G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells.

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in ß-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.

Antiallergic and antihistaminic actions of Ceasalpinia bonducella seeds: Possible role in treatment of asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Seed kernel of the plant Ceasalpinia bonducella Linn (Caesalpiniacaeae) are used for the treatment of asthma in folk medicine and ancient books. AIM OF STUDY: To assess the pharmacological efficacy of the plant in asthma and to confine and describe the synthetic constituents from the seeds that are in charge of the action. MATERIAL AND METHODS: The viability of petroleum ether, ethanol extract and ethyl acetate fraction from ethanol extract of C. bonducella seeds were screened for the treatment of asthma by various methods viz. effect of test drug on clonidine and haloperidol induced catalepsy, milk-induced leukocytosis and eosinophilia, mast cell stabilizing activity in mice and studies on smooth muscle preparation of guinea pig ileum (in-vitro). Column chromatography of active extract was done to pinpoint the active compound followed by structure elucidation by FTIR, GCMS and NMR spectroscopic methods. RESULTS: Ethyl acetate fraction from ethanol extract of C. bonducella seeds exhibited antihistaminic activity at the dose of 50 and 100 mg/kg, inhibited clonidine-induced catalepsy but not haloperidol-induced catalepsy. Ethyl acetate fraction from ethanol extract significantly inhibited increased leukocyte and eosinophil count due to milk allergen and showed maximum protection against mast cell degranulation by clonidine. The results of guinea pig ileum indicated that the compound 2 methyl, 1 hexadecanol isolated from ethyl acetate fraction of ethanol extract relaxed significantly the ileum muscle strips pre-contracted by which suggests the involvement of ß2-agonists on the relaxation of the tissue. All the results are dose dependent. Active ethyl acetate fraction from ethanol extract showed presence of anti-asthmatic compound, 2-methyl, 1-hexadecanol. CONCLUSION: The ethyl acetate fraction from ethanol extract of seeds of the plant C. bonducella can inhibit parameters linked to asthma disease.

QishenYiqi Dripping Pill Improves Heart Failure by Up-Regulation of ß2-Adrenergic Receptor Expression.

BACKGROUND: QishenYiqi Dripping Pill (QYDP) is a Chinese herbal medicine that originally was used for the treatment of coronary artery disease. Recently, QYDP was used as a complementary treatment for heart failure (HF) in China. METHODS: An HF rat model was used to clarify the possible therapeutic effects of QYDP on HF. The HF rats were allocated to two groups, HF and HF+QYDP, while normal rats served as a negative control. Cardiac functions were evaluated echocardiographically and hemodynamically. Cardiac apoptosis and the expression of ß-adrenergic receptors were also investigated. RESULTS: Compared to the HF group, rats in the HF+QYDP group had a significantly higher fraction shortening (p<0.05), ejection fraction (p<0.05), left ventricular systolic pressure (p<0.05), maximum positive derivatives of left ventricular pressure (p<0.05), maximum negative derivatives of left ventricular pressure (p<0.05), and ß2-adrenergic receptor expression (p<0.05), and lower left ventricular end-diastolic pressure (p<0.05) and apoptotic index (p<0.05). CONCLUSIONS: The study results indicated that QYDP could efficiently improve HF, possibly by an inhibition of cardiac apoptosis via the ß2-adrenergic receptor signaling pathway. Hence, QYDP might be a promising candidate drug for HF therapy.

Confirming therapeutic target of protopine using immobilized ß2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized ß2 -adrenoceptor (ß2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized ß2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to ß2 -AR by the 2 methods were (1.00 ± 0.06) × 105 M-1 and (1.52 ± 0.14) × 104 M-1 . The numbers of binding sites were (1.23 ± 0.07) × 10-7 M and (9.09 ± 0.06) × 10-7 M, respectively. These results indicated that ß2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.

Rikkunshito induces gastric relaxation via the ß-adrenergic pathway in Suncus murinus.

