beta-AR Antagonist Research Articles

Sustained β-Adrenergic Stimulation Increased L-Type Ca2+ Channel Expression in Cultured Quiescent Ventricular Myocytes

The abundance of voltage-gated L-type Ca2+ channels is altered by beta-adrenergic receptor (beta-AR) stimulation and by an elevation of the intracellular Ca2+ concentration in cardiac myocytes. In whole animal, chronic beta-AR stimulation or pacing heart results in various changes in the abundance of the channel, but it reduces the beta-AR responsiveness of the L-type channel. Because beta-AR stimulation facilitates the L-type calcium channels, it is difficult in the whole animal to study the effects of beta-AR and Ca2+ influx on the upregulation of the L-type channel independently of each other, which makes the culture of nonbeating adult myocytes an attractive model. We found that culturing quiescent adult rabbit ventricular myocytes with isoproterenol (ISO, 2 microM) for 72 h or more caused a significant increase in the expression of mRNA coding for the L-type channel alpha(1C) subunit by approximately twofold as compared to time-matched controls, and it was followed by a 1.8-fold increase in the Ca2+ current density at 96 h. Somewhat surprisingly, an acute application of 1 microM ISO increased the current amplitude even in ISO-treated cells. The increase in the current density, induced by sustained beta-AR stimulation, was blocked by a beta-AR antagonist, propranolol (10 microM), but not by a Ca2+ antagonist, nitrendipine (10 microM). In addition, the effects were reproduced by forskolin (10 microM), but not by a Ca2+ agonist, Bay-K 8644 (2 microM). Taken together, these results suggest that sustained beta-AR stimulation upregulates L-type channel expression, but does not alter the beta-AR responsiveness of the channel in quiescent myocytes.

Studies on the mechanisms underlying .BETA.-adrenoceptor-mediated relaxation of rat abdominal aorta

Mechanisms underlying beta-adrenoceptor (beta-AR)-mediated vascular relaxation were studied in the isolated rat abdominal aorta. In the endothelium-denuded helical preparations, a non-selective beta-AR agonist isoprenaline elicited a concentration-dependent relaxation. In the absence of beta-AR antagonists, isoprenaline-induced relaxation was not practically affected by an adenylyl cyclase inhibitor SQ 22,536 (300 microM), but was strongly diminished by high-KCl (80 mM). Isoprenaline-induced relaxation in the presence of SQ 22,536 was significantly diminished by iberiotoxin (IbTx, 0.1 microM), but was not affected by 4-aminopyridine (4-AP, 3 mM). Isoprenaline-induced relaxation was not also affected by SQ 22,536 (300 microM) even in the presence of CGP20712A (a beta(1)-selective antagonist) and ICI-118,551 (a beta(2)-selective antagonist) (0.1 microM for each), but was strongly diminished by high-KCl. By contrast, SQ 22,536-resistant, isoprenaline-induced relaxation in the presence of CGP20712A plus ICI-118,551 was not affected by IbTx (0.1 microM), but was inhibited significantly by 4-AP (3 mM). These results suggest that in rat abdominal aortic smooth muscle: 1) both beta(1)-/beta(2)-AR- and beta(3)-AR-mediated relaxations substantially involve cAMP-independent mechanisms; 2) beta(1)-/beta(2)-AR-mediated, cAMP-independent relaxant mechanisms are partly attributed to the large-conductance, Ca (2+)-sensitive K(+) (MaxiK, BK) channel whereas beta(3)-AR-mediated relaxant mechanisms are attributed to K(v) channel.

