Receptors for neurohypophyseal hormones along the rat nephron : 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH92] vasotocin binding in microdissected tubules

• Published in: Pflügers Archiv 1991-04, Vol.418 (3), p.220-227
• Main Authors: AMMAR, A, SCHMIDT, A, SEMMEKROT, B, ROSEAU, S, BUTLEN, D
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Iodine Radioisotopes; Kidney Cortex - metabolism; Kidney Medulla - metabolism; Kidney Tubules, Collecting - metabolism; Kidney Tubules, Distal - metabolism; Kinetics; Male; Nephrons - metabolism; Oxytocin - analogs & derivatives; Oxytocin - metabolism; Rats; Rats, Inbred Strains; Receptors, Angiotensin - metabolism; Receptors, Vasopressin; Vasopressins - metabolism; Vertebrates: urinary system
• ISSN: 0031-6768; 1432-2013
• DOI: 10.1007/BF00370519

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 4-threonine, 8-ornithine, 9-125I-tyrosylamide]vasotocin [125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT] to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4 degrees C, specific binding sites (expressed at 10(-18) mol 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67 +/- 0.49; cortical thick ascending limbs, 2.20 +/- 0.80; cortical collecting ducts, 2.39 +/- 0.86; outer medullary collecting ducts (OMCD), 2.54 +/- 0.53 and inner medullary collecting ducts, 5.33 +/- 0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4 degrees C were 1.06 x 10(7) M-1 min-1 and 1.95 x 10(-2) min-1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:dDAVP greater than AVP greater than d(CH2)5-[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT = AVT = OT greater than d(CH2)5[Tyr(Me)2]AVP = [Thr4, Gly7]OT greater than [Phe2, Orn8]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V2 vasopressin receptors.