Heterotrimeric G protein Gi is involved in a signal transduction pathway for ATP release from erythrocytes

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein G(s) resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the G(i) subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins G(alphai1), G(alphai2), and G(alphai3) but not G(alphao) were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples G(i/o) from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 microM) or Mas-7 (1 microM), which are compounds that directly activate G(i) proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 microM), which is an inactive Mas analog. In separate experiments, Mas (10 microM) but not Mas-17 (10 microM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein G(i) is a component of a signal transduction pathway for ATP release from erythrocytes.     

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.     

Nitric Oxide Causes Glutamate Release from Brain Synaptosomes

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µ M ) produced (4 and 10 nmol/mg of synaptosomal protein) Ca²⁺-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca²⁺-independent glutamate release. Preincubation of synaptosomes with 5 µ M 1 H -[1,2,4]oxadiazole[4,3- a ]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca²⁺-independent glutamate release. Ca²⁺-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.     

Molecular mechanisms of regulatory action of adrenergic receptor agonists on functional activity of adenylyl cyclase signaling system of the ciliates Dileptus anser and Tetrahymena pyriformis

Adenylyl cyclase signaling system (ACS) of the higher eukaryotes involves the following main components: receptor, heterotrimeric G protein, adenylyl cyclase (AC), and protein kinase A. At present, these components have been found in cells of different species of the lower eukaryotes. Hence, the signal transduction through ACS of unicellular eukaryotes may have some features in common with those of the higher eukaryotes. We showed earlier that agonists of adrenergic receptors (ARs) regulate AC activity of ciliates Dileptus anser and Tetrahymena pyriformis. The aim of this work was to study molecular mechanisms of AR ligand action on the functional activity of different components of ACS of the ciliates. It has been shown that beta-AR antagonist [3H]-dihydroalprenolol binds membranes of the ciliates with a comparatively lower affinity than those of the higher eukaryotes (Kd for D. anser was 13.4 nM, for T. pyriformis--27 nM). Beta-AR ligands--agonist (-)-isoproterenol and antagonists propranolol and atenolol in competition manner displace [3H]-dihydroalprenolol with IC50 that are 10-100 times higher than corresponding IC50 of beta-AR of the higher eukaryotes. In the presence of GTP, the right shift of competition curves of [3H]-dihydroalprenolol displacement by isoproterenol was obtained, being most considerable in the case of D. anser. Adrenaline and isoproterenol in a dose-dependent manner stimulated GTP-binding in cell cultures of D. anser and T. pyriformis. Suramin (10(-5) M), the inhibitor of heterotrimeric G proteins, completely blocked effects of these hormones. In D. anser culture, adrenaline and isoproterenol in a dose-dependent manner, stimulated AC activity, and its stimulating effects in the presence of beta-AR blockers vanished (propranolol) or decreased to a great extent (atenolol). At the same time the effects were unchanged in the presence of alpha2-AR antagonists yohimbine and idazoxan. These data show the involvement of G protein-coupled beta-AR in signal transduction induced by AR agonists in D. anser cells. In cell culture of T. pyriformis isoproterenol weakly stimulated AC activity, and its effect was completely blocked by beta-AR blockers. Adrenaline in T. pyriformis cells in a dose-dependent manner inhibited AC activity. Inhibiting effect of hormone was decreased in the presence of alpha2-AR blockers. On the basis of the obtained data we concluded that adrenaline in T. pyriformis cells inhibited AC activity through G protein-coupled receptor, being close to alpha2-AR of vertebrate animals.     

Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V(m)), and endothelial V(m) of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8-37) (1 microM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NNA; 10 microM), an action that was reversed by the addition of L-arginine (100 microM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (K+(ATP)) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), dideoxyadenosine (10 microM), N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 microM) and glibenclamide (10 microM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or V(m). CGRP (100 nM) hyperpolarized the smooth muscle V(m), an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 microM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K+(ATP) channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K+(ATP) channels and resultant hyperpolarization.     

Adenylyl cyclase regulates signal onset via the inhibitory GTP-binding protein, Gi

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G(s) and G(i), respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Galpha(s) and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G(s) (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331). This latter action of ACV would facilitate the rapid onset of signaling via G(s). Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Galpha(i), we investigated whether the receptor-mediated activation of heterotrimeric G(i) was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G(i) such that the amount of peptide required to stimulate G(i) in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) to G(i) was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPgammaS binding to G(i) in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc(-) cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G(i) and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G(i).     