β1-Adrenoceptor Mediated Origin of the Heart Rate Performance Curve Deflection

The deflection of the HR performance curve (HRPC) has been described as an objective marker of submaximal exercise performance. HR response to incremental cycle ergometer exercise is shown to be neither linear nor uniform and a physiological explanation of the deflection phenomenon is lacking. We hypothesized that differences in the beta1-adrenoceptor site are the source of these differences. The aim of the study was to investigate the influence of the highly selective beta1-adrenoceptor (beta1-AR) antagonist bisoprolol (Bi) on the HRPC in young healthy male subjects with different HR response patterns. Sixteen subjects were treated in randomized order with Bi or a placebo (Pl) in two separate trials. HR response during incremental cycle ergometer exercise was compared between the two trials. Blood lactate concentration (La) and ventilatory variables were measured throughout both tests. Bi changed the direction of the HRPC more in subjects with a regular, s-shaped response pattern under placebo than those with a nonregular or linear pattern. The influence of Bi on the HR at the second lactate turn point was significantly related (R = 0.78; P < 0.001) to the pattern of the HRPC in Pl conditions. We suggest that differences between the subjects with regular s-shaped versus nonregular HRPC may be due to differences at the beta1-AR site. The origin of the HRPC deflection is mediated in part by the beta1-AR sensitivity.

Enhanced Inhibition of L-type Ca2+ Current by β3-Adrenergic Stimulation in Failing Rat Heart

beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.

Role of β2-adrenoceptors (ß-AR), but not ß1-, β3-AR and endothelial nitric oxide, in β-AR-mediated relaxation of rat intrapulmonary artery

The aim of this study was to analyze beta-adrenoceptor (beta-AR)-mediated relaxation in rat intralobar pulmonary artery. The relaxant responses of beta-AR agonists were characterized using beta-AR antagonists in prostaglandin F2alpha (PGF2alpha)-precontracted arteries. The role of nitric oxide (NO) and endothelium in beta-AR-mediated relaxation was also investigated. Isoprenaline (a non-selective beta-AR agonist) and salbutamol (a selective beta2-AR agonist) induced vasorelaxation. ICI 118551 (a selective beta2-AR antagonist) antagonized the effect of both isoprenaline and salbutamol (pA2 values of 9.57 and 9.51 respectively). In contrast, atenolol (1 microM) and CGP 20712A (0.1 microM), two beta1-AR antagonists, did not modify the relaxing effect of isoprenaline. The response to isoprenaline obtained in the presence of nadolol (10 microM, a beta1/beta2-AR antagonist) was not further inhibited by SR 59230A (1 microM, a selective beta3-AR antagonist). The non-beta1/beta2-AR agonists studied (BRL 37344, SR 58611A, and CGP 12177A) did not elicit vasorelaxation. Relaxation to isoprenaline and salbutamol was unaffected by L-N(G)-nitro-arginine methyl ester (100 microM, an inhibitor of NO synthase) or after endothelium removal. These results demonstrate the role of beta2-AR in mediating relaxation in rat intralobar pulmonary artery precontracted with PGF2alpha. They indicate that beta2-AR-mediated relaxation in this artery is NO- and endothelium-independent. Furthermore, they do not provide evidence of a relaxant role of either beta1- or beta3-AR in PGF2alpha-precontracted rat intrapulmonary artery.

Characterization of Beta3-Adrenoceptors in Human Internal Mammary Artery and Putative Involvement in Coronary Artery Bypass Management

The aim of the present study was to analyze whether beta3-adrenoceptors (beta3-ARs) were effectively present and functional in the human internal mammary artery (IMA). The beta1- and beta2-adrenoceptors classically mediate the relaxant effects of catecholamines in the vessels. In vitro and in vivo studies performed in various animal species described vasodilating effects due to activation of a third beta-ARs subtype (beta3). Reverse transcription-polymerase chain reaction analysis, Western blot experiments, and pharmacological studies were carried out in human IMA samples harvested from 27 patients undergoing coronary bypass surgery. The beta3-ARs messenger ribonucleic acid and protein were detected in intact IMA, but were absent in endothelium-free samples. This finding was confirmed by immunohistochemical experiments. In organ baths, a beta3-AR agonist, SR 58611A, induced an endothelium-dependent relaxation of phenylephrine-precontracted IMA rings. This vasodilation was not modified by beta1/beta2-AR antagonists, but was greatly altered in the presence of L-748,337, a selective human beta3-AR antagonist. Moreover, the inhibition of nitric oxide (NO) synthases abolished the beta3-adrenergic vasodilation, suggesting the involvement of a NO-signaling pathway. Those results demonstrated the presence of beta3-ARs in the endothelial layer of human IMA. The present work highlights the role of beta3-ARs in vasomotor control of IMA and opens new fields of investigation in coronary bypass graft management, heart failure, and hypertension.

