Catalytic Subunit Of Adenylyl Cyclase Research Articles

PTTH-stimulated ecdysone secretion is dependent upon tyrosine phosphorylation in the prothoracic glands of Manduca sexta

PTTH stimulates ecdysteroid secretion by the insect prothoracic glands. The peptide activates cAMP synthesis in a calcium-dependent manner, ultimately enhancing ecdysteroid synthesis. We have found that PTTH stimulates a rapid increase in tyrosine phosphorylation of at least four proteins in the prothoracic glands of larval Manduca sexta, as seen on Western blots of glandular lysates probed with antibody directed against phosphotyrosine. PTTH-stimulated tyrosine phosphorylation is blocked by an inhibitor of Src family tyrosine kinases, 4-amino-5-(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1). The inhibitor also blocks PTTH-stimulated ecdysone secretion, as well as PTTH-stimulated cAMP synthesis. Direct activation of the catalytic subunit of adenylyl cyclase by forskolin is not affected by PP1. In addition, ecdysteroid secretion stimulated by the cAMP analog dbcAMP is not blocked by PP1. These findings point to an important role for a Src-family tyrosine kinase at a very early step in the PTTH signaling pathway, prior to the activation of adenylyl cyclase.

Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, G i function, and somatostatin receptors in rat exocrine pancreas

To analyze the effect of bombesin on the somatostatin (SS) mechanism of action in the exocrine pancreas, male Wistar rats (250–270 g) were injected intraperitoneally with bombesin (10 μg/kg) three times daily at 8-h intervals for 7 or 14 days. Bombesin attenuated the ability of SS to inhibit forskolin-stimulated adenylyl cyclase activity in pancreatic acinar membranes. However, it did not decrease the ability of forskolin to stimulate the adenylyl cyclase catalytic subunit. The ability of 5′-guanylylimidodiphosphate [Gpp(NH)p] (a nonhydrolyzable GTP analog) to inhibit forskolin-stimulated adenylyl cyclase activity was diminished in pancreatic acinar cell membranes from bombesin-treated rats. Bombesin administration did not affect the ADP-ribosylation of a 41-kDa G protein catalyzed by pertussis toxin. The maximal SS binding capacity of pancreatic acinar membranes from bombesin-treated rats was decreased when compared with controls at the two time periods studied. The bombesin/gastrin-releasing peptide antagonist [ d-Tpi 6,Leu 13ψ(CH 2NH)Leu 14]bombesin (6–14) (RC-3095) (10 μg/kg ip), injected three times daily at 8-h intervals for 7 or 14 days, had a similar effect to that of bombesin on the SS mechanism of action. The combined administration of bombesin and its antagonist RC-3095 had a greater effect on the SS receptor–effector system than when administered separately. The present study indicates that the pancreatic SS receptor–effector system may be regulated by bombesin in vivo.

Alterations of tubulin function caused by chronic antidepressant treatment in rat brain.

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, Gs, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity. 2. Male Sprague-Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what. 3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin-GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats. 4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between Gs and the catalytic subunit of adenylyl cyclase but also tubulin interaction with Gs in the cerebral cortex of the rat.

Short-Term Effects of Postnatal Manipulation on Central β-Adrenoceptor Transmission

Neonatal handling is known to induce long-lasting changes in behavioral and neuroendocrine responses to stress. Since the central noradrenergic system participates in the adaptive responses to stressful conditions we have analyzed the effects of postnatal handling on β-adrenoceptor binding sites and isoprenaline- and forskolin-stimulated cyclic AMP accumulation in cerebral cortex, hippocampus and cerebellum of rats at 1 and 3 months of age. Handled animals showed reduced emotional reactivity and lower ACTH and corticosterone secretion after stress, Binding studies using [3H]CGP12-177 revealed increased β-adrenoceptor binding sites in handled rats in cerebellum and cerebral cortex with no changes in hippocampus, and decreased affinity in all cerebral regions. Handling reduced basal levels of cyclic AMP in hippocampus and cerebellum but not in cerebral cortex. The concentration-response curves of cyclic AMP to isoprenaline were displaced to the right in cerebellum of handled rats without differences in Emax; however, Emax was significantly reduced in cerebral cortex and hippocampus. Direct stimulation of the catalytic subunit of adenylyl cyclase by forskolin reduced the efficiency in hippocampus and cerebellum, but not in cerebral cortex of handled animals. It is concluded that neonatal handling reduces the binding properties of β-adrenoceptor and its primary biochemical responses in the young rat brain, which may account for the reduced responsiveness to stress attained in the handled rats, and may explain the persistence of the effect. The present study emphasizes the role of the central noradrenergic system in modulating the behavioral and neurendocrine responses to neonatal handling.

