Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.     

Greater glycogen utilization during 1- than 2-adrenergic receptor stimulation in the isolated perfused rat heart

Differences in energy metabolism during beta(1)- and beta(2)-adrenergic receptor (AR) stimulation have been shown to translate to differences in the elicited functional responses. It has been suggested that differential access to glycogen during beta(1)- compared with beta(2)-AR stimulation may influence the peak functional response and modulation of the response during sustained adrenergic stimulation. Interleaved (13)C- and (31)P-NMR spectroscopy was used during beta(1)- and beta(2)-AR stimulation at matched peak workload (2.5 times baseline) in the isolated perfused rat heart to monitor glycogen levels, phosphorylation potential, and intracellular pH. Simultaneous measurements of left ventricular (LV) function [LV developed pressure (LVDP)], heart rate (HR), and rate-pressure product (RPP = LVDP x HR) were also performed. The heart was perfused under both substrate-free (SF) conditions and with exogenous glucose (G). The greater glycogenolysis was observed during beta(1)- than beta(2)-AR stimulation with G (54% vs. 38% reduction, P = 0.006) and SF (92% vs. 79% reduction, P = 0.04) perfusions. The greater beta(1)-AR-mediated glycogenolysis was correlated with greater ability to sustain the initial contractile response. However, with SF perfusion, the duration of this ability was limited: excessive early glycogen depletion caused an earlier decline in LVDP and phosphorylation potential during beta(1)- than beta(2)-AR stimulation. Therefore, endogenous glycogen stores are depleted earlier and to a greater extent, despite a slightly weaker overall inotropic response, during beta(1)- than beta(2)-AR stimulation. These findings are consistent with beta(1)-AR-specific PKA-dependent glycogen phosphorylase kinase signaling.     

Targeted disruption of the beta2 adrenergic receptor gene.

beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.     

Nandrolone-pretreatment enhances cardiac beta(2)-adrenoceptor expression and reverses heart contractile down-regulation in the post-stress period of acute-stressed rats

To investigate whether nandrolone decanoate (ND)-pretreatment can modulate (1) beta-adrenoceptor expression and (2) myocardial contractility in response to beta-adrenoceptors stimulation with isoproterenol (ISO), in hearts of both normal and stressed rats. Rats were treated with 15 mg/(kgday) of Deca-Durabolin (ND, 1 ml i.m.) or with vehicle (oil) for 14 days. The day after the last injection, the dose-response to ISO (1 x 10(-8), 5 x 10(-8) and 10(-7)M), was studied in isolated rat hearts harvested from unstressed animals (unstressed+vehicle (control) or unstressed+ND) or from stressed animals (stressed+vehicle or stressed+ND): acute stress protocol consisted in restrain for 1h immediately before sacrifice. ND-pretreatment increased beta(2)-adrenoceptor expression. In baseline conditions all hearts had a similar left ventricular developed pressure (LVDP) and maximum rate of increase of LVDP (dP/dt(max)). In hearts of unstressed+vehicle or unstressed+ND, ISO caused a similar increase in LVDP (+90-100%) and dP/dt(max) (+120-150%). However, hearts of stressed+vehicle animals showed a marked depression of inotropic response to ISO (i.e. for ISO 1 x 10(-8),-55% in LVDP response versus unstressed). Yet, in hearts of stressed+ND-animals the effect of stress was reversed, showing the highest response to ISO (i.e. for ISO 1 x 10(-7), +30% LVDP response versus unstressed). The ND-induced beta(2)-adrenoceptor overexpression does not affect ISO-response in unstressed animals. However, acute stress induces a down-regulation of ISO-response, which is reversed by ND-pretreatment. Since the physiological post-stress down-regulation of adrenergic-response is absent after nandrolone treatment, the heart may be exposed to a sympathetic over-stimulation. This might represent a risk for cardiovascular incidents in anabolic steroid addicts under stressing conditions.     

Gender differences on the effects of aging on cardiac and peripheral adrenergic stimulation in old conscious monkeys

We examined the effects of gender and aging on cardiac and peripheral hemodynamic responses to beta-adrenergic receptor (beta-AR) stimulation in young (male = 5.9 +/- 0.4 yr old and female = 6.5 +/- 0.7 yr old) and old (male = 19.8 +/- 0.7 yr old and female = 21.2 +/- 0.2 yr old) conscious monkeys (Macaca fascicularis), chronically instrumented for measurements of left ventricular (LV) and arterial pressures as well as cardiac output. Baseline LV pressure, the first derivative of LV pressure (LV dP/dt), cardiac index, mean arterial pressure, total peripheral resistance (TPR), and heart rate in conscious monkeys were not different among the four groups. Increases in LV dP/dt in response to 0.1 microg/kg isoproterenol (Iso) were diminished (P < 0.05) in old males (+99 +/- 11%) compared with young males (+194 +/- 18%). In addition, the inotropic responses to norepinephrine (NE) and forskolin (FSK) were significantly depressed (P < 0.05) in old males. Iso-induced reductions of TPR were less (P < 0.05) in old males (-28 +/- 2%) than in young males (-49 +/- 2%). The changes of TPR in response to NE and FSK were also significantly attenuated (P < 0.05) in old males. However, the LV dP/dt responses to BAY y 5959 (15 microg. kg-1. min-1), a Ca2+ channel promotor independent of beta-AR signaling, were not significantly different between old and young males. In contrast to results in male monkeys, LV dP/dt and TPR responses to Iso, NE, and FSK in old females were similar to those observed in young females. Thus both cardiac contractile and peripheral vascular dynamic responses to beta-AR stimulation are preserved in old female but not old male monkeys. This may explain, in part, the reduced cardiovascular risk in the older female population.     

