Chronic muscarinic cholinoceptor stimulation increases adenylyl cyclase responsiveness in rat cardiomyocytes by a decrease in the level of inhibitory G-protein α-subunits

Exposure of neonatal rat cardiomyocytes for 3 days to the muscarinic cholinoceptor agonist carbachol led to a concentration-dependent increase in adenylyl cyclase stimulation by the \-adrenoceptor agonist isoproterenol by up to 115% (at 1 mmol/l carbachol). In addition, direct adenylyl cyclase stimulation by forskolin was increased in carbachol (1 mmol/l)-treated cells by 32010. Pretreatment of the rat cardiomyocytes with pertussis toxin, which enhances adenylyl cyclase activity by a functional inactivation of the inhibitory G-protein (G_i), was performed to investigate the possible role of G_i proteins in carbachol-induced sensitization of adenylyl cyclase stimulation. After pretreatment of the cells with pertussis toxin, the carbachol-mediated increase in forskolin-stimulated adenylyl cyclase activity was lost and the carbachol-mediated increase in \-adrenoceptor-stimulated adenylyl cyclase activity was attenuated. Labelling of the 40 kDa pertussis toxin substrates in cardiomyocyte membranes was decreased by carbachol in a concentration-dependent manner by up to 34010 (at 1 mmol/l carbachol). The number and affinity of \₁-adrenoceptors was unaltered following the chronic carbachol treatment.The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins and increase in adenylyl cyclase activity depend on de-novo protein synthesis. Pseudomonas exotoxin A inhibits peptide chain elongation by ADP-ribosylating elongation factor 2. Treatment of the cells with 1 ng/ml Pseudomonas exotoxin A for 3 days led to a reduction in the subsequent ADP-ribosylation of elongation factor 2 in the cytosol of the heart muscle cells by 57%. Exposure of the cells to 1 mmol/l carbachol for 3 days increased ADP-ribosylation of elongation factor 2 by 40% concomitant with a slight (about 20%) increase in the total protein content of the cardiomyocytes. The partial protein synthesis inhibition by Pseudomonas exotoxin A had no influence on the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins. Similarly, the carbachol-induced increase in adenylyl cyclase responsiveness also remained unaltered by Pseudomonas exotoxin A.The data presented indicate that chronic muscarinic cholinoceptor agonist treatment decreases the level of -subunits of G_i- proteins. This decrease in G_i- subunits is apparently at least in part responsible for the observed increase in adenylyl cyclase responsiveness after chronic carbachol treatment.     

Tumor necrosis factor α up-regulates Giα and Gß proteins and adenylyl cyclase responsiveness in rat cardiomyocytes

Treatment of cultured rat cardiomyocytes in serum-free medium for 48 h with recombinant human tumor necrosis factor α (TNFα) led to a concentration-dependent increase in the level of membrane-inhibitory guanine nucleotide-binding protein (G₁) α-subunits and in pertussis toxin-catalyzed [³²P]ADP ribosylation of 40 kDa proteins. Both G_iα protein subtypes present in rat cardiac myocyte membranes, G_iα40 and G_iα41, were up-regulated by the cytokine, with the maximal increase occurring at 10 U/ml TNFα. In contrast to noradrenaline exposure, which causes a similar, but apparently exclusive, increase in α₁-subunits, treatment with TNFα in addition increased the level of membrane G protein ß₃₆-subunits. Furthermore, while noradrenaline exposure led to a decrease in receptor-dependent and -independent adenylyl cyclase activity, treatment of cardiomyocytes with TNFα caused a concentration-dependent increase in cyclase responsiveness to either forskolin, guanosine 5′-O-(3-thiotriphosphate) or isoproterenol, even though ß-adrenoceptor density was decreased by TNFα. The increase in adenylyl cyclase activity induced by TNFα was completely suppressed when the cells were cocultured with noradrenaline, a condition leading to an additive increase in G_iα level. The data indicate that the cytokine TNFα can potently modulate G protein-mediated signal transduction in rat cardiac myocytes. Although TNFα, like noradrenaline, exposure of the cells increased the level of membrane G_iα proteins, it did not decrease but rather caused an increase in adenylyl cyclase responsiveness. This hypertensitivity may be due to concomitant alterations of other components, e.g. G_ß, G_sα and/or the cyclase itself, of this multi-subunit signal transduction system following TNFα exposure.     

