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Aminergic G protein-coupled receptors (GPCRs) have been a major focus of pharmaceutical research for many years. Due partly to the lack of reliable receptor structures, drug discovery efforts have been largely ligand-based. The recently determined X-ray structure of the [[beta].sub.2]-adrenergic receptor offers an opportunity to investigate the advantages and limitations inherent in a structure-based approach to ligand discovery against this and related GPCR targets. Approximately 1 million commercially available, 'lead-like' molecules were docked against the [[beta].sub.2]-adrenergic receptor structure. On testing of 25 high-ranking molecules, 6 were active with binding affinities docking | GPCR | inverse agonists | library bias | ligand design

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both [[beta].sub.1]-adrenergic and [M.sub.2] muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is ~ 1.2 [micro]M, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both [[beta].sub.1] and [M.sub.2] receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though [[beta].sub.1] and [M.sub.2] receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors. [beta]-adrenergic receptor signaling; muscarinic receptor signaling; live cell imaging; fluorescence resonance energy transfer; biosensors

Mutating Arg-238 to Glu (R238E) in the switch 3 region of a transducin [alpha] (*T[alpha]) in which 27 aa of the GTPase domain have been replaced with those of the [alpha]-subunit of the inhibitory G protein 1 (Gi1[alpha]), was reported to create an [alpha]-subunit that is resistant to activation by GTP[gamma]S, is devoid of resident nucleotide, and has dominant negative (DN) properties. In an attempt to create a DN stimultory G protein a (Gs[alpha]) with a single mutation we created Gs[alpha]-R265E, equivalent to *T[alpha]-R238E. G[alpha]-R265E has facilitated activation by GTP[gamma]S, a slightly facilitated activation by GTP but much reduced receptor plus GTP stimulated activation, and an apparently unaltered ability to interact with receptor as seen in ligand binding studies. Further, the activity profile of Gs[alpha]-R265E is that of an [alpha]-subunit with unaltered or increased GTPase activity. The only change in Gs[alpha] that is similar to that in *T[alpha] is that the apparent affinity for guanine nucleotides is decreased in both proteins. The molecular basis of the changed properties are discussed based on the known crystal structure of Gs[alpha] and the changes introduced by the same mutation in a *T[alpha] (Gt[alpha]*) with only 23 aa from Gi1[alpha]. Gt[alpha]-R238E, with four fewer mutations in switch 3, was reported to show no evidence of DN properties, is activated by GTP[gamma],S, and has reduced GTPase activity. The data highlight a critical role for the switch 3 region in setting overall properties of signal-transducing GTPases. adenylyl cyclase | [beta]-adrenergic receptor | GTP shift | GTPase | crystal

Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of [beta]-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([[Ca].sub.i]) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [[Ca].sub.i] peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to [beta]-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function. transgenic mice; sarcoplasmic reticulum; hypertrophy; Ca signaling; cardiac function; force-frequency relationship; [beta]-adrenergic receptor stimulation

