beta-AR Downregulation Research Articles

Bitransgenesis with β2‐Adrenergic Receptors or Adenylyl Cyclase Fails to Improve β1‐Adrenergic Receptor Cardiomyopathy

Cardiomyopathic effects of beta-adrenergic receptor (betaAR) signaling are primarily due to the beta(1)AR subtype. beta(1)/beta(2)AR and beta(1)/adenylyl cyclase type 5 (AC5) bitransgenic mice were created to test the hypothesis that beta(2)AR or AC5 co-overexpression has beneficial effects in beta(1)AR-mediated cardiomyopathy. In young mice, beta(1)/beta(2) hearts had a greater increase in basal and isoproterenol-stimulated contractility compared to beta(1)/AC5 and beta(1)AR hearts. By 6 months, beta(1)AR and beta(1)/beta(2) hearts retained elevated basal contractility but were unresponsive to agonist. In contrast, beta(1)/AC5 hearts maintained a small degree of agonist responsiveness, which may be due to a lack of beta(1)AR downregulation that was noted in beta(1)- and beta(1)/beta(2) hearts. However, by 9 -months, beta(1), beta(1)/beta(2), and beta(1)/AC5 mice had all developed severely depressed fractional shortening in vivo and little response to agonist. p38 mitogen activated protein kinase (MAPK) was minimally activated by beta(1)AR, but was markedly enhanced in the bitransgenics. Akt activation was only found with the bitransgenics. The small increase in cystosolic second mitochondria-derived activator of caspase (Smac), indicative of apoptosis in 9-month beta(1)AR hearts, was suppressed in beta(1)/AC5, but not in beta(1)/beta(2), hearts. Taken together, the unique signaling effects of enhanced beta(2)AR and AC5, which have the potential to afford benefit in heart failure, failed to salvage ventricular function in beta(1)AR-mediated cardiomyopathy.

Reduction of High-Affinity β2-Adrenergic Receptor Binding by Hyperforin and Hyperoside on Rat C6 Glioblastoma Cells Measured by Fluorescence Correlation Spectroscopy

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

Chronic Direct Stimulation of Adenylyl Cyclase Induces Cardiac Desensitization to Catecholamine and Beta-Adrenergic Receptor Downregulation in Rabbits

Chronic stimulation of beta-adrenergic receptors (betaARs) induces betaAR downregulation. However, it is not known whether continuous activation of adenylyl cyclase without direct stimulation of betaARs leads to receptor downregulation. This study investigated the effects of chronic stimulation of adenylyl cyclase with colforsin, on hemodynamic variables, and on myocardial betaAR density. In all, 55 rabbits received intravenous colforsin (1.6 microg/kg/min, n = 20), isoproterenol (ISO; 0.4 microg/kg/min, n = 16), or saline (n = 19) for two weeks. After chronic drug administration, responses of systolic (Delta% peak LV +dP/dt) and diastolic function (Delta% peak LV -dP/dt), and heart rate (Delta% heart rate), to acute administration of ISO (0.05 to 0.2 microg/kg/min) or colforsin (5 to 20 nmol/kg/min) were decreased compared to those before chronic administration. betaAR density in the colforsin group (69.8 +/- 13.8 fmol/ml protein) was less than that in the saline group (79.8 +/- 15.0 fmol/ml protein, P < 0.05), but was greater than that in the ISO group (56.3 +/- 8.4 fmol/ml protein, P < 0.05). Thus, chronic direct stimulation of adenylyl cyclase elicited systolic and diastolic functional desensitization to betaAR stimulation or adenylyl cyclase stimulation, and myocardial betaAR downregulation.

Changes in lymphocyte glucocorticoid andβ‐adrenergic receptors in veal calves treated with clenbuterol and steroid hormones for growth‐promoting purposes*