Pseudomonas exotoxin A prevents beta-adrenoceptor-induced upregulation of Gi protein alpha-subunits and adenylyl cyclase desensitization in rat heart muscle cells

Exposure of rat heart muscle cells to noradrenaline (1 microM) for 48 hr led to a decrease in the number of beta 1-adrenoceptors of 50% and a concomitant decrease in adenylyl cyclase stimulation by isoprenaline and forskolin of about 60 and 30%, respectively. In addition, the levels of two inhibitory guanine nucleotide-binding protein (Gi protein) alpha-subunits (Gi alpha 40 and Gi alpha 41) were increased in membranes of noradrenaline-treated cells. Evidence is presented that noradrenaline induces this increase by activation of beta-adrenoceptors. First, the noradrenaline action was mimicked by the beta-adrenoceptor agonist isoprenaline. Second, beta-adrenoceptor blockade by timolol but not alpha-adrenoceptor blockade by prazosin prevented the noradrenaline-induced up-regulation of Gi alpha proteins. Furthermore, timolol but not prazosin abolished the noradrenaline-induced down-regulation of beta 1-adrenoceptors and the decreases in receptor-dependent (isoprenaline) and -independent (forskolin) adenylyl cyclase stimulation. The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the noradrenaline-induced up-regulation of Gi alpha subunits depends on increased synthesis of these proteins. This toxin inhibits peptide chain elongation by ADP-ribosylating elongation factor 2. Treatment of rat heart muscle cells with Pseudomonas exotoxin A (1 ng/ml) completely prevented the noradrenaline-induced increase in Gi alpha proteins, measured by both pertussis toxin-catalyzed ADP-ribosylation and immunoblotting with anti-Gi alpha antibodies. Most importantly, Pseudomonas exotoxin A also completely prevented the noradrenaline-induced decrease in forskolin-stimulated adenylyl cyclase activity. Furthermore, the noradrenaline-induced decrease in isoprenaline-stimulated adenylyl cyclase activity was significantly attenuated by the toxin, although the down-regulation of beta 1-adrenoceptors caused by noradrenaline treatment was not affected. The data presented suggest that prolonged activation of beta-adrenoceptors in rat heart muscle cells, in addition to causing a receptor down-regulation, induces the synthesis of Gi alpha proteins, which then apparently mediate a decreased adenylyl cyclase responsiveness. The data, additionally, suggest that the synthesis of Gi alpha proteins is under control of the activity of the adenylyl cyclase system and that altered levels of these proteins may play a major role in long term regulation of signal transduction by this enzyme.     

Carbachol-induced decrease in thyroid cell adenylyl cyclase activity is independent of calcium and phosphodiesterase activation.

The mechanism of adenylyl cyclase desensitization by carbachol, an agent that stimulates polyphosphoinositide hydrolysis, was studied in thyroid cells. Incubation of cultured dog thyroid cells with 10 microM carbachol for 2-4 hr reduced the subsequent thyrotropic hormone (TSH) stimulation of adenylyl cyclase activity of membrane preparations by approximately 40%. This inhibition was reversed by atropine, occurred even in a Ca(2+)-free medium containing ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, and was not reproduced by the Ca2+ ionophore A23187. The carbachol effect was not prevented by simultaneous incubation of cells with either isobutylmethylxanthine, an inhibitor of phosphodiesterase, or H-7, an inhibitor of protein kinase. Pretreatment of cells with pertussis toxin to inactivate the Gi inhibitory protein also failed to affect the carbachol inhibition. Although carbachol did not reduce the basal or the TSH-stimulated cyclase activities when added to membranes directly during the assay, exposure of cells to carbachol for 2-4 hr resulted in long lasting inhibition of TSH-stimulated cyclase activity (for at least 24 hr); recovery was seen by 48 hr after its removal. Carbachol pretreatment had no effect on 125I-TSH binding to membranes but reduced the cyclase stimulation by not only TSH but also cholera toxin, guanosine 5'-O-(3-thio)triphosphate, and forskolin; it also significantly reduced the cholera toxin-mediated AD[32P]-ribosylation of Gs in membranes. These data indicate that carbachol-induced inhibition of adenylyl cyclase occurs beyond the level of TSH receptor binding and that Gs is a possible site of its action. Thus, in dog thyroid cells, carbachol, via muscarinic receptors, can reduce the adenylyl cyclase activity by a process that does not involve Ca2+ or activation of phosphodiesterase.     

