Isoproterenol-stimulated Adenylate Cyclase Activity Research Articles

The role of phospholipase in reduced beta-adrenergic responsiveness in experimental asthma.

This investigation was designed to elucidate the role of phospholipase (PLase) in relation to reduced beta-adrenergic responsiveness in guinea pigs subjected to experimental asthma. In the in vivo experiment, guinea pigs that had developed asthma-like symptoms after exposure to an aerosol of 2% ovalbumin for 7 to 8 min for 10 successive days were used as the experimental asthma group. The control group was exposed to saline. The endogenous PLase activity was determined by high performance liquid chromatography using ditridecanoyl phosphatidylcholine as a substrate. PLase activity of lung membranes in the experimental asthma group was significantly elevated by 50% compared with that in the control group. Phospholipid content of lung membranes in the experimental asthma group was decreased by 11% compared with that in the control group. The experimental asthma group showed a 37% decrease in the number of beta-adrenoceptors in lung membranes and a 54% decrease in isoproterenol-stimulated adenylate cyclase activity in lung membranes compared with the control group. Although forskolin-stimulated adenylate cyclase activity was also reduced by 24%, decreases in forskolin-stimulated activity were less than decreases in isoproterenol-stimulated activity. In the in vitro experiment phospholipids in lung membranes were degraded by pretreatment with 0.1 U of PLase A2. After pretreatment of lung membranes with PLase A2, the number of beta-adrenoceptors was reduced by 25% compared with that in the control group, and adenylate cyclase activity stimulated by isoproterenol and forskolin were also reduced by 67 and 28%, respectively. PLase A2 had a minor effect on forskolin-stimulated activity as compared with isoproterenol-stimulated activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of PKC-ζ in NADPH oxidase–PKCα–Giα axis dependent inhibition of β-adrenergic response by U46619 in pulmonary artery smooth muscle cells

Treatment of bovine pulmonary artery smooth muscle cells (BPASMCs) with U46619 attenuated isoproterenol caused stimulation of adenyl cyclase activity and cAMP production. Pretreatment with SQ29548 (Tp receptor antagonist), apocynin (NADPH oxidase inhibitor) and Go6976 (PKC-α inhibitor) eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with SQ29548 and apocynin prevented U46619 induced increase in NADPH oxidase activity, PKC-α activity and Giα phosphorylation. However, pretreatment with CZI, a PKC-ζ inhibitor, markedly, but not completely, inhibited U46619 induced increase in NADPH oxidase activity, PKC-α activity, Giα phosphorylation and also significantly eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976 inhibited U46619 induced increase in Giα phosphorylation, but not PKC-ζ activity and NADPH oxidase activity. Pretreatment with pertussis toxin eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity without any discernible change in PKC-ζ, NADPH oxidase and PKC-α activities. Transfection of the cells with Tp, PKC-ζ and PKC-α siRNA duplexes corroborate the findings observed with their respective pharmacological inhibitors on the responses produced by U46619. Taken together, we suggest involvement of PKC-ζ in U46619 caused attenuation of isoproterenol stimulated β-adrenergic response, which is regulated by NADPH oxidase–PKCα–Giα axis in pulmonary artery smooth muscle cells.

Role of PKCα−p38MAPK−Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO−) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO− stimulated PKC-α activity and that subsequently increased p38MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p38MAPK inhibitor) eliminated ONOO− caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO− induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p38MAPK (p38MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO− effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO− was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a Gi dependent mechanism. This hypothesis was reinforced by Giα phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of Gi mediated response) to stimulate adenyl cyclase activity upon ONOO− treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (Gi)-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p38MAPK axis dependent phosphorylation of its α-subunit (Giα) in the pulmonary artery smooth muscle cells.

Impaired β-adrenergic receptor signalling in post-resuscitation myocardial dysfunction

