Effects of cyclopentyladenosine on isoproterenol response in adult and senescent cardiac tissue from Fischer 344 rats

To characterize age-related changes in beta-adrenergic responsiveness and to test the hypothesis that an increase in the effects of adenosine contribute to impaired beta-adrenergic responsiveness, Fischer 344 rat right atria (RA), left atria (LA), and left ventricular trabeculae carnae were exposed to the beta-receptor agonist isoproterenol (ISO), followed by four doses of the selective adenosine A(1) receptor agonist cyclopentyladenosine (CPA). Spontaneous contractile rates of adult RA were inhibited more than senescent RA by CPA. Contractility (+dF/dt) of adult LA was reduced more than senescent LA by CPA. Left trabeculae carnae tissue responded weakly to CPA, but senescent tissue was less responsive than adult tissue. Senescent atrial A(1) receptor density was 56% greater than in adult tissue, whereas the density in senescent ventricles was 39% lower than in adult tissue. No significant difference in antagonist affinities (K(d)) of A(1) receptor was observed between adult and senescent atria. In addition, agonist competition curves indicated a significant increase in senescent atrial and a decrease in senescent ventricular tissue in the affinity of agonist for high-affinity A(1) receptors with no difference in dissociation constant (K(i)). No significant age-related differences in atrial or ventricular tissues occurred in either the antagonist affinity (K(d)) or density (B(max)) of the beta-adrenergic receptors. CPA was found to inhibit ISO-stimulated adenylate cyclase activity more in senescent than in adult atrial and ventricular membrane preparations. We conclude that age-related differences in functional response to ISO and CPA, A(1) receptor density, and ISO-stimulated adenylate cyclase activity differ in atrial and ventricular myocardium.     

Beta-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms.

In order to investigate the general cause of beta-adrenergic receptor neuroeffector abnormalities in the failing human heart, we measured ventricular myocardial adrenergic receptors, adrenergic neurotransmitters, and beta-adrenergic receptor-effector responses in nonfailing and failing hearts taken from nonfailing organ donors, subjects with endstage biventricular failure due to idiopathic dilated cardiomyopathy (IDC), and subjects with primary pulmonary hypertension (PPH) who exhibited isolated right ventricular failure. Relative to nonfailing PPH left ventricles, failing PPH right ventricles exhibited (a) markedly decreased beta 1-adrenergic receptor density, (b) marked depletion of tissue norepinephrine and neuropeptide Y, (c) decreased adenylate cyclase stimulation in response to the beta agonists isoproterenol and zinterol, and (d) decreased adenylate cyclase stimulation in response to Gpp(NH)p and forskolin. These abnormalities were directionally similar to, but generally more pronounced than, corresponding findings in failing IDC right ventricles, whereas values for these parameters in nonfailing left ventricles of PPH subjects were similar to values in the nonfailing left ventricles of organ donors. Additionally, relative to paired nonfailing PPH left ventricles and nonfailing right ventricles from organ donors, failing right ventricles from PPH subjects exhibited decreased adenylate cyclase stimulation by MnCl2. These data indicate that: (a) Adrenergic neuroeffector abnormalities present in the failing human heart are due to local mechanisms; systemic processes do not produce beta-adrenergic neuroeffector abnormalities. (b) Pressure-overloaded failing right ventricles of PPH subjects exhibit decreased activity of the catalytic subunit of adenylate cyclase, an abnormality not previously described in the failing human heart.     

Decreased stimulatory guanosine triphosphate binding protein in dogs with pressure-overload left ventricular failure.

Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (Gs) from the cardiac sarcolemma were examined in a canine model of heart failure. The present study is based on our previous investigations that demonstrated both a loss of beta-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labeled the alpha subunit of Gs (Gs alpha) and found a 59% reduction in the level of this protein. Further, a 50% reduction in Gs activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in Gs. These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the beta-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein Gs. Such an abnormality may explain the decreased adrenergic responsiveness of the failing left ventricle.     