Receptor crosstalk: effects of prolonged carbachol exposure on β1-adrenoceptors and adenylyl cyclase activity in neonatal rat ventricular myocytes

Supersensitivity of adenylyl cyclase after exposure to inhibitory agonists is a general means of cellular adaptation. We hypothesized that such crosstalk between muscarinic cholinergic agonists, ₁-adrenoceptors, and adenylyl cyclase may be an important mechanism of cardiac adaptation to interventions that enhance vagal activity. We used primary cultures of neonatal rat ventricular myocytes and measured -adrenoceptors by radioligand binding and adenylyl cyclase activity by a single column method. Carbachol induced a time- and dose-dependent reversible decrease in cell surface ₁-adrenoceptors. The peak effect occurred afte 20 h of exposure to 100 M carbachol which caused a decrease in the maximum number of binding sites for the -adrenoceptor antagonist ³H-CGP-12177 from 42.3±3.4 to 33.0±2.6 fmol/mg protein (n = 12, P < 0.03) without a change in antagonist affinity. Loss of cell surface receptors was prevented by atropine and by the protein kinase C inhibitor H7. The decrease in cell surface receptors was not accompanied by receptor internalization as assessed by equilibrium binding experiments in a cytosolic fraction using ¹²⁵I-iodocyanopindolol. In contrast to the well-known acute inhibitory effects of carbachol on adenylyl cyclase activation, prolonged carbachol exposure preserved (–)-isoprenaline-stimulated adenylyl cyclase activity and enhanced postreceptor stimulated adenylyl cyclase activity. Carbachol did not further enhance adenylyl cyclase activity after pretreatment with pertussis toxin. The protein kinase C inhibitor chelerythrine prevented the carbachol induced enhancement of forskolin-stimulated adenylyl cyclase activity. We conclude that prolonged incubation with carbachol in rat neonatal ventricular myocytes causes a reduction in cell surface ₁-adrenoceptor density. ₁-Adrenoceptor-mediated adenylyl cyclase activity is preserved and postreceptor-mediated adenylyl cyclase activity is augmented. Our data suggest that carbachol-stimulated protein kinase C activity may play a key role in the prolonged muscarinic regulation of adenylyl cyclase activity.This work was supported by a grant from the Veterans Affairs Research Service and by Program Project Grant HL25847 from the National Heart, Lung, and Blood Institute and by the CVRI Training Program in Heart and Vascular Diseases supported by the National Institutes of Health, HL 07192 (A.P.)     

Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein α-subunits

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 μM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of ß-adrenoceptor-independent adenylate cyclase by guanosine-5′-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. G_i, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the M_r ≈ 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified ßγ-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of ßγ-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-G_iα-antibodies demonstrated a marked increase in the amount of G_iα in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-ß-antibodies showed that the level of the ß-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of α-subunits of G_i-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.     

Forskolin blocks carbachol-mediated ion-permeability of chick myotube nicotinic receptors and inhibits binding of 3H-phencyclidine to Torpedo microsac nicotinic receptors