ObjectivePost-resuscitation myocardial dysfunction is a major cause of fatality in patients receiving successful cardiopulmonary resuscitation. The mechanism of post-resuscitation myocardial dysfunction is largely unknown, although is generally considered related to ischaemia occurring during cardiac arrest and resuscitation and/or reperfusion injury after restoration of circulation. A key mechanism responsible for reduced contractile reserves in chronic heart failure is impaired β-adrenergic receptor signalling. Thus, we hypothesised that β-adrenergic receptor signalling is markedly abnormal in the post-resuscitation period following cardiopulmonary resuscitation. MethodsMale landrace domestic pigs were randomised into a sham group (anaesthetised and instrumented, no ventricular fibrillation) or cardiopulmonary resuscitation (CPR) group (ventricular fibrillation) (n=8 per group). Haemodynamic and echocardiographic data were recorded. β-Adrenergic receptor signalling was assessed at 6h after the operation by measuring myocardial adenylate cyclase activity, β-adrenergic receptor density and β-adrenergic receptor kinase expression. ResultsLeft ventricular function in the CPR group was significantly decreased at 6h after restoration of spontaneous circulation. Basal and isoproterenol-stimulated adenylate cyclase activity was blunted in the CPR group compared with the sham group. Total β-AR density was significantly decreased in CPR group compared with the sham group. Myocardial β-adrenergic receptor kinase expression was 2.03-fold greater in the CPR group than in the sham group. Conclusionsβ-Adrenergic receptor signalling is markedly impaired in the post-resuscitation period, which may be a mechanism of post-resuscitation myocardial dysfunction.

Reverse Remodeling With Left Ventricular Assist Devices

Over the last 2 decades, numerous advancements in medical therapies have improved patient outcomes in heart failure (HF). However, a significant number of patients still progress to end-stage HF, in which treatment options are largely limited to cardiac transplantation. As patient demands for transplant continue to exceed the supply of available organs, mechanical assist devices—specifically, the left ventricular assist device (LVAD)—were initially introduced as a bridge to cardiac transplantation. LVADs have 2 important beneficial effects. First, LVADs are placed in parallel to the native left ventricle (LV), causing pressure and volume unloading of the LV. Second, LVADs restore cardiac output and subsequent perfusion to the organs. As a result of these 2 effects, it became evident that some patients had actual improvement in LV function after LVAD placement. The term reverse remodeling was used to describe the improvement in myocardial function that was observed in patients with a seemingly end-stage disease. With reverse remodeling, a new hope for the treatment of HF was born—using LVADs as a bridge to recovery; however, to date, this promise has largely been unrealized. This probably is reflective of the fact that the sequela of mechanical ventricular unloading are quite complex and appear to involve the engagement of competing biological pathways including regression of cardiomyocyte hypertrophy as well as progressive cell atrophy. Although the promise of ventricular recovery still persists, its actualization will await a more comprehensive dissection of these competing biological processes. This review will discuss the beneficial clinical effects of LVAD support as well as review what is known about the cellular and molecular response to mechanical unloading and mechanisms of reverse remodeling. Key research findings have been summarized in the Table. View this table: Table. Summary of Research of LVAD Support on Clinical Effects and the Cellular and Molecular Changes That May Contribute to Reverse …

Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells: Targeting the cAMP signalling cascade

Previous studies have demonstrated that perfluorinated chemicals (PFCs) can affect reproduction by disruption of steroidogenesis in experimental animals. However, the underlying mechanism(s) of this disruption remain unknown. Here we investigated the effects and mechanisms of action of 1H, 1H, 2H, 2H-perfluoro-decan-1-ol (8:2 FTOH) on steroidogenesis using a human adrenocortical carcinoma cell line (H295R) as a model. H295R cells were exposed to 0, 7.4, 22.2 or 66.6 μM 8:2 FTOH for 24 h and productions of progesterone, 17α-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol were quantified by HPLC-MS/MS. With the exception of progesterone, 8:2 FTOH treatment significantly decreased production of all hormones in the high dose group. Exposure to 8:2 FTOH significantly down-regulated cAMP-dependent mRNA expression and protein abundance of several key steroidogenic enzymes, including StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21. Furthermore, a dose-dependent decrease of cellular cAMP levels was observed in H295R cells exposed to 8:2 FTOH. The observed responses are consistent with reduced cellular cAMP levels. Exposure to 8:2 FTOH resulted in significantly less basal (+ GTP) and isoproterenol-stimulated adenylate cyclase activities, but affected neither total cellular ATP level nor basal (−GTP) or NaF-stimulated adenylate cyclase activities, suggesting that inhibition of steroidogenesis may be due to an alteration in membrane properties. Metabolites of 8:2 FTOH were not detected by HPLC-MS/MS, suggesting that 8:2 FTOH was not metabolized by H295R cells. Overall, the results show that 8:2 FTOH may inhibit steroidogenesis by disrupting the cAMP signalling cascade.

