Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

The adenosine derivative, N⁶-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar K_m values of about 0.2 μM GTP. PIA-induced low K_m GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low K_m GTPase by PIA and PGE₁, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE₁ was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Effect of thyroid status on α- and β-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased β-catecholamine responsiveness in hyperthyroid animals is due to a decrease in α-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the α₂-adrenergic inhibition of adenylate cyclase or the α₁-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamster. No effect of hyperthyroidism was found on the K_d of [³H]dihydroegocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to β-catecholamines was enhanced in adipocytes from hyperthyroid hamster, as was lipolysis. However, in adipocytes from hyperthyroid hamster the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on α-catecholamine action but enhances the activation of lipolysis by β-catecholamine agonists.     

Does glucocorticoid deprivation promote the expression of adenosine receptor-sites stimulating adenylate cyclase in rat adipocyte membranes?

The influence of N⁶-phenylisopropyladenosine (PIA) on adenylate cyclase was compared in adipocyte membranes from adrenalectomized and sham operated rats. In the presence of 100 mM sodium, 10 μM GTP and adenosine deaminase, PIA inhibited basal adenylate cyclase activity in sham rats, but elicited biphasic effects in adrenalectomized rats: at concentrations up to 10 nM, PIA first stimulated the enzyme, after which higher concentrations produced inhibition. In the presence of theophylline, these biphasic effects could not be observed. When isoproterenol maximally-stimulated adenylate cyclase was studied, the same biphasic effects of PIA were also observed in adrenalectomized rats, provided that no sodium was added in the assay, since with 100 mM sodium, only inhibition was seen. Finally, the stimulatory but not the inhibitory effect of PIA was prevented by glucocorticoid administration, a phenomenon which suggests that glucocorticoid deprivation may promote the expression of adenosine receptorsites which activate adenylate cyclase and which are normally absent, cryptic or unfunctional in normal adipocytes.     

The effects of adrenalectomy and of hydrocortisone administration on adenylate cyclase activity in rabbit adipose cells (author's transl)]

Adenylate cyclase activity in rabbit adipocyte plasma membranes was studied with special reference to the effects of adrenalectomy and administration of cortisol in vivo. Adrenalectomy was accompanied by an increase in adenylate cyclase activity during basal conditions; cortisol (5 mg/kg body wt., intramuscularly) partly prevents this effect of adrenalectomy. The response of adenylate cyclase to corticotropin, epinephrine and norepinephrine stimulation was higher in the adrenalectomized rabbit than in the sham operated animal. Our in vitro results were in agreement with the striking fat mobilization observed in rabbit plasma after adrenalectomy and with the hypolipemic effects of cortisol we had previously observed in both normal and adrenalectomized rabbit.     

Adipocyte G-proteins and adenylate cyclase. Effects of adrenalectomy.

Steroid hormones modulate the ability of cells to respond to hormones that act via cyclic AMP. In adipocytes of adrenalectomized rats, cyclic AMP accumulation and lipolysis in response to adrenaline are attenuated. However, the mechanism(s) of these effects are poorly understood. The effects of altered glucocorticoid status in vivo on the steady-state amounts of components of the hormone-sensitive adenylate cyclase were analysed in rat adipocytes. beta-Adrenergic receptors were analysed by using radioligand binding and immunoblotting with an anti-receptor antiserum. Neither the amount of radioligand binding nor the amount of beta-adrenergic-receptor peptide (Mr 67,000) was altered by adrenalectomy, whereas treatment of adrenalectomized rats with dexamethasone was found to increase both parameters by more than 25% with respect to the control. Forskolin-stimulated adenylated cyclase activity was unchanged in membranes isolated from adipocytes of adrenalectomized rats, but was decreased (50%) in those from dexamethasone-treated rats. The alpha-subunit of Gs was probed by using cholera-toxin-catalysed ADP-ribosylation. Immunoblotting was used to analyse the steady-state amounts of G-protein beta-subunits (beta-G35/36). Adrenalectomy was associated with decreases in the steady-state amounts of alpha-Gs (30%) and beta-G35/36 (50%). Dexamethasone treatment of adrenalectomized animals partially restored the lipolytic response of adipocytes to adrenaline and the amounts of alpha-Gs, increased the amounts of beta-G35/36 subunits from 50% to 150% of control values, increased beta-adrenergic receptors by more than 25% and decreased adenylate cyclase activity (50%). These results suggest that the steady-state amounts of components of hormone-sensitive adenylate cyclase are differentially regulated by glucocorticoids.     