Differential regulation of right and left ventricular beta-adrenergic receptors in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia secondary to an intracardiac right-to-left shunt alters regulation of the myocardial beta-adrenergic receptor/adenylate cyclase system, we produced chronic hypoxemia in nine newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 wk. Eight lambs served as normoxemic controls. beta-receptor density (Bmax) and ligand affinity (KD) were determined with the radio-ligand [125I]iodocyanopindolol and adenylate cyclase activity determined during stimulation with isoproterenol, sodium fluoride (NaF), and forskolin. During chronic hypoxemia, Bmax decreased 45% (hypoxemic, 180.6 +/- 31.5 vs. control, 330.5 +/- 60.1 fmol/mg) in the left ventricle (exposed to hypoxemia alone) but was unchanged in the right ventricle (exposed to hypoxemia and pressure overload). KD was not different from control in either ventricle. Left ventricular isoproterenol-stimulated adenylate cyclase activity was decreased by 39% (30.0 +/- 4.3% increase vs. 44.1 +/- 9.5% increase) whereas right ventricular adenylate cyclase activity was unchanged. Stimulation of adenylate cyclase with NaF or forskolin was not different from control in either ventricle. Circulating epinephrine was increased fourfold whereas circulating and myocardial norepinephrine were unchanged. These data demonstrate a down-regulation of the left ventricular beta-adrenergic receptor/adenylate cyclase system during chronic hypoxemia secondary to an intracardiac right-to-left shunt.     

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling associated with chronic reduction in beta-adrenergic receptor agonist activation were found. First, there was coordinate downregulation of Gi alpha 2 and Gs alpha mRNA and protein expression in the left ventricle; reduced G-protein content was also found in the right atrium. Second, in the left ventricle, there was a twofold increase in beta-adrenergic receptor-dependent stimulation of adenylyl cyclase and a persistent high affinity state of the beta-adrenergic receptor. Finally, there was a reduction in left ventricular beta-adrenergic receptor kinase activity, suggesting a previously unrecognized association between the degree of adrenergic activation and myocardial beta-adrenergic receptor kinase expression. The heart appears to adapt in response to chronic beta-adrenergic receptor antagonist administration in a manner that would be expected to offset reduced agonist stimulation. The mechanisms for achieving this extend beyond beta-adrenergic receptor upregulation and include alterations in G-protein expression, beta-adrenergic receptor-Gs interaction, and myocardial beta-adrenergic receptor kinase activity.     

Effect of Digoxin on Ventricular Remodeling and Responsiveness of beta-Adrenoceptors in Chronic Volume Overload

BACKGROUND: Digoxin improves baroreflex function and reduces neurohumoral activation in severe heart failure, but it is uncertain how digoxin affects ventricular remodeling and progression to left ventricular dysfunction. In addition, the effect of digoxin in in vitro beta-adrenoceptor density and function, and contractile reserve in vivo is not well understood. METHODS AND RESULTS: To study this, we compared digoxin with placebo treatment in rats with chronic volume overload induced by aortocaval fistula and in sham-operated control animals. Left ventricular end-diastolic cavity dimensions (LVDd) and wall thickness were measured weekly by in vivo transthoracic echocardiography, and left ventricular mass (LVM) and percent fractional shortening (%FS) were calculated. Six weeks after fistula creation, simultaneous echocardiographic and invasive hemodynamic evaluation at rest and in response to incremental dobutamine (1-10 μg/kg/min intravenously) were measured. Myocardial plasma membrane beta-adrenoceptor density and maximal adenylate cyclase responses (V(max)) to isoproterenol, 5'-guanylylimi dodiphosphate, and forskolin were measured in vitro. Volume overload induced progressive increases in LVDd and LVM over the 6-week study period. Percent fractional shortening at rest, and the change in %FS in response to dobutamine stress were dramatically reduced 6 weeks after fistula creation. Although 6-week fistula animals had unchanged beta-adrenoceptor density (B(max)) and binding affinity (K(d)) as compared with controls, maximal adenylate cyclase responses to stimulation in vitro (V(max)) were markedly reduced. Digoxin treatment prevented this loss of responsiveness of adenylate cyclase but did not affect beta-adrenoceptor density or affinity in vitro. Digoxin had no effect on LVDd, LVM, %FS, or the response to dobutamine infusion in vivo. CONCLUSIONS: Although digoxin prevented beta-adrenoceptor desensitization and improved in vitro myocardial adenylate cyclase response, the cardiac response to adrenergic stimulation in vivo was not significantly improved. These results suggest that the role of beta-adrenoceptor desensitization in the progression from volume overload hypertrophy to left ventricular dysfunction and heart failure may be less important than previously thought. Furthermore, although digoxin treatment did produce modest hemodynamic benefits, it did not prevent progressive remodeling in this model.     