Catecholamine refractoriness and their mechanisms in cardiocirculatory shock and chronic heart failure.

In heart failure, a strong sympathetic activation has been observed and is regarded as the cause of beta-adrenergic desensitization in this condition. On the receptor level, there is a down-regulation of beta1-adrenergic receptors. In myocardium of patients on catecholamine treatment, the number of beta-adrenergic receptors can be further reduced. An uncoupling of beta2-adrenoceptors has been related to an increased activity and gene expression of beta-ARK in failing myocardium leading to phosphorylation and uncoupling of receptors. Beta3-adrenoceptors mediate negative inotropic effects, but alterations of these receptors are not known. In addition, an increase of inhibitory G-protein alpha-subunits (Gialpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G-protein alpha-subunits and betagamma-subunits have been observed to be unchanged. In patients with catecholamine-refractory septic or cardiogenic shock, an increase of Gialpha has been observed and related to the reduced effects of catecholamines in these conditions. The discovered mechanisms set the stage for the development of alternative strategies to increase force of contraction like the combination of PDE-inhibitors and catecholamines or Ca2+ sensitizing agents.

Beta-adrenergic signal transduction in the failing and hypertrophied myocardium.

A strong sympathetic activation has been observed in heart failure and is the cause of beta-adrenergic desensitization in this condition. On the receptor level there is downregulation of beta1-adrenergic receptors and uncoupling of beta2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of beta-adrenoceptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. beta3-Adrenoceptors mediate negative inotropic effects, but alterations in these receptors are not known. In addition, an increase in inhibitory G protein alpha subunits (Gi alpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G protein alpha and betagamma subunits, have been observed to be unchanged. Recent evidence shows that increases in Gi alpha also depress adenylyl cyclase in compensated cardiac hypertrophy both in monogenic and polygenic and in secondary hypertension. These increases of Gi alpha can suppress adenylyl cyclase in the absence of beta-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure, these observations indicate that adenylyl cyclase desensitization by Gi alpha may be a pathophysiologically relevant mechanism contributing to the progression from compensated cardiac hypertrophy to heart failure.

A post-receptor defect of adenylyl cyclase in severely failing myocardium from children with congenital heart disease

The aim of this study was to determine whether a defect at the post-receptor level of adenylyl cyclase may also contribute to the decreased effectiveness of cAMP-increasing agents in severely failing patients with congenital heart disease. The severity of congestive heart failure in 31 patients with congenital heart disease was graded by a scoring system which included a description of historical and clinical variables. Patients were divided into a group with no or mild heart failure (score ≤6) and a group with severe heart failure (score >6). β-Adrenoceptor-stimulated adenylyl cyclase activity was significantly decreased by 65% in patients with severe heart failure in comparison to the group of patients with no or mild heart failure. In addition, receptor-independent adenylyl cyclase stimulation by forskolin was reduced by 52% in patients with score >6 compared to patients with score ≤6. This post-receptor defect of adenylyl cyclase was apparently due to a decrease in the activity of catalytic subunit of adenylyl cyclase as adenylyl cyclase stimulation by forskolin in the presence of Mn 2+ which uncouples catalytic subunit from the G proteins, G s and G i, was also significantly diminished in the patients with severe heart failure. In contrast, the level of inhibitory G protein α-subunits was apparently not different in the two groups. In summary, the data indicate that a defect at the catalytic subunit of adenylyl cyclase apparently contributes to the decreased effectiveness of cAMP-increasing agents in severely failing patients with congenital heart disease.