Studies of corticotropin receptors on rat adipocytes

Synthetic [¹²⁵I]-Tyr²³, Phe², Nle⁴-adrenocorticotropin (ACTH)-(1–38) ([¹²⁵I]-ACTH analog) with full biological potency and near theoretical specific radioactivity (1800 ± 75 Ci/mmol) was used to investigate ACTH receptors on isolated rat adipocytes derived from 42-day-old rats. Binding to adipocytes was studied in the presence of 1% bovine serum albumin (BSA) as well as 4% BSA. The interaction of the [¹²⁵I]-ACTH analog with adipocytes was highly specific, rapid, saturable, and reversible. Scatchard analysis of the binding data obtained in medium containing 1% BSA revealed a single class of binding sites with an apparent K_D = 170 ± 11.9 pM. Competition experiments with unlabeled ACTH also yielded a comparable value for the apparent K_D (143 ± 16.5 pm). The number of receptors per adipocyte was quite low (521–841/cell). The stimulation of lipolysis by ACTH was closely correlated with the binding, the apparent K_m being 145–177 pm. At a concentration of 4% BSA in the incubation medium, the binding curve was shifted significantly to the right (apparent K_D = 446 ± 77 pM) and the binding capacity was also significantly enhanced (1663 ± 208/cell) without any change in the apparent K_m for glycerol release (187 ± 7.1 pm).     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH

The isolated intact white adipocyte of the Swiss mouse responds to both ACTH and catecholamines by an elevation of cAMP levels and an increase in lipolysis. However, in the isolated plasma membrane of the mouse adipocyte, adenylate cyclase loses its responsiveness to ACTH but retains its ability to respond to catecholamines. This lack of responsiveness to ACTH by adenylate cyclase of mouse adipocyte plasma membrane can be overcome, at least partially, by addition of GPP (NH)p, an analog of GTP, to the assay medium. The data on mouse adipocyte membrane suggests that the coupling of ACTH receptor to adenylate cyclase is dependent on GTP and that catecholamine-activation of adenylate cyclase is less dependent on this nucleotide. The isolated intact white adipocyte of adult New Zealand rabbit responds to ACTH, but does not (or only weakly) respond to catecholamines. In contrast to the mouse plasma membrane preparation, adenylate cyclase of adipocyte membrane of the rabbit responds to ACTH. And the addition of GPP(NH)P is not required to demonstrate the CTH: sensitive adenylate cyclase activity. The difference between mouse and rabbit adipocyte membrane in the requirement for GPP(NH)P in ACTH action is not readily explained. The lack of catecholamine sensitivity of rabbit membrane enzyme cannot be reversed by addition of GPP(NH)P or adenosine deaminase. These two adenylate cyclase model systems using mouse and rabbit adipocyte plasma membrane may be useful tools for the study of the specificity and mechanism of action of lipolytic hormones such as ACTH and catecholamines.     

Irreversible Antagonism of Beta-Adrenoceptors with Para-Amino-Benzyl-Carazolol Provides Further Evidence for an Atypical Rat Adipocyte Beta-Adrenoceptor

Abstract

Rat adipocytes possess typical beta₁ adrenoceptors that can be identified by ¹²⁵I-cyanopindolol binding but the receptor mediating isoprenaline adenylate cyclase activation possesses properties quite unlike beta₁ or beta₂ receptors. Separation of these sites has been attempted using the photoaffinity antagonist para-amino-benzyl-carazolol. Preincubation of rat reticulocyte and adipocyte membranes with this agent followed by washing induced a concentration-dependent loss of specific ¹²⁵I-cyanopindolol sites in both tissues, though the maximal loss was apparently greater in the reticulocyte. However, the loss of sites in both tissues induced a different effect on isoprenaline-stimulated adenylate cyclase. In the reticulocyte, the loss of specific sites was accompanied by an equivalent fall in the maximal stimulation of adenylate cyclase. In the adipocyte there were no significant effects of receptor site loss on the isoprenaline dose-response curve. It is suggested that this data supports the concept that an atypical beta-adrenoceptor, with relatively low affinity for many antagonists, mediates catecholamine-stimulated adenylate cyclase (and lipolysis) in the adipocyte.     