In order to identify possible peripheral markers of illegal treatments with growth-promoting agents in veal calves, beta-adrenergic receptor (beta-AR) and glucocorticoid receptor (GR) concentrations were measured in lymphocytes of 12 male Friesian crossbred calves (six controls and six treated). The animals received a cocktail of anabolic and re-partitioning agents [17beta-oestradiol: 3 x 10 mg intramuscular (i.m.) doses at 17-day intervals; dexamethasone sodium phosphate: 4 mg/day for 6 days and 5 mg/day for six further days dissolved in milk; and clenbuterol: 20 microg/kg/day dissolved in milk for the last 40 days before slaughter]. Blood samples were collected by venipuncture at different time points and lymphocytes were isolated by density gradient centrifugation. Lymphocyte beta-AR and GR levels were measured by binding assays. Treatment with re-partitioning agents caused a significant down-regulation of lymphocyte beta-ARs 19 days after the beginning of clenbuterol administration and at day 55 (after dexamethasone withdrawal, just before slaughter). This phenomenon was partially reversed at day 50, after dexamethasone administration, at which time a significant decrease in GR concentrations also occurred. For both types of receptors, no significant changes in the dissociation constant values were observed at any time point. Lymphocytes express measurable concentrations of beta-ARs and GRs and the measurement of receptor levels highlights the fluctuation of receptor expression due to the dynamic interaction of the drugs used in combination. Lymphocyte receptor determination could therefore be included in a battery of biological assays to detect illegal treatments with anabolic agents in veal calves in the light of a multivariate approach.

?2 Adrenoceptor gene therapy ameliorates left ventricular dysfunction following cardiac surgery

Heart surgery is associated with impairment of the myocardial beta-adrenoceptor (betaAR) system. Effective therapies for post-operative ventricular dysfunction are limited. Prolonged inotrope exposure is associated with further betaAR down-regulation. Left ventricular (LV) dysfunction and myocardial betaAR impairment were assessed following cardiopulmonary bypass (CPB) and cardioplegic arrest in a pig model. Transfer of the human beta2-adrenoceptor transgene (Adeno-beta2AR) during cardioplegic arrest was then tested as a potential therapy. Five groups of six neonatal piglets were studied. One group did not undergo surgery (Group A). Adeno-beta2AR or phosphate buffered saline (PBS) were delivered via the aortic root during cardioplegic arrest. Groups B (PBS) and C (Adeno-beta2AR) were assessed at 2 days while Groups D (PBS) and E (Adeno-beta2AR) were assessed at 2 weeks from the time of surgery. An LV micromanometer was inserted under sedation to obtain pressure recordings following surgery. betaAR density was measured subsequently. Following cardiac surgery LV betaAR density was reduced (104+/-5.7 vs 135+/-6.1 fmol/mg membrane protein; P=0.007), and, in response to beta agonist stimulation, LV dP/dtmax was reduced (4337+/-405 vs 5328+/-194 mmHg/s; P<0.05) compared to animals which did not undergo surgery. Adeno-beta2AR therapy during cardiac surgery resulted in elevated LV betaAR density (520+/-250.9 fmol/mg) 2 days post-operatively compared to PBS (104+/-5.7 fmol/mg; P=0.002) and compared to the no surgery group (135+/-6.1 fmol/mg; P=0.002). Elevated LV betaAR density was also present at 2 weeks (315+/-74.1 vs 119+/-7.1 fmol/mg; P=0.002). In addition, Adeno-beta2AR therapy enhanced beta agonist stimulated LV dP/dtmax (5348+/-121 vs 4337+/-405 mmHg/s; P<0.05) and heart rate (209+/-6.9 vs 173+/-11.0 bpm; P<0.05), and reduced LVEDP (2.1+/-0.4 vs 6.4+/-1.8 mmHg; P<0.05) compared to PBS treatment. Interestingly, gene delivery was cardiac-selective and beneficial effects on function persisted for 2 weeks. Moreover, beta2AR gene transfer ameliorated LV dysfunction following surgery such that there were no significant differences between non-operated controls and animals treated with Adeno-beta2AR during CPB and cardioplegic arrest. Reduced betaAR density and impaired LV function were present following CPB and cardioplegic arrest. Cardiac-selective beta2AR gene transfer during CPB resulted in amelioration of LV dysfunction after cardiac surgery. Such a technique may offer a new approach to post-operative ventricular support.

