Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland. Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)5(1)[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT.
The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Soloff
M S
MS
Department of Biochemistry, Medical College of Ohio, Toledo 43699.
Fernström
M A
MA
Fernström
M J
MJ
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000032:analysis / Q000187:drug effects; D005260:Female; D007295:Inositol Phosphates / Q000096:biosynthesis; D007679:Kidney Medulla / Q000378:metabolism; D007774:Lactation / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000032:analysis* / Q000166:cytology / Q000187:drug effects; D008670:Metals; D009928:Organ Specificity; D010121:Oxytocin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism; D010904:Pituitary Gland, Posterior / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D011945:Receptors, Angiotensin / Q000032:analysis*; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D014667:Vasopressins / Q000037:antagonists & inhibitors*
D007295:Inositol Phosphates; C055830:KB 5-21; D008024:Ligands; D008670:Metals; D010455:Peptides; D011945:Receptors, Angiotensin; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D014667:Vasopressins; D001127:Arginine Vasopressin; D010121:Oxytocin