β2-Adrenoceptor, Gs and adenylate cyclase coupling in purified detergent-resistant, low density membrane fractions

Membrane rafts and caveolae are specialized microdomains of the cell membrane that form physical platforms for compartmentalization of signalling molecules. Here, we intended to gain insight into the consequences of caveolar localization in G protein-coupled receptor function. We analysed beta(2)-adrenoceptor signalling in purified CRLDF (caveolin-rich low density fractions) of beta(2)-adrenoceptor-overexpressing HEK-293 cells. beta(2)-adrenoceptor and Gs immunoreactivities and forskolin-stimulated adenylate cyclase activity were all detected in CRLDF obtained by the conventional raft purification method that uses Triton X-100 solubilization. However, Triton X-100 caused a complete loss of the functional coupling between beta(2)-adrenoceptor, Gs and adenylate cyclase. Therefore, we developed an optimized purification method based on n-octyl-beta-d-glucopyranoside solubilization, where the functional properties of beta(2)-adrenoceptor, Gs and adenylate cyclase were preserved in the CRLDF. Using this method, we showed that isoproterenol-stimulated adenylate cyclase activity was similar in CRLDF and bulk membrane preparations of HEK-293 cells that overexpress beta(2)-adrenoceptor or beta(2)-adrenoceptor-Gs fusion. Accordingly, treatment of cells with methyl-beta-cyclodextrin, a caveola-disrupting agent, did not affect beta(2)-adrenoceptor-induced cAMP response. Likewise, these responses were insensitive to caveolin 1 and 2 overexpression. On the other hand, methyl-beta-cyclodextrin treatment did decrease beta(2)-adrenoceptor-induced ERK phosphorylation. However, the latter effect of methyl-beta-cyclodextrin could be attributed to a non-specific effect rather than its ability to disrupt membrane microdomains. We showed that localization in the raft microdomains did not affect the signalling efficiency of beta(2)-adrenoceptor-Gs-adenylate cyclase pathway, and that methyl-beta-cyclodextrin may inhibit signalling by directly affecting the signalling system independently of its caveola-disrupting property.

Acute β-blockade prevents myocardial β-adrenergic receptor desensitization and preserves early ventricular function after brain death

Beta-adrenergic receptor desensitization through activation of the G protein-coupled receptor kinase 2 is an important mechanism of early cardiac dysfunction after brain death. We hypothesized that acute beta-blockade can prevent myocardial beta-adrenergic receptor desensitization after brain death through attenuation of G protein-coupled receptor kinase 2 activity, resulting in improved cardiac function. Adult pigs underwent either sham operation, induction of brain death, or treatment with esmolol (beta-blockade) for 30 minutes before and 45 minutes after brain death (n = 8 per group). Cardiac function was assessed at baseline and for 6 hours after the operation. Myocardial beta-adrenergic receptor signaling was assessed 6 hours after operation by measuring sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, and G protein-coupled receptor kinase 2 expression and activity. Baseline left ventricular preload recruitable stroke work was similar among sham, brain death, and beta-blockade groups. Preload recruitable stroke work was significantly decreased 6 hours after brain death versus sham, and beta-blockade resulted in maintenance of baseline preload recruitable stroke work relative to brain death and not different from sham. Basal and isoproterenol-stimulated adenylate cyclase activities were preserved in the beta-blockade group relative to the brain death group and were not different from the sham group. Left ventricular G protein-coupled receptor kinase 2 expression and activity in the beta-blockade group were markedly decreased relative to the brain death group and similar to the sham group. Beta-adrenergic receptor density was not different among groups. Acute beta-blockade before brain death attenuates beta-adrenergic receptor desensitization mediated by G protein-coupled receptor kinase 2 and preserves early cardiac function after brain death. These data support the hypothesis that acute beta-adrenergic receptor desensitization is an important mechanism in early ventricular dysfunction after brain death. Future studies with beta-blocker therapy immediately after brain death appear warranted.

