Isoprenaline-stimulated Adenylyl Cyclase Research Articles

Functional β2-adrenoceptors in rat left atria: effect of foot-shock stress.

Altered sensitivity to the chronotropic effect of catecholamines and a reduction in the β1/β2-adrenoceptor ratio have previously been reported in right atria of stressed rats, human failing heart, and aging. In this report, we investigated whether left atrial inotropism was affected by foot-shock stress. Male rats were submitted to 3 foot-shock sessions and the left atrial inotropic response, adenylyl cyclase activity, and β-adrenoceptor expression were investigated. Left atria of stressed rats were supersensitive to isoprenaline when compared with control rats and this effect was abolished by ICI118,551, a selective β2-receptor antagonist. Schild plot slopes for the antagonism between CGP20712A (a selective β1-receptor antagonist) and isoprenaline differed from unity in atria of stressed but not control rats. Atrial sensitivity to norepinephrine, as well as basal and forskolin- or isoprenaline-stimulated adenylyl cyclase activities were not altered by stress. The effect of isoprenaline on adenylyl cyclase stimulation was partially blocked by ICI118,551 in atrial membranes of stressed rats. These findings indicate that foot-shock stress equally affects inotropism and chronotropism and that β2-adrenoceptor upregulation contributes to the enhanced inotropic response to isoprenaline.

Inhibition of Na+/H+‐exchanger with sabiporide attenuates the downregulation and uncoupling of the myocardial β‐adrenoceptor system in failing rabbit hearts

Chronic heart failure (HF) is characterized by left ventricular (LV) structural remodeling, impaired function, increased circulating noradrenaline (NA) levels and impaired responsiveness of the myocardial beta-adrenoceptor (betaAR)-adenylyl cyclase (AC) system. In failing hearts, inhibition of the sodium/proton-exchanger (NHE)-1 attenuates LV remodeling and improves LV function. The mechanism(s) involved in these cardioprotective effects remain(s) unclear, but might involve effects on the impaired betaAR-AC system. Therefore, we investigated whether NHE-1 inhibition with sabiporide (SABI; 30 mg kg(-1) day(-1) p.o.) might affect myocardial betaAR density and AC activity in relation to changes in LV end-diastolic diameter (LVEDD) and LV systolic fractional shortening (LVS-FS) after 3 weeks of rapid LV pacing in rabbits. After 3 weeks of rapid LV pacing LVEDD was significantly increased (Shams 17+/-0.2 mm, n=9 vs 3 wksHF 20+/-0.5 mm, n=8; P<0.05) and LVS-FS decreased (Shams 31+/-1%, n=9 vs 3 wksHF 10+/-1%, n=8; P<0.05). SABI treatment significantly improved LV function independent of whether rabbits were treated after 1 week of pacing (3 wksHF+2 wksSABI (n=7): LVEDD 18+/-1 mm; LVS-FS 16+/-4%) or before pacing (3 wksHF+3wksSABI (n=9): LVEDD 18+/-1 mm; LVS-FS 18+/-6%). After 3 weeks of rapid LV pacing, SABI treatment significantly attenuated increases in serum NA content (Shams 0.83+/-0.19, 3 wksHF 2.68+/-0.38, 3 wksHF+2 wksSABI 1.22+/-0.32, 3 wksHF+3wksSABI 1.38+/-0.33 ng ml(-1)). Moreover, betaAR density (Shams 64+/-5, 3 wksHF 38+/-3, 3 wksHF+2 wksSABI 48+/-4, 3 wksHF+3 wksSABI 55+/-3 fmol mg(-1) protein) and responsiveness (isoprenaline-stimulated AC activity. (Shams 57.6+/-4.9, 3 wksHF 36.3+/-6.0, 3 wksHF+2 wksSABI 56.9+/-6.0, 3 wksHF+3 wksSABI 54.5+/-4.8 pmol cyclic AMP mg(-1) protein(-1) min(-1)) were significantly improved in SABI-treated rabbits. From the present data we cannot address whether the improved betaAR-AC system permitted improved LV function and/or whether the improved LV function resulted in less activation of the sympathetic nervous system and by this in a reduced stimulation of the betaAR-AC system. Accordingly, additional studies are needed to fully establish the cause-and-effect relationship between NHE-1 inhibition and the restoration of the myocardial betaAR system.

Overexpression of wild-type Galpha(i)-2 suppresses beta-adrenergic signaling in cardiac myocytes.

