Cholera and pertussis toxins modify regulation of glucose transport activity in rat adipose cells: evidence for mediation of a cAMP-independent process by G-proteins.

Adenylyl cyclase in rat adipose cells is stimulated by ligands for Rs receptors (e.g. isoproterenol) and inhibited by ligands for Ri receptors (e.g. adenosine). In contrast, Rs receptors mediate inhibition and Ri receptors mediate augmentation of insulin-stimulated glucose transport activity by a process independent of changes in cellular cAMP-dependent protein kinase activity [Kuroda M., Honnor R. C., Cushman S. W., Londos C. and Simpson I. A. (1987) J. biol. Chem. 262, 245-253]. The present study examines the possible role of G-proteins in the regulation of insulin-stimulated glucose transport activity by Rs and Ri receptors. First, conditions were established that permit intoxication of isolated rat adipocytes by cholera and pertussis toxins without compromising cell integrity. Effectiveness of toxin treatment was monitored by examining adenylyl cyclase activity in isolated plasma membranes. Secondly, neither toxin interfered with the ability of a maximal concentration insulin to initiate the glucose transport response. Thirdly, pertussis toxin eliminated the augmenting effects of adenosine on insulin-stimulated glucose transport activity, but enhanced the inhibitory effects of isoproterenol. Findings with ligands for other Ri receptors (nicotinic acid and prostaglandin E2) mirrored those with adenosine. Finally, cholera toxin elicited a modest depression of transport activity, and only in the absence of an Ri ligand (e.g. adenosine). Furthermore, in contrast to the enhanced stimulation of adenylyl cyclase by isoproterenol and GTP, cholera toxin eliminated the inhibitory effect of isoproterenol on transport activity. The augmentative effects of adenosine on transport activity were unchanged. Measurements of (-/+cAMP) cAMP-dependent protein kinase activity ratios reinforce the notion that modulation of glucose transport activity is independent of changes in cAMP. We conclude that regulation of glucose transport activity by Rs and Ri receptors is mediated by the G-proteins, Gs and Gi (or other toxin substrates), respectively. Inasmuch as such regulation occurs at the plasma membrane and appears to be cAMP-independent, it is suggested that glucose transporters may be direct targets for receptor: G-protein interactions.     

Evidence that metabolically active synaptosomes lack functional cyclic AMP-dependent protein kinase.

Cyclic adenosine monophosphate (cAMP)-mediated signal transduction was evaluated in synaptosomes prepared from rat brain cortex. Adenylate cyclase was responsive to known adenylate cyclase stimulators including peptides (CRH and VIP), catecholamines (norepinephrine and isoproterenol) and ligands that directly stimulate adenylate cyclase (forskolin). Cyclic AMP accumulation also increased approximately 2 to 3-fold, but none of the agonists was able significantly to activate cyclic AMP-dependent protein kinase (A-kinase) in cortical synaptosomes. However, in parallel studies with slices prepared from rat brain cortex, adenylate cyclase activity, cAMP accumulation and A-kinase activity were all stimulated by CRH, VIP, norepinephrine, isoproterenol and forskolin. These data suggest that, in intact synaptosomes, either the cellular machinery which facilitates binding of cAMP to the regulatory subunit of A-kinase is missing or the cAMP produced by adenylate cyclase is not accessible to A-kinase.     

cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators

The relationship between cAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios greater than 0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A-kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the cAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC50 for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligand-free environment achieved with adenosine deaminase; insulin IC50 values were approximately 120 and 600 pM, respectively. Such data establish a link between insulin actions in modifying cAMP concentrations and in modifying events apparently independent of changes in cAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells

