βAR Density Research Articles

Regulation of Myocardial β-Adrenergic Receptor Function in Adult and Neonatal Rabbits

β-Adrenergic receptor (βAR) desensitization is the decrease in response following sustained agonist stimulation by catecholamines. While developmental changes in βAR response have been well documented in the mammalian heart, much less is known regarding the regulation of βAR function in immature hearts. The purpose of the present study was to determine whether there are developmental differences in myocardial βAR desensitization. We used an isolated heart preparation to examine the βAR-mediated inotropic response before and after sustained exposure to 1 μM isoproterenol in adult and neonatal rabbits. We also assayed the adenylyl cyclase activity and performed radioligand-binding studies to determine βAR characteristics in adult and neonatal ventricular tissues with and without exposure to isoproterenol. Both adult and neonatal rabbit hearts showed a concentration-dependent increase in systolic function, namely, isovolumic left ventricular developed pressure (LVDP) and maximal positive dP/dt of LVDP (dP/dt_max) in response to isoproterenol. Adults, however, showed a significantly greater response than neonates. After sustained exposure to isoproterenol, the subsequent βAR-mediated responses in LVDP and dP/dt_max were significantly attenuated in adults, but much less so in neonates. The adenylyl cyclase activity in response to isoproterenol was significantly different between adult, but comparable in neonatal tissues both exposed or not to isoproterenol. The total βAR density was higher in neonatal than in adult tissues without isoproterenol exposure, but there was no significant change in βAR density in either group following isoproterenol exposure. In addition, isoproterenol exposure increased the amount of the inhibitory G protein in adult, but not neonatal tissues. Our results suggest that there were developmental differences in myocardial βAR functional responses in βAR desensitization.

Adrenergic receptor function in panic disorder II. Neutrophil β 2 receptors: G s protein coupling, effects of imipramine treatment and relationship to treatment outcome

Panic attacks are associated with increased autonomic symptoms, suggesting increased β 2-adrenergic receptor (β 2AR) function in PD. Tricyclic antidepressants downregulate βAR function. Previous studies on βAR function in PD, however, are inconsistent. We recently found increased βAR coupling and density in neutrophils of symptomatic drug-free PD patients. This study evaluated βAR coupling to G s protein in 28 controls, 25 drug-free PD patients and 8 PD imipramine-treated patients. PD patients had significantly higher coupling and receptor density, particularly in the high-conformational state. Differences were more pronounced in patients with less depressive symptomatology. Treatment with imipramine was associated with decreased βAR coupling and density in the high-conformational state. Several βAR binding parameters were related to severity of anxiety symptoms and treatment outcome. Antidepressants downregulate βAR density and induce uncoupling from G s protein in PD. Future studies may investigate βAR coupling in relationship to treatment outcome and the role of βAR kinase in PD.

Enhanced contractility and decreased beta-adrenergic receptor kinase-1 in mice lacking endogenous norepinephrine and epinephrine.

Elevated circulating norepinephrine (NE) has been implicated in causing the profound beta-adrenergic receptor (betaAR) downregulation and receptor uncoupling that are characteristic of end-stage human dilated cardiomyopathy, a process mediated in part by increased levels of beta-adrenergic receptor kinase (betaARK1). To explore whether chronic sustained NE stimulation is a primary stimulus that promotes deterioration in cardiac signaling, we characterized a gene-targeted mouse in which activation of the sympathetic nervous system cannot lead to an elevation in plasma NE and epinephrine. Gene-targeted mice that lack dopamine beta-hydroxylase (dbh-/-), the enzyme needed to convert dopamine to NE, were created by homologous recombination. In vivo contractile response to the beta1AR agonist dobutamine, measured by a high-fidelity left ventricular micromanometer, was enhanced in mice lacking the dbh gene. In unloaded adult myocytes isolated from dbh-/- mice, basal contractility was significantly increased compared with control cells. Furthermore, the increase in betaAR responsiveness and enhanced cellular contractility were associated with a significant reduction in activity and protein level of betaARK1 and increased high-affinity agonist binding without changes in betaAR density or G-protein levels. Mice that lack the ability to generate NE or epinephrine show increased contractility associated primarily with a decrease in the level of betaARK1 protein and kinase activity. This animal model will be valuable in testing whether NE is required for the pathogenesis of heart failure through mating strategies that cross the dbh-/- mouse into genetically engineered models of heart failure.

Low- and high-level transgenic expression of beta2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galphaq-overexpressing mice.