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.     

Enhanced in vivo and in vitro contractile responses to beta(2)-adrenergic receptor stimulation in dogs susceptible to lethal arrhythmias.

The response to beta-adrenergic receptor (beta-AR) stimulation was evaluated in both isolated cardiomyocytes (video edge detection) and the intact animal (echocardiography) in dogs either susceptible (S) or resistant (R) to ventricular fibrillation induced by a 2-min coronary occlusion during the last minute of exercise. In the intact animal, velocity of circumferential fiber shortening (Vcf) was evaluated both before (n = 27, S = 12 and R = 15) and after myocardial infarction. Before infarction, increasing doses of isoproterenol provoked similar contractile and heart rate responses in each group of dogs. Either beta(1)-AR (bisoprolol) or beta(2)-AR (ICI-118551) antagonists reduced the isoproterenol response, with a larger reduction noted after the beta(1)-AR blockade. In contrast, after infarction, isoproterenol induced a significantly larger Vcf and heart rate response in the susceptible animals that was eliminated by beta(2)-AR blockade. The single-cell isotonic shortening response to isoproterenol (100 nM) was also larger in cells obtained from susceptible compared with resistant dogs and was reduced to a greater extent by beta(2)-AR blockade in the susceptible dog myocytes (S, -48%, n = 6; R, -15%, n = 9). When considered together, these data suggest that myocardial infarction provoked an enhanced beta(2)-AR response in susceptible, but not resistant, animals.     

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.     

Enhanced isoproterenol-induced cardiac hypertrophy in transgenic rats with low brain angiotensinogen

We have previously shown that a permanent deficiency in the brain renin-angiotensin system (RAS) may increase the sensitivity of the baroreflex control of heart rate. In this study we aimed at studying the involvement of the brain RAS in the cardiac reactivity to the beta-adrenoceptor (beta-AR) agonist isoproterenol (Iso). Transgenic rats with low brain angiotensinogen (TGR) were used. In isolated hearts, Iso induced a significantly greater increase in left ventricular (LV) pressure and maximal contraction (+dP/dt(max)) in the TGR than in the Sprague-Dawley (SD) rats. LV hypertrophy induced by Iso treatment was significantly higher in TGR than in SD rats (in g LV wt/100 g body wt, 0.28 +/- 0.004 vs. 0.24 +/- 0.004, respectively). The greater LV hypertrophy in TGR rats was associated with more pronounced downregulation of beta-AR and upregulation of LV beta-AR kinase-1 mRNA levels compared with those in SD rats. The decrease in the heart rate (HR) induced by the beta-AR antagonist metoprolol in conscious rats was significantly attenuated in TGR compared with SD rats (-9.9 +/- 1.7% vs. -18.1 +/- 1.5%), whereas the effect of parasympathetic blockade by atropine on HR was similar in both strains. These results indicate that TGR are more sensitive to beta-AR agonist-induced cardiac inotropic response and hypertrophy, possibly due to chronically low sympathetic outflow directed to the heart.     

The putative beta4-adrenergic receptor is a novel state of the beta1-adrenergic receptor

The atypical beta3-adrenergic receptor (AR) agonist CGP-12177 has been used to define a novel atypical beta-AR subtype, the putative beta4-AR. Recent evaluation of recombinant beta-AR subtypes and beta-AR-deficient mice, however, has established the identity of the pharmacological beta4-AR as a novel state of the beta1-AR protein. The ability of aryloxypropanolamine ligands like CGP-12177 to independently interact with agonist and antagonist states of the beta1-AR has important implications regarding receptor classification and the potential development of tissue-specific beta-AR agonists.     

Cardiac responsiveness to beta-adrenergic stimulation in experimental anemia.