The role of Galpha(i)-2 overexpression in desensitization of beta-adrenergic signaling in heart failure is controversial. An adenovirus-based approach was used to investigate whether overexpression of Galpha(i)-2 impairs beta-adrenergic stimulation of adenylyl cyclase (AC) activity and cAMP levels in neonatal rat cardiac myocytes (NRCM) and cell shortening of adult rat ventricular myocytes (ARVM). Infection of NRCM with Ad5Galpha(i)-2 increased Galpha(i)-2 by 50-600% in a virus dose-dependent manner. Overexpression was paralleled by suppression of GTP- and isoprenaline-stimulated AC by 10-72% (P<0.001) in a PTX-sensitive manner. Isoprenaline-stimulated shortening of Ad5Galpha(i)-2-infected ARVM was attenuated by 34% (P<0.01). Ad5Galpha(i)-2/GFP (Galpha(i)-2, green fluorescent protein; bicistronic) was constructed to monitor transfection homogeneity and target Galpha(i)-2 overexpression to levels found in heart failure. At Galpha(i)-2 levels of 93% above control, isoprenaline-stimulated AC activity and cAMP levels were reduced by 17% and 40% (P<0.02), respectively. Beta1- and beta2-adrenergic stimulation was reduced similarly. Our results suggest that (a) the Galpha(i)-2 system exhibits tonic inhibition of stimulated AC in cardiac myocytes, (b) Galpha(i)-2-mediated inhibition is concentration-dependent and occurs at Galpha(i)-2 levels seen in heart failure, and (c) Galpha(i)-2-mediated inhibition affects both beta1- and beta2-adrenergic stimulation of AC. The data argue for an important, independent role of the Galpha(i)-2 increase in heart failure.

Evidence for a short form of RGS3 preferentially expressed in the human heart.

RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase-activating proteins (GAP) for G protein alpha-subunits. The existence of mRNAs of different size for some of the RGS proteins, e.g. RGS3, suggests that these proteins may exist in isoforms due to alternative splicing. We therefore investigated RGS3 mRNA and protein expression in different human tissues. Ribonuclease protection assays and Northern blot analysis showed two specific mRNAs for RGS3 (RGS3L, RGS3S) in human myocardium, suggesting an additional, N-terminally truncated form of approximately 168 aa. When expressed as a recombinant protein RGS3S was recognized at approximately 23 kDa by an antipeptide antiserum originally raised against an RGS2 sequence. In membranes of human tissues this antiserum detected specific signals for RGS3L (approximately 70 kDa), RGS2 (approximately 30 kDa) and a 25-kDa protein, most likely RGS3S. Both RGS3S mRNA and the 25 kDa protein were abundant in human heart, whereas expression in liver, brain and myometrium was much weaker. To characterize RGS3S functionally, single turnover GTPase, adenylyl cyclase (AC) and phospholipase C (PLC) activities were determined. Both recombinant RGS3S and RGS16 increased Pi release from Galphai1 by about 150% and increased GTP- and GTP plus isoprenaline-stimulated AC activity by 20-30% in human left ventricular myocardial membranes. Additionally, both RGS proteins reduced basal and endothelin-stimulated PLC activity in these membranes by about 40%. We conclude that an additional truncated form of RGS3 is expressed in the human heart. As described for the full-length protein, RGS3S negatively regulates the activity of Gi/o- and Gq-, but not Gs-subfamily members.

Functional Coupling of Overexpressed β1-Adrenoceptors in the Myocardium of Transgenic Mice

To assess functional and cellular effects of myocardial β1-adrenoceptor overexpression, alterations of the β-adrenergic signal transduction pathway and contractile function in transgenic mice with atrial overexpression of the human β1-adrenoceptor were investigated. Radioligand binding experiments confirmed a 5- to 6-fold increase in β-adrenoceptor density and a 2.7-fold increase in high-affinity binding sites in atria of transgenic mice. Dose–response curves for isoprenaline-induced force of contraction showed unchanged maximum effects but significantly increased pD2values. Basal, MnCl2- and isoprenaline-stimulated adenylyl cyclase activities did not significantly differ, whereas the Gpp(NH)p and forskolin effect tends to be reduced in transgenic mice. The level of Giα (pertussis toxin-catalyzed ADP-ribosylation) was unchanged, whereas the bioactivity of Gsα (reconstitution experiments into S49 cyc−cell membranes) was reduced by about 19% in the transgenic group. These results suggest that overexpressed β1-adrenoceptors act as functional spare receptors. In addition, increased β1-adrenoceptor density is associated with a decrease in Gsα-activity.

Decreased β-adrenoceptor-stimulated adenylyl cyclase activity in lymphocytes from Alzheimer's disease patients

Previous studies have shown that in Alzheimer's disease post-mortem brain there are disruptions of both β1-adrenoceptor-G-protein coupling and G-protein stimulation of adenylyl cyclase activity. Decreased β-adrenoceptor stimulated adenylyl cyclase activity has also been shown in Alzheimer's disease primary skin fibroblasts. In the present study, we determined the regulation of adenylyl cyclase in Alzheimer's disease patients using an easily accessible tissue source, namely peripheral blood lymphocytes. β-Adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were investigated in lymphocytes from 12 Alzheimer's disease and 12 carefully matched and selected control subjects. No significant differences were found in basal or forskolin-stimulated enzyme activities between Alzheimer's disease and control lymphocytes. In contrast, isoprenaline-stimulated adenylyl cyclase activities were significantly lower in the Alzheimer's disease groups, as compared to controls. These results indicate that there is a widespread disruption of β-adrenoceptor-G-protein-enzyme coupling in different tissues from Alzheimer's disease patients, and that adenylyl cyclase disturbances previously reported in Alzheimer's disease brain do not occur as a consequence of disease pathology or of terminal illness.