BACKGROUND: Rikkunshito (RKT) is a gastroprotective herbal medicine. In this study, we investigated the role of RKT in the relaxation of the gastric body (fundus and corpus) and antrum. METHODS: We used Suncus murinus, a unique small model animal with similar gastrointestinal motility to humans and dogs. RKT was added at 0.1, 1.0, and 5.0 mg/mL to induce relaxation in vitro; the outcome measure was the intensity of relaxation. The number of spontaneous antral contractions in the absence or the presence of RKT was also counted. KEY RESULTS: Rikkunshito induced the relaxation of the gastric body and antrum and decreased the number of spontaneous antral contractions in a dose-dependent manner. The responses to RKT (1.0 mg/mL) were not affected by pretreatment with atropine, N-nitro-l-arginine methyl ester, ritanserin, or ondansetron. On the other hand, timolol almost completely reversed the relaxation induced by RKT (1.0 mg/mL) on the gastric body and antrum and the occurrence of the spontaneous antral contractions. Both butoxamine, a ß(2) -adrenoreceptor antagonist, and L 748337, a ß(3) -adrenoreceptor antagonist, but not CGP 20712, a ß(1) -adrenoreceptor antagonist, significantly reversed the RKT-induced (1.0 mg/mL) gastric relaxation. CONCLUSIONS & INFERENCES: These results indicate that RKT stimulates and modulates gastric relaxation through ß(2) - and ß(3) -adrenergic, but not ß(1) -adrenergic, pathways in S. murinus.

Effects of an n-butanol extract from the stem of Tinospora crispa on blood pressure and heart rate in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for control of blood pressure, as an antipyretic, for cooling down the body temperature and for maintaining good health. AIM OF THE STUDY: To investigate the effects and mechanisms of action of an n-butanol extract from the stems of Tinospora crispa (T. crispa extract) on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: Air-dried stems of T. crispa were extracted with water, followed by partitioned extract with chloroform, ethyl acetate, and finally by n-butanol. The n-butanol soluble part was evaporated under reduced pressure and lyophilization to obtain a crude dried powder (T. crispa extract). The effects and mechanisms of the T. crispa extract on blood pressure and heart rate were studied in anesthetized normal and reserpinized rats in vivo in the presence of different antagonists. RESULTS: T. crispa extract (1-100 mg/kg, i.v.) caused a decrease in mean arterial blood pressure (MAP) and this effect was inhibited by propranolol, phentolamine, atenolol and/or the ß(2)-antagonist ICI-118,551, but not by atropine or hexamethonium. In reserpinized rats, the T. crispa extract had a dual effect: reduction in hypotensive activity, followed by a small increase in blood pressure. The decrease in MAP in reserpinized rat was slightly potentiated by phentolamine, but inhibited by propranolol or ICI-118,551 only if atenolol and phentolamine were also present. The increase in MAP was potentiated by propranolol and ICI-118,551, but was inhibited by phentolamine. The T. crispa extract had a dual effect on heart rate in the normal rat: a small transient decrease, followed by an increase in heart rate. The positive chronotropic effect of T. crispa extract was inhibited by propranolol, phentolamine and atenolol, but not by ICI-118,551, atropine or hexamethonium. Reserpine potentiated the positive chronotropic effect of the T. crispa extract and this effect was inhibited by propranolol, atenolol and ICI-118,551, but not by phentolamine. CONCLUSIONS: From these results we suggest that T. crispa extract possesses at least three different cardiovascular-active components that act directly via (1) ß(2)-adrenergic receptors to cause a decrease in blood pressure, and ß(1)- and ß(2)-adrenergic receptors to cause an increase in heart rate, (2) α-adrenergic receptors to cause an increase in blood pressure and heart rate, and (3) a non-adrenergic and non-cholinergic pathway to cause a decrease in MAP and heart rate. These findings provide scientific support for the tradition of using this plant to modify the actions of the human cardiovascular system.

Label-free integrative pharmacology on-target of drugs at the ß(2)-adrenergic receptor.

We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the ß(2)-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

Investigation of beta(2)-adrenoceptor subtype selectivity and organ specificity for bedoradrine (KUR-1246), a novel tocolytic beta-adrenergic receptor stimulant.