Norepinephrine Induces Lipolysis in β1/β2/β3-Adrenoceptor Knockout Mice

Catecholamines are major stimulants of adipose tissue metabolism. Norepinephrine and epinephrine act through three subtypes of beta-adrenoceptors (beta-AR) expressed in the adipocytes. The aim of this work was to study the mechanisms of lipid mobilization in beta1/beta2/beta3-AR triple-knockout (beta-less) mice. Glycerol and nonesterified fatty acids released from isolated adipocytes were measured as an index of lipolytic activity. There was no difference between the two genotypes for basal lipolysis and lipolytic response to corticotropin or to agents acting at the adenylyl cyclase and protein kinase A levels. The lipolytic response to norepinephrine and beta-AR agonists was blunted in beta-less mice. However, a residual low-affinity lipolytic effect was observed in the presence of catecholamines and beta3-AR agonists but not of beta1- or beta2-AR agonists. cAMP levels were increased by a beta-AR agonist in white and brown adipocytes of beta-less mice. The residual lipolytic effect was blocked by beta-AR antagonists. It was mediated neither by alpha1- or alpha2-AR nor dopaminergic, serotonergic, and histaminergic by receptors. Bioinformatic analyses do not provide evidence for a fourth beta-AR. We conclude that the residual lipolytic effect observed in beta-less mice can be attributed to an unknown Gs-protein-coupled receptor with low affinity for catecholamines.

β1 Adrenergic Receptor-Mediated Enhancement of Hippocampal CA3 Network Activity

Norepinephrine is an endogenous neurotransmitter distributed throughout the mammalian brain. In higher cortical structures such as the hippocampus, norepinephrine, via beta adrenergic receptor (AR) activation, has been shown to reinforce the cognitive processes of attention and memory. In this study, we investigated the effect of beta1AR activation on hippocampal cornu ammonis 3 (CA3) network activity. AR expression was first determined using immunocytochemistry with antibodies against beta1ARs, which were found to be exceptionally dense in hippocampal CA3 pyramidal neurons. CA3 network activity was then examined in vitro using field potential recordings in rat brain slices. The selective betaAR agonist isoproterenol caused an enhancement of hippocampal CA3 network activity, as measured by an increase in frequency of spontaneous burst discharges recorded in the CA3 region. In the presence of alphaAR blockade, concentration-response curves for isoproterenol, norepinephrine, and epinephrine suggested that a beta1AR was involved in this response, and the rank order of potency was isoproterenol > norepinephrine = epinephrine. Finally, equilibrium dissociation constants (pK(b)) of subtype-selective betaAR antagonists were functionally determined to characterize the AR subtype modulating hippocampal CA3 activity. The selective beta1AR antagonists atenolol and metoprolol blocked isoproterenol-induced enhancement, with apparent K(b) values of 85 +/- 36 and 3.9 +/- 1.7 nM, respectively. In contrast, the selective beta2AR antagonists ICI-118,551 and butoxamine inhibited isoproterenol-mediated enhancement with apparent low affinities (K(b) of 222 +/- 61 and 9268 +/- 512 nM, respectively). Together, this pharmacological profile of subtype-selective betaAR antagonists indicates that in this model, beta1AR activation is responsible for the enhanced hippocampal CA3 network activity initiated by isoproterenol.

