Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes.
Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.
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D000241:Adenosine / Q000494:pharmacology; D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004822:Epididymis; D005033:Ethylmaleimide / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008297:Male; D010121:Oxytocin / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000378:metabolism*; D018045:Receptors, Oxytocin
D007328:Insulin; D011945:Receptors, Angiotensin; D018045:Receptors, Oxytocin; D010121:Oxytocin; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D010447:Peptide Hydrolases; D000241:Adenosine; D005033:Ethylmaleimide; D004229:Dithiothreitol