Age-associated reductions in cardiac beta1- and beta2-adrenergic responses without changes in inhibitory G proteins or receptor kinases.

While an age-associated diminution in myocardial contractile response to beta-adrenergic receptor (beta-AR) stimulation has been widely demonstrated to occur in the context of increased levels of plasma catecholamines, some critical mechanisms that govern beta-AR signaling must still be examined in aged hearts. Specifically, the contribution of beta-AR subtypes (beta1 versus beta2) to the overall reduction in contractile response with aging is unknown. Additionally, whether G protein-coupled receptor kinases (GRKs), which mediate receptor desensitization, or adenylyl cyclase inhibitory G proteins (Gi) are increased with aging has not been examined. Both these inhibitory mechanisms are upregulated in chronic heart failure, a condition also associated with diminished beta-AR responsiveness and increased circulatory catecholamines. In this study, the contractile responses to both beta1-AR and beta2-AR stimulation were examined in rat ventricular myocytes of a broad age range (2, 8, and 24 mo). A marked age-associated depression in contractile response to both beta-AR subtype stimulation was observed. This was associated with a nonselective reduction in the density of both beta-AR subtypes and a reduction in membrane adenylyl cyclase response to both beta-AR subtype agonists, NaF or forskolin. However, the age-associated diminutions in contractile responses to either beta1-AR or beta2-AR stimulation were not rescued by inhibiting Gi with pertussis toxin treatment. Further, the abundance or activity of beta-adrenergic receptor kinase, GRK5, or Gi did not significantly change with aging. Thus, we conclude that the positive inotropic effects of both beta1- and beta2-AR stimulation are markedly decreased with aging in rat ventricular myocytes and this is accompanied by decreases in both beta-AR subtype densities and a reduction in membrane adenylate cyclase activity. Neither GRKs nor Gi proteins appear to contribute to the age-associated reduction in cardiac beta-AR responsiveness.     

Overexpression of myocardial Gsalpha prevents full expression of catecholamine desensitization despite increased beta-adrenergic receptor kinase.

Inotropic and chronotropic responses to catecholamines in young adult transgenic mice overexpressing myocardial Gsalpha are enhanced. One might predict that over the life of the animal, this chronically enhanced beta-adrenergic receptor stimulation would result in homologous catecholamine desensitization. To test this hypothesis, old transgenic Gsalpha mice and age-matched controls were studied physiologically in terms of responsiveness of left ventricular function (ejection fraction) to isoproterenol in vivo and in vitro in terms of beta-adrenergic receptor signaling. Old transgenic mice still responded to isoproterenol with augmented (P < 0.05) left ventricular ejection fraction (+44+/-3%) compared with age-matched controls (+24+/-1%). Although total beta-adrenergic receptor density was reduced in the old transgenic mice, and G protein receptor kinase 2 (beta-adrenergic receptor kinase) levels were increased, the fraction of receptors binding agonist with high affinity as well as isoproterenol- and G protein-stimulated adenylyl cyclase activities were enhanced. Thus, classical catecholamine desensitization is not effective in attenuation of persistently enhanced responses to sympathetic stimulation in mice overexpressing myocardial Gsalpha. To support this conclusion further, experiments were performed with chronic isoproterenol, which elicited effective desensitization in wild-type controls, but failed to elicit desensitization in overexpressed Gsalpha mice. The results of this study suggest that the lack of protective desensitization mechanisms may be responsible in part for the dilated cardiomyopathy which develops with chronic sympathetic stress over the life of these animals.     