Beta-adrenergic Receptor Antagonism Preserves Myocardial Function After Brain Death in a Porcine Model

Cardiac dysfunction after brain death decreases the already limited number of potential donors for cardiac transplantation. Acute beta-adrenergic receptor (betaAR) desensitization after the brain death-associated catecholamine surge is an important mechanism. We hypothesized that acute betaAR antagonism could improve myocardial function after brain death by preserving betaAR signaling. Pigs were randomly assigned to three study groups (n = 5): sham; brain death; and brain death with betaAR antagonist (200 microg/kg/min esmolol), 30 minutes before brain death until 45 minutes after brain death. Functional data were collected for 6 hours after brain death and tissues procured. Compared with baseline, pre-load recruitable stroke work (PRSW), a pre-load-independent measure of systolic function (21.4 +/- 7.5 vs 43.3 +/- 6.8, slope of regression line during vena caval occlusion, p < 0.001), diastolic function (Tau, 101 +/- 54.7 vs 36.4 +/- 5.4 ms, p = 0.03) and systemic oxygen delivery (151 +/- 79.7 vs 298 +/- 78.7 ml/min, p < 0.001) deteriorated in untreated animals at 6 hours after brain death. In contrast, betaAR antagonist maintained baseline systolic function (PRSW, 37.8 +/- 5.6 vs 38.2 +/- 4.7, slope of regression line during vena caval occlusion, p = 0.92), diastolic function (Tau, 32.6 +/- 5.1 vs 48.5 +/- 28.3 ms, p = 0.57) and oxygen delivery (427 +/- 116 vs 397 +/- 98.8 ml/min, p = 0.36) at 6 hours after brain death. betaAR antagonist preserved betaAR signaling, as demonstrated by similar left ventricular (LV) basal (55.4 +/- 32.8 vs 58.8 +/- 10.9 pmol/mg/min, p = 0.40) and isoproterenol-stimulated (125 +/- 70.5 vs 124 +/- 52.0 pmol/mg/min, p = 0.49) adenylate cyclase activity at 6 hours after brain death, upon comparing betaAR antagonist and sham treatment groups. Both LV basal and isoproterenol-stimulated adenyl cyclase activity were higher with betaAR antagonist (25.9 +/- 4.8 pmol/mg/min, p = 0.03) than with untreated brain death (55.6 +/- 17.3 pmol/mg/min, p = 0.02). Beta-adrenergic receptor antagonism before brain death preserves cardiac function by preventing betaAR desensitization. This therapy in potential donors might increase the number of organs available for transplantation.

Role of the β-adrenergic receptor kinase in myocardial dysfunction after brain death

Significant cardiac dysfunction after brain death leading to exclusion from procurement for cardiac transplantation is seen in up to 25% of potential organ donors in the absence of structural heart disease. The cause includes uncoupling of the myocardial beta-adrenergic receptor signaling system. The mechanism, however, has not yet been described. This study investigates our hypothesis that brain death causes acute activation of the betaAR kinase and leads to desensitization of myocardial beta-adrenergic receptors and impaired ventricular function.Adult pigs underwent a sham operation or induction of brain death by means of subdural balloon inflation (n = 8 in each group). Cardiac function was assessed by using sonomicrometry at baseline and for 6 hours after the operation. beta-Adrenergic receptor signaling was assessed at 6 hours after the operation by measuring myocardial sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, beta-adrenergic receptor kinase expression, and activity.Induction of brain death led to significantly decreased left ventricular systolic and diastolic function. Basal and isoproterenol-stimulated adenylate cyclase activity was blunted in the brain dead group compared with the sham-operated group (28.3 +/- 4.3 vs 48.3 +/- 7.6 pmol of cyclic adenosine monophosphate.mg(-1) x min(-1) [P = .01] and 54.8 +/- 9.6 vs 114.5 +/- 18 pmol of cyclic adenosine monophosphate x mg(-1) x min(-1) [P < .02]). There was no difference in beta-adrenergic receptor density between the brain dead and sham-operated groups. Myocardial beta-adrenergic receptor kinase expression was 3-fold greater in the brain dead versus sham-operated groups, and membrane beta-adrenergic receptor kinase activity was 2.5-fold greater in the brain dead group compared with that seen in the sham-operated group.Induction of brain death leads to significant left ventricular dysfunction in this porcine model. Cardiac beta-adrenergic receptors are clearly uncoupled after brain death, and our data suggest that the mechanism is acute increase of myocardial beta-adrenergic receptor kinase activity, leading to beta-adrenergic receptor desensitization and ventricular dysfunction.

G protein and adenylate cyclase complex-mediated signal transduction in the rat heart during sepsis.