Dimeric tubulin-stimulated adenylyl cyclase activity is augmented after long-term amitriptyline treatment.

We have investigated altered association of tubulin dimers interacting with G proteins and modulating adenylyl cyclase (AC) as a result of long-term amitriptyline (AMT) treatment. Gpp(NH)p-stimulated, but not basal or manganese-stimulated, AC activity was significantly augmented in cortex membranes prepared from rats chronically treated with AMT. The enhancement of AC activity by Gpp(NH)p-liganded tubulin (tubulin-Gpp(NH)p) was significantly higher in chronically AMT-treated rats than in control rats. Moreover, in cortex membranes from controls, tubulin-Gpp(NH)p prepared from chronically AMT-treated rats was more effective to activate AC activity than tubulin-Gpp(NH)p from controls. Immunoblotting and photoaffinity guanine nucleotide binding procedures showed no significant differences in the amount and the function of G proteins between controls and AMT-treated groups. It is suggested that long-term AMT treatment causes alteration in the functional interaction between tubulin and G protein, and this modification may participate in enhanced coupling of Gs to the catalytic subunit of AC induced by the chronic antidepressant treatment.

Effect of dimaprit and cimetidine on the somatostatinergic system in the rat frontoparietal cortex

A recent study carried out by this laboratory demonstrated that exogenous histamine increases the somatostatin (SS) receptor/effector system in the rat frontoparietal cortex (Puebla and Arilla, 1995). In the present study we examined the participation of the H2-histaminergic system in this modulation by use of the H2-receptor agonist and antagonist dimaprit and cimetidine, respectively. Dimaprit administration [20 micrograms/rat, intracerebroventricularly (ICV)] to rats 2 hours before decapitation increased the number of SS receptors in the frontoparietal cortex without changing the affinity constant. Pretreatment with cimetidine (20 micrograms/rat, ICV) prevented the dimaprit-induced changes in SS binding in the frontoparietal cortex, whereas cimetidine alone (20 micrograms/rat, ICV) had no observable effect on this parameter. The in vitro addition of dimaprit or cimetidine to frontoparietal cortex membranes from untreated rats did not markedly affect the SS binding characteristics. Somatostatin caused a significantly higher inhibition of basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity in frontoparietal cortex membranes from dimaprit-treated rats than in controls, an effect that was prevented by pretreatment with cimetidine. No significant differences, however, were detected for the basal or FK-stimulated AC enzyme activity in the control, dimaprit-, and/or cimetidine-treated groups, which suggests no impairment of the AC catalytic subunit. In addition, the functional activity of the guanine nucleotide-binding inhibitory protein Gi, as measured by the capacity of the stable GTP analogue 5'-guanylylimidodiphosphate [Gpp(NH)p] to inhibit FK-stimulated AC activity, was not altered by dimaprit. Thus, the increased SS-mediated inhibition of AC activity observed in the dimaprit-treated rats may be caused by the increase in the number of SS receptors. Neither dimaprit nor cimetidine affected somatostatinlike immunoreactivity (SSLI) content. The present results, together with the fact that SS and histamine have been shown to influence locomotor activity and nociception in a similar manner, suggest that some of the neurotransmitter effects of SS may be modulated by histamine via H2-histaminergic receptors.

Role of G-proteins in altered beta-adrenergic responsiveness in the failing and hypertrophied myocardium.

In the heart and other tissues, beta-adrenergic desensitization occurs during treatment with catecholamines. In heart failure, a strong sympathetic activation has been observed and is the cause of beta-adrenergic desensitization in this condition. On the receptor level, there is a downregulation of beta 1-adrenergic receptors as well as an uncoupling of beta 2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of beta-ARK2 in failing myocardium leading to phosphorylation and uncoupling of receptors. In addition, an increase of inhibitory G-protein alpha-subunits (Gi alpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G-protein alpha-subunits and beta gamma-subunits have been observed to be unchanged. Recently, evidence has been raised that increases of Gi alpha also depress adenylyl cyclase in compensated cardiac hypertrophy in monogenic and polygenic as well as in secondary hypertension. These increases of Gi alpha can suppress adenylyl cyclase in the absence of beta-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure these observations indicate that adenylyl cyclase desensitization by Gi alpha could be a pathophysiologically relevant mechanism to contribute to the progression from compensated cardiac hypertrophy to heart failure.

