Kinetic and pharmacological characterization of vasopressin membrane receptors from human kidney medulla: Relation to adenylate cyclase activation

Membranes from the medullopapillary portions of three human kidneys unsuitable for transplantation were prepared and stored in liquid nitrogen. Specific vasopressin binding sites were detected on these membranes and characterized using tritiated lysine vasopressin. The biological activities of vasopressin and of structurally related peptides were estimated on the same membrane preparations by measuring dose-dependent adenylate cyclase activation. The population of vasopressin binding sites was homogeneous and displayed the following characteristics: equilibrium dissociation constant K D 27 ± 7 nM (for lysine vasopressin); maximal specific binding capacity 0.7±0.1 pmol [ 3H]LVP bound/mg protein. Maximal adenylate cyclase activation by lysine vasopressin was 5.4 times the basal value of 50 ± 4 pmol cAMP formed/5 min per mg protein. Half-maximal activation (K act) was obtained at a concentration of 34 ± 5 nM. Both lysine vasopressin binding and hormone-induced adenylate cyclase activation were sensitive to guanyl nucleotides. 5′-Guanylyl imidodiphosphate reduced K act by a factor of 3 and increased K D by a factor of about 8. K D and K act were determined for a series of peptides including natural vasopressins, oxytocin and their structural analogues which were found to have enhanced or reduced antidiuretic activities in the rat. The concentration range of the K D and K act values covered a concentration range of more than three orders of magnitude. The most active peptide was the natural arginine vasopressin (K D 4.4 ± 0.7 nM; K act 3.1 ± 1.6 nM). There was a highly significant correlation between paired K D and K act values. The two vasopressin structural analogues [1-(β- mercapto-β, β-cyclopentamethylene propionic acid), 4-valine, 8-D-arginine]vasopressin and [1-β-mercapto-β, β-cyclopenthamethylene propionic acid), 2-O-ethyltyrosine, 4-valine]arginine vasopressin, behaved like competitive inhibitors of vasopressin-induced adenylate cyclase activation (K i = 65 and 59 nM respectively). Freezing the membranes in liquid nitrogen reduced the affinity of vasopressin receptors but fully preserved their selectivity towards vasopressin structural analogues. It is concluded that the vasopressin binding and/or adenylate cyclase assays might be useful for estimating several aspects of the pharmacological activities of vasopressin structural analogues in man.