Changes in the protein level of various subunits of GTP-binding protein and the activity of adenylate cyclase in the rat heart during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The protein levels of various subunits of GTP-binding protein were determined by Western blot analysis. The activity of adenylate cyclase was measured based on the rate of formation of cAMP from [alpha-32P]ATP. The results show that protein levels of G alphas and G beta remained stable during the early and the late phases of sepsis. The protein levels of G alpha i-2 and G alpha i-3 remained relatively unaltered during the early phase of sepsis, but they were increased by 46.5% (P < 0.05) and 61.3% (P < 0.01), respectively, during the late phase of sepsis. The basal adenylate cyclase activity remained unchanged during the early phase while it was decreased by 25.7% (P < 0.05) during the late phase of sepsis. The isoproterenol-stimulated adenylate cyclase activity was unchanged during early sepsis while it was decreased by 44.6% (P < 0.01) during late sepsis. These data demonstrate that during the late hypodynamic phase of sepsis, myocardial G alpha i-2 and G alpha i-3 protein levels were increased and the increases were coupled with a reduction in adenylate cyclase activity. Because GTP-binding proteins mediate sympathetic control of cardiac function, the present findings may have a pathophysiological significance in contributing to the understanding of the pathogenesis of cardiac dysfunction during the late stage of sepsis.

Effects of angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on beta-adrenoceptor signaling in heart failure produced by myocardial Infarction in rabbits: reversal of altered expression of beta-adrenoceptor kinase and G i alpha.

Both angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 (AT1) receptor blockers have been demonstrated to improve symptoms and prognosis in heart failure (HF). We compared the effects of ACE inhibition and AT1 receptor blockade on myocardial beta-adrenoceptor desensitization in rabbits with HF established 3 weeks after myocardial infarction (MI) with left circumflex coronary artery ligation. Rabbits with MI were randomized to no treatment, the ACE inhibitor temocapril (0.5 mg/kg/day) or AT1 receptor blocker valsartan (3 mg/kg/day). Echocardiographic examinations showed that, relative to rabbits with untreated MI, rabbits receiving temocapril or valsartan had a limitation of cardiac remodeling and prevention of the development of systolic dysfunction. Circulating plasma norepinephrine levels that were markedly elevated in MI animals were strongly inhibited by temocapril or valsartan therapy. beta-Adrenoceptor density, beta-adrenoceptor proportion showing high-affinity agonist binding, and basal and isoproterenol-stimulated adenylate cyclase activities were significantly reduced in MI rabbits. These defects were similarly reversed by temocapril or valsartan. Importantly, as found in human HF, myocardial protein levels of beta-adrenoceptor kinase 1 and G(i alpha) were significantly elevated in MI rabbits, suggesting that these molecules are contributing to the defects in myocardial beta-adrenoceptor signaling. The expression levels of these molecules were normalized equally by both treatments. The results suggest that pharmacologically different interventions in the renin-angiotensin system can equivalently improve the derangements in the beta-adrenoceptor signaling system in the failing heart. This may be important for the beneficial effects of these agents in HF.

Characterization of Adenosine A1 Receptors in Human Proximal Tubule Epithelial (HK-2) Cells

Adenosine A1 receptors (A1ARs) modulate various aspects of renal functions, such as hormone release, hemodynamics and tubular absorption. Here we set up to demonstrate the expression of A1ARs in an immortalized cell line (HK-2) derived from normal adult human proximal tubule. We also examined the mechanism whereby A1ARs signal in HK-2 cells and their potential role in renal physiology such as sodium-dependent phosphate transport. Ligand binding assay of A1ARs was performed using plasma membranes of HK-2 cells and a selective high-affinity A1AR radioligand [3H]DPCPX. HK-2 cells in 96-well plates were treated with various agents (forskolin, adenosine receptor agonists, and antagonists) to activate or inhibit adenylate cyclase. Intracellular cyclic AMP accumulation was measured using cAMP flashplates. mRNA levels of adenosine receptors in HK-2 cells was determined by real-time PCR technique. Sodium-dependent phosphate transport across cell membrane was measured after 15-minute incubation of phosphorus-33 in transport buffer with HK-2 cells at room temperature. In HK-2 cells, A1ARs were expressed at a density of 211 +/- 74 fmol/mg membrane proteins. [3H]DPCPX bound to A1ARs on HK-2 cell membranes with Kd of 8.3 +/- 2.2 nM. Activation of A1ARs inhibited isoproterenol-stimulated adenylate cyclase activity through pertussis toxin-sensitive Gi protein in HK-2 cells. Coexpression of adenosine A2a receptors at a seemingly lower level than A1ARs was revealed by synergistically activating adenylate cyclase with forskolin. Real-time RT-PCR further demonstrated the expression of both A1AR and A2aAR in HK-2 cells. Sodium-dependent phosphate transport was augmented by activation of A1ARs in HK-2 cells. A1ARs are expressed in human proximal tubule epithelial (HK-2) cells and modulate sodium-dependent phosphate transport.