The counterregulatory action of catecholamines on insulin-stimulated glucose transport and its relation to glucose transporter phosphorylation were studied in isolated rat adipose cells. Plasma membranes exhibiting reduced glucose transport activity were prepared as described previously (Joost, H. G., Weber, T. M., Cushman, S. W., and Simpson, I. A. (1986) J. Biol. Chem. 261, 10033-10036) from cells treated with insulin, and subsequently with isoproterenol and adenosine deaminase. In these membranes, transporter affinity for cytochalasin B binding was significantly reduced (KD = 133.5 +/- 14 versus 89.8 +/- 11 nM, means +/- S.E.) with no change in number of sites or immunoreactivity of the transporter on Western blots. Reconstituted plasma membrane transport was significantly lower with isoproterenol treatment (0.50 +/- 0.12 versus 0.97 +/- 0.27 nmol/mg protein/10 s). In contrast, transport activity reconstituted from corresponding intracellular transporters (from low density microsomes) was unchanged (5.4 +/- 2.2 versus 6.9 +/- 1.2 nmol/mg protein/10 s). Thus, the intrinsic activity change of the transporter produced by catecholamines appears to reflect a structural modification that is confined to the plasma membrane and not recycled into the intracellular compartment. In cells equilibrated with [32P]phosphate, neither insulin nor isoproterenol induced [32P]phosphate incorporation into the glucose transporter immunoprecipitated from plasma membranes. Conversely, phorbol 12-myristate 13-acetate stimulated significant incorporation of [32P]phosphate into the glucose transporter in insulin-stimulated cells without any change in plasma membrane transport activity or transporter concentration. Thus, the phosphorylation state of the glucose transporter does not seem to be involved in either signaling transporter translocation or triggering changes in transporter intrinsic activity.     

Glycogen synthesis stimulation by adenylate cyclase inhibition in rat epididymal adipocytes

The effects of adenylate cyclase inhibition on the transport of glucose and fructose and their incorporation into glycogen were investigated in order to assess the extent to which lowered cAMP levels can take part in the various components of glycogen synthesis regulation in isolated rat epididymal adipocytes. The dose-response characteristics of (R)-N-(2-phenylisopropyl)adenosine (PIA), a potent and specific adenylate cyclase inhibitor, on glycogen synthesis were compared with those effectively inhibiting lipolysis, a measure of functional cAMP levels. PIA had no effect on basal glucose or fructose transport but stimulated glucose and fructose incorporation into glycogen. Their respective incorporation was 10 and 69% of that achieved in the presence of insulin. These effects of PIA were shown to be in part the result of increased glycogen synthase I activity. PIA was 20% as effective as insulin in this action. Thus, were insulin to lower cAMP levels and/or inhibit cAMP-dependent protein kinase, this action would be irrelevant to glucose transport but would contribute to the stimulation of glycogen metabolism. However, an additional mechanism(s) involving neither increased glucose transport nor lowered cAMP levels is required to account for the full action of insulin. Fat cells in the absence of medium glucose and in the presence of 10(-7) M PIA and adenosine deaminase constitute a system functionally depleted of cAMP where this mechanism can be studied in isolation.     

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK1L) grown in defined media

LLC-PK1L cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PK1L cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

Heterologous desensitization of pigeon erythrocyte adenylate cyclase by the catalytic subunit of cAMP-dependent protein kinase

Preincubation of pigeon erythrocyte plasma membranes with the catalytic subunit of cAMP-dependent protein kinase results in the desensitization of erythrocyte adenylate cyclase. The adenylate cyclase activity measured in the presence of 10 microM isoproterenol and 50 microM GTP-gamma-S decreases by 40% after 10 min incubation; that in the presence of 50 microM GTP-gamma-S by 35% (20 min). The decrease of the adenylate cyclase activity is due to the prolongation of the lag phase of the enzyme activation in the presence of a hydrolysis-resistant GTP analog and to the drop in activity in the steady state of the activation. The heterologous desensitization of adenylate cyclase induced by cAMP-dependent protein kinase is also coupled with the decrease of the number of beta-adrenoreceptors capable of acquiring a high affinity for the agonists in the absence of guanyl nucleotides. The effect of the catalytic subunit on adenylate cyclase is fully compatible with the process of the enzyme desensitization in erythrocytes treated with isoproterenol or cAMP.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

Hormone-sensitive adenylate cyclase of prolactin-producing rat pituitary adenoma (GH4C1) cells: molecular organization