Calcineurin, a [Ca.sup.2+] -regulated protein phosphatase, links myocardial [Ca.sup.2+] signaling with hypertrophic gene transcription. Calcineurin abundance increases in pressure-overload hypertrophy and may reduce agonist-mediated phospholamban (PLB) phosphorylation to underlie blunted [beta]-adrenergic receptor ([beta]-AR) responsiveness in hypertension. This hypothesis was tested by measuring the effects of calcineurin inhibition on changes in cardiac contractility caused by [beta]-adrenergic stimulation in spontaneously hypertensive rats (SHR). Female SHR (age: 7 mo) and age-matched female Wistar-Kyoto rats (WKY) were studied. Heart weight-to-body weight ratio (P < 0.01) and systolic blood pressure (P < 0.01) were greater in SHR compared with WKY and were associated with increased myocardial calcineurin mRNA (CnA[beta]) and activity (P < 0.05). [beta]-AR stimulation with isoproterenol (Iso) increased calcineurin activity (P < 0.05) in both WKY and SHR hearts, and this activity was suppressed with cyclosporin A (CsA) treatment. In SHR, CsA improved left ventricular whole heart and isolated myocyte [beta]-AR responsiveness by normalizing PLB phosphorylation at [Ser.sup.16] and [Thr.sup.17] (P < 0.05). These CsA-induced, PLB-mediated effects were associated with an augmentation in cardiomyocyte peak [Ca.sup.2+] and a reduced rate (time constant of isovolumic pressure relaxation, tau) and magnitude of diastolic [Ca.sup.2+] during [beta]-AR stimulation. In conclusion, CsA normalized the blunted [beta]-AR responsiveness associated with hypertension, in part, by mitigating calcineurin activity while improving PLB phosphorylation and subsequent sarcoplasmic reticulum [Ca.sup.2+] regulation. myocardium; hypertrophy; phosphatase; protein kinase A; calcium calmodulin kinase II

We previously showed that lysozyme (Lzm-S), derived from leukocytes, caused myocardial depression in canine sepsis by binding to the endocardial endothelium to release nitric oxide (NO). NO then diffuses to adjacent myocytes to activate the cGMP pathway. In a canine right ventricular trabecular (RVT) preparation, Lzm-S also decreased the inotropic response to field stimulation (FSR) during which the sympathetic and parasympathetic nerves were simulated to measure the adrenergic response. In the present study, we determined whether the pathway by which Lzm-S decreased FSR was different from the pathway by which Lzm-S reduced steady-state (SS) contraction. Furthermore, we determined whether the decrease in FSR was due to a decrease in sympathetic stimulation or enhanced parasympathetic signaling. In the RVT preparation, we found that the inhibitory effect of Lzm-S on FSR was prevented by NO synthase (NOS) inhibitors. A cGMP inhibitor also blocked the depressant activity of Lzm-S. However, in contrast to the Lzm-S-induced decline in SS contraction, chemical removal of the endocardial endothelium by Triton X-100 to eliminate endothelial NO release did not prevent the decrease in FSR. An inhibitory G protein was involved in the effect of Lzm-S, since FSR could be restored by treatment with pertussis toxin. Atropine prevented the Lzm-S-induced decline in FSR, whereas [[beta].sub.1]- and [[beta].sub.2]-adrenoceptor function was not impaired by Lzm-S. These results indicate that the Lzm-S-induced decrease in FSR results from a nonendothelial release of NO. NO then acts through inhibitory G protein to enhance parasympathetic signaling. adrenergic [beta]-receptors; septic shock; myocardial depression; sympathetic response

Reperfusion after a brief period of cardiac ischemia can lead to potentially lethal arrhythmias. Importantly, there are sex-related differences in cardiac physiology and in the types and severity of cardiac arrhythmias. Therefore, we tested the hypothesis that gonadal hormones influence the susceptibility to reperfusion-induced sustained ventricular tachycardia (VT), as well as the response to [beta]-adrenergic receptor blockade. Male and female intact and gonadectomized rats were instrumented, and arterial pressure, temperature, ECG, and cardiac output were recorded. In addition, a snare was placed around the left main coronary artery. Tension was applied to the snare for determination of susceptibility to sustained VT produced by 3 rain of occlusion and reperfusion of the left main coronary artery in conscious rats. Reperfusion culminated in sustained VT in 77% (10 of 13 susceptible) of female rats and 56% (9 of 16 susceptible) of male rats (P > 0.05, male vs. female). [beta]-Adrenergic receptor blockade prevented sustained VT in females only [1 of 9 susceptible females (11%) vs. 6 of 9 susceptible males (67%), P < 0.05]. Ovariectomy did not significantly reduce the susceptibility to reperfusion arrhythmias [5 of 9 susceptible (56%)]. In sharp contrast, orchidectomy significantly increased the susceptibility to reperfusion arrhythmias [9 of 9 susceptible (100%)]. Finally, [beta]-adrenergic receptor blockade prevented sustained VT in ovariectomized females [0 of 4 susceptible (0%)] and orchidectomized males [0 of 7 susceptible (0%)], but the protective effect of [beta]-blockade was due to a reduction in heart rate in males only. Thus gonadal hormones influence the susceptibility to reperfusion-induced arrhythmias, as well as the effects and mechanisms of [beta]-adrenergic receptor blockade. cardiovascular risks; arrhythmia; sex differences