Inhibition of receptor-localized PI3K preserves cardiac beta-adrenergic receptor function and ameliorates pressure overload heart failure.

beta-Adrenergic receptor (betaAR) downregulation and desensitization are hallmarks of the failing heart. However, whether abnormalities in betaAR function are mechanistically linked to the cause of heart failure is not known. We hypothesized that downregulation of cardiac betaARs can be prevented through inhibition of PI3K activity within the receptor complex, because PI3K is necessary for betaAR internalization. Here we show that in genetically modified mice, disrupting the recruitment of PI3K to agonist-activated betaARs in vivo prevents receptor downregulation in response to chronic catecholamine administration and ameliorates the development of heart failure with pressure overload. Disruption of PI3K/betaAR colocalization is required to preserve betaAR signaling, since deletion of a single PI3K isoform (PI3Kgamma knockout) is insufficient to prevent the recruitment of other PI3K isoforms and subsequent betaAR downregulation with catecholamine stress. These data demonstrate a specific role for receptor-localized PI3K in the regulation of betaAR turnover and show that abnormalities in betaAR function are associated with the development of heart failure. Thus, a strategy that blocks the membrane translocation of PI3K and leads to the inhibition of betaAR-localized PI3K activity represents a novel therapeutic approach to restore normal betaAR signaling and preserve cardiac function in the pressure overloaded failing heart.

A Quantitative Analysis of mRNA Expression of α1 and β-adrenoceptor Subtypes and Their Functional Roles in Human Normal and Obstructed Bladders

We compared the expression level of alpha 1-adrenoceptor (AR) subtype mRNA to that of beta-AR subtype mRNA in control and obstructed human bladders, and examined whether alpha 1-AR mediated contraction and beta-AR mediated relaxation of human detrusor muscle are altered by bladder outlet obstruction. A real-time quantitative reverse transcriptase-polymerase chain reaction based method was used to quantify alpha1 and beta-AR subtype mRNA expression. The contractile response to alpha-AR agonists and the relaxant effect of beta-AR agonists were examined using an isometric contraction technique. The mRNA of alpha 1a, alpha 1b, alpha 1d, beta 1 and beta 2-AR mRNAs was expressed at low levels, while beta 3-AR was the most highly expressed subtype at the mRNA level in control and obstructed bladders. The expression level of alpha 1 and beta-AR subtype mRNA was not significantly different between the 2 groups. The mean contractile response to 10(-4) M phenylephrine +/- SEM was only 4.4% +/- 1.4% and 5.2% +/- 1.4% of the 10(-7) M carbachol induced contraction in the control and obstructed groups, respectively. Contractile responses to phenylephrine were not significantly increased in obstructed bladder. Isoproterenol and the beta 3-AR selective agonist L755,507 relaxed human detrusor muscle in a concentration dependent manner (10(-9) to 10(-4) M). However, the beta 1/beta 2-AR agonist dobutamine and the beta 2-AR selective agonist clenbuterol did not produce relaxation at a concentration of 10(-9) to 10(-5) M in the 2 groups. These findings indicate that neither up-regulation of alpha1-ARs nor down-regulation of beta-ARs occurs and relaxation mediating beta 3-ARs are by far predominant in the human obstructed bladder. Therefore, it is not likely that bladder alpha1-ARs are responsible for detrusor overactivity and storage symptoms in patients with benign prostatic obstruction.

Exercise restores beta-adrenergic vasorelaxation in aged rat carotid arteries.

Aging is associated with alterations in beta-adrenergic receptor (beta-AR) signaling and reduction in cardiovascular responses to beta-AR stimulation. Because exercise can attenuate age-related impairment in myocardial beta-AR signaling and function, we tested whether training could also exert favorable effects on vascular beta-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the beta-AR agonist isoproterenol (10-10-3 x 10-8 M), the alpha2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). beta-AR density and cytoplasmic beta-AR kinase (beta-ARK) activity were tested on pooled carotid arteries. beta-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. beta-AR, alpha2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored beta-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced beta-AR number and increased beta-ARK activity. Training counterbalanced these phenomena and restored beta-AR density and beta-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs beta-AR vasorelaxation in rat carotid arteries through beta-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in beta-ARs and beta-ARK. Our data support an important role for beta-ARK in vascular beta-AR vasorelaxation.

Regulation of fetal cardiac and hepatic beta-adrenoceptors and adenylyl cyclase signaling: terbutaline effects.

