ß-blockers augment L-type Ca2+ channel activity by targeting spatially restricted ß2AR signaling in neurons.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of ß-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate ß2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated ß2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant ß2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant ß2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which ß-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Computational study on the different ligands induced conformation change of ß2 adrenergic receptor-Gs protein complex.

ß2 adrenergic receptor (ß2AR) regulated many key physiological processes by activation of a heterotrimeric GTP binding protein (Gs protein). This process could be modulated by different types of ligands. But the details about this modulation process were still not depicted. Here, we performed molecular dynamics (MD) simulations on the structures of ß2AR-Gs protein in complex with different types of ligands. The simulation results demonstrated that the agonist BI-167107 could form hydrogen bonds with Ser203(5.42), Ser207(5.46) and Asn293(6.55) more than the inverse agonist ICI 118,551. The different binding modes of ligands further affected the conformation of ß2AR. The energy landscape profiled the energy contour map of the stable and dissociated conformation of Gαs and Gßγ when different types of ligands bound to ß2AR. It also showed the minimum energy pathway about the conformational change of Gαs and Gßγ along the reaction coordinates. By using interactive essential dynamics analysis, we found that Gαs and Gßγ domain of Gs protein had the tendency to separate when the inverse agonist ICI 118,551 bound to ß2AR. The α5-helix had a relatively quick movement with respect to transmembrane segments of ß2AR when the inverse agonist ICI 118,551 bound to ß2AR. Besides, the analysis of the centroid distance of Gαs and Gßγ showed that the Gαs was separated from Gßγ during the MD simulations. Our results not only could provide details about the different types of ligands that induced conformational change of ß2AR and Gs protein, but also supplied more information for different efficacies of drug design of ß2AR.

Acebutolol and alprenolol metabolism predictions: comparative study of electrochemical and cytochrome P450-catalyzed reactions using liquid chromatography coupled to high-resolution mass spectrometry.

A comparative study of the electrochemical conversion and the biotransformation performed by the cytochrome P450 (CYP450) obtained by rat liver microsomes has been achieved to elucidate the oxidation mechanism of both acebutolol and alprenolol. For this purpose, a wide range of reactions such as N-dealkylation, O-dealkoxylation, aromatic hydroxylation, benzyl hydroxylation, alkyl hydroxylation, and aromatic hydroxylation have been examined in this study, and their mechanisms have been compared. Most of the results of the electrochemical oxidation have been found to be in accordance with those obtained by incubating acebutolol and alprenolol in the presence of CYP450, i.e., N-dealkylation, benzyl hydroxylation, and O-dealkoxylation reactions catalyzed by liver microsomes were found to be predicted by the electrochemical oxidation. The difficulty for the electrochemical process to mimic both aromatic and alkyl hydroxylation reactions has also been discussed, and the hypothesis for the absence of aromatic hydroxylated and alkyl hydroxylated products, respectively, for alprenolol and acebutolol, under the anodic oxidation has been supported by theoretical calculation. The present study highlights the potential and limitation of coupling of electrochemistry-liquid chromatography-high-resolution mass spectrometry for the study of phase I and phase II reactions of acebutolol and alprenolol.

Influence of lipid composition on the structural stability of g-protein coupled receptor.

ß2 Adrenergic receptor (ß2AR) is a kind of G-protein coupled receptors (GPCRs) which transduce a wide range of extracellular signals into intracellular messages responsible for the regulation of diverse cell functions. Because of their functional ubiquity, GPCR is one of the most important drug targets in pharmaceutical industry. Although recent crystallographic studies provided both the active and the inactive states of some families of GPCRs, the influence of lipid composition of bilayer membrane on their activation is still poorly understood. In this work, we address the influence of lipid composition on the structural stability of GPCR, performing molecular dynamics simulations of three kinds of states: apo-, and agonist epinephrine-, or antagonist alprenolol-bound ß2AR. These three kinds of ß2ARs were embedded in four types of lipid membranes: (i) pure palmitoyl-oleoyl-phosphatidyl-choline (POPC), (ii) POPC/cholesterol (CHL), (iii) POPC/CHL/GM1 (GM1 ganglioside), (iv) POPC/palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE)/CHL/sphingomyeline (SM). The side chains of Lys267(6.29) and Asp331(7.58) showed different conformations among the three states in all types of lipid membranes. The distances between Lys267(6.29) and Asp331(7.58) of apo- and alprenolol-bound ß2ARs are smaller than that of the epinephrine-bound ß2AR. In contrast, ß2ARs in POPC/CHL bilayer were unstable in which the salt bridge; i.e., ionic lock, was not formed between Arg131(3.50) and Glu268(6.30). We have also examined the distribution of lipid molecules. A stable hydrophobic interaction between CHL and ß2AR was observed at transmembrane helix5 in POPC/CHL/GM1 and POPC/POPE/CHL/SM membranes. These results suggest that the lipid composition strongly affects the conformation of GPCR and essentially concerns the GPCR activation.

