β2-肾上腺素受体在新生大鼠心肌成纤维细胞中的分布及其促增殖作用

为了明确β-肾上腺素受体(AR)亚型在新生大鼠心肌成纤维细胞中的分布及其在成纤维细胞增殖反应中的作用,采用放射配体结合实验和[3H]-thymidine掺人法检测了新生大鼠心肌成纤维细胞的β-AR密度和DNA合成速率。结果显示,在培养心肌细胞和心肌成纤维细胞中β-AR密度(Bmtax)和解离常数(Kp)无显著性差异;竞争抑制曲线分析结果提示,心肌成纤维细胞对CGP 20712A和ICI ll8551单位点拟合均显著优于两位点拟合(P＜0．01),表现为对选择性β1-AR拮抗剂CGP 20712A的低亲和性(IC50值：10．1μmol/L)和对选择性β2-AR拮抗剂ICI 118551的高亲和性(IC50值：0.147μmol/L)。异丙肾上腺素(ISO)促心肌成纤维细胞增殖作用可被ICI 118551和心得安(非选择性β-AR拮抗剂)完全抑制,而CGP20812A则无此作用。上述结果提示,在培养心肌成纤维细胞中β-AR亚型占绝对优势,并且ISO引起的心肌成纤维细胞增殖反应是由β2-AR介导的。     

beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists.     

Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes

Catecholamine-induced and beta-adrenergic receptor (beta-AR)-mediated thermogenesis in skeletal muscle is a significant component of whole-body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of beta-AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6-24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9-cis retinoic acid, and mimicked by isoproterenol and salbutamol (beta2-AR agonists), but abolished by propranolol and ICI-118,551 (beta2-AR antagonists). Pharmacological and mRNA analyses revealed the existence of beta2-AR, but not beta1- and beta3-ARs, in L6 myotubes. These results suggested that catecholamines up-regulate UCP2 and UCP3 expression through direct action on the beta2-AR in skeletal muscle.     

Chronotropic response to (+/-)-CGP12177 in right atria of stressed rats

Foot-shock stress changes the sensitivity of the rat right atria to beta1- and beta2-adrenoceptor (AR) agonists. We investigated whether the same stress protocol also changes the atrial sensitivity to the non conventional agonist, (+/-)-CGP12177. Concentration-response curves to (+/-)-CGP12177, a beta1- and beta2-adrenoceptor antagonist with agonist properties at the putative beta4-adrenoceptors, were obtained in the absence and presence of propranolol (200 nM or 2 microM), CGP20712A 10 nM plus ICI118,551 50 nM, or CGP20712A (1 microM or 3 microM), in right atria from rats submitted to three daily foot-shock sessions (120 mA pulses of 1.0 s duration applied at random intervals of 5-25 s over 30 min) and killed after the third session. The pD2 for (+/-)-CGP12177 was not influenced by foot-shock stress. The stimulant effect of (+/-)-CGP12177 was resistant to blockade by 200 nM and 2 microM (+/-)-propranolol, and to combined blockade by CGP20712A and IC1118,551. However, in right atria from stressed rats given 200 nM propranolol, the concentration-response curve to the agonist was shifted 2.0-fold to the right. CGP20712A shifted the concentration-response curve to (+/-)-CGP12177 to the right by 4.6- (1 microM) and 19-fold (3 microM) in atria of control rats, and by 2.2- (1 microM) and 43-fold (3 microM) in atria of stressed rats. Maximum response to CGP12177 was not affected by propranolol or CGP20712A in concentrations ranging from 0.1 nM to 10 microM. These results show that the chronotropic effect of (+/-)-CGP12177 is mediated by atypical beta4-adrenoceptors. In constrast with to beta1-and (or) beta2-AR, this receptor is resistant to the effects of foot-shock stress, suggesting that the putative beta4-AR is a different receptor from a low affinity state of beta1-adrenoceptor, as previously proposed, unless both proposed isoforms of beta1-adrenoceptor show independent stress-induced behavior.     

Coupling of beta-adrenergic receptors to cardiac L-type Ca2+ channels: preferential coupling of the beta1 versus beta2 receptor subtype and evidence for PKA-independent activation of the channel.