OBJECTIVES: The aim of this study was to evaluate the beta-adrenergic receptor (beta-AR) selectivity, organ specificity and efficacy of delaying the onset of spontaneous delivery of bedoradrine (KUR-1246), a novel uterine relaxant. METHODS: beta-AR selectivity was evaluated in terms of the amount of cyclic adenosine monophosphate produced by bedoradrine, ritodrine and isoprenaline in Chinese hamster ovary cells expressing human beta(1)-, beta(2)-AR or beta(3)-AR. Inhibition of contractions of the atrium, trachea and proximal colon by bedoradrine were compared with those of the uterus in pregnant rats using an organ bath method. Finally, the delaying effect of bedoradrine on spontaneous labor was evaluated by an in vivo study using term pregnant rats. RESULTS: EC(50) values of bedoradrine for cyclic adenosine monophosphate production in Chinese hamster ovary cells via beta(1)-, beta(2)- and beta(3)-AR were 2400 +/- 30, 2.9 +/- 0.10 and 363 +/- 3 nmol/L, respectively, indicating that bedoradrine had 832- and 126-fold higher selectivity for beta(2)-AR than for beta(1)- and beta(3)-AR. EC(50) values of bedoradrine for the uterus, atrium, trachea and proximal colon were 1.01 +/- 0.27, 2300 +/- 356, 1610 +/- 299 and 219 +/- 23.5 nmol/L, respectively. Thus, bedoradrine was 2280-, 1590- and 217-fold more specific for the uterus than for the atrium, trachea and proximal colon, respectively. Bedoradrine delayed the spontaneous delivery of 21-day-pregnant rats in a dose-dependent manner. CONCLUSIONS: Bedoradrine is a promising drug for the treatment of preterm labor in obstetrical practice because it has better selectivity for beta(2)-AR and specificity for the uterus than currently used agents and may effectively delay spontaneous delivery.

Beta2-adrenoceptor-mediated tracheal relaxation induced by higenamine from Nandina domestica Thunberg.

The fruit of Nandina domestica Thunberg (ND, Berberidaceae) has been used to improve cough and breathing difficulties in Japan for many years, but very little is known about the constituent of ND responsible for this effect. We have recently reported that the crude extract from ND (NDE) inhibits histamine- and serotonin-induced contraction of isolated guinea pig trachea, and the inhibitory activity was not explained by nantenine, a well-known alkaloid isolated from ND. To explore other constituent(s) of NDE with tracheal smooth muscle relaxant activity, we fractionated NDE and assessed the pharmacological effects of the fractions using isolated guinea pig tracheal ring preparations. NDE was introduced into a polyaromatic absorbent resin column and stepwise eluted to yield five fractions, among which only the 40 % methanol fraction was active in relaxing tracheal smooth muscle precontracted with histamine. Further separation of the 40 % methanol fraction with high-performance liquid chromatography yielded multiple subfractions, one of which was remarkably active in relaxing histamine-precontracted trachea. Chemical analysis with a time-of-flight mass spectrometer and nuclear magnetic resonance spectrometer identified the constituent of the most active subfraction as higenamine, a benzyltetrahydroisoquinoline alkaloid. The potency and efficacy of the active constituent from NDE in relaxing trachea were almost equivalent to synthetic higenamine. In addition, the effect of the active constituent from NDE was competitively inhibited by the selective beta (2)-adrenoceptor antagonist ICI 118,551. These results indicate that the major constituent responsible for the effect of NDE is higenamine, which probably causes the tracheal relaxation through stimulation of beta (2) adrenoceptors.

Beta2-adrenoreceptor ligands regulate osteoclast differentiation in vitro by direct and indirect mechanisms.

Pharmacological modulators of beta-adrenoceptors can influence bone mineral density and fracture risk in humans. Studies reported that beta-adrenoceptor ligands stimulate bone resorption by enhancing the expression of RANK-L, whereas the mechanisms by which beta-adrenoreceptors regulate bone formation are poorly understood. Here we show that beta2-adrenoceptor is predominantly expressed by bone cells, although low levels of beta1- and beta3-adrenoceptors were detectable. Noradrenaline and the selective beta2-adrenoceptor agonists isoprenaline and salmeterol stimulated osteoclast formation and bone resorption in BM osteoblast co-cultures and increased expression of RANK-L by osteoblasts. All three ligands enhanced RANK-L induced osteoclast formation and increased osteoclast multinuclearity. There was no significant effect of noradrenaline or isoprenaline on osteoblast growth, differentiation or function. These findings confirm the importance of the sympathetic nervous system in the regulation of bone mass, and demonstrate that pharmacological agonists of beta2-adrenoceptors directly and indirectly stimulate osteoclast formation, but have no direct effect on osteoblast growth, differentiation or function.

Stimulatory effect of Crocus sativus (saffron) on beta2-adrenoceptors of guinea pig tracheal chains.