Development of glucagon sensitivity in neonatal rat liver.

The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied in the rat. With a partially purified liver membrane preparation, fetal adenylate cyclase was less responsive to glucagon than the enzyme from neonatal or adult livers. Similar results were obtained in gently prepared liver homogenates, suggesting that destruction of essential components of the fetal liver membrane did not account for the relative unresponsiveness of the adenylate cyclase enzyme to glucagon. Investigation of other factors that might account for diminished fetal hepatic responsiveness to glucagon indicate (a) minimal glucagon degradation by fetal membranes relative to 8-day or adult tissue; and (b) available adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMP formation with NaF in fetal liver membranes. These results indicate that neither enhanced glucagon degradation nor adenylate cyclase enzyme deficiency accounts for the relative insensitivity of the fetal hepatic adenylate cyclase system to glucagon. In early neonatal life, hepatic adenylate cyclase responsiveness to glucagon rapidly developed and was maximal 6 days after birth. These changes were closely paralleled by a fivefold increase in glucagon binding and the kinetically determined Vmax for cyclic AMP formation. These observations suggest that (a) fetal hepatic unresponsiveness to glucagon may be explained by a limited number of glucagon receptor sites; (b) during the neonatal period, the development of glucagon binding is expressed primarily as an increase in adenylate cyclase Vmax; (c) the ontogenesis of hepatic responsiveness to glucagon may be important in the resolution of neonatal hypoglycemia.     

Effects of acute ischemia in the dog on myocardial blood flow, beta receptors, and adenylate cyclase activity with and without chronic beta blockade.

We ligated the left anterior descending coronary artery for 1 or 2 h in 31 purebred beagles. We did not detect any changes in beta-adrenergic receptor density or affinity when normal and ischemic zones were compared, either in the subendocardium or in the subepicardium. In the ischemic zones, there was a significant decline in all measures of adenylate cyclase activity, including activity mediated by the beta-adrenergic receptor. By contrast, after chronic beta-adrenergic blockade (1.5 mg/kg propranolol i.v. twice daily for 7 d), there was an increase in adenylate cyclase activity stimulated by (-)-isoproterenol relative to adenylate cyclase activity stimulated by guanyl-5'imidodiphosphate (GppNHp) in both normal and ischemic tissue, suggesting that one effect of chronic beta blockade may be to enhance coupling between the stimulatory guanine nucleotide regulatory protein (Gs) and the beta-adrenergic receptor, despite a reduction in the number or function of Gs units. Chronic beta blockade also led to up regulation of beta-adrenergic receptor density in subepicardial regions. After 20 min of reperfusion following 2 h of ischemia, adenylate cyclase activity tended to return to control levels, particularly in the subepicardium, where (-)-isoproterenol-stimulated adenylate cyclase activity was not different from normal myocardium. We conclude that chronic beta-adrenergic blockade may have beneficial effects during prolonged episodes of myocardial ischemia by preserving signal transduction mediated by the beta-adrenergic receptor.     