Selective stimulation of [[beta].sub.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].sub.2]-AR stimulation on L-type [Ca.sup.2+] current ([I.sub.Ca,L]) during postnatal development. [I.sub.Ca,L] was measured (37[degrees]C; either [Ca.sup.2+] or [Ba.sup.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.sup.2+] transients were measured concomitantly by dialyzing the cell with indo-1. Activation of [[beta].sub.2]-ARs (with either 100 nM zinterol or 1 [micro]M isoproterenol in the presence of the [[beta].sub.1]-AR antagonist, CGP20712A) stimulated [I.sub.Ca,L] twofold in newborns but not in adults. The [[beta].sub.2]-AR-mediated increase in [Ca.sup.2+] transient amplitude in newborns was due exclusively to the augmentation of [I.sub.Ca, L]. Zinterol increased the rate of inactivation of [I.sub.Ca.L] and increased the [Ca.sup.2+] flux integral. The [[beta].sub.2]-AR inverse agonist, ICI- 118551 (500 nM), but not the [[beta].sub.1]-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 [micro]M), PKI 6-22 amide (10 [micro]M), and Rp-cAMP (100 [micro]M), all failed to prevent the response to zinterol but completely blocked responses to selective [[beta].sub.1]-AR stimulation of [I.sub.Ca,L] in newborns. Our results demonstrate that in addition to the conventional [[beta].sub.1]-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel [[beta].sub.2]-AR-mediated, PKA-insensitive pathway that stimulates [I.sub.Ca,L]. This striking developmental difference plays a major role in the age-related differences in inotropic responses to [[beta].sub.2]-AR agonists. development; protein kinase A; adenosine 3',5'-cyclic monophosphate

Previous studies demonstrated an enhanced [[beta].sub.2]-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on [[beta].sub.1]-AR and [[beta].sub.2]-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue [beta]-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because [[beta].sub.2]-ARs are located in caveolae, caveolin-3 was also quantified. [[beta].sub.1]-AR gene expression decreased (~5-fold), [[beta].sub.2]-AR gene expression was not changed, and the ratio of [[beta].sub.2]-AR to [[beta].sub.1]-AR gene expression was significantly increased in susceptible compared with resistant dogs. [[beta].sub.1]-AR protein decreased (~50%) and [[beta].sub.2]-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned [[beta].sub.1]-AR gene expression to levels seen in resistant animals but did not alter [[beta].sub.2]-AR protein levels in susceptible dogs. These data suggest that [[beta].sub.2]-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves [[beta].sub.1]-AR gene expression, thereby restoring a more normal [beta]-AR balance. [beta]-adrenergic receptors; myocardial infarction; sympathetic nervous system

For many years, [beta]-adrenergic receptor antagonists ([beta]-blockers or [beta]AR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, [beta]-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different [beta]-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective [beta]AR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant [beta]AR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in [beta]2 adrenergic receptor ([beta]2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating [G.sub.s]-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptor's cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (it) recruitment of [beta]-arrestin to the [beta]2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant [[beta]2AR.sup.T68F,Y132G,Y219A] ([[beta]2AR.sup.TYY]) and abolished by [beta]-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from [G.sub.s], is nonetheless able to stimulate [beta]-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic [beta]2AR ligands. [beta]-adrenergic receptor | antagonist | ERK 1/2 | scaffold | internalization