Inhibition of βARK1 Restores Impaired Biochemical β-Adrenergic Receptor Responsiveness but does not Rescue CREBA133 Induced Cardiomyopathy

A. D. Eckhart, R. C. Fentzke, J. Lepore, R. Lang, H. Lin, R. J. Lefkowitz, W. J. Koch and J. M. Leiden. Inhibition of βARK1 Restores Impaired Biochemical β-Adrenergic Receptor Responsiveness but does not Rescue CREBA133 Induced Cardiomyopathy. Journal of Molecular and Cellular Cardiology (2002) 34, 669–677. The myocardial β-adrenergic receptor (βAR) system plays a key role in dysfunctional signaling and physiology of the failing heart. Recently we described a murine model of dilated cardiomyopathy (DCM) produced by cardiac-specific expression of a dominant negative form of the CREB transcription factor (CREBA133 mice). CREBA133 mice display abnormalities within the βAR signaling system including loss of inotropic reserve. Rapid desensitization of βARs is mediated by the βAR kinase (βARK1), which is upregulated during heart failure. Inhibition of βARK1 activity in the heart via expression of a peptide inhibitor (βARKct) has been shown to enhance myocardial function and to “rescue” several animal models of heart failure. To determine the role of βAR dysfunction in the progression of DCM in the CREBA133 mice, we interbred them with mice expressing the βARKct. Concurrent expression of the βARKct peptide and CREBA133 in mouse hearts resulted in the normalization of elevated βARK1 levels. This biochemical change resulted in partial restoration of isoproterenol-stimulated adenylate cyclase activity as well as improvement in fractional shortening in response to βAR stimulation. Interestingly, the progression of DCM and premature mortality was not altered. Therefore, the pathogenesis of DCM in CREBA133 mice does not appear to involve abnormal βAR signaling as a key element in its pathological progression and accordingly, the restoration of βAR signaling is not sufficient to prevent the development and progression of all forms of heart failure.

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha.

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.

Agonist-independent Tonic Inhibitory Influence of Gion Adenylate Cyclase Activity in Rabbit Ventricular Myocardium and its Removal by Pertussis Toxin:a Role of Empty Receptor-mediated Giactictivitation

We evaluated whether Gihas a tonic inhibitory influence on myocardial adenylate cyclase (AC) in an agonist-independent way, and, if so, whether this is attributable to substantial coupling between agonist-free, empty inhibitory receptors and Gi. Rabbits received pertussis toxin (PTX, 10μg/kg i.v.) 40 h before preparing ventricular myocardial membranes, which was associated with virtually completein vivoADP-ribosylation and inactivation of the 41-kDa substrate. Pretreatment with PTX had no influence on basal AC activity but significantly enhanced AC activity elicited by 100μmGTP. Furthermore, it markedly increased AC activity stimulated with 5′-guanylyl imidodiphosphate (GppNHp) and isoproterenol through a wide range of concentrations of these stimulants. These findings indicate that Gihas a tonic influence on the stimulatory effects of guanine nucleotides andβ-adrenoceptor stimulation on AC, even in the absence of the inhibitory receptor agonists. The muscarinic receptor antagonists atropine and AF-DX 116 significantly enhanced isoproterenol-stimulated AC activity, as PTX pretreatment did, except that statistically significant increasing effects of these antagonists on GppNHp-stimulated AC activity was observed only at higher concentrations of GppNHp. The enhancement by atropine was not detected in PTX-pretreated membranes. The selectiveβ2-adrenoceptor antagonist ICI 118,551 did not modify the stimulatory effects of guanine nucleotides and isoproterenol on AC in either control or PTX-pretreated membranes, excluding the possible involvement ofβ2-adrenoceptors in tonic activation of Gi. We conclude that Giis tonically activated by agonist-free, empty muscarinic receptors, which leads to attenuation of Gs-mediated orβ-adrenoceptor-mediated activation of AC. The potentiating effect of PTX pretreatment on GppHNp-stimulated AC activity may be at least partially due to the direct action of PTX on the Giheterotrimeric complex, independently of the coupled receptors.