Impairment of adenylyl cyclase signal transduction in mecobalamin-deficient rats

This study examined alterations in the β-adrenoceptor-G s-adenylyl cyclase system in cerebral cortex membranes from vitamin B 12-deficient rats fed a diet lacking vitamin B 12 (mecobalamin) for 15 weeks. Basal, 5'-guanylylimidodiphosphate (GppNHp)-, isoproterenol-, and forskolin-stimulated adenylyl cyclase activities were significantly reduced in mecobalamin-deficient rats compared with those in control rats. However, no significant differences were observed in the amount and function of G s, estimated by immunoblotting and guanine nucleotide photoaffinity labeling, respectively, or in the densities and the dissociation constants of β-adrenoceptors, estimated by [ 125I] pindolol binding, between control and the deficient rats. These results indicate that vitamin B 12 deficiency results in the impairment of the coupling among the β-adrenoceptor, G s, and the catalytic subunit of adenylyl cyclase, and in dysfunction of the catalytic subunit of the enzyme, suggesting that vitamin B 12 participates in the regulation of neuronal adenylyl cyclase signal transduction.

Adrenergic desensitization in left ventricle from streptozotocin diabetic swine

Patients with diabetes mellitus that exhibit cardiac pump failure display compromised stroke volume, ejection fraction, and slower rates of rise and fall of left ventricular (LV) dP/dt in the absence of ischemic injury. We hypothesized that diabetic cardiomyopathy may involve decrements in adrenergic sensitivity, with specific molecular alterations in the beta-adrenergic receptor (beta AR)- G protein- adenylyl cyclase (AC) signal transduction system. We assessed the effects of 3 months of streptozotocin-induced diabetes (125 mg/kg i.v.; DIAB, n = 10) on myocardial signal transduction in mini-pigs. DIAB were hyperglycemic compared to controls (CON, n = 10; 20.92 +/- 2.64 v 5.24 +/- 0.35 mM glucose), and had lower fasting insulin levels (6.46 +/- 0.97 v 13.68 +/- 3.91 microU/ml). Transmural LV free wall homogenates from DIAB exhibited similar beta AR density as CON, but decreased cAMP production (pmol cAMP/mg prot.min) using these pharmacological stimulators: 10 microM Isoproterenol plus 100 microM GTP (74 +/- 5 v 97 +/- 11); 100 microM Gpp(NH)p (116 +/- 7 v 161 +/- 17); 10 mM fluoride ion (266 +/- 16 v 324 +/- 25). No differences between DIAB and CON were observed when stimulated by 100 microM forskolin (440 +/- 20 v 429 +/- 33), suggesting no alterations in the catalytic subunit of AC. In DIAB, quantitative immunoblotting indicated slightly depressed levels of Gs (552 +/- 44 v 630 +/- 59 pmol/g ww; NS), but a significant redistribution of alpha s from the sarcolemma to the cytosol (32.7 +/- 0.82% v 25.9 +/- 1.7%). Significantly elevated levels of cardiac Gi were seen in DIAB homogenates compared to CON ventricles (2326 +/- 145 v 1522 +/- 181 pmol/g ww), with no alpha i subunit redistribution. We conclude that despite maintained beta AR density, receptor-dependent and G protein-dependent stimulation of AC is depressed so that streptozotocin-induced diabetic LV is affected by increased cardiac Gi, redistribution of Gs alpha to the cytosol, and an increase in the Gi/Gs ratio. These results help explain depressed catecholamine responsiveness and cardiac performance exhibited by diabetic patients.