Summary Forskolin, a commonly used adenylate cyclase activator, was found to inhibit reversibly the carbachol-induced ion-translocating capacity of the nicotinic acetylcholine receptor (nAChR) on chick myotubes in a dose- (IC₅₀ = 20 M) and time-dependent manner. This effect was not correlated to increases in cellular cAMP. Forskolin, at a concentration (50 M) that totally blocked the carbachol-induced ⁸⁶Rb influx, caused no change in carbachol or -bungarotoxin binding to chick myotube nAChR in situ. In contrast, in the presence of carbachol, forskolin inhibited (IC₅₀ = 10 M) the binding of ³H-phencyclidine, a putative nAChR ion-channel ligand, to Torpedo microsac nAChR. Inhibition of ³H-phencyclidine binding in the absence of carbachol was not complete. Membrane leakage studies on myotubes, measuring ³H-efflux from 2-deoxy-d(1-³H)-glucose loaded cells and electrophysiological measurements of membrane properties supported the interpretation that forskolin induced decreases in plasma membrane permeability. In conclusion, forskolin blocks the carbachol-mediated increase in permeability of the nAChR channel by (1) binding to the ion-channel (open state) and (2) generally perturbing the plasma membrane function possibly by interfering with the protein-lipid interface. Send offprint requests to Johan Haggblad     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction

In gallbladder smooth muscle, carbachol interacts with M₃ receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [³H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates and contraction were measured at various times (0–90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 g/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [³²P]ADP-ribosylation of a –40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates. The time course of [³H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [³H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [³H]inositol trisphosphates and on contraction were abolished by atropine. Pretreatment with pertussis toxin resulted in ADP-ribosylation of a 40/41 kDa protein from gallbladder cell membranes but did not affect the concentration-response or time course of carbachol-induced contraction. These results indicate that carbachol-induced contraction of gallbladder smooth muscle cells is accompanied by the activation of inositol phosphate turnover and does not involve a pertussis toxin-sensitive G-protein.This article is based in part on the doctoral thesis of Burkhard Mackensen at the Faculty of Medicine, University of Hamburg, Germany. Some of the results were presented at the meeting of the American Gastroenterological Association (AGA) in San Francisco 1992 (von Schrenck et al. 1992) Correspondence to: T. von Schrenck at the above address     

Distinct pathways for β-adrenoceptor-induced up-regulation of muscarinic acetylcholine receptors and inhibitory G-protein α-subunits in chicken cardiomyocytes

Summary Exposure of cardiomyocytes from chicken embryos for 3 days to the -adrenoceptor agonist, isoproterenol, lead to a down-regulation of -adrenoceptors by about 70% and to a decrease in isoproterenol-stimulated adenylyl cyclase activity by about 40% (homologous desensitization). In addition, the isoproterenol treatment induced an increase in the level of muscarinic acetylcholine receptors by about 30% and an increase in pertussis toxin-catalyzed ADP-ribosylation of two about 40 kDa proteins, most probably -subunits of the inhibitory G-protein (G_i), by about a factor of two (heterologous desensitization). The purpose of the present study was to characterize the role of -adrenoceptor-dependent and -independent mechanisms in heterologous desensitization of adenylyl cyclase. Therefore, the effect of pretreatment with the -adrenoceptor antagonist, propranolol, with the partial agonists, celiprolol and xamoterol, and with the -adrenoceptor-independent adenylyl cyclase activators, prostaglandin E₁ and forskolin, on -adrenoceptors, muscarinic acetylcholine receptors and pertussis-toxin-catalyzed ADP-ribosylation of G-protein -subunits was studied.Pretreatment of the cardiomyocytes for 3 days with xamoterol or celiprolol, but not with propranolol, induced a small decrease in -adrenoceptor number and in isoproterenol-stimulated adenylyl cyclase activity by about 15–20%. Exposure to prostaglandin E₁ and forskolin lead to a more pronounced decrease in -adrenoceptor binding and in isoproterenol-mediated adenylyl cyclase stimulation by about 40–60% (heterologous desensitization). An increase in the level of muscarinic acetylcholine receptors, similar to that induced by isoproterenol exposure, was only observed after pretreatment with the partial agonists, celiprolol and xamoterol, but not after pretreatment with the -adrenoceptor-independent agonists, prostaglandin E₁ and forskolin, nor after pretreatment with propranolol. In contrast, prostaglandin E₁ and forskolin exposure lead to a similar increase in pertussis toxin-catalyzed ADP-ribosylation of about 40 kDa G-proteins as isoproterenol exposure whereas treatment with propranolol, celiprolol and xamoterol had no or only a very small effect on pertussis toxin substrates. In summary, the data suggest that, similar as shown for homologous desensitization, cyclic AMP-dependent and -independent mechanisms are also involved in heterologous desensitization of adenylyl cyclase stimulation. The -adrenoceptor-induced upregulation of muscarinic acetylcholine receptors and of the -subunits of pertussis toxin-sensitive G-proteins, most probably of G_i, seem to be mediated via distinct pathways. Send offprint requests to C. Reithmann at the above address     