Adenylate cyclase and beta adrenergic receptor development in the mouse heart

We have demonstrated previously a postnatal peak for the beta adrenergic receptor in the heart and detected the appearance of a beta adrenergic receptor before an (-)-isoproterenol inducible increase in heart rate. The present study examined 1) agonist displaceable [3H] dihydroalprenolol (DHA) binding in the neonatal and adult mouse heart and 2) adenylate cyclase in fetal, neonatal and adult mouse heart. 3[H]DHA binding displaceable by (-)-isoproterenol gave a similar Ki from 1 day neonate through adult. Similar to the result found for antagonist displacement binding, there was a dramatic increase in the agonist displaceable [3H] DHA binding postnatally. The maximum was achieved in 2 weeks and then gradually declined to adult level. Cyclase activity (basal, (-)-isoproterenol- and NaF- stimulated) paralleled beta adrenergic receptor increases before birth. However, no early postnatal peak was present. In the 13 day fetal mouse heart, there is no (-)-isoproterenol increase in heart rate, but beta adrenergic receptor (13 +/- 4% of adult) and (-)-isoproterenol-stimulated adenylate cyclase activity (15 +/- 5% of adult) are present. It is concluded that 1) no significant difference exists between the agonist and antagonist displaceable [3H] DHA binding during development, 2) adenylate cyclase activity increases significantly during the last third of pregnancy in parallel with the beta adrenergic receptor, 3) both the beta adrenergic receptor and adenylate cyclase activity can be detected before the heart rate responses and 4) total adenylate cyclase activity does not increase in parallel with the early postnatal beta adrenergic receptor peak.     

Effects of pressure overload, left ventricular hypertrophy on beta-adrenergic receptors, and responsiveness to catecholamines.

Pressure overload left ventricular (LV) hypertrophy was produced by banding the ascending aorta of puppies and allowing them to grow to adulthood. LV free wall weight per body weight increased by 87% from a normal value of 3.23 +/- 0.19 g/kg. Hemodynamic studies of conscious dogs with LV hypertrophy and of normal, conscious dogs without LV hypertrophy showed similar base-line values for mean arterial pressure, heart rate, and LV end-diastolic pressure and diameter. LV systolic pressure was significantly greater, P less than 0.01, and LV stroke shortening was significantly lss, P less than 0.01, in the LV hypertrophy group. In both normal and LV hypertrophy groups, increasing bolus doses of norepinephrine or isoproterenol produced equivalent changes in LV dP/dt. beta-adrenergic receptor binding studies with [3H]-dihydroalprenolol ( [3H]DHA) indicated that the density of binding sites was significantly elevated, P less than 0.01, in the hypertrophied LV plasma membranes (111 +/- 8.8, n = 8), as compared with normal LV (61 +/- 5.6 fmol/mg protein, n = 11). The receptor affinity decreased, i.e., disassociation constant (KD) increased, selectively in the LV of the hypertrophy group; the KD in the normal LV was 6.8 +/- 0.7 nM compared with 10.7 +/- 1.8 nM in the hypertrophied LV. These effects were observed only in the LV of the LV hypertrophy group and not in the right ventricles from the same dogs. The plasma membrane marker, 5' -nucleotidase activity, was slightly lower per milligram protein in the LV hypertrophy group, indicating that the differences in beta-adrenergic receptor binding and affinity were not due to an increase in plasma membrane protein in the LV hypertrophy group. The EC50 for isoproterenol-stimulated adenylate cyclase activity was similar in both the right and left ventricles and in the two groups. However, maximal-stimulated adenylate cyclase was lower in the hypertrophied left ventricle. Plasma catecholamines were similar in the normal and hypertrophied groups, but myocardial norepinephrine was depressed in the dogs with LV hypertrophy (163 +/- 48 pg/mg) compared with normal dogs (835 +/- 166 pg/mg). Thus, severe, but compensated LV hypertrophy, induced by aortic banding in puppies, is characterized by essentially normal hemodynamics in adult dogs studied at rest and in response to catecholamines in the conscious state. At the cellular level, reduced affinity and increased beta-adrenergic receptor number characterized the LV hypertrophy group, while the EC50 for isoproterenol-stimulated adenylate cyclase activity was normal. By these mechanisms, adequate responsiveness to catecholamines is retained in conscious dogs with severe LV hypertrophy.     

Concanavalin A amplifies both beta-adrenergic and muscarinic cholinergic receptor-adenylate cyclase-linked pathways in cardiac myocytes.

