Promotional role for glucocorticoids in the development of intracellular signalling: enhanced cardiac and renal adenylate cyclase reactivity to beta-adrenergic and non-adrenergic stimuli after low-dose fetal dexamethasone exposure.

Fetal exposure to high doses of glucocorticoids, as used to aid lung maturation in the therapy of Respiratory Distress Syndrome, causes growth retardation and interference with development of beta-adrenergic receptor-mediated cell signalling. The current study examined whether lower levels of steroids might instead play a positive trophic role in receptor transduction. Pregnant rats were given dexamethasone at or below the threshold for growth impairment (0.05-0.2 mg/kg) on gestational days 17, 18 and 19, and the beta-receptor-mediated stimulation of adenylate cyclase activity was evaluated in membrane preparations from heart and kidney. The enzymatic response to isoproterenol was compared with effects on: (1) basal (unstimulated) adenylate cyclase, (2) adenylate cyclase stimulation mediated by forskolin, which bypasses the beta-receptor, and (3) development of beta-receptor binding capabilities, assessed with [125I]pindolol. In the heart, prenatal exposure to dexamethasone produced a dose-dependent enhancement of beta-receptor-mediated stimulation of adenylate cyclase activity; however, both basal and forskolin-stimulated activity were also increased and beta-receptor binding was relatively unaffected. These results suggest that enhanced responsiveness was occurring at the level of the cyclase itself, rather than by effects on receptors or their G-protein coupling to enzyme activity. Promotional effects on adenylate cyclase were detectable at the low dose of dexamethasone, without any evidence of growth impairment. Furthermore, the effects displayed selectivity for age and tissue: adults treated with dexamethasone did not show the effect, and the heart was more sensitively affected than was the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of postnatal beta-adrenergic receptor/adenylate cyclase development by prenatal agonist stimulation and steroids: alterations in rat kidney and lung after exposure to terbutaline or dexamethasone

Glucorticoids and adrenergic stimulation are both thought to control the development of beta-adrenergic receptors/responses. In the current study, rats were exposed to dexamethasone or terbutaline during late gestation and the development of beta-receptor binding capabilities and adenylate cyclase activity evaluated in membrane preparations from kidney and lung. Prenatal dexamethasone exposure produced postnatal adrenergic hyperreactivity of kidney adenylate cyclase; the effect resulted from increases in the enzyme itself, as both basal adenylate cyclase and forskolin-stimulation of the enzyme were also increased by dexamethasone. Similarly, prenatal terbutaline exposure evoked increases in basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase in the kidney. In the lung, dexamethasone produced an initial postnatal deficit in basal adenylate cyclase and deficient responsiveness to isoproterenol, but the deficit resolved shortly after birth. Terbutaline selectively promoted the ability of isoproterenol to stimulate lung adenylate cyclase in the first few days after birth, without alterations in basal adenylate cyclase; this was followed by a period of prolonged subsensitivity of both basal and isoproterenol-stimulated activity. Although dexamethasone and terbutaline also caused significant changes in development of beta-receptor binding capabilities, in neither tissue could these effects account for the direction or magnitude of the changes in adenylate cyclase reactivity. Thus, glucocorticoids and beta-agonists can participate in the programming of development of postsynaptic reactivity by exerting actions upon post-receptor coupling mechanisms.     

Prenatal terbutaline treatment: tissue-selective dissociation of perinatal changes in beta-adrenergic receptor binding from regulation of adenylate cyclase activity.