Evidence for cocaine and methylecgonidine stimulation of M(2) muscarinic receptors in cultured human embryonic lung cells

1. Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M(2) muscarinic receptors. 2. A concentration-dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 microM carbachol (P < 0.01), 3.1 fold for 1 microM cocaine (P < 0.01), and 7.8 fold for 1 microM MEG (P < 0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M(2) blocker methoctramine. 3. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 microM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 microM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 microM significantly attenuated or abolished the cocaine-induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine-induced reduction of cyclic AMP production. 4. Western blot analysis showed that HEL299 cells specifically express M(2) muscarinic receptors without detectable M(1) and M(3). Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M(2) protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. 5. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug-induced stimulation of M(2) muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.     

A1 adenosine receptors and muscarinic cholinoceptors in myocardial ischemia

The regulation of cardiac A₁ adenosine receptors and M₂ muscarinic cholinoceptors was investigated in ischemic rat hearts. Ischemia was induced in isolated, perfused hearts either by stop (stop-flow) or by reduction (low-flow) of perfusion flow. Receptor densities and affinities were determined by radioligand binding. The mRNA concentrations of the receptors and of control messages were measured by quantitative polymerase chain reactions (PCR). Second messenger coupling of the receptors was evaluated by measuring their inhibition of adenylate cyclase activity.Up to 60 min of stop-flow ischemia and 6 h of lowflow ischemia, cardiac A₁ adenosine receptor density and affinity, and adenosine receptor-mediated inhibition of adenylate cyclase, did not change significantly, compared to non-ischemic hearts. Receptor down-regulation, however, could be induced by perfusion with the A₁ receptor agonist R-phenyl-isopropyl-adenosine (R-PIA) during normal flow. After 6 h of perfusion with R-PIA (0.1 mol/l), A₁ adenosine receptor density was reduced. Agonist-induced receptor down-regulation was not found after perfusion with R-PIA in low-flow ischemia. The density and the affinity of muscarinic cholinoceptors were not affected during stop-flow ischemia up to 1 h either, whereas the density was down-regulated to 75% of controls (P<0.05) after 6 h of low-flow ischemia. This intervention also reduced inhibition of adenylate cyclase via muscarinic cholinoceptors. In non-ischemic hearts, perfusion with carbachol (10 µmol/l) suppressed receptor densities to 72% of control values.No significant changes in the concentration of A₁ adenosine receptor or M₂ cholinoceptor mRNAs occurred during normal flow, stop-flow and low-flow ischemia. Likewise, agonist stimulation with R-PIA or carbachol during normal flow did not change the respective receptor mRNA concentrations significantly. Conclusion: Although a down-regulation of A₁ adenosine receptor density was demonstrated after receptor agonist perfusion with normal flow, adenosine did not affect the density or functional activity of cardiac A₁ adenosine receptors in the ischemic myocardium. In contrast, muscarinic cholinoceptor density and function was down-regulated after prolonged ischemia. The lack of an agonist-induced down-regulation of A₁ adenosine receptors in the presence of decreasing activity of m-cholinoceptors suggests a growing importance of the adenosine system in myocardial ischemia.     