Concanavalin A (Con A) is a tetrameric plant lectin that disrupts plasma membrane-cytoskeletal interactions and alters plasma membrane fluidity. We used Con A as a probe to explore beta-adrenergic and muscarinic cholinergic receptor-mediated regulation of cAMP in intact neonatal rat ventricular myocytes. Preincubation with Con A, 0.5 micrograms/ml, attenuated 1 microM (-)-norepinephrine (NE)-induced downregulation of beta-adrenergic receptors and resulted in a 50% augmentation of cAMP accumulation stimulated by 1 microM NE. Con A also augmented forskolin (1-10 microM)-stimulated cAMP accumulation by an average of 37% (P less than 0.05); however, Con A preincubation had no effect on basal or cholera toxin-stimulated cAMP content. The muscarinic cholinergic agonist carbachol (1-100 microM) decreased 1 microM NE-stimulated cAMP generation by an average of 32% (n = 7, P less than 0.05); preincubation with Con A further enhanced the inhibitory effect of carbachol by 18% (n = 7, P less than 0.05). Carbachol (1 microM) for 2 h decreased muscarinic cholinergic receptor density in whole cells by 33%; preincubation with Con A prevented this receptor downregulation. Con A pretreatment did not affect (-)-isoproterenol- or forskolin-stimulated adenylate cyclase activity in cell homogenates, suggesting that an intact cytoarchitecture is necessary for Con A to augment cAMP formation. We conclude that Con A, through its modulation of beta-adrenergic and muscarinic cholinergic receptor signaling, amplifies both stimulatory and inhibitory adenylate cyclase-linked pathways in intact neonatal ventricular myocytes. These data suggest the possibility that plasma membrane-cytoskeletal interaction is an important regulator of transmembrane signaling because interference with this interaction results in alterations in cAMP accumulation mediated by both beta-adrenergic- and muscarinic cholinergic-adenylate cyclase pathways.     

Desensitization of adenylate cyclase and down regulation of beta adrenergic receptors after in vivo administration of beta agonist

In vitro incubation of cells with catecholamines leads to both down regulation of beta adrenergic receptor number and desensitization of agonist-stimulated adenylate cyclase activity. These same parameters, down regulation of beta adrenergic receptor number and desensitization of adenylate cyclase activity were assessed in rat lung membranes after in vivo administration of metaproterenol, a beta-2 selective agonist. In vivo treatment with metaproterenol leads to: 1) reduced beta adrenergic receptor number; 2) reduced isoproterenol-stimulated adenylate cyclase activity; 3) unaffected NaF or 5'-guanylylimidodiphosphate-stimulated adenylate cyclase activity; and 4) reduced affinity of the receptor for isoproterenol similar to the affinity observed in the presence of 5'-guanylylimidodiphosphate. The date suggest that in vivo metaproterenol administration results in an uncoupled receptor-adenylate cyclase complex. The effects of in vivo administration of the glucocorticoid, methylprednisolone, to metaproterenol-pretreated animals were also assessed. Glucocorticoid treatment was associated with 1) increased beta adrenergic receptor number in rats in which the receptors have been down regulated, 2) increased isoproterenol responsiveness in agonist-desensitized rats and 3) no effect on agonist affinity in desensitized animals. These data suggest that the restoration of agonist responsiveness by glucocorticoids in the catecholamine refractive state is not simply a reversal of receptor down regulation or adenylate cyclase desensitization.     

Loss of high affinity cardiac beta adrenergic receptors in dogs with heart failure.

We studied the alterations in myocardial beta-adrenergic receptor-adenylate cyclase activity and muscarinic receptor density in a canine model of left ventricular (LV) failure. LV failure was characterized by a doubling of LV weight/body weight ratio (3.3 +/- 0.1 to 6.9 +/- 0.4 g/kg) and an elevation of LV end-diastolic pressure, 32 +/- 4.5 mmHg, compared with 7.7 +/- 0.6 mmHg in normal dogs. Despite a 44% increase in receptor density as measured by antagonist binding studies with [3H]dihydroalprenolol, there was a twofold decrease in receptor affinity, i.e., an increase in the dissociation constant (Kd) (5.6 +/- 0.7 to 12 +/- 1.6 nM) in heart failure. Agonist displacement of [3H]dihydroalprenolol binding with isoproterenol in the presence and absence of 5'-guanylylimidodiphosphate [Gpp(NH)p] demonstrated a striking loss of high affinity binding sites in heart failure (51 +/- 16 to 11 +/- 5%). Beta-Adrenergic receptor-mediated stimulation of adenylate cyclase and maximal stimulation with Gpp(NH)p or sodium fluoride was reduced in heart failure. There was a concomitant marked, P less than 0.01, reduction in muscarinic receptor density (242 +/- 19 vs. 111 +/- 20 fmol/mg). Thus, while muscarinic receptor density fell, beta-adrenergic receptor density actually increased in LV failure. However, a larger portion of the beta-adrenergic receptors are not functionally coupled to the GTP-stimulatory protein (Ns), as evidenced by a decrease in the fraction of receptors that bind agonist with high affinity.     