The role of prenatal beta-receptor stimulation in development of adrenergic reactivity was examined by administering the beta-agonist, terbutaline, to pregnant rats on gestational days 17, 18 and 19. On gestational day 20, liver membrane beta-receptor binding capabilities showed the depression characteristic of down-regulation, but heart and kidney receptor binding were essentially normal. Basal adenylate cyclase activity in the fetal liver membrane preparation was unchanged by terbutaline exposure and enzymatic reactivity to beta-adrenergic stimulation showed only a slight lowering; forskolin stimulation, however, was markedly increased in the terbutaline group. By postnatal day 2, receptor binding had returned to normal in the liver and remained at control levels in the other two tissues. Responsivity of adenylate cyclase to beta-receptor stimulation was markedly elevated in heart and kidney membranes; the effect represented an alteration at the level of the cyclase itself, rather than the receptor, since both basal activity and forskolin stimulation of the enzyme showed equivalent enhancement. These data thus suggest that early beta-adrenergic stimulation promotes cellular reactivity by fostering the development of membrane transduction mechanisms, rather than through effects on the receptor ligand binding site per se.     

Thyroid hormone differentially regulates development of beta-adrenergic receptors, adenylate cyclase and ornithine decarboxylase in rat heart and kidney.

In mature animals, thyroid hormone produces parallel up-regulation of beta-adrenergic receptor binding sites and their linkage to adenylate cyclase; during development, these same processes may be critical in establishing the set-point for subsequent adrenergic reactivity. In the current study, we administered triiodothyronine to neonatal rats for the first five days postpartum and evaluated [125I]pindolol binding capabilities and adenylate cyclase activity in membrane preparations from heart and kidney. In the heart, hyperthyroidism elicited an initial increase in receptor density, with subsequent deficits and an eventual return to normal values by young adulthood. In contrast, the ability of isoproterenol, a beta-adrenergic agonist, to stimulate adenylate cyclase was enhanced regardless of whether receptor numbers were increased or decreased; the same effects were also present for basal adenylate cyclase activity and non-receptor-mediated stimulation by forskolin. Enhanced cyclase activity involved both increases in the magnitude of response as well as accelerated onset of the postweaning peak of enzyme activity, results which suggest a direct impact of thyroid status on the ontogenetic expression of adenylate cyclase itself. The kidney, which possesses less efficient beta-receptor coupling to adenylate cyclase in the neonate, was less drastically affected by triiodothyronine for either beta-receptor binding sites or enzyme activity. As we had previously shown that neonatal hyperthyroidism uncouples beta-receptors from growth-related enzymes, such as ornithine decarboxylase, we also evaluated whether the promotion of adenylate cyclase responses was mechanistically linked to effect on ornithine decarboxylase; administration of cyclic AMP analogs to 5 days-old rats led to inhibition of the enzyme in the heart, whereas the same treatment in 9 days-old animals was ineffective. These data suggest that thyroid hormone differentially regulates the development of beta-receptors as well as adenylate cyclase and ornithine decarboxylase, with preferential effects on tissues, such as the heart, that already possess efficient linkage of the receptors to cell transduction mechanisms at birth.     

Interaction of the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane with the beta-receptor adenylate cyclase system in Chang liver cell membranes

Interaction of the insecticide 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) with beta-receptor binding and adenylate cyclase activity of biological membranes has been studied. Following exposure of cultured Chang liver cells to DDT, maximal binding of the catecholamine antagonist [125I]-iodohydroxybenzylpindolol (HYP) to isolated cell membranes was decreased by 30% whereas the dissociation constant remained unchanged. Both basal activity and maximal isoproterenol-stimulated activity of adenylate cyclase were not altered. The isoproterenol concentration required for half-maximal stimulation of the enzyme was increased about 2-fold as was the agonist concentration required for half-maximal displacement of the antagonist HYP at the beta-receptor binding site. Thus, coupling efficiency of hormone-stimulated adenylate cyclase activity was not influenced by the presence of DDT in these membranes. The data show that interaction of DDT with the beta-receptor adenylate cyclase complex is restricted to the receptor component. Enzyme activity is directly linked to changes of agonist binding at the beta-receptor. Interference of DDT with signal transduction via 'fluidization' of membrane lipids has not been detected.     

Fetal dexamethasone exposure accelerates development of renal function: relationship to dose, cell differentiation and growth inhibition.