A MODULATING ROLE OF PITUITARY-ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3',5'-monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.
The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3'.5'-nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Specific and saturable binding of albumin to rat adipocytes: Modulation by epinephrine and possible role in free fatty acid transfer

Specific and saturable binding of ¹²⁵I-bovine albumin to rat adipocytes in suspension was observed (apparent K_d 2.09 ± 0.52 × 10^?6 M; 8.58 ± 2.49 × 10⁶ sites per cell; mean ± SEM). The binding was rapid and reversible for at least 10 min, suggesting that endocytosis of albumin was minor under assay conditions. Pre-incubation of cells with epinephrine bitartrate caused an apparent increase in number and decrease in affinity of the adipocyte binding sites for albumin. These findings suggest that a specific and saturable interaction of albumin with the adipocyte surface may play a role in the cellular uptake and release of free fatty acids.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15–18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [³H]ACTH (11–24) and [³H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [³H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (K _d 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [³H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (K _i 2.3 ± 0.2 nM), whose [³H] labeled derivative binds to rat adrenocortical membranes (K _d 2.1 ± 0.1 nM) with a high affinity. The specific binding of [³H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (K _i 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Glucocorticoid effects on gastric peroxidase activity

The peroxidase activity in rat gastric mucosa is inhibited after administration of glucocorticoids. The synthetic steroid dexamethasone is more potent than the naturally occurring steroids, such as cortisone or corticosterone. Almost complete inhibition of the enzyme occurs after 24 h with a single dose of 100 μg dexamethasone/120 g body weight. Other mitochondrial enzyme activities, like monoamine oxidase, succinic dehydrogenase and Mg²⁺-ATPase, remain unaltered under the same experimental condition. Submaxillary peroxidase and thyroid peroxidase activity are not inhibited by dexamethasone. Gastric peroxidase activity is increased 200–250% on the 6th day after adrenalectomy. This effect is blocked by the administration of dexamethasone. In fact, the enzyme becomes more sensitive to dexamethasone after adrenalectomy, since it is inhibited by more than 90% at the dose of 25 μg/120 g body weight. The inhibition by dexamethasone in normal animals is reversible. The enzyme is also inhibited after the administration of a single dose of ACTH. The apparent K_m of the enzyme for H₂O₂ is not altered after dexamethasone treatment or after adrenalectomy. The increase in enzyme activity following adrenalectomy is not blocked by actinomycin D or by α-amanitin, but is prevented by puromycin or cycloheximide. After administration of dexamethasone, the iodide concentration process in the gastric mucosa is not affected, but the organification of iodide is significantly diminished.     

Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5′-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the K_a was 0.05 μM. 5′-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, K_D 0.07 μM. Beta adrenergic amines accelerated the rate of 5′-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ≈ soterenol ≈ salbutamol > epinephrine ? norepinephrine. Catecholamine activation was antagonized by propranolol and the β₂ antagonist butoxamine; the β₁ antagonist practolol was inactive. [³H]Dihydroalprenolol bound to the membranes and binding was antagonized by β adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the β₂ type.     

Dexamethasone-dependent expression of beta 1-24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3-F442A cells

When 3T3-F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes. Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600-fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1-24) analog. In contrast, 3T3-F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4-fold by ACTH(1-24). The stimulation of the adenylate cyclase activity by GTP gamma S remained unchanged (about 20-25-fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone. Binding studies with 125I-ACTH revealed that specific cellular binding could be evidenced in dexamethasone-treated cells while control adipocytes did not exhibit any specific binding of 125I-ACTH. These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3-F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.