Chronic nonocclusive coronary artery constriction in rats. Beta-adrenoceptor signal transduction and ventricular failure.

To determine the effects of chronic coronary artery constriction on the relationship between cardiac function and regulation of beta-adrenoceptor signal transduction, the left main coronary artery was narrowed in rats and the animals were killed 5 mo later. An average reduction in coronary luminal diameter of 44% was obtained and this change resulted in an increase in left ventricular end-diastolic pressure and a decrease in positive and negative dP/dt. Significant increases in left and right ventricular weights indicative of global cardiac hypertrophy were observed. Radioligand binding studies of beta-adrenoreceptors, agonist-stimulated adenylate cyclase activity, and ADP ribosylation of 45-kD substrate by cholera toxin were all depressed in the failing left ventricle. In contrast, in the hypertrophic non-failing right ventricle, beta-adrenoreceptor density was preserved and receptor antagonist affinity was increased. In spite of these findings at the receptor level, agonist stimulated cyclic AMP generation was reduced in the right ventricular myocardium. The quantity of the 45-kD substrate was also decreased. In conclusion, longterm nonocclusive coronary artery stenosis of moderate degree has profound detrimental effects on the contractile performance of the heart in association with marked attenuation of adrenergic support mechanisms.     

Noncoordinate regulation of cardiac Gs protein and beta-adrenergic receptors by a physiological stimulus, chronic dynamic exercise.

We used a physiological stimulus, chronic dynamic exercise, in pigs to examine resultant changes in chronotropic responsiveness to catecholamine and biochemical features of cardiac beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein, GS. Long-term treadmill running resulted in a substantial (44%) down-regulation of right atrial beta-adrenergic receptors, but the dose of isoproterenol yielding a 50% maximal increase in heart rate was decreased by 57% (from 0.07 +/- 0.03 to 0.03 +/- 0.01 microgram/kg; P less than 0.02) despite this decrease in receptor number. This disparity between receptor number and physiological responsiveness suggested altered signal transduction. We therefore quantitated GS in myocardial membranes obtained before and after chronic exercise in a competitive ELISA based on an antipeptide antibody developed to the alpha S portion of GS. We found a 42% increase in the amounts of GS in right atrial membranes (from 11.4 +/- 0.8 to 16.2 +/- 2.0 pmol/mg; P less than 0.05) and a 76% increase in the amounts of GS in left ventricular membranes (from 15.6 +/- 2.6 to 27.4 +/- 5.2 pmol/mg; P = 0.02) after chronic running. These data suggest that in the heart physiological perturbations can result in changes in the levels of GS, that GS and beta-adrenergic receptor number are not coordinately regulated, and that GS may contribute to altered adrenergic responsiveness independently of changes in beta-adrenergic receptor number.     

Characterization of beta-adrenergic receptor linked to adenylate cyclase in a human cancer cell line (COLO 16).