Cardiac reactivity of beta-adrenergic stimulation was assessed by isoproterenol dose-reponse curves (dose range 0.025-0.4 mug/kg) before and 1 h after the rapid induction of anemia in dogs anesthetized with halothane:N2O:O2. Anemai (hematocrit = 16 +/- 4%) was induced by an isovolumic exchange transfusion with Dextran 70, and was followed by significant increments in cardiac output (+57 +/- 9%), max dP/dt of the left ventricle (+37 +/- 7%), and in peak acceleration of blood flow in the ascending aorta (+46 +/- 13%). Anemia was associated with a significant reduction of the chronotropic responses to all but the lowest dose of isoproterenol. The simultaneously determined inotropic responses (max dP/dt) where the same before and after the induction of anemia. The responses in terms of peak acceleration of aortic blood flow tended to be greater in the anemic than in the control phase, at all dose levels used. These findings indicate that in rapidly induced experimental anemia the heart is capable of responding to marked degrees of beta-adrenergic stimulation, representing a more than two-fold increase in the dP/dt.     

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.     

Spinal vasopressin modulates the reflex cardiovascular response to static contraction.

Recent evidence has demonstrated that arginine vasopressin (AVP) may modulate primary afferent activity of nociceptors in the dorsal horn of the spinal cord. Because nociceptors are group III and IV afferents, spinal AVP also may modulate the activity of group III and IV afferents that cause reflex cardiovascular responses to muscle contraction. Thus, we compared the pressor (mean arterial pressure), myocardial contractile (dP/dt), and heart rate (HR) responses to electrically induced static contraction of the cat hindlimb before and after lumbar intrathecal (IT) injection (L1-L7) of AVP (n = 9), the V1 receptor antagonist d(CH2)5Tyr(Me)AVP (n = 6), the V2 receptor antagonist d(CH2)5[D-Ile2,Ile4,Ala-NH2(9)]AVP (n = 6), and the V2 agonist [Val4,D]AVP (n = 8). After IT injection of AVP (0.1 or 1 nmol) the pressor and contractile responses to static contraction were attenuated by 55 and 44%, respectively. HR was unchanged. Forty-five to 60 min after AVP injection, the contraction-induced pressor and contractile responses were restored to control levels. V1 receptor blockade augmented contraction-induced increases in mean arterial pressure (36%) and dP/dt (49%) but not HR. V2 receptor blockade had no effect on the cardiovascular response to contraction, whereas selective V2 stimulation attenuated the dP/dt (-20%) and HR (-33%) responses but not the pressor response. These results suggest that AVP attenuates the reflex cardiovascular response to contraction by modulating sensory nerve transmission from contracting muscle primarily via a V1 receptor mechanism in the lumbar spinal cord.     

beta2-Adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle.

High doses of the beta2-adrenergic receptor (AR) agonist clenbuterol can induce necrotic myocyte death in the heart and slow-twitch skeletal muscle of the rat. However, it is not known whether this agent can also induce myocyte apoptosis and whether this would occur at a lower dose than previously reported for myocyte necrosis. Male Wistar rats were given single subcutaneous injections of clenbuterol. Immunohistochemistry was used to detect myocyte-specific apoptosis (detected on cryosections via a caspase 3 antibody and confirmed with annexin V, single-strand DNA labeling, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). Myocyte apoptosis was first detected at 2 h and peaked 4 h after clenbuterol administration. The lowest dose of clenbuterol to induce cardiomyocyte apoptosis was 1 microg/kg, with peak apoptosis (0.35 +/- 0.05%; P < 0.05) occurring in response to 5 mg/kg. In the soleus, peak apoptosis (5.8 +/- 2%; P < 0.05) was induced by the lower dose of 10 microg/kg. Cardiomyocyte apoptosis was detected throughout the ventricles, atria, and papillary muscles. However, this damage was most abundant in the left ventricular subendocardium at a point 1.6 mm, that is, approximately one-quarter of the way, from the apex toward the base. beta-AR antagonism (involving propranolol, bisoprolol, or ICI 118551) or reserpine was used to show that clenbuterol-induced myocardial apoptosis was mediated through neuromodulation of the sympathetic system and the cardiomyocyte beta1-AR, whereas in the soleus direct stimulation of the myocyte beta2-AR was involved. These data show that, when administered in vivo, beta2-AR stimulation by clenbuterol is detrimental to cardiac and skeletal muscles even at low doses, by inducing apoptosis through beta1- and beta2-AR, respectively.     

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.     

Molecular modeling of SWR-0342SA, a beta3-selective agonist, with beta1- and beta3-adrenoceptor

The molecular dynamics (MD) simulations study in the formation of the complex between compound SWR-0342SA and beta-ARs suggested that upon binding SWR-0342SA stimulates receptor activation through residues network (Asp104, Leu335 in beta(1)-AR; Asp117, Ser209, Leu303, Ser191 in beta(3)-AR) in an active conformation state. The models suggest that the structural origin of the selectivity of SWR-0342SA to beta(3)-AR vs. beta(1)-AR comes from the following results: (a) the tight interaction between the agonist and the TMs 3, 5, 6 and 2 nd EC loop. Asp117 interacts with the cationic amino group of the agonist molecule. (b) Additional contacts are done with Ser209, Leu303 and Ser191. These results are in good agreement with the binding affinities (pKi values) of SWR-0342SA to beta-AR family expressed in recombinant mammalian cells.     

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.     

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.