To study the mechanism(s) of the relaxant effects of Crocus sativus (Iridaceae), the stimulatory effect of aqueous-ethanolic extracts of this plant and one of its constituent, safranal was examined on beta-adrenoceptors in tracheal chains of guinea pigs. The beta(2)-adrenergic stimulatory was tested by performing the cumulative concentration-response curves of isoprenaline-induced relaxation of pre-contracted isolated guinea pig tracheal chains. The studied solutions were included two concentrations of aqueous-ethanolic extract from Crocus sativus (0.1 and 0.2g%), safranal (1.25 and 2.5 microg), 10nM propranolol, and saline. The study was done in two different conditions including: non-incubated (group 1, n=9) and incubated tissues with 1 microM chlorpheniramine (group 2, n=6). The results showed clear leftward shifts in isoprenaline curves obtained in the presence of only higher concentration of the extract in group 1 and its both concentrations in group 2 compared with that of saline. The EC(50) (the effective concentration of isoprenaline, causing 50% of maximum response) obtained in the presence of both concentrations of the extract (0.17+/-0.06 and 0.12+/-0.02) and safranal (0.22+/-0.05 and 0.22+/-0.05) in group 1 and only in the presence of two concentrations of the extract (1.16+/-0.31 and 0.68+/-0.21) in group 2 was significantly lower compared to saline (1.00+/-0.22 and 4.06+/-1.04 for groups 1 and 2, respectively) (p<0.05-0.001). The maximum responses obtained in the presence of both concentrations of the extract and safranal in group 1 were significantly lower than that of saline (p<0.005 for all cases). All values (CR-1=(EC(50) obtained in the presence of active substances/EC(50), obtained in the presence of saline)-1) obtained in the presence of higher concentrations of extract in group 1, its both concentrations and higher concentration of safranal in group 2 were negative and there were significant differences in this value between propranolol and those obtained in the presence of extract and safranal (p<0.05 to p<0.001). The results indicated a relatively potent stimulatory effect of the extract from Crocus sativus on beta(2)-adrenoceptors which is partially due to its constituent, safranal. A possible inhibitory effect of the plant on histamine (H(1)) receptors was also suggested.

Beta1- and beta2-adrenoceptors in basolateral nucleus of amygdala and their roles in consolidation of fear memory in rats.

It is known that beta-adrenoceptor (AR) in the basolateral nucleus of amygdala (BLA) plays an essential role in fear memory formation. However, the cellular and subcellular distributions of beta1- and beta2-ARs in the BLA and their roles in fear memory formation are poorly understood. Here, we report that both beta1- and beta2-ARs are predominantly expressed in BLA neurons but not in astrocytes. beta1-AR is distributed in the cell membrane and cytoplasm of neurons, whereas beta2-AR is localized not only in the cell membrane and cytoplasm but also in the nucleus. Intra-BLA infusion of the beta1-AR antagonist metoprolol and atenolol or the beta2-AR antagonist ICI118551 and butoxamine produces a severe deficit in 24-h auditory fear memory, leaving 1-h memory intact. Western-blot analysis reveals that the protein level of cytoplasmic beta1-AR significantly increases 2- and 4-h postconditioning, whereas that of cytoplasmic or nuclear beta2-AR is unchanged. The present results indicate that beta1- and beta2-ARs in the BLA have differential subcellular localizations and both are required for the consolidation of auditory fear memory.

The discovery of indole-derived long acting beta2-adrenoceptor agonists for the treatment of asthma and COPD.

The design and profile of a series of indole containing long acting beta(2)-adrenoceptor agonists is described. Evaluation of these analogues using an in vitro guinea pig trachea tissue model demonstrates that analogues within this series have salmeterol-like duration of action with potential for long duration of action in humans.

The effect of thyme extract on beta2-receptors and mucociliary clearance.