Fetal exposure to high doses of glucocorticoids slows cellular development and impairs organ performance, in association with growth retardation. Nevertheless, low doses of glucocorticoids may enhance cell differentiation and accelerate specific functions. The current study examined this apparent paradox in the developing rat kidney, using doses of dexamethasone that span the threshold for growth impairment: 0.05 or 0.2 mg/kg given on gestational days 17, 18 and 19. At the lower dose, which did not significantly retard body growth, the postnatal development of tubular reabsorptive capabilities for sodium, potassium, osmotic particles, water and urea was accelerated. These effects were less notable at the higher dose, which caused initial body growth impairment. The selectivity toward promotion of tubular function was evidenced by the absence of effect of either dose of dexamethasone on development of glomerular filtration rate. Because of the wide spectrum of dexamethasone's effects on tubular function, we also assessed fetal kidney adenylate cyclase as a means of detecting altered cell differentiation in the prenatal period during which dexamethasone was given. Either glucocorticoid dose increased the total adenylate cyclase catalytic activity (assessed with forskolin). Thus, the net effect of fetal dexamethasone exposure on development of renal excretory capabilities probably represents the summation of promoted cell differentiation and slowed development consequent to growth retardation. At low dose levels, the former effect predominates, leading to enhanced functional development, whereas higher doses that interfere with general growth and development can offset the direct promotional effect.     

Ontogenetic development of isoproterenol subsensitivity of myocardial adenylate cyclase and beta-adrenergic receptors in spontaneously hypertensive rats

[3H]Dihydroalprenolol binding and adenylate cyclase activity in the myocardial membranes of Kyoto Wistar normotensive rats and spontaneously hypertensive rats were compared at various stages of postnatal development ranging from 2 to 36 weeks. Basal as well as agonist-stimulated myocardial adenylate cyclase activity was consistently decreased in spontaneously hypertensive rats as compared to normotensive rats as early as 2 weeks of age with significant differences (P < 0.05) observed after 6 weeks of age. When results were expressed as percent stimulation over the basal activity, only isoproterenol plus GTP-stimulated enzyme activity was reduced by 25--30% in spontaneously hypertensive rats, suggesting a specific loss of stimulation by isoproterenol in hypertensive animals. The number of [3H]dihydroalprenolol binding sites of KD for dihydroalprenolol binding were comparable between spontaneously hypertensive and normotensive rats at 3, 6 and 12 weeks of age. The competition of isoproterenol with [3H]dihydroalprenolol for the specific binding sites showed that the affinity of isoproterenol binding was decreased 3--4-fold in spontaneously hypertensive compared with normotensive rats. With postnatal development in age, basal as well as agonist-stimulated activities decreased progressively in both spontaneously hypertensive and normotensive rats. Similarly, the number of [3H]dihydroalprenolol binding sites decreased with the development in age, whereas affinity of dihydroalprenolol binding increased up to 12 weeks of age. These results therefore suggest that adenylate cyclase activity and the number of beta-adrenergic receptors in rat heart, decrease with age and that in hypertension, specific decrease in isoproterenol stimulation of cyclase appears at all stages of development.     

Two site binding of bepridil and modulation of adenylate cyclase in cardiac sarcolemmal membranes

A preparation of cardiac sarcolemmal membranes is described. These membranes exhibit 9-24-fold purification of (Na+ + K+)-ATPase, potassium-stimulated nitrophenolphosphatase, 5'-nucleotidase, adenylate cyclase, sialic acid content, and beta-receptor number. Sarcolemmal membranes have two classes of binding sites for the calcium entry blocker, bepridil, 70 X 10(12) high-affinity sites/mg, Kd 25-40 nM; and 30 X 10(15) low-affinity sites/mg, Kd 54-70 microM. Binding of bepridil to these sites appears responsible for inhibition of isoprenaline-stimulated and activation of fluoride-stimulated adenylate cyclase. Since basal adenylate cyclase activity is not influenced, bepridil must act not at the catalytic site, but by altering the interactions between beta-receptor and catalytic and regulatory components of adenylate cyclase.     