Oxidant Stress Enhances Adenylyl Cyclase Activation

The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H2O2) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H2O2 caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED50, 44 mumol/L to a maximum of 166% of control activity; n = 4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H2O2 were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H2O2 alone, 149 +/- 13%; H2O2 + tyrphostin A9, 100 +/- 9%; H2O2 + tyrphostin A1, 171 +/- 21%; n = 4). H2O2 comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166 +/- 17% of control, n = 5) and sodium fluoride (177 +/- 18% of control, n = 5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase-mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation.

Protein kinase C regulation of the adenylyl cyclase system in rat prostatic epithelium.

In the context of the crosstalk between transmembrane signalling pathways, we studied the loci within the stimulatory receptor/Gs protein/adenylyl cyclase system at which protein kinase C (PKC) exerts regulatory effects in rat prostatic epithelial cells. The treatment of cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in an impairment of the stimulation of adenylyl cyclase activity in terms of both potency, as seen with both vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP-27), and efficacy, as seen with the beta-adrenergic agonist isoproterenol. This inhibitory effect of PMA could be prevented by cell incubation with pertussis toxin but not with cholera toxin, pointing to a Gi- but not Gs-dependent mechanism. This hypothesis was reinforced by ADP-ribosylation experiments that showed a low extent of alpha i with pertussis toxin but no change of alpha s with cholera toxin, as well as by the observation of the loss of the ability of low Gpp[NH]p doses to inhibit forskolin-stimulated adenylyl cyclase activity (a measure of Gi function) after cell treatment with PMA. However, the phorbol ester did not modify the adenylyl cyclase catalytic subunit, as shown by experiments on direct stimulation of the enzyme by forskolin. Whatever the exact mechanisms, the results support a crosstalk between the PKC and the adenylyl cyclase systems in rat prostatic epithelial cells in terms of an impairment of adenylyl cyclase stimulation, due presumably to phosphorylation of both membrane receptors (coupled to Gs) and Gi protein, but not of Gs protein or the adenylyl cyclase itself.

Transforming growth factor‐β1 modulates adenylyl cyclase signaling elements and epidermal growth factor signaling in cardiomyocytes

Studies presented in this report were designed to investigate the effects of transforming growth factor-beta 1 (TGF-beta 1) on epidermal growth factor (EGF)-mediated stimulation of cAMP accumulation in cardiac myocytes and elucidate the mechanism(s) involved in this modulation. TGF-beta 1 (20 pM) treatment of cardiac myocytes, in a time-dependent manner, decreased the ability of EGF (100 nM) to increase cAMP accumulation. Significant attenuation of EGF-elicited cAMP accumulation was observed 2 h after exposure to TGF-beta 1 and 18 h after addition of TGF-beta 1, the ability of EGF to increase cAMP accumulation was completely obliterated. TGF-beta 1 neither decreased immunoprecipitable EGF receptors in membranes from cardiomyocytes nor altered the specific binding of [125I]EGF to cardiomyocyte membranes. However, TGF-beta 1 decreased the ability of EGF to phosphorylate membrane proteins on tyrosine residues. TGF-beta 1 treatment of cardiomyocytes also decreased the ability of forskolin to augment cAMP accumulation in intact cells and stimulate adenylyl cyclase activity. Similarly, in membranes of TGF-beta 1-treated cells, neither isoproterenol nor EGF stimulated adenylyl cyclase activity. Interestingly, as assessed by the ability of A1F4- to stimulate adenylyl cyclase, TGF-beta 1 did not alter the coupling between Gs and catalytic subunits. Likewise, TGF-beta 1 did not alter the functional activity of the inhibitory regulatory element of the system, Gi. Western analysis of cellular proteins revealed that TGF-beta 1 did not alter the amounts of Ga alpha, Gi alpha 2, and Gi alpha 3. We conclude that TGF-beta 1 attenuates EGF-elicited cAMP accumulation in cardiomyocytes, in part, by decreasing the EGF receptor kinase function and that TGF-beta 1-mediated alterations in the activity of adenylyl cyclase catalytic subunit also contribute toward the regulation of adenylyl cyclase by various agonists.