A1 adenosine receptors expressed in CHO-cells couple to adenylyl cyclase and to phospholipase C

A₁ adenosine receptors are in general coupled to inhibition of adenylyl cyclase, but have more recently been reported to be capable of also activating phospholipase C. The present study was done in order to investigate whether these different effects can be elicited by a single A₁ receptor, or whether A₁ receptor subtypes have to be invoked. The cDNA of a rat brain A₁ adenosine receptor was stably expressed in CHO-cells, resulting in clones with varying receptor densities; a clone expressing 1.9 pmol receptors/mg membrane protein was used for further characterization. The ligand binding properties of the expressed receptors were typical for the rat A₁ adenosine receptor. A₁ receptor agonists caused a concentration-dependent inhibition of adenylyl cyclase activity in the membranes, with maximal inhibition by 70%. A₁ receptor stimulation also caused concentration-dependent stimulation of inositol phosphate generation in these cells, with maximal effects of 300%. Both adenylyl cyclase inhibition and enhancement of inositol phosphate generation were essentially abolished after pretreatment of the cells with pertussis toxin. These results indicate that a single A₁ adenosine receptor can couple to two effector pathways, and that both effectors are activated via pertussis toxin sensitive G proteins.Abbreviations CHA N⁶-cyclohexyladenosine - CPA N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxamidoadenosine - R-PIA R-N⁶-phenylisopropyladenosine - IP₁ inositol monophosphates - IP₂ inositol bisphosphates - IP₃ inositol trisphosphates - PCR polymerase chain reaction Correspondence to: M. J. Lohsc at the above address     

The indirect negative inotropic effect of carbachol in beta1-adrenoceptor antagonist-treated human right atria

To find out whether indirect negative inotropic effects of carbachol (i.e. decreases in force of contraction that had been stimulated by cyclic AMP-increasing agents) might differ dependent on the agonist employed to increase contractile force in isolated human right atrium, we studied effects of carbachol on atria prestimulated with noradrenaline, terbutaline, histamine and serotonin. All four agonists increased right atrial adenylyl cyclase activity and contractile force, whereby increases for terbutaline, histamine and serotonin, but not for noradrenaline, were significantly larger in right atria from beta(1)-adrenoceptor antagonist-treated vs. non-beta(1)-adrenoceptor antagonist-treated patients. Carbachol (10(-8)-10(-3) M) concentration-dependently decreased agonist-stimulated contractile force: maximum decrease was not significantly different within the four agonists. pD(2) values for carbachol, however, were higher in atria from non-beta(1)-adrenoceptor antagonist-treated vs. beta(1)-adrenoceptor antagonist-treated patients.We conclude that, in isolated human right atria, carbachol-induced indirect negative inotropic effect is not dependent from the agonist employed to increase (via cyclic AMP accumulation) contractile force. However, in atria from beta(1)-adrenoceptor antagonist-treated patients, carbachol-induced indirect negative inotropic effect is attenuated.     

Pertussis toxin-sensitive G protein but not NO/cGMP pathway mediates the negative inotropic effect of carbachol in adult rat cardiomyocytes

Previous studies have shown that muscarinic inhibition of cardiac contractility is mediated by either activation of nitric oxide (NO)/guanosine 3',5'-cyclic monophosphate (cGMP) pathway or stimulation of inhibitory G protein (G(i)). However, it still remains controversial as to whether NO/cGMP pathway or G(i) protein or both mediate(s) the negative inotropic effect of muscarinic agonists in adult ventricular myocytes. In the present study that involves the use of adult rat ventricular myocytes, the muscarinic agonist, carbachol, inhibited beta-adrenergic (isoproterenol) stimulation of contractility (cell shortening) by 82% and increased cGMP levels by 49% within 6 min. Pretreatment of myocytes with soluble guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ) or NO synthase inhibitor (N(G)-monomethyl-L-arginine, L-NMMA) for 30 min blocked carbachol-induced increases in cGMP levels. However, neither ODQ nor L-NMMA pretreatment had any effect on carbachol inhibition of isoproterenol-induced contractility. In addition, carbachol did not attenuate increases in myocyte contractility induced by forskolin (a direct activator of adenylyl cyclase) or 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a cell-permeable cAMP analog which activates cAMP-dependent protein kinase). Pretreatment of myocytes with G(i) protein inhibitor, pertussis toxin (PTX, 1 microg/ml), for 18-20 h abolished carbachol inhibition of isoproterenol-induced contractility. Furthermore, in ventricular myocytes isolated 3 days after in vivo treatment of rats with PTX (3 microg/100 g, i.p.), there was a complete loss of the negative inotropic effect of carbachol. These data indicate that pertussis toxin-sensitive G protein but not NO/cGMP pathway is required for muscarinic inhibition of beta-adrenoceptor-mediated increases in contractility in adult rat ventricular myocytes.     