UNLABELLED: Thyme is a broncholytic und secretomotoric agent. Thus, our aim was to investigate the influence of a thyme extract on beta (2)-receptors in competition binding experiments and relaxation experiments on rat uteri and trachea. Furthermore, the influence of the extract on respiratory clearance was of interest. Binding experiments were performed using purified rat lung membranes with the beta(2)-receptor ligand [(125)I]-CYP {[(125)I]-(+/-)-Iodocyanopindolol}. The transport of the fluorescence dye rhodamin 123 concerning ciliary action in the tracheal area of a mouse was investigated using a microdialysis technique. The thyme extract reduces only slightly [(125)I]-CYP binding and amplifies the displacement of [(125)I]-CYP by propranolol (non-specific beta-receptor antagonist): the displacement curve in the concentration range representing beta (2)-receptors (nM) is shifted to the left. Thyme extract had relaxing effects on organs possessing beta (2)-receptors (uterus and trachea). The propranolol-induced antagonism to isoprenaline is reverted concentration-dependently by the extract. A duplication of the rate of ciliary clearance by the extract was observed. IN CONCLUSION: 1) There is evidence for an influence of a thyme extract on beta (2)-receptors by both binding studies and biological effects: As can be derived from the shift of the propranolol displacement curve (nM), ingredients of the thyme extract slightly interact with beta (2)-receptors in rat lung tissue. This effect is indirect since no full range competition curve was reached. 2) An at least indirect interaction with beta (2)-receptors in rat uteri and trachea is revealed by a decreased antagonism of propranolol on the relaxing effect of isoprenaline by the plant extract. 3) An additional mechanism is presumed because at high extract concentrations isoprenaline-induced relaxation is complete, whereas the displacement of propranolol at beta (2)-receptors is only weak. 4) Thyme extract has an indirect (modulatory) effect on the beta (2)-receptor system. 5) Mucociliary clearance is improved in vivo. Its mechanism has still to be elucidated.

The role of the new beta-blockers in treating cardiovascular disease.

The beta-adrenergic receptor blockers play an important role in the management of cardiovascular disease, including hypertension and chronic heart failure. However, concerns regarding safety and tolerability with currently available agents can limit their use. The beta-blockers vary with regard to several pharmacologic properties, including beta1/beta2 selectivity, intrinsic sympathomimetic activity, and, with the newest beta-blockers, vasodilation. These pharmacologic differences may result in clinically important differences in tolerability and hemodynamic properties. Nebivolol is a novel beta-blocker with both a greater degree of selectivity for beta1-adrenergic receptors than other agents in this class and an ability to stimulate endothelial nitric oxide production, leading to vasodilation and other potential clinical effects. Published randomized, controlled, multicenter studies with nebivolol have shown that once-daily treatment significantly reduces systolic and diastolic blood pressure in patients with mild-to-moderate hypertension, compared with placebo, in a dose-dependent manner, and is well tolerated, with an adverse event profile similar to that of placebo. When compared with other beta-blockers as well as with other antihypertensive classes of agents in head-to-head trials, nebivolol demonstrated similar antihypertensive efficacy and a lower incidence of adverse events. Nebivolol has also been shown to significantly reduce morbidity and mortality in a large population of elderly patients with chronic heart failure, independent of left ventricular ejection fraction. Nebivolol is currently available in Europe for the management of hypertension and is expected to be available soon in the United States.

Does the beta2-agonist clenbuterol help to maintain myocardial potential to recover during mechanical unloading?

OBJECTIVE: Chronic mechanical unloading induces left ventricular (LV) atrophy, which may impair functional recovery during support with an LV-assist device. Clenbuterol, a beta2-adrenergic receptor (AR) agonist, is known to induce myocardial hypertrophy and might prevent LV atrophy during LV unloading. Furthermore, beta2-AR stimulation is reported to improve Ca2+ handling and contribute to antiapoptosis. However, there is little information on the effects of clenbuterol during LV unloading. METHODS AND RESULTS: We investigated LV atrophy and function after LV unloading produced by heterotopic heart transplantation in isogenic rats. After transplantation, rats were randomized to 1 of 2 groups (n=10 each). The clenbuterol group received 2 mg.kg(-1).d(-1) of the drug for 2 weeks; the control group received normal saline. The weight of unloaded control hearts was 48% less than that of host hearts after 2 weeks of unloading. Clenbuterol significantly increased the weight of the host hearts but did not prevent unloading-induced LV atrophy. Papillary muscles were isolated and stimulated, and there was no difference in developed tension between the 2 groups. However, the inotropic response to the beta-AR agonist isoproterenol significantly improved in the clenbuterol group. The mRNA expression of myocardial sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) and fetal gene shift (myosin heavy chain [MHC] mRNA isozyme) was also significantly improved by clenbuterol treatment. There was no difference in beta1-AR mRNA expression between the 2 groups. In contrast, beta2-AR mRNA was significantly decreased in the clenbuterol-treated, unloaded heart. This indicates that clenbuterol may downregulate beta2-ARs. In the evaluation of apoptosis, mRNA expression of caspase-3, which is the central pathway for apoptosis, tended to be better in the clenbuterol group. CONCLUSIONS: During complete LV unloading, clenbuterol did not prevent myocardial atrophy but improved gene expression (SERCA2a, beta-MHC) and beta-adrenergic responsiveness and potentially prevented myocardial apoptosis. However, chronic administration of clenbuterol may be associated with downregulation of beta2-ARs.