Galpha(s) is palmitoylated at the N-terminal glycine

Covalent lipid attachments are essential co- and post-translational modifications for signalling proteins. Galpha(s), the alpha-subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring Galpha(s) to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly(2)). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by Galpha(i) are downregulated to <10%. Thereby, Gly(2)-palmitoylation of Galpha(s) relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Galpha(i) more difficult.     

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.     

G proteins immunodetection and adrenergic transduction pathways in the liver of Anguilla anguilla

G proteins are members of a highly conserved superfamily of GTPases, which includes heterotrimeric (alpha, beta, gamma) proteins acting as critical control points for transmembrane signaling. In ectothermal vertebrates, knowledge about these proteins is scarce, and our work provides the first demonstration that G(s), G(q), and G(i) proteins are all present in the liver of a fish. G(q)alpha subunits of about 42 kDa have been identified in European eel (Anguilla anguilla) liver membranes, supporting previous reports about the existence of hormone transduction pathways coupled to inositol 1,4,5-trisphosphate/Ca(2+) enhancement in fish hepatocytes. Although two G(s)alpha proteins of about 45 and 52 kDa have been reported in mammals, a single isoform of approximately 45 kDa has been recognized in eel liver. G(s)alpha and G(q)alpha proteins are involved in the epinephrine transduction pathway, leading to cAMP and Ca(2+) intracellular increments, respectively. Interestingly, both messengers significantly stimulated glucose release from eel hepatocytes but with a different time course. In fact, the Ca(2+)-dependent glucose output preceded the cAMP-mediated release by about 7 min. G(i)alpha subunits of about 40 kDa were also immunodetected, suggesting the presence of hormone receptors leading to adenylyl cyclase inhibition in eel liver; however, alpha(2)- adrenoreceptor ligands were ineffective on both enzyme activity and glucose release.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

Molecular mechanisms of nitric oxide-induced growth arrest and apoptosis in fetal pulmonary arterial smooth muscle cells

Superoxide plays an important role in pulmonary arterial smooth muscle cell (SMC) proliferation and survival. The rapid reaction between superoxide and nitric oxide (NO) to form peroxynitrite suggests that endothelium-derived NO may influence adjacent SMC growth. To investigate this possibility, we determined the dose-dependent effects of NO on the proliferation and viability of pulmonary arterial SMC isolated from fetal lambs (FPASMC). Using fluorescence microscopy we found a dose-dependent decrease in superoxide levels in FPASMC treated with the NO donor spermine NONOate. This was associated with an increase in peroxynitrite-mediated protein nitration. At doses between 50 and 250 microM, spermine NONOate attenuated serum-induced FPASMC proliferation resulting in a G(0)/G(1) cell cycle arrest. This process involved a decrease in levels of cyclin A and an increase in the nuclear localization of p21 and p27. Furthermore, 500 microM spermine NONOate decreased viable cell number by inducing programmed cell death: FPASMC treated with 500 microM spermine NONOate displayed a loss of mitochondrial membrane potential, followed by caspase activation and DNA fragmentation. These data suggest that NO inhibits superoxide-induced proliferation of FPASMC and at higher doses induces apoptosis. NO donors may therefore prove to be useful therapeutic tools to treat diseases resulting from excessive proliferation of vascular smooth muscle.     

Erythrocytes of humans with cystic fibrosis fail to stimulate nitric oxide synthesis in isolated rabbit lungs

Erythrocytes (red blood cells) of either rabbits or healthy humans are required to demonstrate the participation of nitric oxide (NO) in the regulation of pulmonary vascular resistance in the isolated rabbit lung. The property of the erythrocyte that is responsible for the stimulation of NO synthesis was reported to be the ability to release ATP in response to physiological stimuli, including deformation. Moreover, a signal transduction pathway that relates mechanical deformation of erythrocytes to ATP release has been described, and the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a component, i.e., erythrocytes of individuals with CF do not release ATP in response to deformation. Here, we investigated the hypothesis that, in contrast to those of healthy humans, erythrocytes of humans with CF fail to stimulate endogenous NO synthesis in the isolated rabbit lung. We report that CFTR is a component of the membranes of both rabbit and human erythrocytes. The addition of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 muM) produced increases in vascular resistance in isolated rabbit lungs perfused with physiological salt solution (PSS) containing erythrocytes of healthy humans, but L-NAME was without effect when the lungs were perfused with PSS alone or PSS containing erythrocytes of CF patients. These results provide support for the hypothesis that, in CF, a defect in ATP release from erythrocytes could lead to decreased endogenous pulmonary NO synthesis and contribute to pulmonary hypertension.     

Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.     

A selective phosphodiesterase 3 inhibitor rescues low PO2-induced ATP release from erythrocytes of humans with type 2 diabetes: implication for vascular control

Erythrocytes, via release of ATP in areas of low oxygen (O(2)) tension, are components of a regulatory system for the distribution of perfusion in skeletal muscle ensuring optimal O(2) delivery to meet tissue needs. In type 2 diabetes (DM2), there are defects in O(2) supply to muscle as well as a failure of erythrocytes to release ATP. The goal of this study was to ascertain if a phosphodiesterase 3 (PDE3) inhibitor, cilostazol, would rescue low O(2)-induced ATP release from DM2 erythrocytes and, thereby, enable these cells to dilate isolated erythrocyte-perfused skeletal muscle arterioles exposed to decreased extraluminal O(2). Erythrocytes were obtained from healthy humans (HH; n = 12) and humans with DM2 (n = 17). We determined that 1) PDE3B is similarly expressed in both groups, 2) mastoparan 7 (G(i) activation) stimulates increases in cAMP in HH but not in DM2 erythrocytes, and 3) pretreatment of DM2 erythrocytes with cilostazol resulted in mastoparan 7-induced increases in cAMP not different from those in HH cells. Most importantly, cilostazol restored the ability of DM2 erythrocytes to release ATP in response to low O(2). In contrast with perfusion with HH erythrocytes, isolated hamster retractor muscle arterioles perfused with DM2 erythrocytes constricted in response to low extraluminal PO(2). However, in the presence of cilostazol (100 μM), DM2 erythrocytes induced vessel dilation not different from that seen with HH erythrocytes. Thus rescue of low O(2)-induced ATP release from DM2 erythrocytes by cilostazol restored the ability of erythrocytes to participate in the regulation of perfusion distribution in skeletal muscle.     

Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism

Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.     

RACK1 regulates specific functions of Gbetagamma

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.     

Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system

While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).     

Tumor necrosis factor alpha stimulates adenylyl cyclase activity in human myometrial cells

Cytokines such as tumor necrosis factor alpha (TNFalpha) have been implicated in amniotic fluid infections and preterm and term labor. The underlying mechanisms are incompletely understood. In some smooth muscle cells, TNFalpha affects function of the beta-adrenergic/adenylyl cyclase pathway. The present study was performed to examine the effects of chronic TNFalpha exposure on adenylyl cyclase activity in cell cultures of human myometrium. Chronic TNFalpha exposure led to a dose- and time-dependent increase in basal-, GTP-, NaF-, and forskolin-stimulated adenylyl cyclase (AC) activity. The increase in AC activity was not mediated by changes in the expression of the heterotrimeric G proteins G(s)alpha or G(i)alpha as determined by immunoblotting. In addition, increases in AC activity occurred in the presence of indomethacin, indicating that these changes were not provoked by TNFalpha-induced changes in prostaglandin production. The present results suggest that TNFalpha-induced increases in AC activity in human myometrial cells obtained from the lower uterine segment occur at the level of G-protein/AC interaction or at the level of the AC enzyme itself.     

Polylysine activates membrane-bound adenylyl cyclase from Xenopus laevis oocytes through the Gs transducing protein.

1. The activity of the adenylyl cyclase found in the membranes of Xenopus laevis can be affected by polylysine and other polycations. 2. The activity of the catalytic subunit measured with forskolin is inhibited by polylysine and polyarginine at concentrations above 10 microM and by spermine above 3 mM. 3. The adenylyl cyclase activity stimulated by GTP-gamma-S or F- through the stimulatory G protein (Gs) can be increased by polylysine, polyornithine and spermine but not by polyarginine. 4. Polylysine stimulation of Gs dependent activity is due to the increase in the apparent affinity for GTP-gamma-S and to a lowering of the requirement for Mg2+ concentration.