Functional Characterization of the β-Adrenergic Receptor Subtypes Expressed by CA1 Pyramidal Cells in the Rat Hippocampus

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

Isoproterenol Opens K+ ATPChannels via a β2-Adrenoceptor-linked Mechanism in Sertoli Cells from Immature Rats

In the present study, we investigated the mechanism by which isoproterenol hyperpolarises membrane potential (MP) in Sertoli cells from seminiferous tubules of 15-day-old rat testes. Modification of MP and resistance (R0) was analysed using conventional intracellular glass microelectrodes. Isoproterenol (2 x 10(-6) M) induced an immediate and significant hyperpolarisation in the Sertoli-cell membrane. The beta2-AR antagonist, butoxamine (1 x 10(-6) M), nullified isoproterenol action. The effect of the beta1 antagonist, metoprolol (1 x 10(-6) M), was light and non-significant. Sulphonylurea glibenclamide inhibition of the K+(ATP) channels suppressed isoproterenol action, and testosterone, while depolarising Sertoli-cell MP closing the K+(ATP) channels through the PLC/PIP2 pathway, reduced beta-AR agonist-induced hyperpolarisation. Also, polycations LaCl3 and spermine reversed isoproterenol's hyperpolarisation effect, probably depolarising the membrane potential through ionic interaction neutralising the action of isoproterenol on K+(ATP) channels. Adenylate cyclase agonist forskolin (0.1 microM) rapidly hyperpolarised Sertoli-cell MP, mimicking the isoproterenol effect. These effects indicate that isoproterenol's action on K+(ATP) channel probably involves the known signalling cascade beta-AR/Gs/AC/cAMP/PKA. These results suggest that the isoproterenol-induced hyperpolarisation is mediated by the opening of K+(ATP) channels in Sertoli cells. This beta-adrenergic hyperpolarisation might play a physiological role in the modulation of MP.

The roles of α‐ and β‐adrenoceptor stimulation in myocardial ischaemia

beta-Adrenoceptor (AR) ligands have been the mainstay of cardiovascular therapy for decades, with beta-AR antagonist being used for hypertension, angina and myocardial infarction and adrenaline in use for cardiopulmonary resuscitation for nearly 100 years. Ischaemia of the heart through coronary artery occlusion causes cell injury and death through necrosis and apoptosis. Reperfusion of the ischaemic myocardium results in cardiac dysfunction and infarction. Stimulation of alpha- and beta-ARs in the ischaemic heart have variable and inconsistent effects depending on when the agonist is applied. This review describes the different effects of stimulation of the three established beta-AR subtypes (beta(1)-, beta(2)- and beta(3)-ARs) either before ischaemia (preconditioning) or during ischaemia and reperfusion of the heart (postconditioning). Brief periods of ischaemia preceding a major ischaemic episode can have a protective effect against post-ischaemia-reperfusion damage, known as ischaemic preconditioning. This review considers the role of endogenous catecholamines released during preconditioning and the nature of the adrenoceptor subtypes that mediate these effects. The clinical significance of this to the use of beta-AR antagonists is considered. The transduction pathways and effects on apoptosis of the cardioprotective and deleterious effects of AR activation are considered. This commentary reviews the literature and attempts to bring together a unified synopsis of the effects of adrenoceptor stimulation in myocardial ischaemia and the potential clinical relevance.

Beta-adrenoceptor subtype dependence of chronotropy in mouse embryonic stem cell-derived cardiomyocytes.