Actions of isoproterenol in frog proximal tubules

In the present work, we compared biochemical and electrophysiological actions of isoproterenol on frog proximal tubular cells by using tubule suspensions and giant entities obtained by cell fusion. Isoproterenol (ISO) dose-dependently stimulated cAMP production in tubule suspension and depolarized the “giant cell” membrane. Both effects were triggered by β receptor occupancy, but strongly differed in their concentration-dependency, since depolarization occurred with an ISO concentration as low as 10 −12 mol/l whereas cAMP accumulation could be seen only with more than 10 −8 mol/l ISO. ISO-induced membrane depolarization was mimicked by forskolin which directly stimulated the catalytic subunit of adenylyl cyclase. In both isoproterenol- and forskolin-stimulated giant cells, membrane depolarization was accompanied by a decrease in membrane conductance, and both effects were inhibited by tetraethylammonium (TEA) and 4-aminopyridine (4-AP). On the other hand, ISO- and forskolin-induced cAMP production were not affected by TEA. The present data thus show that isopoterenol produces two independent effects in frog proximal tubule: it depolarizes the cell membrane by blocking a K + conductance and activates adenylyl cyclase.

31P NMR investigation of energy metabolism in perifused MMQ cells.

The MMQ cell line is a unique prolactin-secreting rat pituitary cell line. MMQ cells entrapped in agarose gel threads are metabolically active, as determined by the uptake and phosphorylation of creatine and the maintenance of high energy phosphates for over 15 h. Forskolin activates the catalytic subunit of adenylyl cyclase and, in MMQ cells, elevates the level of cAMP and stimulates prolactin secretion. 31P NMR spectroscopy was used to investigate the energy metabolism of the MMQ cells during stimulation by forskolin. The ability to measure small changes in the energy status of these cells was enhanced by increasing the PCr levels in the cells. Administration of forskolin to the perifused MMQ cells resulted in acute, reversible, and dose-dependent changes in the 31P NMR spectra of the cells within 12 to 24 min of the beginning of forskolin exposure. Several lines of evidence indicate that the changes observed in the MMQ cells are the composite result of the interaction of forskolin with adenylyl cyclase and the plasma membrane glucose transporter. Also, preincubation of the MMQ cells with the dopamine agonist, bromocriptine, attenuates the forskolin-stimulated decrease in the PCr resonance by approximately 50%. This attenuation indicates that the forskolin-stimulated changes in energy metabolism are probably related to the prolactin secretion process.

G proteins in rat liver proliferation during cholestasis.

Liver proliferation appears to be dually regulated, in part by cyclic AMP levels. Here we studied the alterations in the stimulatory action of cholera toxin and other agents on the adenylyl cyclase system, as well as the status of Gs and Gi protein subunits during the liver proliferation that follows bile duct ligation in rats. The stimulatory effects of glucagon and vasoactive intestinal peptide (which act through membrane receptors) or guanosine 5'-[beta gamma-imido]triphosphate (which interacts with G proteins) and forskolin (which directly activates the adenylyl cyclase catalytic subunit) on liver adenylyl cyclase activity were blunted in cholestasis. The results indicated an impairment in the stimulatory interaction between the alpha s subunit of Gs protein and the adenylyl cyclase catalytic subunit. Indeed, we observed an important decrease in the stimulation of adenylyl cyclase activity by cholera toxin in cholestasis that was accompanied by a reduced extent of [32P]ADP ribosylation of alpha s protein catalyzed by cholera toxin, as revealed by the poor labeling of the 42,000 Da band in liver membranes from cholestatic rats. However, there was no change in the amount of alpha s or beta proteins as measured with immunoblotting techniques. Experiments on [32P]ADP ribosylation of alpha i subunits of Gi proteins indicated an impairment in liver membranes from cholestatic rats, whereas Western blotting for the detection of alpha i subunits showed decreased alpha i3 and increased alpha i2 levels in this condition. Further efforts are needed to better understand the molecular mechanisms underlying the relationship between the observed divergent expression of Gs and Gi proteins and liver cell proliferation in the cholestatic liver.