1. A human cancer cell line (COLO 16) derived originally from an epidermal squamous cell carcinoma was found to possess adenylate cyclase responsiveness to beta-adrenergic agonists. 2. The adenylate cyclase response was characterized with respect to activation constants (KA) for various beta-adrenergic agonists and inhibition constants (Ki) for antagonists. 3. Intact cells responded with dose-dependent increases in production of cyclic adenosine 3':5'-monophosphate. 4. Properties of the beta-adrenergic receptor were evaluated by using the specific binding of [3H]propranolol to cell membranes. Specific binding was saturable, with KD 5.79 nmol/l and binding sites 0.68 pmol/mg of protein. 5. Competition for binding to cell membranes was shown by beta-adrenergic agonists and antagonists and was stereospecific. There was close agreement between the affinity of these various agents on adenylate cyclase and receptor binding. 6. It is likely that the beta-adrenergic receptor-linked adenylate cyclase in COLO 16 cells represents persistence in a cancer cell line of a receptor present normally in epidermal cells.     

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

Objective

Post-resuscitation myocardial dysfunction is a major cause of fatality in patients receiving successful cardiopulmonary resuscitation. The mechanism of post-resuscitation myocardial dysfunction is largely unknown, although is generally considered related to ischaemia occurring during cardiac arrest and resuscitation and/or reperfusion injury after restoration of circulation. A key mechanism responsible for reduced contractile reserves in chronic heart failure is impaired β-adrenergic receptor signalling. Thus, we hypothesised that β-adrenergic receptor signalling is markedly abnormal in the post-resuscitation period following cardiopulmonary resuscitation.

Methods

Male landrace domestic pigs were randomised into a sham group (anaesthetised and instrumented, no ventricular fibrillation) or cardiopulmonary resuscitation (CPR) group (ventricular fibrillation) (n = 8 per group). Haemodynamic and echocardiographic data were recorded. β-Adrenergic receptor signalling was assessed at 6 h after the operation by measuring myocardial adenylate cyclase activity, β-adrenergic receptor density and β-adrenergic receptor kinase expression.

Results

Left ventricular function in the CPR group was significantly decreased at 6 h after restoration of spontaneous circulation. Basal and isoproterenol-stimulated adenylate cyclase activity was blunted in the CPR group compared with the sham group. Total β-AR density was significantly decreased in CPR group compared with the sham group. Myocardial β-adrenergic receptor kinase expression was 2.03-fold greater in the CPR group than in the sham group.

Conclusions

β-Adrenergic receptor signalling is markedly impaired in the post-resuscitation period, which may be a mechanism of post-resuscitation myocardial dysfunction.     

Thyroid hormone regulates ontogeny of beta adrenergic receptors and adenylate cyclase in rat heart and kidney: effects of propylthiouracil-induced perinatal hypothyroidism.

In mature animals, thyroid hormone is permissive for beta adrenergic receptor expression and adrenergic control of adenylate cyclase. To determine if endogenous thyroid hormones play a similar role in the development of receptors and transduction mechanisms, we administered propylthiouracil perinatally to rat dams and pups from gestational day 17 through postnatal day 5. Circulating thyroid hormones were completely suppressed through postnatal day 10 and then rose to only slightly subnormal values by the 3rd to 4th postnatal week. In the heart, hypothyroidism completely suppressed the initial development of beta adrenergic receptor binding sites, with recovery paralleling the return of thyroid hormone levels. In contrast, development of basal and isoproterenol-stimulated adenylate cyclase activity showed more lasting deficiencies with a delayed onset corresponding to general growth impairment; however, forskolin-stimulated adenylate cyclase developed in a nearly normal pattern. Effects on development of renal beta receptors and adenylate cyclase were of smaller magnitude and comprised only the delayed onset phase; receptor deficiencies appeared after 10 days and adverse effects on adenylate cyclase were limited to the isoproterenol-sensitive component, consisting of a shift of the ontogenetic peak to later ages. Endogenous thyroid hormones thus contribute two distinct factors to beta receptor/adenylate cyclase development: they are obligatory for cardiac beta receptor development, but also, in parallel with general effects on growth and development, serve to program the ontogeny of transduction factors linking the receptors to adenylate cyclase. The predominance of propylthiouracil effects on isoproterenol-stimulated adenylate cyclase but not on enzymatic responses to forskolin suggests that thyroid hormones may be controlling the development of regulatory G-proteins.