Cardiomyocytes derived from embryonic stem cells (ESCM) have potential both as an experimental model for investigating cardiac physiology and as a source for tissue repair. For both reasons it is important to characterise the responses of these cells, and one of the key modulators of contraction is the beta-adrenergic system. We therefore undertook a detailed study of the response of the spontaneous beating rate of ESCM to beta-adrenoceptor (betaAR) stimulation. Embryoid bodies (EBs) were generated from murine ES line E14Tg2a by the hanging drop method, followed by plating. Spontaneously beating areas were seen starting from 9-14 days after differentiation: the experiments described here were performed on EBs between developmental day 19 and 48. Beating cell layers were seeded with charcoal to allow tracking of movement by a video-edge detection system. Experiments were performed in physiological medium containing 1 mM Ca2+ at 37 degrees C. Isoprenaline (Iso) increased beating rate with an EC50 value of 52 nM. Iso (0.3 microM) increased basal rate from 67 +/- 7 beats per minute (bpm) to 138 +/- 18 bpm, P < 0.001, n = 22. At earlier developmental time points the response to Iso was not maintained through 5 min exposure; this spontaneous desensitisation only being observed before day 36. A repeat application of Iso after a wash period of 20 min produced reproducible effects on beating rate. Subtype dependence of the betaAR response was determined by comparing an initial response with a second in the presence of selective beta1- or beta2AR antagonists. In the presence of the specific beta1AR-blocker CGP 20712A (300 nM) the increase in rate with Iso was reduced from 207 +/- 42% of basal to 128 +/- 13%, P < 0.01. With the beta2AR-blocker ICI 118,551 (50 nM) there was no significant change in Iso response. Exposure to the muscarinic agonist, carbachol (10 microM), inhibited the increase in frequency mediated by isoprenaline, but had mixed stimulatory and inhibitory effects on basal rate. This study extends the characterisation of ESCM as a preparation for studying receptor pharmacology, and indicates that the beta1AR is the predominant subtype mediating increases in contraction rate in murine ESCM.

Autocrine catecholamine biosynthesis and the beta-adrenoceptor signal promote pigmentation in human epidermal melanocytes.

Earlier it has been shown that human proliferating/undifferentiated basal keratinocytes hold the full capacity for autocrine catecholamine synthesis/degradation and express beta2-adrenoceptors (beta2-AR). In this report, we show that human melanocytes also express all of the mRNA and enzymes for autocrine synthesis of norepinephrine but fail to produce epinephrine. So far, it was established that human melanocytes express alpha1-AR which are induced by norepinephrine yielding the inosine triphosphate diacylglycerol signal. The presence of catecholamine synthesis and the beta2-AR signal escaped definition at that time. Using RT-PCR, immunofluorescence and radioligand binding with the beta2-AR antagonist (-)-[3H]CGP 12177, we show here that human melanocytes express functional beta2-AR (4230 receptors per cell) with a Bmax at 129.3 and a KD of 3.19 nM but lack beta1-AR expression. beta2-AR stimulation with epinephrine 10(-6) M and salbutamol 10(-6)-10(-5) M yielded a strong cyclic adenosine monophospate (cAMP) response in association with upregulated melanin production. Taken together these results indicate that the biosynthesis and release of epinephrine (10(-6) M) by surrounding keratinocytes can provide the cAMP response leading to melanogenesis in melanocytes via the beta2-AR signal. Moreover, the discovery of this catecholaminergic cAMP response in melanocytes adds a new source for this important second messenger in melanogenesis.

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation.

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.

Attenuation of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced rhabdomyolysis with alpha1- plus beta3-adrenoreceptor antagonists.

1. Studies were designed to examine the effects of alpha(1) (alpha(1)AR)- plus beta(3)-adrenoreceptor (beta(3)AR) antagonists on 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced hyperthermia and measures of rhabdomyolysis (creatine kinase (CK)) and renal function (blood urea nitrogen (BUN) and serum creatinine (sCr)) in male Sprague-Dawley rats. 2. MDMA (40 mg x kg(-1), s.c.) induced a rapid and robust increase in rectal temperature, which was significantly attenuated by pretreatment with the alpha(1)AR antagonist prazosin (100 microg x kg(-1), i.p.) plus the beta(3)AR antagonist SR59230A (5 mg x kg(-1), i.p.). 3. CK levels significantly increased (peaking at 4 h) after MDMA treatment and were blocked by the combination of prazosin plus SR59230A. 4. At 4 h after MDMA treatment, BUN and sCr levels were also significantly increased and could be prevented by this combination of alpha(1)AR- plus beta(3)AR-antagonists. 5. The results from this study suggest that alpha(1)AR and beta(3)AR play a critical role in the etiology of MDMA-mediated hyperthermia and subsequent rhabdomyolysis.