Preserved β-adrenoceptor-mediated adenylyl cyclase activity despite receptor and postreceptor dysfunction in acute myocardial ischemia

To examine responses of the β-adrenoceptor guanine nucleotide protein (G protein)/adenylyl cyclase complex to acute myocardial ischemia, we measured adenylyl cyclase activity stimulated at the β-adrenoceptor and postreceptor levels and compared crude homogenates prepared from ischemic and nonischemic rabbit myocardium obtained after 30 minutes of coronary artery occlusion. Basal adenylyl cyclase activity was unchanged, but enzyme activity stimulated by the guanosine triphosphate analog guanyl-5′ -imidodiphosphate (GppNHp) at 10 μmol/L was depressed 63% by ischemia ( n = 16, p = 0.001). In contrast, adenylyl cyclase activity stimulated by 1 μmol/L (-)-isoproterenol in the presence of 10 μmol/L GppNHp was not significantly reduced ( n = 10), a finding that indicates relative preservation of β-adrenoceptor-mediated adenylyl cyclase activity in ischemia. The ratio of (-)-isoproterenol-stimulated to GppNHp-stimulated adenylyl cyclase activity increased fourfold in ischemic myocardium ( n = 6, p = 0.001), consistent with more efficient β-adrenergic signal transduction via less functional stimulatory G protein (G s). These data could not be explained by augmented β-adrenoceptor density or agonist affinity or by a reduction in inhibitory G protein-mediated inhibition of adenylyl cyclase. Forskolin (1 mmol/L) and Mn 2+ (1 mmol/L), agents that directly stimulate the catalytic subunit of adenylyl cyclase, each increased enzyme activity significantly more in ischemic than in nonischemic myocardium. We conclude that preservation of (-)-isoproterenol-mediated adenylyl cyclase activity during acute myocardial ischemia in the rabbit results at least in part from enhanced function of the catalytic subunit of adenylyl cyclase. In addition, preserved β-adrenergic signal transduction, despite a decrease in β-adrenoceptor density, agonist affinity, and G s function, suggests more efficient signal amplification between the β-adrenoceptor and G s during acute myocardial ischemia.

Age-related changes in beta-adrenergic neuroeffector systems in the human heart.

Aging decreases cardiac beta-adrenergic responsiveness in model systems and in humans in vivo. The purpose of this study was to comprehensively evaluate the age-related changes in the beta-receptor-G protein-adenylyl cyclase complex in nonfailing human hearts. Twenty-six nonfailing explanted human hearts aged 1 to 71 years were obtained from organ donors and subjected to pharmacological investigation of beta-adrenergic neuroeffector systems. When the population was subdivided into the 13 youngest and 13 oldest subjects, total beta-receptor density assessed by maximum [125I]ICYP binding (beta max) was reduced in older hearts by 37% in left ventricles and 31% in right ventricles (both P < .05), and the downregulation was confined to the beta 1 subtype (r = .78 left ventricle beta 1 density versus donor age). Older donor hearts exhibited a 3- to 4-fold rightward shift of ICYP-isoproterenol (ISO) competition curves and demonstrated 43% fewer receptors in a high-affinity agonist binding state (P < .05). Older hearts exhibited decreased adenylyl cyclase stimulation by ISO, by zinterol (beta 2-agonist), and by the G protein-sensitive probes forskolin, Gpp(NH)p, and NaF. In contrast, there was no change in response to manganese, a specific activator of the adenylyl cyclase catalytic subunit. Toxin-catalyzed ADP ribosylation in membranes prepared from older versus younger hearts revealed a 29% to 30% reduction (P < .05) with cholera toxin (Gs) but no difference with pertussis toxin (Gi). The systolic contractile response of isolated right ventricular trabeculae to ISO was decreased by 46%, with a 10-fold increase in ISO EC50 in older relative to younger donor hearts. There is a profound decrease in cardiac beta-adrenergic responsiveness with aging. This occurs by multiple mechanisms including downregulation and decreased agonist binding of beta 1-receptors, uncoupling of beta 2-receptors, and abnormal G protein-mediated signal transduction.