Terbutaline (Ter), a beta(2)-adrenergic agonist used in preterm labor, stimulates fetal beta-adrenoceptors (beta-ARs). We administered Ter to pregnant rats on gestational days 17-20 and examined beta-ARs and adenylyl cyclase (AC) signaling in heart and liver. Ter produced less downregulation of cardiac beta-ARs than in adults, despite a higher proportion of the beta(2)-subtype, and failed to elicit desensitization of the receptor-mediated AC response. AC stimulants acting at different points indicated an offsetting of homologous desensitization at the level of the beta-AR by heterologous sensitization at the level of AC: induction of total AC catalytic activity and a shift in the catalytic profile or AC isoform. In fetal liver, Ter produced downregulation of beta-ARs, in keeping with the predominance of the beta(2)-subtype; hepatic receptor downregulation was equivalent in fetus and adult. Nevertheless, there was still no desensitization of beta-AR-mediated AC responses and again AC was induced. Our results indicate that, unlike in the adult, fetal beta-AR signaling is not desensitized by beta-agonists and, in fact, displays heterologous sensitization, thus sustaining responses during parturition. At the same time, the inability to desensitize beta-AR AC responses may lead to disruption of cardiac, hepatic, or neural cell development as a consequence of tocolytic therapy with beta-agonists.

β2-Adrenergic Receptor Down-regulation: EVIDENCE FOR A PATHWAY THAT DOES NOT REQUIRE ENDOCYTOSIS

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.

Beta-adrenergic receptor-dependent and -independent stimulation of adenylate cyclase is impaired during severe sepsis in humans.

a) To investigate the functional consequences of sepsis on the beta-adrenergic signal transduction in human circulating lymphocytes; b) to appreciate sepsis-associated catecholamine and cytokine release. Experimental, comparative study. Research laboratory in a university hospital. Healthy controls (n = 10); critically ill patients who were not septic (n = 7); septic patients with severe sepsis or septic shock (n = 11). Experiments were carried out using freshly isolated peripheral blood mononuclear cells (PBMC). We measured beta-adrenergic receptor (betaAR) number and affinity, and intracellular cAMP content at baseline and after the pharmacological stimulation of each component of the beta-adrenergic complex: betaAR with isoproterenol, Gs-protein with sodium fluoride (NaF), adenylate cyclase with forskolin. Catecholamine (adrenaline, noradrenaline) and cytokine (TNFalpha, IL-1alpha, IL-1beta, IL-6) serum levels were measured. In both septic and non-septic patients we observed a similar 40 % down-regulation of betaARs compared to controls, and a reduced basal and isoproterenol-stimulated cAMP accumulation (p < 0.05). The cAMP production elicited by NaF or forskolin was lower in septic patients than in the controls (p < 0.01). Forskolin-stimulated cAMP accumulation was significantly lower in septic patients than it was in non-septic ones (p < 0.001). Catecholamine serum concentrations were increased in the two patient groups without any significant difference. Elevated cytokine serum levels were detected in 45% of the septic patients (versus 14% of non-septic patients p < 0.05). Patients presenting with severe sepsis or septic shock have extended postreceptor defects of the beta-adrenergic signal transduction. This finding suggests a heterologous desensitization of adenylate cyclase stimulation.

Physical activity, skeletal muscle beta-adrenoceptor changes and oxidative metabolism in experimental chronic heart failure.

In chronic heart failure (CHF), changes in sympathetic nervous activity and skeletal muscle metabolism contribute to a limitation in the capacity for exercise. The aim of this study was to investigate the potential relationships between physical deconditioning, skeletal muscle beta-adrenoceptor (beta-AR) characteristics and muscle metabolic changes in rats with coronary ligation-induced experimental CHF. Muscle beta-AR and norepinephrine levels were assessed in rats with CHF that had been treated with propranolol at 28 mg/kg/day and compared with rats with CHF that had not been treated and those that had undergone sham operations. The soleus muscle was investigated because of its predominantly oxidative fibre-type composition. Measurements of spontaneous locomotion activity were carried out using telemetry. After 85 days, muscle energetic phosphate levels were assessed using 31P-magnetic resonance spectroscopy. The phosphocreatine resynthesis rate was decreased in the untreated CHF rats (15 +/- 3 vs 33 +/- 5 mmol L-1 min-1 in the sham-operated rats, p < 0.05), but this had been partially reversed in the rats given propranolol (22 +/- 3 mmol L-1 min-1, non-significant (NS) when compared with the sham-operated rats). Spontaneous activity did not differ among the three groups of animals. Soleus beta-adrenoceptor density was decreased in rats with CHF (8.8 +/- 3.0 fM/mg of protein vs 22.0 +/- 7.0 fM/mg of protein in the sham-operated rats, p < 0.05) and normalized in the propranolol-treated rats (31.9 +/- 7.0 fM/mg of protein, NS vs the sham-operated rats; p < 0.05 vs the untreated rats with CHF). Unchanged spontaneous activity in the rats with CHF suggests that physical deconditioning could not account for the muscle metabolic changes. Changes in skeletal muscle energy metabolism were accompanied by changes in beta-AR density, occurring in typically oxidative beta-AR-rich muscles, reversible after beta-blocker therapy and therefore suggestive of beta-AR downregulation.