Beta1- and beta2-adrenergic receptors (beta-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels (CaCh). To investigate the contribution of each beta-AR subtype in AC and CaCh coupling, we stably expressed cardiac CaCh alpha1 and beta2 subunits along with either beta1-AR or beta2-AR in CHW fibroblasts. Co-expression of either beta-AR with CaCh subunits conferred responsiveness of AC and CaCh to isoproterenol (ISO), which was not observed in non-transfected cells. ISO-promoted cAMP formation occurred at a lower EC50 through the beta2-AR than through the beta1-AR (0.13 +/- 0.01 vs. 0.6 +/- 0.14 nM). In contrast, activation of CaCh was more efficacious via the beta1-AR than the beta2-AR (EC50 for CaCh activation = 238 +/- 33 vs. 1057 +/- 113 nM). Pre-treatment with pertussis toxin (PTX) had no effect upon the responsiveness of either cAMP formation or CaCh activation through either receptor. We conclude (1) that beta1-ARs exhibit preferential coupling to CaCh activation, versus that observed for the beta2-AR; (2) that this preferential coupling cannot be explained solely by cAMP-dependent processes; and (3) that the relative attenuation of beta2-AR-promoted CaCh activation is not due to receptor coupling to PTX-sensitive G proteins. Thus, it is likely that other subtype-specific, cAMP-independent coupling of the beta-AR to CaCh is present.     

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)     

G(i)-dependent suppression of beta(1)-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs

It has been suggested that there is a preferential coupling in heart muscle between the inhibitory G protein (G(i)) and the beta(2)-subtype of the beta-adrenergic receptor (beta-AR), since pertussis toxin (which inactivates G(i)) reveals latent beta(2)-ARs in rat and mouse myocytes. We have previously shown that guinea pigs treated with norepinephrine (NE) for 7 days have myocytes that are desensitized to beta-AR-agonist stimulation, and that pertussis toxin restores these responses. The purpose of the present investigation was to determine whether pertussis toxin specifically upregulated beta(2)-ARs in myocytes from NE-treated guinea pigs. The sole beta-AR subtype in control guinea pig myocytes was confirmed as beta(1)-AR by radioligand binding, single-cell autoradiography, and concentration-response curves to isoproterenol in contracting myocytes. In contrast, a minor pool of beta(2)-ARs was observed in rat myocytes by use of the same methods. NE treatment decreased the maximum isoproterenol response (relative to high Ca(2+)) from 0.89 +/- 0.06 to 0.58 +/- 0.08 (n = 7, P < 0.01) and the pD(2) (-log EC(50)) from 8.8 +/- 0.2 to 7.5 +/- 0.2 (n = 7, P < 0.01). Pertussis toxin treatment increased the isoproterenol-to-Ca(2+) ratio to 0.88 +/- 0.04 (n = 6, P < 0.05) and the pD(2) to 8.6 +/- 0.3 (P < 0.01). This was not mediated by increases in either number or function of beta(2)-ARs. G(i) is therefore able to modulate beta(1)-AR responses in guinea pig myocytes.     

Overexpression of beta2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.     

Expression of human alpha 2-adrenergic receptors in adipose tissue of beta 3-adrenergic receptor-deficient mice promotes diet-induced obesity

Catecholamines play an important role in controlling white adipose tissue function and development. beta- and alpha 2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte alpha 2/beta-AR balance in obesity, and it has been proposed that increased alpha 2-ARs in adipose tissue with or without decreased beta-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte alpha 2/beta-AR balance was genetically manipulated in mice. Human alpha 2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (-/-) or heterozygous (+/-) for a disrupted beta 3-AR allele. Mice expressing alpha 2-ARs in fat, in the absence of beta 3-ARs (beta 3-AR -/- background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of alpha2-ARs, the absence of beta 3-ARs, and a high fat diet. Of note, obese alpha 2-transgenic beta 3 -/- mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased alpha 2/beta-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (alpha 2 and beta 3-AR) and diet interact to influence fat mass.     

Synthesis and first in vivo evaluation of new selective high affinity beta1-adrenoceptor radioligands for SPECT based on ICI 89,406

The results of cardiac biopsies suggest that myocardial beta1-adrenoceptor (AR) density is reduced in patients with chronic heart failure, while changes in cardiac beta2-ARs vary. A technique for visualization and quantification of beta1-AR populations rather than total beta-AR densities in the human heart would be of great clinical interest. Molecular imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET), with appropriate radiopharmaceuticals offer the possibility to assess beta-AR density noninvasively in humans, but to date, neither a SPECT nor a PET-radioligand is clinically established for the selective imaging of cardiac beta1-ARs. The aim of this study was to design a high affinity selective beta1-AR radioligand for the noninvasive in vivo imaging of cardiac beta1-AR density in man using SPECT. Based on the well-known selective beta1-AR antagonist, ICI 89,406, both the racemic iodinated target compound 11a and the (S)-enantiomer 15a were synthesized. Competition studies using the nonselective AR ligand, [(125)I]iodocyanopindolol ([(125)I]ICYP), and ventricular membrane preparations from mice showed that 11a and 15a possess higher beta1-AR affinities (up to 265-fold) and beta1-AR selectivities (up to 245-fold) than ICI 89,406. Encouraged by these results, the radioiodinated counterparts of racemic 11a (11b: (125)I, 11c: (123)I) and (S)-configurated 15a (15b: (125)I, 15c: (123)I) were synthesized. The target compounds were evaluated in rats. Biodistribution and metabolism studies in rats indicated that there is a specific heart uptake of 11b-c and especially 15b-c accompanied by rapid metabolism of the radioligands. Therefore, radioiodinated 11c and 15c appeared to be unpromising SPECT-radioligands for assessing beta1-ARs in vivo in the rat. However, the rat may metabolize beta-AR ligands more rapidly than other species as demonstrated for (S)-[(11)C]CGP 12177, a radioligand structurally related to 11a-c and 15a-c. Therefore further studies in a different animal model will be carried out.     

Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different beta-adrenoceptor subtypes

The three known subtypes of beta-adrenoreceptors (beta(1)-AR, beta(2)-AR, and beta(3)-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the beta-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the beta(3)-adrenoreceptor gene (beta(3)-AR KO) were treated with vehicle or the beta(1)/beta(2)-AR selective antagonist, propranolol (20 microgram/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 microgram/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and beta(3)-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in beta(3)-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from beta(3)-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the beta(3)-AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the beta(1)/beta(2)-ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.     

The beta1-adrenoceptor site activated by CGP12177 varies in behavior according to the estrous cycle phase and stress

The aim of this work was to assess whether stress and estrous cycle phases affected the beta1-adrenoceptor (beta1-AR) site activated by CGP12177 in the right atria of rats. The chronotropic response to CGP12177 in the absence or presence of antagonists was determined in atria from rats submitted to one daily foot-shock session for 3 consecutive days. Blood was collected for hormonal assays. The pD2 for CGP12177 in atria from females was lower than in atria from males and was unaltered by stress or the estrous cycle. Propranolol (200 nM) or CGP20712A (3 microM) shifted the concentration-response curves to CGP12177 to the right in control and stressed estrus or control diestrus rats. Atria from stressed diestrus rats were resistant to blockade by propranolol or CGP20712A, indicating that the effect of beta-adrenoceptor antagonists on the response to CGP12177 is influenced by estrous cycle phases. The stress-induced increase in serum corticosterone levels was independent of the estrous cycle or gender, but the estradiol/progesterone ratio was affected differently in the two groups of female rats. In the diestrus group, serum estradiol levels decreased after the first foot-shock session and remained low until the day of sacrifice, whereas in the estrus group the serum levels of estradiol did not decrease after stress and peaked on the second day, which corresponded to proestrus. These data do not indicate whether there is a direct or indirect effect of stress hormones and (or) sex steroids on cardiac beta1-AR sensitivity. However, they do show that the classic and low-affinity binding sites of the beta1-AR are independently regulated and that the beta1-AR atypical site affinity for antagonists depends on the estrous cycle.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Regulation of the black bullhead hepatic beta-adrenoceptors

Black bullhead catfish (Ameiurus melas) were exposed to air for 1 h to examine the effect of an acute stress on the distribution and function of the hepatic beta-adrenoceptors (beta-ARs). Air exposure significantly reduced both adrenaline (ADR)- and noradrenaline (NADR)-stimulated glucose production in isolated hepatocytes with no effect on either receptor affinity (K(d)) or number of binding sites (B(max)). A 24 h exposure of isolated hepatocytes to the beta-agonist isoproterenol also had no significant impact on either binding parameter. Competition studies using selective agonists and antagonists suggest that the hepatic beta-AR in this species is pharmacologically beta(2)-like. However in addition to the beta(2)-AR, molecular evidence provides support for the existence of hepatic beta-ARs that phylogenetically group with the beta(3)-ARs and the beta(1)-ARs. Despite the presence of several potential phosphorylation sites in the third intracellular loop and cytoplasmic tail of the bullhead beta(2)-AR, no significant changes were observed in the binding parameters. While physiological data supports the presence of only a single subtype, molecular data supports the existence of multiple beta-AR subtypes in this species. The mechanisms thought to regulate mammalian beta-ARs exist in the bullhead ARs reported here but these mechanisms are not as effective in this fish system as in mammals.     

Inhibition of spontaneous beta 2-adrenergic activation rescues beta 1-adrenergic contractile response in cardiomyocytes overexpressing beta 2-adrenoceptor

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.     

Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes.

