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In Xenopus laevis oocytes progesterone is able to inhibit directly the plasma membrane adenylate cyclase activity and induce reinitiation of meiotic maturation. To determine whether progesterone inhibition is mediated by the inhibitory guanine nucleotide‐binding regulatory component of adenylate cyclase, Ni, the effect of the Bordetella pertussis toxin (IAP) and limited proteolysis on progesterone action in oocytes was investigated. Treatment of oocyte membranes with islet activating protein (IAP) in the presence of [32P]NAD led to incorporation of radiolabel into a 41 000‐dalton membrane protein. However, exposure of isolated oocytes to 100 ng/ml IAP for up to 24 h, or oocyte membranes with concentrations of toxin as high as 100 micrograms/ml, had no effect on either progesterone inhibition of adenylate cyclase or induction of maturation. Similarly, limited alpha‐chymotrypsin proteolysis of oocyte membranes failed to modify progesterone‐induced inhibition of adenylate cyclase. In contrast, inhibition of human platelet adenylate cyclase by epinephrine, acting via a GTP‐dependent, alpha 2‐adrenergic receptor‐mediated pathway, is almost completely abolished by both IAP treatment and limited proteolysis of platelet membranes. These data indicate that unlike attenuation of platelet enzyme activity, the inhibition of adenylate cyclase in oocyte membranes by progesterone does not occur via a classical Ni‐mediated pathway.

Adrenergic receptors exist as two subtypes, a and ß, in various tissues. In rat liver, catecholamines exert the same effect on glycogenolysis and neoglucogenesis via either a or ß control. In this review we report on the modifications in the balance between a- and ß-adrenoreceptors induced as the result of adrenalectomy and extrahepatic cholestasis. Following adrenalectomy, the number of ß-adrenergic receptors in rat liver, measured using specific radioligands, increases twofold, with no change in the a-adrenoreceptors. In extrahepatic cholestasis we measured a fourfold increase in the number of ß-sites and a two- to threefold decrease in the number of a-sites. Moreover, this modification in the balance between a- and ß-adrenoreceptors was concomitant with the apparition of a mixed a- and ß-adrenergic regulation of glycogenolysis in cholestatic rats, whereas this process is purely or almost purely a-adrenergic in normal rats.

In order to purity the a-adrenergic receptor labeled with [3H]POB, antibodies against the irreversible a-adrenergic antagonist, phenoxyben/amine (POB), have been raised in rabbits immunized with POB conjugated to immunoglobulin. The antigen was prepared by first reacting POB with the sulfhydryl group of cysteine, after which the POB complex was coupled to thyroglobulin in the presence of a water soluble carbodiimide. POB-specific antibodies were first detected 10 days after the first booster injection. These antibodies display a high degree of specificity. Slight cross-reactivity occurred only with compounds structurally related to POB, such as dibenamine and nitrogen mustards, while catecholamines and a-adrenergic compounds not structurally related to POB did not cross-react, even when present in 100,000-fold excess. Moreover, the antibodies are still capable of recognizing POB once bound to a-adrenergic receptor. When membranes, prelabeled with [3H]POB, are solubilized with digitonin and incubated with anti-POB antiserum, the radioactivity can be specifically precipitated upon addition of goat anti-rabbit ?-globulin serum. Thus these antibodies may be a useful tool in the purification of the a-adrenergic receptor of rat liver covalently linked to POB.

1. Adenylate cyclase (EC 4.6.1.1) activity was characterized in human liver, and its subcellular distribution compared with that of three other potential enzyme markers of the pericellular membrane: leucine aminopeptidase (EC 3.4.11.1), γ-glutamyltransferase (EC 2.3.2.2) and 5′-nucleotidase (EC 3.1.3.5). Although these three enzyme activities were detected in each of the subcellular fractions studied, 85% of the total adenylate cyclase activity was found in the 1000 g pellet (‘nuclear’ fraction) with a threefold increase in specific activity as compared with the homogenate. No adenylate cyclase activity existed in the 150 000 g supernatant fraction. 2. In the ‘nuclear’ fraction, adenylate cyclase activity was increased in a dose-dependent fashion by glucagon with a half-maximal stimulation at 10 nmol/l and a maximal four- to seven-fold increase at 1 μmol/l. Catecholamines activated adenylate cyclase 2·5- to three-fold, with an order of potency (protokylol > isoprenaline > adrenaline > noradrenaline) typical of a β2-adrenoreceptor. Prostaglandin E1 and NaF also stimulated cyclase two- and four-fold respectively. Insulin, serotonin, dopamine, thyroid-stimulating hormone and ACTH had no effect. Adenosine provoked a weak inhibition at 0·1 mmol/l. Finally guanosine triphosphate and 5′-guanylyl imidodiphosphate induced a marked increase in basal activity, four- and eight-fold respectively, but both reduced the relative increase in enzyme activity due to glucagon or adrenaline. 3. Cyclase from foetal liver (12–16 weeks old) and cirrhotic adult liver appeared to behave similarly to that from normal liver; however, foetal cyclase was more active, and cirrhotic enzyme less active than normal adult liver. Both systems responded to catecholamines via a β2-adrenoreceptor. 4. These results validate the use of rat liver adenylate cyclase as a tool for pharmacological and physiological studies.

Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.