Transgenic overexpression of Galphaq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed beta-adrenergic receptor (betaAR) function. The role of betaAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the betaAR kinase (betaARK), which acts to inhibit the kinase, or concomitant overexpression of the beta2AR at low (approximately 30-fold, Galphaq/beta2ARL), moderate (approximately 140-fold, Galphaq/beta2ARM), and high (approximately 1,000-fold, Galphaq/beta2ARH) levels above background betaAR density. Expression of the betaARK inhibitor had no effect on the phenotype, consistent with the lack of increased betaARK levels in Galphaq mice. In marked contrast, Galphaq/beta2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and alpha-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized beta2AR/AC signal cannot be excluded. Higher expression of receptors in Galphaq/beta2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With approximately 1,000-fold overexpression, the majority of Galphaq/beta2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of betaAR signaling is deleterious in heart failure, selective enhancement by overexpressing the beta2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

The myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF) rats.

-Responsiveness to beta-adrenergic stimulation is reduced in the failing human myocardium. This results principally from reduced beta-adrenergic receptor (betaAR) density, elevated beta-adrenergic receptor kinase 1 (betaARK1) levels, and functional uncoupling of remaining receptors. The temporal nature of changes in the human myocardial beta-adrenergic system relative to onset of symptomatic heart failure (HF) has been difficult to discern. A relatively new model of HF, the spontaneously hypertensive heart failure (SHHF) rat spontaneously and reproducibly develops left ventricular hypertrophy (LVH) and progresses to HF, thus enabling longitudinal studies to examine the cellular and molecular bases for hypertension-induced cardiac hypertrophy and subsequent HF. The purpose of this study was to examine age-dependent changes in the betaAR system in this model. Lean male SHHF rats at 3, 7, 14, and 20 months were compared with age-matched Sprague-Dawley (SD) control rats ([C]; 4 animals/group). At all ages the SHHF rats had elevated blood pressures and left ventricular end-diastolic pressure relative to the SD control rats (P<0.05). Compared with age-matched SD control rats, LVH was evident by 3 months in SHHF rats; 20-month-old SHHF rats had significantly greater LVH compared with the other SHHF rat groups. beta-adrenergic responsiveness (maximal heart rate to isoproterenol) was reduced only in 20-month-old SHHF rats. betaARK1 protein levels and activity were elevated at 14 months (162+/-10% and 195+/-20% C, respectively), and betaARK1 protein remained elevated at 20 months (140+/-14% C). In contrast, G protein-coupled receptor kinase 5, a second receptor kinase in the heart, remained unchanged at all ages. betaAR density did not change with age in the SD control rats and was similar in the SHHF rats until 20 months of age when the receptor number was reduced (30+/-1%). These data indicate that cardiac dysfunction is coincident with reduced betaAR density. Importantly, cardiac dysfunction was preceded by elevated betaARK1 levels and activity, thus suggesting that betaARK1 may be a precipitating factor in the transition from hypertension-induced compensatory cardiac hypertrophy to HF. Furthermore, these results indicate that the SHHF rat is a powerful model for use in examination of the mechanisms involved in alterations of beta-adrenergic signaling that occur in human HF.

Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.

Heart failure is accompanied by severely impaired beta-adrenergic receptor (betaAR) function, which includes loss of betaAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of betaAR function is agonist-stimulated receptor phosphorylation by the betaAR kinase (betaARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in betaAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of betaARK1 or the beta2AR were mated into a genetic model of murine heart failure (MLP-/-). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP-/- and MLP-/-/beta2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP-/-/betaARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP-/-/betaARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP-/- mice but less than controls. Importantly, heightened betaAR desensitization in the MLP-/- mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the betaARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal betaAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit betaARK1 as a novel mode of therapy.

REDUCTION IN CANINE CARDIAC ??-ADRENERGIC RECEPTOR (??AR) DENSITY FOLLOWING CHRONIC ENDURANCE EXERCISE

90 The goal of this project was to test the hypothesis that the increase in sympathetic tone associated with chronic, repetitive, endurance training results in a decreased density of βARs expressed in the myocardium. Mixed-breed dogs of either sex were either sedentary (cage-rested) or performed a nine week progressive treadmill exercise program (exercised). Half of the animals in each group received oral doses of the short-acting βAR antagonist, timolol, either daily (sedentary animals) prior to or during each bout of exercise. At the end of the training period, βAR density in myocardial microsomal membranes prepared from left ventricular wall tissue were determined using radioligand binding with 125I-cyanopindolol(125I-CYP). Specific 125I-CYP binding decreased (p<0.05) by≅40% (149 ± 34 fmol/mg protein) in myocardial membranes obtained from exercise animals compared to receptor density measured in membranes from untrained animals (250 ± 40 fmol/mg protein) with no apparent change in receptor affinity for the radioligand. βAR density measured in membranes from animals that received timolol treatment during exercise was not significantly different from sedentary controls. No significant change was observed in the ratio of β1 to β2-adrenergic receptors measured by competitive inhibition of 125I-CYP binding with the subtype-selective antagonists ICI-118,551 and atenolol. These data suggest that one adaptation that accompanies the remodeling of the myocardium during chronic exercise training is a downregulation of the βAR density.

