Structural requirements of the oxytocin receptor in rat uterus. Free-Wilson analysis in a series of competitive oxytocin inhibitors.

Published and newly calculated pA2-values of 147 neurohypophyseal hormone analogues (7 positions varied) acting as inhibitors of oxytocin on isolated rat uterus in vitro have been subjected to fractionation according to the method by Free and Wilson which was slightly modified for this purpose. The computation was carried out in several steps. After each step, substances with outlying pA2-values were eliminated. The reduced group containing 73-79% of the original substances displayed a high degree of additivity of side chain contributions (SCC). This group seems to follow the "participation" rule as formulated by Free and Wilson. Analysis of the group of eliminated substances and of the resulting SCC-spectrum (level diagram) enabled us to draw some conclusions concerning the structural requirements of receptor binding: i) The intact ring structure is necessary for the peptide-receptor interaction: linear peptides or peptides with an extended ring are always outliers; ii) Carba analogues (substitution with CH2 in the disulfide ring) display better affinities than peptides with an S-S ring; D-Arg8 substitution decreases the binding affinity; iii) Considerably better additivity is achieved when peptides are divided into subgroups with vasopressin-like and oxytocin-like features; populations of receptors more specific for vasopressin and for oxytocin, respectively, can be assumed. Estimates of the "true" receptor-peptide dissociation constants can be obtained by summation of the corresponding SCC's in each investigated position. The value obtained for oxytocin is identical with the medium affinity binding site on myometrial cells, and not with the high affinity site. A nonlinear relationship exists between SCC's computed from pA2-values for magnesium-free and magnesium-containing (0.5 mM) media but no evidence speaks in favor of a Mg-potentiating effect on receptor binding.

Antihaemophilic effect of vasopressin, deamino-(D-arginine8)-vasopressin and adrenaline in sheep: proposal for an in vivo assay system.

Female sheep were used to assay antihaemophilic (factor VIII enhancing) activity of arginine vasopressin, deamino-(D-arginine8)-vasopressin (DDAVP) and adrenaline. The time course of the response was biphasic, two surges of factor VIII being observed. DDAVP was found to be the most potent of the substances investigated. Its optimal dose was 1 microgram kg-1 body wt (i.v.). It is suggested that a similar procedure can be employed to search for new peptides with anti-haemophilic action.

Binding of oxytocin to uterine cells in vitro. Occurrence of several binding site populations and reidentification of oxytocin receptors.

Myometrial and endometrial cells of sheep, rat, and calf in monolayer cell culture display at least three populations of binding sites for oxytocin, with dissociation constants (Kd) of approximately 5 X 10(-9), 4 X 10(-7), and greater than 10(-5) mol/liter, respectively. Binding of the tritium-labeled oxytocin (concentration range, 10(-11) to 5 X 10(-4) M) to the first two sites is displaceable by cold oxytocin. The ratio of binding capacities of the high to medium affinity site appears to average 1:18. Dissociation rate constants for these sites (22 degrees C) are roughly 10(-4) and 2 X 10(-3) s-1, respectively. The capacity of the low affinity site varies in individual cell preparations and is between 5 and 66 times that of the medium affinity site. The low affinity binding sites may not be fully saturable and may follow a nonasymptotic binding isotherm. Logarithms of Kd and binding capacity for individual binding sites are linearly correlated. The coexistence of the three sites was also proven by cluster analysis based on similarities between Kd, binding capacity, and Hill coefficient. Only minor systematic species and cell type differences occur in these properties. The value of Kd for the oxytocin receptor in rat myometrium, derived recently from a stepwise irreversible inhibition of uterotonic response to oxytocin, is close to 2.5 X 10(-7) mol/liter. Additional pharmacological data (pA2 values of structural analogues of oxytocin acting as competitive inhibitors) also reveal a Kd value of 3 X 10(-7). It is, therefore, concluded that the receptors for oxytocin in rat myometrium are identical with the medium affinity site.