G and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P-4 secretion by LLCs and SLCs. However, FK 33-824 compl etely abolished the stimulatory effect of hCG on P-4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP relea se (p < 0.01) by LLCs and a decrease in cGMP secretion by SLCs(p < 0.05). T he effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secr etion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output. only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influ ence of Fit 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [H-3]-myo-inositol (100 mu Ci/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supp lemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absen ce of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled i nositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptide s affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs. (C) 1999 Elsevier Science B.V. All rights reserved.