Myocardial presynaptic and postsynaptic autonomic dysfunction in hypertrophic cardiomyopathy.

Although hypertrophic cardiomyopathy (HCM) is genetically determined, several other factors, including autonomic dysfunction, may play a role in the phenotypic expression. A recent study using positron emission tomography with [11C]CGP 12177 ([11C]CGP) demonstrated that beta-adrenoceptor (betaAR) density is reduced in HCM and is correlated with disease progression. This present study tested the hypothesis that this downregulation is associated with reduced catecholamine reuptake (uptake 1) by myocardial sympathetic nerve terminals leading to increased local norepinephrine concentration. Myocardial presynaptic catecholamine reuptake was assessed by measuring the volume of distribution (Vd) of the catecholamine analogue [11C]hydroxyephedrine ([11C]HED) in 9 unrelated HCM patients aged 45+/-15 years. The maximum number of binding sites (Bmax) for myocardial betaAR density was measured in 13 unrelated HCM patients aged 40+/-12 years using the nonselective beta blocker [11C]CGP. Six patients were studied with both [11C]HED and [11C]CGP. Comparison was made with two groups of healthy control subjects for each ligand ([11C]HED, n=10, aged 35+/-8 years; [11C]CGP, n=19, aged 44+/-16 years). Myocardial Vd of [11C]HED (33.4+/-4.3 mL/g tissue) and betaAR density (7.3+/-2.6 pmol/g tissue) were significantly reduced in HCM patients compared with control subjects (71.0+/-18.8 mL/g tissue, P<.001, and 10.2+/-2.9 pmol/g tissue, P=.008, respectively). These results are consistent with our hypothesis that myocardial betaAR downregulation in HCM is associated with an impaired uptake-1 mechanism and hence increased local catecholamine levels.

Effect of exercise on erythrocyte beta-adrenergic receptors and plasma concentrations of catecholamines and thyroid hormones in Thoroughbred horses.

The effects of exercise stress on erythrocyte beta-adrenergic receptor characteristics and plasma concentrations of adrenaline, noradrenaline and thyroid hormones were studied in Thoroughbred racehorses during rest and after exercise. Five minutes after a maximal speed race of 1200 +/- 200 m (mean +/- s.d.), both plasma adrenaline and noradrenaline concentrations increased with respect to basal values (from 2.48 +/- 0.15 to 3.83 +/- 0.27 and from 2.13 +/- 0.11 to 3.53 +/- 0.27 nmol/l respectively). The increment of adrenaline was greater in high performance (HP) as compared to low performance (LP) horses (76.9 vs. 43.5%), in accordance with the contribution of the adrenal medulla in the sympathoadrenal response to exercise. Triiodothyronine (T3), but not thyroxine (T4) levels increased 5 min after exercise (from 55.6 +/- 2.9 to 81 +/- 3.7 ng/dl and from 0.67 +/- 0.04 to 0.70 +/- 0.05 micrograms/dl respectively). No differences were observed in basal values of thyroid hormones or in the percentage of T3 increment, when comparing HP vs. LP horses. Erythrocyte membranes obtained 5 min after racing showed decreased concentrations of beta-adrenergic receptors (beta-AR) and dissociation constant as compared to basal values (50.1 +/- 7.0 vs. 95.7 +/- 12.0 fmol/mg protein and 0.97 +/- 0.24 vs. 2.04 +/- 0.3 nmol/l respectively). This temporal pattern suggest that the observed changes in beta-AR characteristics could be mediated by catecholamines, but not by thyroid hormones, in this model. This down regulation of beta-AR may act as a protecting mechanism preventing the erythrocytes from the decrease in membrane fluidity known to be provoked by adrenergic agonists. The accomplished study showed that, in the Thoroughbred horse, there is a homeostatic response to race stress, characterised by a sudden increase in plasma catecholamines and T3 and a parallel decrease in beta-AR concentration on the erythrocyte membrane. In this way the racing horse could rapidly adjust its metabolism to the exercise stress, but at the same time override one possible undesirable side-effect caused by these hormonal changes. Further studies will be required to establish performance-related differences occurring in endocrine changes.