Hormonal activation and inhibition of the GH4Cl1 cell adenylate cyclase complex is delineated. In the presence of the guanyl nucleotide GTP, enzyme activity was enhanced twofold by thyroliberin, sixfold by vasoactive intestinal peptide (VIP), twofold by prostaglandin E2 and twofold by isoproterenol. The diterpene, forskolin, increased, the activity 14-fold. In the presence of high GTP (400 microM) and NaCl (150 mM) concentrations, somatostatin inhibited (ED50 = 0.5 microM) the cyclase activity by 40%. In the presence of 10 microM somatostatin, the ED50 values (5 nM) for thyroliberin- and VIP-stimulated adenylate cyclase activities were shifted to 20 nM. Forskolin-elicited activation was, however, not affected by somatostatin. Cholera-toxin and pertussis-toxin pretreatment of the enzyme brought about some 20-fold and twofold activation, respectively. Inhibition by somatostatin was abolished upon pre-exposure to pertussis toxin. Mild alkylation by N-ethylmaleimide increased basal and hormone-activated adenylate cyclase while somatostatin again failed to express its inhibitory potential. Further alkylation caused a gradual decline and convergence of hormone-modulated cyclase activities towards zero. The N-ethylmaleimide-induced attenuation of thyroliberin-elicited activity was paralleled by a decrease in [3H]thyroliberin binding. Trifluoperazine and an anti-calmodulin serum reduced basal and net thyroliberin-, VIP- and forskolin-enhanced cyclase activities by some 30%, 100%, 70% and 80%, respectively. The Vmax of basal and thyroliberin-stimulated adenylate cyclase was diminished by 65%, leaving the apparent Km values (7.2 mM and 2.6 mM, respectively) for Mg2+ unaltered. Finally, the phorbol ester 12-O-tetra-decanoyl-phorbol 13-acetate (TPA) doubled the activity. This effect was counteracted by the protein kinase C inhibitor, polymyxin B, while thyroliberin-enhanced adenylate cyclase remained unaffected. In summary, we have described an adenylate cyclase with stimulatory (Rs) and inhibitory (Ri) receptors coupled to a calmodulin-sensitive holoenzyme through the Gs and Gi type of GTP-binding proteins. The ratio of the Gs to Gi is high. It appears that the GH4C1 cell adenylate cyclase is also activated by protein kinase C by interference with Gi. Apparently, thyroliberin activates the cyclase both directly through Gs and indirectly via protein kinase C stimulation.     

Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine

The mechanism of modulation of insulin-stimulated glucose transport activity in isolated rat adipose cells by lipolytic and antilipolytic agents has been examined. We have measured glucose transport activity in intact cells with 3-O-methylglucose and in plasma membranes with D-glucose, and the concentration of glucose transporters in plasma membranes using a cytochalasin B binding assay. In intact cells, isoproterenol reduced insulin-stimulated transport activity by 60%. This effect was lost after cooling and washing the cells with homogenization buffer, and neither the concentration of glucose transporters nor transport activity in the plasma membranes differed from control. However, treatment of cells with KCN prior to homogenization preserved the isoproterenol effect through the fractionation procedure. Plasma membranes from these cells contained an unchanged number of transporters (31 +/- 7, mean +/- S.E., versus 31 +/- 4 pmol/mg of protein in controls) but transported glucose at a reduced rate (19 +/- 6 versus 48 +/- 9 pmol/mg of protein/s). Conversely, incubation of intact cells in the presence of adenosine stimulated plasma membrane glucose transport activity compared to that in the absence of adenosine (44 +/- 6 versus 36 +/- 6 pmol/mg of protein/s). Kinetic studies of isoproterenol-inhibited glucose transport in plasma membranes revealed a 60% decrease in Vmax (2900 +/- 350 versus 7200 +/- 1000 pmol/mg of protein/s) and a small increase in Km (15.1 +/- 1 versus 13.0 +/- 0.6 mM). These data indicate that modifications of glucose transport activity produced by lipolytic and antilipolytic agents in intact adipose cells can be fully retained in plasma membranes isolated under appropriate conditions. Furthermore, the effects of these agents occur through a modification of the glucose transporter intrinsic activity.     