Nebivolol increases arterial distensibility in vivo.

Arterial stiffness is a key determinant of cardiovascular risk in hypertensive patients. beta-Blockers appear to be less effective than other drugs in improving outcome in hypertensive patients, and a potential explanation may be that beta-blockers are less effective in reducing arterial stiffness. The aim of this study was to assess the direct effect of beta-blockade on pulse wave velocity (PWV), a robust measure of arterial distensibility, using a local, ovine, hind-limb model. In addition, we hypothesized that the vasodilating beta-blocker nebivolol, but not atenolol, would increase arterial distensibility in vivo. All studies were conducted in anesthetized sheep. PWV was recorded in vivo using a dual pressure-sensing catheter placed in the common iliac artery. Intraarterial infusion of nebivolol reduced PWV by 6+/-3% at the higher dose (P<0.001), but did not alter mean arterial pressure (change of -1+/-3 mm Hg, P=0.1). In contrast, atenolol had no effect on PWV (P=0.11) despite a small drop in mean pressure (change of -5+/-3 mm Hg, P<0.01). Infusion of glyceryl trinitrate led to a dose-dependent fall in PWV, and 2 nmol/min produced a similar reduction in PWV to the higher dose of nebivolol (500 nmol/min). The effect of nebivolol on PWV was significantly attenuated during coinfusion of N(G)-monomethyl-L-arginine (P=0.003) and also during coinfusion of butoxamine (P=0.02). These results demonstrate that nebivolol, but not atenolol, increases arterial distensibility. This effect of nebivolol is mediated through the release of NO via a beta2 adrenoceptor-dependent mechanism. Thus, nebivolol may be of benefit in conditions of increased large artery stiffness, such as isolated systolic hypertension.

The beta2-adrenergic receptor specifically sequesters Gs but signals through both Gs and Gi/o in rat sympathetic neurons.

Beta(2)-adrenergic receptors (beta(2)-AR) and CB1 cannabinoid receptors share the property of being constitutively active. The CB1 cannabinoid receptor can also sequester G(i/o) proteins; however, it is not known whether the beta(2)-AR can also sequester G proteins. Beta(2)-ARs were heterologously expressed in rat superior cervical ganglion neurons by microinjection of cDNA and studied using the patch-clamp technique. The beta-AR agonist isoproterenol increased the Ca(2+) current 25.9+/-1.6% in neurons microinjected with 100 ng/microl beta(2)-AR cDNA but was without effect on control neurons. Pretreatment with cholera toxin (CTX) abolished the effect of isoproterenol, indicating coupling via G(s) proteins. In neurons microinjected with 200 ng/microl beta(2)-AR cDNA, isoproterenol had the opposite effect of inhibiting the Ca(2+) current 36.5+/-2.0%. Inhibition of the Ca(2+) current was sensitive to pertussis toxin, indicating beta(2)-AR coupling to G(i/o) proteins. Pretreatment with CTX resulted in a greater 54+/-3.8% inhibition of the Ca(2+) current, indicating that G(s) coupling masks the full effect of G(i/o) coupling. Expression of beta(2)-ARs abolished signaling by G(s)-coupled receptors for vasoactive intestinal polypeptide (VIP). VIP inhibited the Ca(2+) current 49.5+/-0.5% in control neurons but had no effect in neurons expressing beta(2)-ARs. In contrast, expression of beta(2)-ARs had no effect on signaling by the G(i/o)-coupled alpha(2)-adrenergic receptor. This study demonstrates that the beta(2)-AR couples to both G(s) and G(i/o) proteins but specifically sequesters G(s) proteins, preventing their interaction with another G(s)-coupled receptor. beta(2)-adrenergic receptors thus have the potential to prevent other G(s)-coupled receptors from transducing their biological signals.

Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology.

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.