Reoxygenation after severe hypoxia induces cardiomyocyte hypertrophy in vitro: activation of CREB downstream of GSK3beta.

In vivo, left ventricular remodeling after myocardial infarction involves hypertrophy generally attributed to increased cardiac workload. We hypothesized that hypoxia/reoxygenation directly induces cardiomyocyte hypertrophy and studied several participating kinases and transcription factors in isolated cardiomyocytes. Hypoxia for 6 h followed by 42 h reoxygenation induced cardiomyocyte hypertrophy assessed by 3H leucine incorporation and immunohistochemistry. Inhibition of reactive oxygen species (ROS), serine/threonine kinase AKT, and ERK abolished reoxygenation-induced hypertrophy. In addition, a beta2-adrenergic receptor (beta2-AR) antagonist, as well as Gi inhibitor pertussis toxin, blocked reoxygenation-induced hypertrophy. Hypoxia for 6 h increased transcription factors CREB, NF-kappaB, and GATA DNA binding activities. However, only CREB DNA-binding was sustained during reoxygenation. Inhibition of PI3-kinase, ERK, and PKA abrogated reoxygenation-induced CREB DNA-binding without affecting CREB serine-133 phosphorylation. These same pathways were found to regulate hypoxia/reoxygenation-induced GSK3beta kinase activity and CREB serine-129 de-phosphorylation. GSK3beta mutants resistant to phosphorylation blocked the stimulation of CRE-dependent transcription induced by hypoxia/reoxygenation. Transfection of cardiomyocytes with a dominant-negative mutant of CREB abrogated hypoxia/reoxygenation-induced hypertrophy. We suggest that hypoxia/reoxygenation induces cardiomyocyte hypertrophy through CREB activation. Inactivation of GSK3beta by hypoxia/reoxygenation, possibly integrating PI3-kinase and ERK pathways downstream of beta2-AR and ROS, is a prerequisite for CRE-dependent transcription. Transient hypoxia may contribute to cardiac hypertrophy in ischemic heart disease independent of cardiac workload.

Effects of acute and chronic administration of beta-adrenoceptor ligands on airway function in a murine model of asthma.

The clinical effects of treatment with beta-adrenoceptor (beta-AR) agonists and antagonists in heart failure vary with duration of therapy, as do the effects of beta-AR agonists in asthma. Therefore, we hypothesized that chronic effects of "beta-blockers" in asthma may differ from those observed acutely. We tested this hypothesis in an antigen (ovalbumin)-driven murine model of asthma. Airway resistance responses (Raw) to the muscarinic agonist methacholine were measured by using the forced oscillation technique. In comparison with nontreated asthmatic mice, we observed that: (i) The beta-AR antagonists nadolol or carvedilol, given as a single i.v. injection (acute treatment) 15 min before methacholine, increased methacholine-elicited peak Raw values by 33.7% and 67.7% (P < 0.05), respectively; when either drug was administered for 28 days (chronic treatment), the peak Raw values were decreased by 43% (P < 0.05) and 22.9% (P < 0.05), respectively. (ii) Chronic treatment with nadolol or carvedilol significantly increased beta-AR densities in lung membranes by 719% and 828%, respectively. (iii) Alprenolol, a beta-blocker with partial agonist properties at beta-ARs, behaved as a beta-AR agonist, and acutely reduced peak Raw value by 75.7% (P < 0.05); chronically, it did not alter Raw. (iv) Salbutamol, a beta-AR partial agonist, acutely decreased peak Raw by 41.1%; chronically, it did not alter Raw. (v) None of the beta-blockers produced significant changes in eosinophil number recovered in bronchoalveolar lavage. These results suggest that beta-AR agonists and beta-blockers with inverse agonist properties may exert reciprocating effects on cellular signaling dependent on duration of administration.