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.     

Ontogeny of fetal adenylate cyclase; mechanisms for regulation of beta-adrenergic receptors

Transmembrane second messenger signalling systems regulate differentiation, growth and homeostatic responses during fetal development. The beta-adrenergic adenylate cyclase system is the best studied of these and has been used as a model to investigate the control of developmental processes. In tissues such as lung, heart and parotid, beta-adrenergic responsiveness of adenylate cyclase increases during development. In the developing fetal lung beta-receptor concentration increases during gestation or after glucocorticoid treatment, but cannot fully explain enhanced adrenergic responsiveness. To probe developmental and hormonal effects on beta-receptor function, we asked if advancing gestation or glucocorticoid treatment alters beta-receptor-Gs interactions in fetal rabbit lung membrane particulates. Before 25 days gestation, 1-isoproterenol competes for 3H-dihydroalprenolol (DHA), a radiolabelled beta-antagonist, with a single low affinity, later in gestation, high and low affinities of isoproterenol for the beta-receptor are present which can be shifted to the lower affinity by addition of guanyl nucleotide. High affinity binding is precociously induced in 25 days--fetal lung particulates as early as 3 h after maternal betamethasone treatment, but beta-adrenoreceptor concentration in treated fetuses was increased over controls only after 24 h of treatment. Cholera toxin catalyzed ADP ribosylation of membrane particulates showed cholera toxin substrate (Gs) was not altered by glucocorticoid treatment. Stimulation of adenylate cyclase activity with isoproterenol (100mM) and GTP (100mM) resulted in no incremental increase over that produced by GTP (100mM) alone in glucocorticoid treated or control particulates, either early or late in gestation. These data demonstrate that beta-receptor-Gs interactions are not sufficient to produce full agonist responses. Although both beta-adrenergic receptors and Gs are present in fetal rabbit lung early in gestation, interaction of these two adenylate cyclase components appears subsequently. This developmental event can be rapidly induced by maternal betamethasone treatment.     

Possible Involvement of Inhibitory GTP Binding Regulatory Protein in α2-Adrenoceptor-Mediated Inhibition of Adenylate Cyclase Activity in Cerebral Cortical Membranes of Rats

Influences of alpha 2-adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet-activating protein and NAD resulted in a significant increase in basal activity as well as in GTP- or forskolin/GTP-induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet-activating protein and NAD. It is suggested that alpha 2-receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.     

Epidermal growth factor, a modulator of luteal adenylate cyclase. Characterization of epidermal growth factor receptors and its interaction with adenylate cyclase system in bovine luteal cell membrane.

The epidermal growth factor (EGF) binding sites on bovine luteal cell membrane have been characterized in detail, and evidence has been obtained for a direct stimulatory effect of EGF on membrane-associated adenylate cyclase activity. The membrane fraction prepared showed the presence of high affinity (Ka = 1.2 +/- 0.7 x 10(-11) M-1), specific, and saturable EGF receptors of Mr = 170,000. The EGF receptors underwent rapid autophosphorylation and down-regulation following treatment of the cells with EGF. Treatment of the cells with 4 beta-phorbol 12-myristate 13-acetate resulted in a diminished binding of 125I-EGF to the receptors. When luteal cells were preincubated with EGF, both basal and forskolin-stimulated adenylate cyclase activity was increased severalfold. This enhancement of the adenylate cyclase activity was dependent upon the duration of the exposure to EGF and on the concentration of the growth factor. An optimal enhancement was observed when the cells were preincubated with 10 ng/ml EGF for 10-15 min. Furthermore, when the membrane fraction prepared from luteal cells was preincubated in vitro with EGF, a similar dose-related and time-dependent increase in basal, as well as forskolin-stimulated, adenylate cyclase activity was observed. These results demonstrate that luteal cell adenylate cyclase activity is finely regulated by EGF. Such a direct interaction between EGF and membrane-associated adenylate cyclase has not been previously recognized.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Multiple effects of N, N' dicyclohexyl carbodiimide on the beta-adrenergic receptor--adenylate cyclase system in frog erythrocytes.