Evidence against a regulation of Na+/K(+)-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca(2+)-exchange in cardiac sarcolemmal membranes.

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (Gi 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 Gi proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na+/Ca(2+) 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+/Ca(2+) exchange and Na+/K(+)-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A1 (Maximum number of receptors, Bmax 0.033 pmol/mg) and muscarinic M2 (Bmax 2.9 pmol/mg) receptors and contained Gi2 and Gi3, two Gi protein isoforms, and G0, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A1-selective agonist, (-)-N6-(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+/Ca(2+) 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, (-)-N6-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na+/Ca(2+) exchange and Na+/K(+)-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.

Receptor crosstalk: effects of prolonged carbachol exposure on ?1-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, beta 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 beta-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 beta 1-adrenoceptors. The peak effect occurred after 20 h of exposure to 100 microM carbachol which caused a decrease in the maximum number of binding sites for the beta-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 beta 1-Adrenoceptor density. beta 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.

Guanine-nucleotide binding regulatory proteins as targets for novel drugs

Summary:Guanine-nucleotide regulatory binding proteins (G-proteins) serve to transduce information from agonist-bound receptor complexes to either effector enzymes or ion-channels. Drugs that perturb the function of G-proteins may do so by one of four mechanisms, (i) They may exert negative intrinsic activity toward the G-protein. For instance, we have shown that incubation of isolated plasma-membranes with the β-adrenoceptor blocking drug sotalol blocked both GTP-stimulated and isoprenaline-stimulated adenylyl cyclase. This suggests that the empty β-adrenoceptor is capable of tonically stimulating Gsα, and therefore adenylyl cyclase; that is, empty β-adrenoceptors promote GDP-GTP exchange, (ii) They may perturb the GDP-GTP exchange reaction. For instance, certain PDE inhibitors, including SKF 94836 and rolipram, stimulate a marked increase in the pertussis toxin-catalysed NAD+-dependent ADP-ribosylation of Giα. This effect is similar to that of GDP, which promotes stabilisation of the αβγ holomer of Gi. The effect of these PDE inhibitors is completely reversed by GppNHp, which triggers afly dissociation by binding to the guanine-nucleotide binding domain of the G-protein. PDE inhibitors may serve as a class of drugs which perturb GDP-GTP exchange, (iii) They may trigger uncoupling of receptor-G-protein complexes. For instance, the polycationic drug mastoparan binds to the C-terminal end of the G-protein and mimics the effect of receptor activation by promoting GTP-γ-S binding, a reduction in pertussis toxin-catalysed ADP-ribosylation, and inhibition of adenylyl cyclase activity. Other agents, such as polyanionic drugs, bind to the receptor to promote uncoupling of receptor-mediated activation of certain G-proteins. (iv) They may alter the cross-talk mechanisms that operate between different receptor signalling systems. For instance, protein kinase C promotes the phosphorylation and inactivation of Gi. This leads to an unopposed stimulation of adenylyl cyclase via Gsand, therefore, enhanced sensitivity to agents such as glucagon. Protein kinase C inhibitors may be usefully exploited to modulate these processes which appear to be abberant in certain disease states.

Effect of paroxetine, a selective 5-hydroxytryptamine uptake inhibitor, on β-adrenoceptors in rat brain: Autoradiographic and functional studies

Quantitative receptor autoradiography was used to investigate the effects of paroxetine (8.3 mg kg ), amitriptyline (26 mg kg ) and desipramine (17 mg kg ), administered daily in the drinking water for 21 days, on the number of β 1- and β 2-adrenoceptors in the cortex of the rat. In addition, the effect of these drugs on the function of β-adrenoceptors was examined by measuring noradrenaline- and isoprenaline-stimulated production of cyclic AMP in slices of cortex. Paroxetine did not alter the number of cortical β 1, or β 2-adrenoceptors nor did it induce any functional changes in β-adrenoceptor-linked adenylyl cyclase. In contrast, desipramine caused a significant reduction in the density of β 1-adrenoceptors and in the sensitivity of both noradrenaline and isoprenaline-stimulated adenylyl cyclase. Although amitriptyline significantly reduced the number of β 1-adrenoceptors in cortical membranes, no such changes could be detected by autoradiography. It is apparent from these and other studies, that the ability of antidepressants to down-regulate central β-adrenoceptors is not a property shared by all antidepressants. In particular, the more potent and selective inhibitors of the uptake of 5-HT, such as paroxetine, appear to be devoid of effects on this receptor system.