Tumor necrosis factor alpha up-regulates Gi alpha and G beta proteins and adenylyl cyclase responsiveness in rat cardiomyocytes.

Treatment of cultured rat cardiomyocytes in serum-free medium for 48 h with recombinant human tumor necrosis factor alpha (TNF alpha) led to a concentration-dependent increase in the level of membrane-inhibitory guanine nucleotide-binding protein (Gi) alpha-subunits and in pertussis toxin-catalyzed [32P]ADP ribosylation of 40 kDa proteins. Both Gi alpha protein subtypes present in rat cardiac myocyte membranes, Gi alpha 40 and Gi alpha 41, were up-regulated by the cytokine, with the maximal increase occurring at 10 U/ml TNF alpha. In contrast to noradrenaline exposure, which causes a similar, but apparently exclusive, increase in alpha i-subunits, treatment with TNF alpha in addition increased the level of membrane G protein beta 36-subunits. Furthermore, while noradrenaline exposure led to a decrease in receptor-dependent and -independent adenylyl cyclase activity, treatment of cardiomyocytes with TNF alpha caused a concentration-dependent increase in cyclase responsiveness to either forskolin, guanosine 5'-O-(3-thiotriphosphate) or isoproterenol, even though beta-adrenoceptor density was decreased by TNF alpha. The increase in adenylyl cyclase activity induced by TNF alpha was completely suppressed when the cells were cocultured with noradrenaline, a condition leading to an additive increase in Gi alpha level. The data indicate that the cytokine TNF alpha can potently modulate G protein-mediated signal transduction in rat cardiac myocytes. Although TNF alpha, like noradrenaline, exposure of the cells increased the level of membrane Gi alpha proteins, it did not decrease but rather caused an increase in adenylyl cyclase responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells

Summary In the human T -cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E₂ receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects.Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as G_i2 and G_i3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment.In conclusion, activation of protein kinase C by phorbol esters reveals a G_i-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A₂ receptor stimulated CAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive G_i-protein. Send offprint requests to I. van der Ploeg at the above address     

Reconstitution of muscarinic receptor-mediated inhibition of adenylyl cyclase

Inhibition of bovine brain calmodulin-sensitive adenylyl cyclase was examined in a system consisting of the reconstituted purified porcine atrial muscarinic acetylcholine receptor, the purified inhibitory guanine nucleotide-binding protein (Gi), and the partially purified stimulatory guanine nucleotide-binding protein.adenylyl cyclase complex. Under conditions where Gi existed mainly as the Gi.GDP complex, adenylyl cyclase was selectively preactivated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Addition of carbachol formed the receptor.carbachol complex, which catalyzed the exchange of GDP bound to Gi for GTP gamma S, initiating Gi-mediated inhibition of adenylyl cyclase. Adenylyl cyclase activated by calcium plus calmodulin was more sensitive to inhibition by carbachol than either unstimulated adenylyl cyclase or adenylyl cyclase activated by GTP gamma S or forskolin. Studies using the resolved subunits of Gi showed that the beta gamma subunit could inhibit adenylyl cyclase activated by GTP gamma S or calcium plus calmodulin, as well as the unactivated enzyme. The alpha subunit of Gi inhibited adenylyl cyclase only when adenylyl cyclase was activated by calcium plus calmodulin. Possible explanations for these results are discussed.     