Intermittent Warm Blood Cardioplegia Preserves Myocardial β-Adrenergic Receptor Function

Background. Left ventricular dysfunction is frequently observed in patients after hypothermic cardioplegic arrest, and often inotropic intervention is necessary for patients to be successfully weaned from cardiopulmonary bypass (CPB). A myocardial β-adrenergic receptor (βAR) desensitization has been noted to occur after hypothermic CPB in patients undergoing coronary artery bypass grafting. This randomized study was undertaken to determine the effect of cardioplegic solution temperature on cardiac βARs. Methods. Two groups of patients (20 patients in each) scheduled for elective coronary artery bypass grafting underwent CPB with either intermittent warm or cold blood cardioplegia. The density of the βARs, the proportion of β 1- to β 2-adrenergic receptors, and the βAR coupling capacity to adenylate cyclase were determined in specimens of the right atrial tissue at baseline, during CPB, and after discontinuation of CPB. Plasma concentrations of catecholamines were also measured in both arterial and coronary sinus samples. Results. In both cardioplegia groups, no significant modification in either the βAR density or the proportion of β 1- to β 2-adrenergic receptors was detected. However, a significant decrease in adenylate cyclase activity after stimulation with isoproterenol was observed in the cold blood cardioplegia group during CPB ( p < 0.01) and 30 minutes after its discontinuation ( p < 0.05). Moreover, a significant decrease in adenylate cyclase activity during CPB was detected in this group after stimulation with sodium fluoride ( p < 0.05), but this pattern was found to be completely reversed by 30 minutes after discontinuation of CPB. No modification in the basal or stimulated adenylate cyclase activity was observed in the warm blood cardioplegia group during or after CPB. Conclusions. Our results confirm the finding from previous studies of a cardiac βAR desensitization after hypothermic cardioplegic arrest, and provide evidence of the advantages of intermittent warm blood cardioplegia in preserving the autonomic sympathetic function of the heart.

Interleukins Impair Beta-Adrenergic Receptor Adenylate Cyclase (βAR-AC) System Function in Human Airway Epithelial Cells

Inflammatory cells release a variety of cytokines including interleukins-1β (IL-lβ) and interleukin-2 (IL-2) into the airway in asthma. In other cell systems, IL-lβ and IL-2 exert cell type-specific effects on beta-adrenergic receptor-adenylate cyclase (βAR-AC) structure and function. However, the effects of IL-1β and IL-2 on the βAR-AC system expressed by human airway epithelial cells is unknown. The present study examined the effects of human recombinant IL-1β and IL-2 on βAR density (Bmax), ligand affinity binding (Kd), the ability of catecholamines to stimulate cAMP production, β2AR gene expression (mRNA), and the content of the inhibitory G protein (Gi).

Desensitization and Selective Down-Regulation of Rat Cardiac β1-Adrenoceptors by Prolonged In Vivo Infusion of T-0509, a β1-Adrenoceptor Full Agonist

We studied the effects of prolonged infusion of a selective β1-adrenoceptor (β1AR) full agonist, T-0509 [( —)-(R)-1-(3,4-dihydroxyphenyI)-2-[(3,4-dimethoxyphenethyl)amino]ethanol hydrochloride], with regard to its inotropic effect in vivo and cardiac βAR density. The results were compared with those for isoproterenol. Continuous infusion of isoproterenol at doses of 2.5-40 μg/kg/hr, s.c. for 6 days shifted the dose-response curves of isoproterenol (i.v.) for LVdP/dtmax to the right and increased the ED50 values up to fourfold. Isoproterenol infusion at 40μg/kg/hr reduced the density of both β1- and β2ARs by 36% and 43% respectively, in left ventricular membranes. Following 6-day infusion of T-0509 at doses sufficient to induce a positive inotropic effect (5-40 μg/kg/hr), the ED50 value of T-0509 (i.v.) for LVdP/dtmax was also increased up to fourfold. In contrast to isoproterenol, infusion of T-0509 caused selective down-regulation of β1/ARs by 30% without changing the number of β2ARs. These results indicate that long-term application of a selective β1AR full agonist causes desensitization to its inotropy in vivo, with subtype-selective down-regulation of β1ARs in cardiac ventricles.