Treatment of frog erythrocytes with N,N' dicyclohexylcarbodiimide (DCCD) leads to a loss of catecholamine stimulated adenylate cyclase activity without any decrease in fluoride or PGE1 stimulated cyclase. However, the concentrations of the reagent which inhibit catecholamine sensitive adenylate cyclase activity are 10 fold lower than those which inhibit specific [3H]dihydroalprenolol ([3H]DHA) beta-adrenergic receptor binding. By contrast binding of the readiolabeled beta-adrenergic agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) is considerably more sensitive than antagonist binding to the effects of DCCD. The data suggest that low concentrations of the reagent may modify the effector portion of the beta-adrenergic receptor leading to functional uncoupling of the beta-receptor adenylate cyclase system. At higher concentrations of the reagent the ligand bidning site of the beta-receptor appears also to be altered.     

Inhibition of adenylate cyclase by arsenite and cadmium: evidence for a vicinal dithiol requirement.

The effect of sodium arsenite and cadmium chloride on adenylate cyclase activity was examined in turkey erythrocyte membranes. Sodium arsenite was a weak inhibitor of adenylate cyclase -7mM produced only 60% inhibition. Its effect, however, was greatly potentiated by equimolar 2,3 dimercaprol- wherein 0.7 mM sodium arsenite inhibited 100% with an apparent Ki of 0.1 mM. Equimolar mercaptoethanol was less effective in potentiating sodium arsenite inhibition. Thus 0.7mM sodium arsenite in the presence of equimolar mercaptoethanol inhibited adenylate cyclase 56%. Excess 2,3 dimercaprol reversed inhibition by sodium arsenite or cadmium chloride. Sodium arsenite or cadmium chloride inhibited all forms of adenylate cyclase activity tested, including nonhormonal stimulation. Equimolar sodium arsenite and dimercaprol, at concentrations that caused 100% inhibition of adenylate cyclase activity, reduced the binding of the beta-receptor specific ligand iodohydroxybenzylpindolol by less than 15%. These results suggest that turkey erythrocyte membranes contain closely juxtaposed thiol groups and that interaction of such groups with arsenate interferes with the catalytic function of adenulate cyclase.     

Ethanol Does Not Modify Opiate-Mediated Inhibition of Striatal Adenylate Cyclase

Ethanol increases the activity of "basal," guanine nucleotide- and dopamine-stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D-Ala2,D-Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor-coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.     

Regulation of beta-adrenergic receptor-mediated processes in fetal rat lung: selective desensitization caused by chronic terbutaline exposure

Fetal lung beta-receptors become effectively coupled to lung fluid reabsorption and enzymes involved in surfactant synthesis on the day before birth, a period when circulating catecholamine levels are high. Accordingly, we examined the effects of repeated maternal terbutaline exposure on beta-receptor binding capabilities and beta-receptor-mediated processes in the fetal rat lung. Administration of terbutaline to pregnant rats on gestational day 17-20 produced significant reductions in beta-receptor binding to membrane preparations. Similarly, beta-receptor-mediated stimulation of adenylate cyclase activity and ornithine decarboxylase activity showed marked desensitization in the terbutaline-exposed fetuses. However, the linkage of beta-receptors to lung fluid reabsorption and phosphatidic acid phosphatase, an enzyme involved in surfactant synthesis, did not desensitize with chronic terbutaline pretreatment; both of these processes displayed the normal onset of responsiveness on gestational day 21 in the treated animals, as well as a normal magnitude of response. Hence, beta-receptor-mediated events in the developing lung may be differentially regulated during exposure to agonists, allowing the selective expression or depression of function when circulating catecholamine levels are high.     