Adenylyl cyclase and G protein receptor kinase expression during development of heart failure.

We examined alterations in left ventricular (LV) G protein receptor kinase (GRK) and adenylyl cyclase (AC) isoform expression during the development of pacing-induced congestive heart failure (CHF). AC isoform and GRK expression were assessed 4 (mild CHF) and 28 (severe CHF) days after initiation of pacing. LV beta-adrenergic receptor (beta-AR) number and G protein content were unchanged by mild CHF. LV AC isoform mRNA content was unaltered by mild CHF, but there were increases in total GRK activity (P < 0.01), total GRK5 protein content (P < 0.04), and GRK5 mRNA (P = 0.003); total GRK2 protein content and GRK2 mRNA were unchanged. Mild CHF was associated with decreased beta-AR coupling (P < 0.01) and reduced beta-AR stimulation of AC (P < 0.05). Severe CHF was associated with LV beta-AR downregulation (P = 0.0001) and uncoupling (P < 0.001) and marked generalized reduction of AC activity (mean P = 0.01). LV ACVI isoform mRNA content was reduced (P = 0.002), but ACII and ACV isoform mRNA contents were unaffected. Persistent elevations in LV total GRK activity (P < 0.01), total GRK5 protein content (P < 0.001), and GRK5 mRNA (P = 0.01) were found; in contrast, total GRK2 protein content was unchanged and GRK2 mRNA was reduced (P = 0.02). These studies indicate that increased GRK activity is an early charge in heart failure that predates alterations in AC isoform expression. Impaired hormonal stimulation of AC, associated with beta-AR uncoupling, may result from increased GRK5 expression. AC downregulation is isoform specific and accompanies severe but not mild CHF.

Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells.

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.

Effects of clenbuterol as a repartitioning agent onβ-Adrenoceptor concentrations in heart, bronchi and brain of veal calves

The effects induced by dietary clenbuterol (20 micrograms kg-1 body weight day-1 for 40 days) on beta-adrenergic receptor (beta-AR) subtypes in the heart, bronchial smooth muscles and the CNS of veal calves were investigated using a binding method. Clenbuterol exposure caused a significant (P < 0.05, P < 0.01, P < 0.001) decrease in beta 1-AR and beta 2-AR in both cardiac atria and ventricles of treated animals (excluding the beta 2-AR of the right atrium). Similarly, a significant (P < 0.01, P < 0.001) down-regulation of beta-AR subtypes in bronchial smooth muscles of treated calves was observed. In the CNS (cerebral cortex, hippocampus, hypothalamus and cerebellum) the down-regulation was limited to beta 2-AR, with the exception of the hippocampus in which both beta 1-AR and beta 2-AR concentrations were significantly (P < 0.05; P < 0.01) reduced. Scatchard analysis of the binding of the beta-AR antagonist, (-) [3H]CGP 12177, revealed that the down-regulation of beta-AR was not associated with any modification in binding affinity, as Kd values were unaffected by clenbuterol treatment. Data obtained indicated that prolonged clenbuterol exposure induced a remarkable beta-AR down-regulation in the heart, bronchi and brain of veal calves.

Cardiac beta-adrenergic signaling pathway alteration in isoproterenol-induced cardiac hypertrophy in male Sprague-Dawley rats.

Isoproterenol was continuously administered to rats at a rate of 2.5 microg/kg/min for 7 days via subcutaneously implanted osmotic minipumps. This treatment induced cardiac hypertrophy with increases in heart rate and systolic blood pressure. Beta-adrenoceptor (beta-AR) density decreased in hypertrophied hearts, but the affinity for 125I-cyanopindolol did not change. The beta-AR density decrease did not accompany the change in expression of beta1-AR mRNA. In hypertrophied hearts, adenylyl cyclase activities in the steady state, and stimulated with isoproterenol, forskolin, and manganese, decreased but there was no change in the expression of adenylyl cyclase type V mRNA. The results suggest that beta-AR downregulation and adenylyl cyclase desensitization in isoproterenol-induced cardiac hypertrophy are caused by post-transcriptional changes.