Columnar activity regulates astrocytic beta-adrenergic receptor-like immunoreactivity in V1 of adult monkeys.

Recent results indicate that astrocytic beta-adrenergic receptors (beta AR) participate in noradrenergic modulation of synaptic activity. In this study, we sought to examine whether neural activity can, in turn, regulate astrocytic beta AR. To address this question, an antiserum that recognizes beta-adrenergic receptors (beta AR) specifically in astrocytes was used to assess the distribution of the receptors across ocular dominance columns in V1 of two monocular and four visually intact adult monkeys. Cytochrome oxidase histochemistry (CO) was used to identify the position of the cortical laminae and of the ocular dominance columns receiving visual inputs from the intact and enucleated eyes. This stain revealed the expected pattern within V1 of monocular monkeys--i.e. darker and lighter bands of equal widths (ca. 500 microns) spanning laminae 4-6, each associated with larger and smaller blobs, respectively, in lamina 2/3. Alignment of CO sections with adjacent sections stained for astrocytic beta AR by the immunoperoxidase method revealed intense beta AR-like immunoreactivity (beta AR-li) in the superficial laminae, a slightly weaker staining in the infragranular laminae and weakest staining in lamina 4C. Within lamina 4C, a prominent striped pattern was evident. The darker bands of the stripe closely matched widths and positions of the lighter CO columns associated with the enucleated eye. On the other hand, immunocytochemical staining for the astrocytic intermediate filament protein, GFAP, within V1 of monocular monkeys revealed no inter-columnar difference in the density of astrocytic cell bodies or processes. Nissl stain also revealed no overt inter-columnar differences in cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed recovery of lymphocyte beta-adrenoceptors in thyroidectomized aging patients.

We previously found that the beta-adrenoceptor (beta AR) density of lymphocytes from thyroidectomized adult women shows a significant decrease early after surgery, followed by a slower recovery kinetics. In the present article, we posed the question whether the kinetics of lymphocyte beta ARs from thyroidectomized patients could be dependent upon the age of the donors. Thus, postsurgical decrease and the following recovery of beta AR density were studied in thyroidectomized female patients of different ages, along with the time course of triiodothyronine (T3) and thyroxine (T4) levels. The kinetics were analyzed using a mathematical model fitting experimental data. Results show that parallel statistically significant falls in beta AR density and T3 levels are present in every age group tested from 1 to 3 days following surgical intervention. No statistically significant changes have been observed in T4 levels. The recovery of beta AR density shows progressively slower kinetics with increasing patient age. In particular, a parameter estimating the time required to regain 50% of the fall in beta AR density increases exponentially with advancing age. T3 levels show a recovery trend quite similar to that of beta AR density, but the kinetics is slower and does not present any statistically significant age-related changes. The kinetics of T4 levels are completely different, showing a continuous slowly decreasing trend. Thus, the beta AR fall can be accounted for only by a decrease in T3 levels. On the contrary, the comparison of T3 and beta AR kinetics suggests that receptor recovery is also mediated by other regulatory factors. T4 levels do not appear to have any direct role in both fall and recovery of beta ARs.

Short- and long-term effects of reserpine on rat cardiac beta-adrenoceptors

1. 1. In order to explore the mechanism of reserpine-induced supersensitivity, we examined the effects of various doses of reserpine ( R) on rat cardiac β-adrenoceptors (βAR). 2. 2. 24 hr after a single administration of R (1 mg/kg, i.p.) βAR were up-regulated, while with higher doses (2.5–5 mg/kg) βAR density was not changed but βAR coupling, evaluated by the proportion of high-affinity agonist binding states, was impaired. 3. 3. Long-term effects of R (2.5 mg/kg/day, 2 days) included βAR down-regulation and uncoupling 24 hr treatment (day 1), followed by up-regulation at day 7, while all parameters were similar to controls at day 14. 4. 4. It appears that R induces either an increase or a decrease of cardiac βAR density and coupling, dependant on the dose and the time at which βAR are measured after treatment.