A new mechanism of inhibiting adenylate cyclase involving a beta-adrenergic receptor]

The previously unknown mechanism of adenylate cyclase activity inhibition by catecholamines has been found. It is realized through a beta-adrenoreceptor in the smooth muscle of fresh-water mollusc Anodonta cygnea. As to its ligand-binding characteristics (one class of binding sites with Kd = 0.35 + 0.06 nM, a competitive series of ligands substitution: isoproterenol greater than adrenalin greater than propranolol greater than noradrenaline greater than serotonin = dopamine greater than phentolamine) as well as to negative regulation of the GTP affinity this receptor is similar to beta-adrenoreceptors of higher vertebrates. The dose-dependent inhibiting effect (to 50-60%) of isoproterenol and noradrenaline on the basal, GTP- and serotonin-stimulated activity of adenylate cyclase and cAMP level which is removed only by beta-adrenergic blockers is shown in vitro and in vivo. It is concluded that inhibition of adenylate cyclase activity by catecholamines in the muscular tissue of the mollusc is realized via beta-adrenoreceptor.     

The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Mechanism of catabolite repression in Escherichia coli bacteria: interaction between transport proteins and adenylate cyclase

The mechanism of catabolite repression caused by sugar transported via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) and stipulated by the decrease of the adenylate cyclase activity was studied. It was demonstrated that the sensitivity of the adenylate cyclase and beta-galactosidase synthesis to methyl-L-D-glucoside (MeGlc) or sorbitol is correlated with the content and activity of glucose (EIIGlc) or mannitol enzyme II of the PTS, correspondingly. Under anaerobic conditions the cells become insensitive to catabolic repression caused by MeGlc and the adenylate cyclase activity does not decrease in the presence of the sugar despite the increased rate of MeGlc transport. The adenylate cyclase activity of the mutant with the Tn5 transposone inserted into the ptsG gene does not change in the presence of MeGlc, while the activity of adenylate cyclase and the differential rate of beta-galactosidase synthesis increase in these bacteria. The data obtained confirm the hypothesis on the "catabolite signal" which is generated when the substrate binds to its transporter, i. e. adenylate cyclase reacts to the conformational changes in the transporter being complexed with it. The strength of this complex depends on the affinity of adenylate cyclase for the transporter and on the value of the membrane potential, delta mu H+ A model is proposed, which explains the necessity of factor IIIGlc for EIIGlc binding to adenylate cyclase.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.     

Desensitization of beta-adrenoreceptors and its influence on manifestations of the antiradiation effect of isoproterenol

A study was made of the function of beta-adrenoreceptors and Chinese hamster fibroblast cAMP during their desensitization to isoproterenol. Desensitization of adenylate cyclase was demonstrated to lead to the reduction of both the cAMP response to isoproterenol and isoproterenol capacity to protect the cells from ionizing radiation. This did not entail any changes in the number of beta-adrenoreceptors or in the dissociation constant of the beta-antagonist. It is assumed that desensitization provokes functional dissociation of the beta-receptor and adenylate cyclase. Intact adenylate cyclase is an absolute must for realization of the antiradiation potency of isoproterenol.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

Cultured vascular smooth muscle cells from porcine coronary artery possess A1 and A2 adenosine receptor activity

This study tested the hypothesis that an A1 adenosine receptor capable of inhibiting adenylate cyclase activity is present in porcine coronary vascular smooth muscle cells. In the absence of blockade of the A2 adenosine receptor, the A1 adenosine receptor agonists phenylisopropyladenosine (PIA) and cyclopentyladenosine (CPA) (10(-9) M) failed to inhibit Gpp(NH)p stimulated adenylate cyclase activity. However, after blockade of the A2 adenosine receptor with 30 nM CGS 15943A, cyclopentyladenosine (10(-9) M) inhibited Gpp(NH)p stimulated adenylate cyclase activity by 27 +/- 3% (4.3 +/- 0.7, Mean +/- SEM; pmoles/min/mg vs 5.9 +/- 0.8, P less than .05). The data demonstrate that both A1 and A2 adenosine receptors are present in coronary vascular smooth muscle. The results indicate that adenosine may mediate both vasodilation and vasoconstriction in the coronary circulation via A2 and A1 adenosine receptors, respectively.