The relationship between beta-adrenoceptor regulation and beta-adrenergic responsiveness in hepatocytes. Studies on acquisition, desensitization and resensitization of isoproterenol-sensitive adenylate cyclase in primary culture

The role of beta-adrenoceptor regulation in the mechanisms controlling beta-adrenergic responsiveness in hepatocytes was explored, using primary monolayer cultures. When plated in vitro, these cells gradually acquire a strong catecholamine-sensitive adenylate cyclase activity and an enhanced ability to bind the beta-adrenoceptor ligand [125I]iodocyanopindolol (125ICYP). Examination of the time course showed that the increase in the number of 125ICYP binding sites was detectable within 1-2 h of culturing and slightly preceded the elevation of isoproterenol-responsive activity. Then the responsiveness rose steeply and between about 5-24 h it closely followed the increase in beta-receptor binding. Addition of isoproterenol (10 microM) to cells after 20 h of culturing caused a rapid homologous desensitization of the adenylate cyclase (50% after about 5 min). This was paralleled by a down-regulation of beta-adrenoceptors measured both in membrane particles and in total cell lysates. Removal of isoproterenol led to a resensitization of the adenylate cyclase, which was rapid and protein-synthesis-independent after a brief (10-min) desensitization, or slow and cycloheximide-sensitive after prolonged (4-h) exposure to the agonist. In both cases an up-regulation of the 125ICYP binding paralleled the recovery from refractoriness. In contrast, no concurring changes in 125ICYP binding were measured when the beta-adrenoceptor-linked adenylate cyclase activity was enhanced by pretreatment with pertussin toxin (islet-activating protein, IAP) or was desensitized by exposure of the cells to glucagon or 8-bromo-cAMP; however, these modulations of the adenylate cyclase were nonselective, since the pretreatments with IAP, glucagon or 8-bromo-cAMP affected both isoproterenol-sensitive and glucagon-sensitive activities. The results suggest that, in hepatocytes, regulation at the beta-adrenoceptor level is a major determinant for both short-term and long-term selective changes of the beta-adrenergic responsiveness.     

Attenuation of chick heart adenylate cyclase by muscarinic receptors after pertussis toxin treatment

Pertussis toxin selectively modifies the function of Ni, the inhibitory guanine nucleotide binding protein of the adenylate cyclase complex. In chick heart membranes, guanine nucleotide activation of Ni resulted in a decrease in the apparent affinity of the muscarinic receptor for the agonist oxotremorine, inhibition of basal adenylate cyclase activity, and the attenuation of adenylate cyclase by oxotremorine. Treatment of chicks with pertussis toxin caused the covalent modification of 80-85% of cardiac Ni. After this treatment Gpp(NH)p had no effect on muscarinic receptor affinity and GTP stimulated basal adenylate cyclase activity. In contrast, the GTP-dependent attenuation of adenylate cyclase caused by muscarinic receptors was unaffected.     

The role of surface charge and hydrophobic interaction in the activation of rat liver adenylate cyclase

Rat liver plasma membranes were incubated either with procaine, lidocaine or tetracaine to study the binding of glucagon to receptors and the responses of adenylate cyclase to glucagon or fluoride. Procaine treatment increased the glucagon and fluoride activation of the cyclase and the stimulation was concentration-dependent; this compound seemed to act at the G/F unit level since changes in the glucagon binding were not observed and the basal activity was not modified. Tetracaine inhibited the adenylate cyclase activity in the order glucagon greater than basal greater than fluoride; it seems that tetracaine acted at the receptor unit level since it reduced the binding affinity. Tetracaine at high concentration (10 mM) also inhibited the fluoride stimulation of the Lubrol PX-solubilized enzyme; apparently the anesthetic acts on the G/F unit and this would indicate the component is still bound to the catalytic unit. The solubilized enzyme is not longer stimulated by procaine. These data suggested that the F component site of the G/F units is in some aspects different to the G component and more resistant to the detergent. The results of this work allowed a clear distinction among the different components of the glucagon-stimulated adenylate cyclase system and showed the importance of surface charge and hydrophobic interactions as regulatory mechanisms.