Post-natal evolution of rat cardiac beta-adrenoceptors

Cardiac β-adrenoceptors (βAR) were studied using membranes prepared at birth (day 0) and at days 7, 10, 15, 21, 30, 45 and 60. Saturation experiments using the antagonist ligand ( 125I)-iodocyanopindolol (ICYP) allowed the determination of βAR number (Bmax) and ICYP dissociation constant (K d), while (-) isoproterenol competition curves of ICYP binding, performed in the absence or presence of Gpp(NH)p (10 −4M), were used to measure the relative proportions of high and low affinity states of the βAR for the agonist and to assess the ability of βAR to couple with the GTP-binding protein. Rat cardiac βAR evolved at 3 distinct periods: during the first period (days 0–10), the receptor density and ICYP K d were half that of adults, and βAR were present only in an homogenous high affinity state. The second period (days 15–21) was characterized by a progressive increase in βAR number and ICYP K d, while analysis of (-)isoproterenol competition curves indicated that βAR were poorly coupled to the GTP-binding protein. In the third period (days 30–60), ICYP Bmax and K d were respectively 53.9±1.2 fmoles/mg proteinand 106.4±2.9 pM, while analysis of (-)isoproterenol competition curves showed the existence of high and low affinity binding states in equal proportions in the absence Gpp(NH)p, and of one homologous low affinity state of the receptor in its presence. These data indicate that βAR follow a post-natal evolution marked by an increase in βAR density concomittant with a decrease in affinity toward the antagonist ligand ICYP, accompanied by the progressive appearance of a poorly-coupled βAR. However, the number of efficiently coupled receptors was found to be similar in adult and newborn rats.

348 ONTOGENY OF THE β-ADRENERGIC RECEPTOR IN RABBIT PLACENTA

β-Adrenergic stimulation of the placenta causes increased glycogenolysis and cAMP production. These effects are presumably mediated through the β-adrenergic receptor (βAR). In this study the βAR in rabbit placenta was characterized and shown to increase with gestational age. Binding of the β-adrenergic antagonist dihydroalprenolol, (-)[3H]DHA, to rabbit placental membranes was saturable to a single class of sites, KD=1.67±.07(nM), n=11 and Bmax=326±33 fmoles/mg. Agonist competition for the βAR was performed in the presence of the non-hydrolyzable GTP analog Gpp(NH)p (required for Hill coefficient = 1) and showed the potency order (-)iso ≫ (-)epi > (-)norepi characteristic of the β2 subtype. However, competition experiments with subtype specific agents metoprolol (β1) and zinterol (β2) indicated that the rabbit placenta contained approximately equal populations of βAR subtypes (51% β1, 49% β2). Rabbit placenta βAR/DNA increased 5.3 fold from day 16 to day 27 of gestation (410±120 fmoles/mg DNA to 2184±354 p<.001). During this interval protein/DNA (25±1.8mg/mg DNA to 34.2± 4.1) and DNA (4.0±.2mg/gm tissue to 3.5±.2) changed less markedly. KD did not change with gestation. (−)[3H]DHA binding was coupled with catecholamine stimulated adenylate cyclase (1.5 fold stimulation, p<.01). Rabbit placenta has two βAR (β1 and β2) in high density coupled to adenylate cyclase. Maximal βAR density occurs in late gestation coincident with the known catecholamine surge in fetal rodents suggesting a regulatory role for catecholamines in placental metabolism.

338 EFFECT OF ISOXSUPRINE ON β-ADRENERGIC RECEPTORS AND ADENYLATE CYCLASE IN FETAL RABBIT CARDIAC MEMBRANES

Isoxsuprine (ISX) has been used in mothers with premature labor. To examine the effect of chronic exposure of ISX on fetal myocardium, eight doses of ISX (3 mg/kg) were injected intramuscularly into pregnant rabbits between 24-27 days gestation. At 28 days, fetal cardiac membranes were examined for β-adrenergic receptors (βAR) by (-)[3H]dihydroalprenolol (DHA) binding and assayed for adenylate cyclase (AC) activity. There was no difference in mean fetal heart weights in saline and ISX treated groups. (-)[3H]DHA binding sites were rapid (t½<30 sec.), reversible (t½<90 sec.) and saturable with high affinity and low capacity. β-adrenergic agonists and antagonists competed for these sites and had characteristics of β1-subtype of βAR. Chronic ISX treatment led to a decrease in the mean ± SEM of maximal (-)[3H]DHA bound from 126±7 fmoles/mg (n=6) to 64% of the control value, 81±8 fmoles/mg (n=6 (p<.01), with no change in affinity (Kd=1.8±0.2 vs. 2.4±0.3 nM). In addition, AC stimulation by (-) isoproterenol (10 μM) was reduced from 66.1±4.2 pmoles cAMP/mg/15 min. (n=5) to 62% of the control response, 41.2±4.1 pmoles cAMP/mg/15 min. (n=5)(p<.01) with no change in the basal activity in fetal cardiac membranes (24.5±1.9 vs. 24 ±0.9 pmoles cAMP/mg/15 min.). Thus, chronic ISX exposure of rabbit fetuses downregulated the βAR density in fetal cardiac membranes at 28 days and was accompanied by diminution of the response of AC to isoproterenol stimulation.