Hormonal inhibition of adenylate cyclase. A crucial role for Mg2+.

In several adenylate cyclase systems (anterior pituitary gland, human platelets, adipocytes, rat liver membranes), inhibitory hormones were shown to reduce basal adenylate cyclase activity by decreasing the "apparent affinity" of those systems for Mg2+ activation, without modifying the Vmax of the reaction. In the absence of hormones, the Mg2+ dose-activation curves were monophasic, whereas in the presence of hormones a clear heterogeneity was revealed. Therefore, inhibitory hormones induced a right-hand shift in the Mg2+ dose-activation curve. This hormonal effect was concentration-dependent. In human platelets, the inhibition of prostaglandin E1-stimulated adenylate cyclase by norepinephrine was also due to a decrease in the apparent affinity for Mg2+. In anterior pituitary gland, when Mg2+ was substituted by Mn2+, similar results were obtained. Thus, dopamine produced its inhibition by decreasing the apparent affinity for Mn2+ both under basal and vasoactive intestinal peptide-stimulated conditions. At Mg2+ or Mn2+ concentrations high enough to obtain saturation of the low apparent affinity state, hormone-induced inhibition was not observed. In anterior pituitary gland and in human platelet membranes, Na+ was not required in order to observe adenylate cyclase inhibition by catecholamines. In adipocytes and rat liver membranes, however, Na+ was required. In both systems, GTP was able to transform adenylate cyclase to a low Mg2+ apparent affinity state. Na+ was able to reverse (in a dose-dependent manner) the system to a high Mg2+ apparent affinity state. Once in this state, hormones were shown to inhibit adenylate cyclase activity by reverting the enzyme to a low apparent affinity state for Mg2+.

?(1)- and ?(2)-adrenergic receptors in rat corpus striatum.

Rat corpus striatum contained ?(2)-adrenergic receptor which were labelled with [(3)H]clonidine (95 +/- 6 fmol/mg protein). The affinity of these receptors (K(d) = 1.3 to 3.6 nM) was similar to that found in cerebral cortex. Five days after kainic lesions, the number of ?(2)-adrenergic receptors had dropped by half, suggesting that their location might be neuronal. One month after lesions, the number of ?(2)-adrenergic receptors had risen to that of the controls and was higher after two months. This increase would suggest a glial localization of the ?(2)-adrenergic receptor. We have previously described the presence of ?(2)-adrenergic receptors in primary astrocyte cultures (Ebersolt et al., 1981). Rat corpus striatum contained less ?(1)-adrenergic receptors than ?(2)-adrenergic receptors. They were labelled with [(3)H]prazosin (28 +/- 1.9 fmol/mg protein) and were only slightly altered 5 days after kainic acid lesions (?20%). In addition to these classical ?(1)-adrenergic receptors, rat corpus striatum also contained [(3)H]WB4101 binding sites having high affinity for WB4101 (2-5 nM) and norepinephrine (1 ?M) but a very low affinity for prazosin (4.4 ?M). The exact nature of these sites remains unknown.

Adenylate cyclase inhibition by hormones. The Mg2+ hypothesis.

In washed anterior pituitary membranes, there is enough GTP to occupy Ns and therefore to obtain activation of adenylate cyclase by vasointestinal peptide. GTP concentrations needed to obtain adenylate cyclase inhibition by dopamine (above 5 X 10- M) stimulate the adenylate cyclase. The dopamine effect is a blockade of this stimulation. We propose that at least in this system, Ni does not inhibit but stimulates the adenylate cyclase and that inhibitory hormones block this stimulation. We also demonstrate in several adenylate cyclase systems that hormones produced adenylate cyclase inhibition by lowering their Mg affinity A general model for adenylate cyclase activation and inhibition is proposed.