Protein kinase C regulates functional coupling of beta1-adrenergic receptors to Gi/o-mediated responses in cardiac myocytes.

The effect of protein kinase C (PKC) activation on beta1-adrenergic receptor (beta1-AR) regulation of the cardiac L-type Ca2+ current (ICa,L) was studied using the whole-cell patch clamp technique. Treatment of guinea pig ventricular myocytes with phorbol-12,13-dibutyrate (PDBu) caused a significant decrease in ICa,L sensitivity to stimulation by submaximal beta1-AR activation using isoproterenol (Iso). This decrease in sensitivity was also associated with the ability of higher concentrations of Iso to directly inhibit the stimulatory response. PDBu treatment produced similar effects on H2 histamine receptor-mediated ICa,L responses. In the presence of PDBu, higher concentrations of Iso inhibited the histamine stimulated ICa,L, and this effect was blocked by a selective beta1-AR antagonist. Higher concentrations of histamine also inhibited the Iso stimulated ICa,L, and this effect was blocked by a selective H2 receptor antagonist. The effects of PDBu were blocked by the PKC inhibitor bisindolylmaleimide I, and they were not mimicked by the inactive phorbol ester 4alpha-phorbol-12,13-didecanoate. The inhibitory effects of Iso and histamine were significantly reduced when Gi/o mediated responses were blocked with pertussis toxin. These results suggest that PKC promotes coupling of cardiac beta1-adrenergic and H2 histamine receptors to Gi/o mediated inhibitory responses.

Delayed but sustained induction of mitogen-activated protein kinase activity is associated with beta-adrenergic receptor-mediated morphological differentiation of astrocytes.

Astroglial beta-adrenergic receptors (beta-ARs) are functionally linked to regulate cellular morphology. In primary cultures, the beta-AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process-bearing, mature stellate cells by 48 h, an effect that can be blocked by the beta-AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP-induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal-regulated kinase; p-ERK) level: an initial decline in p-ERK level followed by a sustained induction at 12-24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p-ERK. A long-lasting activation of p-ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.

The third intracellular loop and the carboxyl terminus of beta2-adrenergic receptor confer spontaneous activity of the receptor.

It is well established that the beta2-adrenergic receptor (beta2-AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related beta1-AR subtype. To identify the potential domain(s) of beta2-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (beta1/beta2-Li3) or the carboxyl terminus (beta1/beta2-CT) or both domains (beta1/beta2-Li3CT) of beta1-AR are replaced by the corresponding parts of the beta2-AR. Using adenoviral gene transfer, we individually expressed these beta1/beta2-AR chimeras in mouse cardiomyocytes lacking both native beta1-AR and beta2-AR (beta1/beta2 double knockout), and examined their possible spontaneous activities. Overexpression of these beta1/beta2-AR chimeras markedly elevated basal cAMP accumulation and myocyte contractility in the absence of agonist stimulation compared with those infected by a control adenovirus expressing beta-galactosidase or an adenovirus expressing wild type beta1-AR. These effects were fully reversed by a beta2-AR inverse agonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 5 x 10-7 M), regardless of inhibition of Gi with pertussis toxin, but not by a panel of beta1-AR antagonists, including [2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate (CGP20712A), betaxolol, bisoprolol, and metoprolol. Furthermore, we have shown that the C-terminal postsynaptic density 95/disc-large/ZO-1 (PDZ) motif of beta1-AR is not responsible for the lack of beta1-AR spontaneous activation, although it has been known that the beta1-AR PDZ motif prevents the receptor from undergoing agonist-induced trafficking and Gi coupling in cardiomyocytes. Taken together, the present results indicate that both the third intracellular loop and the C terminus are involved in beta2-AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.