Hypoxia-induced downregulation of beta-adrenergic receptors in rat heart.

To test the desensitization hypothesis of cardiac beta-adrenergic receptors (beta-AR) in chronic hypoxia, the effect of 1, 3, 7, 15, and 21 days of exposure to hypobaric hypoxia (380 Torr) was evaluated in Wistar rats. Exposure to hypoxia for 1-15 days did not induce any change in right and left ventricular beta-AR density (Bmax) determined with [125I]iodocyanopindolol or in antagonist affinity. After 21 days, Bmax decreased by 24% in the left ventricle. In contrast, no change in beta-AR was shown in the right hypertrophied ventricle. Agonist affinity in the left ventricle was not altered, as shown by the analysis of displacement curves of isoproterenol (normoxia 185 +/- 26 nM, hypoxia 170 +/- 11 nM). Moreover, there was no significant decrease in adenylate cyclase activity (pmol.mg-1.min-1) in the left ventricle. In the right ventricle, a 21-day exposure to hypoxia led to a decrease in basal and maximal activity when stimulated by isoproterenol. A decrease in tissue norepinephrine content was observed after 7 days of hypoxia. In conclusion, these data support the beta-AR downregulation hypothesis as one of the mechanisms of myocardial adaptation to high altitude occurring after 2-3 wk of exposure to hypoxia. The regulation pathways of beta-AR may differ between left nonhypertrophied and right hypertrophied ventricles. No evidence of profound abnormality of signal transduction was shown.

Desensitization of myocardial beta-adrenergic receptors during cardiopulmonary bypass. Evidence for early uncoupling and late downregulation.

Cardiopulmonary bypass (CPB), a process routinely used during cardiac surgery, is a potent stimulant to the release of endogenous catecholamines. Hence, we tested the hypothesis that CPB results in myocardial beta-adrenergic receptor (beta AR) desensitization. We obtained canine transmyocardial left ventricular biopsies before, during (155 minutes), and after CPB (pre-CPB, CPB, and post-CPB, respectively) and determined beta AR density, proportion of beta 1AR to beta 2AR, and beta AR coupling capacity to adenylyl cyclase. Beta AR density was stable at 112 +/- 14 fmol/mg (pre-CPB) and 103 +/- 9 fmol/mg (CPB) but decreased post-CPB to 84 +/- 7 fmol/mg. The ratio of beta 1AR to beta 2AR (determined by two-site fit for [125I]-iodocyanopindolol competition binding with the beta 1AR selective antagonist ICI89.406) remained constant throughout (60 +/- 3: 40 +/- 3 pre-CPB, 55 +/- 3: 44 +/- 3 CPB, and 61 +/- 2: 39 +/- 2 post-CPB), revealing that both beta 1AR and beta 2AR subtypes were downregulated. A different pattern was noted in the functional properties of these receptors during CPB. Decreased maximal isoproterenol-stimulated adenylyl cyclase activity (252 +/- 14 to 216 +/- 12 pmol/30 min/mg), submaximal isoproterenol-stimulated adenylyl cyclase activity (183 +/- 10 to 157 +/- 11 pmol/30 min/mg), and zinterol-stimulated adenylyl cyclase activity (187 +/- 11 to 159 +/- 11 pmol/30 min/mg, a measure of beta 2AR subtype activation) were noted during CPB, at the time when weaning from CPB takes place. However, this desensitized pattern was found to be completely reversed by 30 minutes post-CPB, with adenylyl cyclase activities returning to pre-CPB levels or slightly higher. Control dogs that did not receive CPB showed no change in beta AR density or adenylyl cyclase activity. These data suggest that myocardial beta AR desensitization does occur during CPB in healthy, nonischemic canine myocardium and that this pattern is reversed 30 minutes after discontinuation of CPB. In addition, a slower process of beta AR downregulation persists after discontinuation of CPB. Because successful weaning from CPB is a critical process during myocardial surgery, these findings have potentially important implications in the management of such patients.