In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K(+) current (I(K,ATP)). In addition, beta-adrenergic receptor (beta-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of beta-AR stimulation acts via beta(1)- and/or beta(2)-ARs and whether it is mediated via NO signaling. 0.1 microM ISO plus ICI 118,551 (ISO-beta(1)-AR stimulation) or ISO plus atenolol (ISO-beta(2)-AR stimulation) both increased L-type Ca(2+) current (I(Ca,L)) markedly, but only ISO-beta(2)-AR stimulation mediated ACh-induced activation of I(K,ATP). 1 microM zinterol (beta(2)-AR agonist) also increased I(Ca,L) and mediated ACh-activated I(K,ATP). Inhibition of NO synthase (10 microM L-NIO), guanylate cyclase (10 microM ODQ), or cAMP-PKA (50 microM Rp-cAMPs) attenuated zinterol-induced stimulation of I(Ca,L) and abolished ACh-activated I(K,ATP). Spermine-NO (100 microM; an NO donor) mimicked beta(2)-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 microM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of I(Ca,L). Measurements of intracellular NO ([NO](i)) using the fluorescent indicator DAF-2 showed that ISO-beta(2)-AR stimulation or zinterol increased [NO](i). L-NIO (10 microM) blocked ISO- and zinterol-induced increases in [NO](i). ISO-beta(1)-AR stimulation failed to increase [NO](i). Inhibition of G(i)-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO](i). Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i). We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling. Only beta(2)-ARs acting exclusively via NO signaling mediate ACh-induced activation of I(K,ATP). NO signaling also contributes to beta(2)-AR stimulation of I(Ca,L). The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.     

Conditioning of beta(1)-adrenoceptor effect via beta(2)-subtype on L-type Ca(2+) current in canine ventricular myocytes

We investigated the roles of beta(1)- and beta(2)-receptors (beta-AR) in adrenergic enhancement of L-type Ca(2+) current (I(CaL)) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-I(CaL) relationship (CR), respectively. alpha(1)-AR inhibition with prazosin and beta(2)-AR stimulation with zinterol or l-epinephrine shifted the CR of l-norepinephrine leftward. Zinterol (50 nM) and l-epinephrine (10 nM), but not prazosin, altered the biphasic CR of l-norepinephrine to a monophasic CR. Zinterol and l-epinephrine applied after l-norepinephrine had no effect on I(CaL). beta(2)-AR inhibition with ICI-118551 reduced the E(max) of isoproterenol and l-norepinephrine by 60% and abolished the augmentation of l-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the I(CaL) response to beta(1)-AR stimulation but abolished the enhancement via beta(2)-AR. Zinterol augmented the enhancement of I(CaL) by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective beta(2)-AR stimulation does not increase I(CaL) but enhances adenylyl cyclase activity when stimulated via beta(1)-AR and with forskolin. beta(2)-AR activity preconditions adenylyl cyclase for beta(1)-AR stimulation.     

Pharmacological evidence for beta2-adrenoceptor in right atria from stressed female rats.

The purpose of the present study was to demonstrate a physiological response to TA2005, a potent m-adrenoceptor (beta2-AR) selective agonist, in right atria isolated from stressed female rats under the influence of the estrous cycle. We obtained concentration-response curves to the agonist in the presence and in the absence of selective antagonists in right atria isolated from female rats submitted to three daily foot-shock sessions (30 min duration, 120 pulses of 1.0 mA, 1.0 s, applied at random intervals of 5-25 s) and sacrificed at estrus or diestrus. Our results showed that the pD2 values of TA2005 were not influenced by estrous cycle phase or foot-shock stress. However, in right atria from stressed rats sacrificed during diestrus, the concentration-response curve to TA2005 was biphasic, with a response being obtained at concentrations of 0.1 nM, whereas during estrus no response was observed at doses lower than 3 nM. ICI 1118,551, a beta2-AR antagonist, abolished the response to nanomolar concentrations of TA2005 in right atria from stressed rats at diestrus, with no changes in agonist pD2 values in right atria from control rats (7.47 +/- 0.09, p > 0.05) but a 3-fold decrease in pD2 values of TA2005 in right atria from foot shock stressed rats (7.90 +/- 0.07, p < or = 0.05). Concentration-response curves to TA2005 in the presence of ICI118,551 were best fitted by a one-site model equation. The beta1-AR antagonist, CGP20712A, shifted to the right only the second part of the concentration-response curves to the agonist, unmasking the putative beta2-AR-mediated response to the agonist in tissues isolated from stressed rats at diestrus. Under this condition, concentration-response curves to the agonist were best fitted by a two-site model equation. pD2 and maximum response of TA2005 interaction with beta1- and putative beta2-adrenoceptor components were calculated. Schild analyses gave a pK(B) value for CGP20712A that was typical for the interaction with beta1-AR in each experimental group. pK(B) values for ICI118,551 could not be obtained in stressed rats sacrificed at diestrus since Schild plot slopes were lower than 1.0. In right atria from control rats, ICI118,551 pK(B) values were similar to reported values for the interaction of the antagonist with beta1-AR. These results confirm that a heterogenous beta-AR population mediating the chronotropic response to catecholamines can be demonstrated in right atria from foot shock stressed female rats sacrificed at diestrus. The stress-induced response seems to be mediated by the beta2-AR subtype. Right atria from rats sacrificed during estrus are protected against stress-induced alterations on the homogeneity of beta-AR population.