HIGH-LEVEL EXPRESSION OF RAT D1A DOPAMINE RECEPTOR cDNA IN MOUSE FIBROBLAST LTK- CELLS BY n-BUTYRATE

1. In order to develop a simple, efficient system for the high-level expression of dopamine receptors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D_1A dopamine receptor cDNA in mouse fibroblast LTK^- cells as compared with those of n-butyrate on endogenous D₁ receptor levels in opossum kidney cells. 2. In the transfected LTK^- cell membranes with pRc/CMV-D_1A receptor cDNA, a selective D₁ dopamine antagonist, [³H]-SCH 23390, exhibited a K₄ of 0.9 ± 0.1 nmol/L and a B_max of 0.35 ± 0.05 pmol/mg protein (n= 5). 3. Addition of n-butyrate (2–10 mmol/L) to the culture medium for 48 h dose-dependently increased the D_1A receptor level up to 1.5 ± 0.3 pmol/mg protein (n= 7), although the K₄ values were not affected. The increase in receptor level was accompanied by an elevation of selective D₁ agonist-induced adenylyl cyclase activity. 4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D₁ receptor levels or fendoldopam-induced adenylyl cyclase activity in opossum kidney cells. 5. These results suggest n-butyrate is a useful tool for obtaining high-level expression of D_1A dopamine receptor cDNA in mouse fibroblast LTK^- cells.     

G-Protein-coupled receptors in HL-60 human leukemia cells

• 1.1. HL-60 human leukemia cells are a widely employed model system for the analysis of signal transduction processes mediated via regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins). HL-60 promyelocytes are pluripotent and can be differentiated into neutrophilic or monocytic cells.
• 2.2. HL-60 cells express formyl peptide-, complement C5a-, leukotriene B₄ (LTB₄)- and platelet-activating factor receptors, receptors for purine and pyrimidine nucleotides, histamine H₁- and H₂-receptors, β₂-adrenoceptors and prostaglandin receptors.
• 3.3. The major G-proteins in HL-60 cells are pertussis toxin (PTX)-sensitive G_i-proteins (G_i2 > G_i3). G_s-proteins and G-proteins of the G_q-family (e.g., G₁₆) are expressed, too.
• 4.4. G-protein-regulated effector systems in HL-60 cells are adenylyl cyclase and phospholipase C-β₂ (PLC-β₂) and, possibly, phospholipase D (PLD), nonselective cation (NSC) channels and NADPH oxidase.
• 5.5. The expression of signal transduction pathways in HL-60 cells strongly depends on the differentiation state of cells.
• 6.6. Formyl peptides, via G_i-proteins, mediate activation of PLC, PLD, NSC channels, NADPH oxidase and azurophilic granule release and are referred to as full secretagogues. In dibutyryl cAMP (Bt₂cAMP)-differentiated HL-60 cells, C5a and LTB₄ are partial and incomplete secretagogues, respectively. There are substantial differences in the G_i-protein activations induced by formyl peptides, C5a and LTB₄.
• 7.7. In HL-60 promyelocytes, purine and pyrimidine nucleotides mediate activation of PLC and NSC channels largely via PTX-insensitive G-proteins and induce functional differentiation. In Bt₂cAMP-differentiated HL-60 cells, they additionally activate PLD, NADPH oxidase and granule release via PTX-sensitive and -insensitive pathways. ATP and UTP are partial secretagogues. Multiple types of receptors (i.e., P_2Y- and P_2U-receptors and pyrimidinocyeptors) may mediate the effects of nucleotides in HL-60 cells.
• 8.8. Bt₂cAMP- and 1α,25-dihydroxycholecalciferol-differentiated HL-60 cells express H_l-receptors coupled to G_i-proteins and PTX-insensitive G-proteins. In the former cells, histamine mediates activation of PLC and NSC channels, and in the latter, activation of NSC channels. Histamine is an incomplete secretagogue in these cells.
• 9.9. HL-60 promyelocytes express H₂-receptors coupled to adenylyl cyclase, PLC, and NSC channels. There are substantial differences in the agonist/antagonist profiles of H₂-receptor-mediated cAMP formation and rises in cytosolic Ca²⁺ concentration, indicative of the involvement of different H₂-receptor subtypes. H₂-receptors mediate functional differentiation of HL-60 cells.
• 10.10. Certain cationic-amphiphilic histamine receptor ligands (i.e., 2-substituted histamines, lipophilic guanidines, and a histamine trifluoromethyl-toluidide derivative) show stimulatory effects in HL-60 cells that are attributable to receptor-independent activation of G_i-proteins.