Involvement of Protein Kinase C in Hypoxia-Induced Desensitization of the β-Adrenergic System in Human Endothelial Cells

In order to better understand the mechanisms whereby oxygen deficiency promotes blunting of the endothelial β-adrenergic receptor (β-AR) system we examined the effects of hypoxia on β-AR, adenylate cyclase (AC) activity and phosphoinositide turnover in cultures of human pulmonary artery and umbilical vein endothelial cells. 1 hr of hypobaric hypoxia (290 mm Hg) increased basal levels of inositol mono-, bis-, and tris-phosphate to those following histamine stimulation under normoxia. Basal and isoproterenol-stimulated AC activities were lowered after 1 hr of hypoxia. β-AR density was decreased after 2–3 hrs of hypoxia and after treatment of EC with histamine, platelet activating factor or phorbol myristate acetate (PMA). In protein-kinase C (PK-C)-downregulated EC, neither hypoxia nor PMA influenced β-AR density or AC activity. Hypoxia-induced desensitization of β-AR signal transduction in EC may involve hypoxia-stimulated phosphoinositide turnover and subsequent PK-C activation.

Myocardial β-Adrenergic Receptor Function and High-Energy Phosphates in Brain Death– Related Cardiac Dysfunction

Cardiac failure remains an important problem after heart transplantation and may be associated with events that occur during brain death (BD) before transplantation. In this study, cardiac function is studied after BD, and biochemical evaluation of myocardial high-energy phosphates and the beta-adrenergic receptor system is presented. The hearts of 17 mongrel dogs (23 to 31 kg) were instrumented with flow probes, micromanometers, and ultrasonic dimension transducers to measure ventricular pressure and volume relationships. In a validated canine BD model, systolic right and left ventricular (RV/LV) function was analyzed by load-insensitive measurements during caval occlusion (preload-recruitable stroke work, PRSW). The beta-adrenergic receptor (BAR) density, adenylate cyclase (AC) activity, and myocardial ATP and creatine phosphate (CP) were measured before and 6 to 7 hours after BD. Results are expressed as mean +/- SEM (*P < .05 versus baseline, paired two-tailed Student's t test). Myocardial function deteriorated significantly from baseline PRSW (RV, 22 +/- 1 erg x 10(3); LV, 75 +/- 4 erg x 10(3)) by 37 +/- 10% for the RV (P < .001) and 22 +/- 7% for the LV (P < .001). BAR density increased from 282 +/- 42 to 568 +/- 173 fmol/mg for the RV and from 291 +/- 64 to 353 +/- 56 fmol/mg for the LV. Isoproterenol-stimulated AC activity was also significantly enhanced after BD. ATP and CP, however, remained unchanged after BD compared with baseline values before BD. BD causes significant systolic biventricular dysfunction. The loss of ventricular function after BD was more prominent in the right ventricle and may contribute to early postoperative RV failure in the recipient. These injuries occurred despite BAR system upregulation after BD. Global myocardial ischemia is unlikely, since ATP and CP remained normal before and after BD.

Refractory hypothalamic adenylate cyclase in anorectic tumor-bearing rats: implications for NPY-induced feeding

Although isoproterenol stimulated adenylate cyclase activity in hypothalamic membranes taken from freely-feeding, food-restricted or nonanorectic tumor-bearing rats, the response was greatly reduced in anorectic tumor-bearing rats. The addition of NPY to the membrane preparation inhibited adenylate cyclase activity in hypothalamus taken from freely-feeding and food-restricted rats, but NPY-inhibitory activity was significantly reduced in both groups of tumor-bearing rats. These results suggest that cyclic AMP formation is refractory in anorectic tumor-bearing rats, and that NPY-induced inhibition of hypothalamic adenylate cyclase is reduced in tumor-bearing rats prior to the onset of significant anorexia. Therefore, NPY-induced feeding may be reduced in tumor-bearing organisms due to a dysfunction in the cyclic AMP second messenger system.

P1-173 - Agonist-independent inhibition by Gi of isoproterenol-stimulated adenylate cyclase activity in rabbit myocardial membranes

P1-173 - Agonist-independent inhibition by Gi of isoproterenol-stimulated adenylate cyclase activity in rabbit myocardial membranes