Enantioselective biodegradation of pharmaceuticals, alprenolol and propranolol, by an activated sludge inoculum.

Biodegradation of chiral pharmaceuticals in the environment can be enantioselective. Thus quantification of enantiomeric fractions during the biodegradation process is crucial for assessing the fate of chiral pollutants. This work presents the biodegradation of alprenolol and propranolol using an activated sludge inoculum, monitored by a validated enantioselective HPLC method with fluorescence detection. The enantioseparation was optimized using a vancomycin-based chiral stationary phase under polar ionic mode. The method was validated using a minimal salts medium inoculated with activated sludge as matrix. The method was selective and linear in the range of 10-800 ng/ml, with a R²>0.99. The accuracy ranged from 85.0 percent to 103 percent, the recovery ranged from 79.9 percent to 103 percent, and the precision measured by the relative standard deviation (RSD) was <7.18 percent for intra-batch and <5.39 percent for inter-batch assays. The limits of quantification and detection for all enantiomers were 10 ng/ml and 2.5 ng/ml, respectively. The method was successfully applied to follow the biodegradation of the target pharmaceuticals using an activated sludge inoculum during a fifteen days assay. The results indicated slightly higher biodegradation rates for the S-enantiomeric forms of both beta-blockers. The presence of another carbon source maintained the enantioselective degradation pattern while enhancing biodegradation extent up to fourteen percent.

Kinetic capillary electrophoresis with mass-spectrometry detection (KCE-MS) facilitates label-free solution-based kinetic analysis of protein-small molecule binding.

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.

Ligand-dependent perturbation of the conformational ensemble for the GPCR ß2 adrenergic receptor revealed by HDX.

Mechanism of G protein-coupled receptor (GPCR) activation and their modulation by functionally distinct ligands remains elusive. Using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry, we examined the ligand-induced changes in conformational states and stability within the beta-2-adrenergic receptor (ß(2)AR). Differential HDX reveals ligand-specific alterations in the energy landscape of the receptor's conformational ensemble. The inverse agonists timolol and carazolol were found to be most stabilizing even compared with the antagonist alprenolol, notably in intracellular regions where G proteins are proposed to bind, while the agonist isoproterenol induced the largest degree of conformational mobility. The partial agonist clenbuterol displayed conformational effects found in both the inverse agonists and the agonist. This study highlights the regional plasticity of the receptor and characterizes unique conformations spanning the entire receptor sequence stabilized by functionally selective ligands, all of which differ from the profile for the apo receptor.

Pathway and mechanism of drug binding to G-protein-coupled receptors.

How drugs bind to their receptors--from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or "pose"--has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the ß(1)- and ß(2)-adrenergic receptors, initially making contact with a vestibule on each receptor's extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug-receptor binding and unbinding rates.

Involvement of ß-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release.

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G(αo) -mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G(o) protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous ß-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using ß-adrenergic receptor antibody, and vesicle swelling experiments using ß-adrenergic agonists and antagonists, demonstrate the presence of functional ß-adrenergic receptors at the SV membrane. Since a recent study shows vH(+) -ATPase to be upstream of AQP-6 in the pathway leading from G(αo) -mediated swelling of SV, participation of an endogenous ß-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.

Microcalorimetric and zeta potential study on binding of drugs on liposomes.

In this work, isothermal titration calorimetry (ITC) combined with zeta potential measurements was used to study the binding and partitioning of three beta-blockers, alprenolol, labetalol and propranolol, and the local anaesthetic tetracaine into liposomes. The thermodynamic parameters of enthalpy, entropy, the Gibbs energy and the binding constant were determined using the one site model. Furthermore, the binding constants corrected for the electrostatic contribution were used to assess the partition coefficients for the drugs. Also, the effect of the concentration, ionic strength, temperature and membrane curvature on the interaction was included in the evaluation.

Functional rescue of beta-adrenoceptor dimerization and trafficking by pharmacological chaperones.

Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the beta(1)-adrenergic receptor (beta(1)AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the beta(1)AR as they did not prevent heterodimerization with the beta(2)AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic beta-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised beta(1)AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes.

Effects of dexamethasone and colostrum feeding on mRNA levels and binding capacities of beta-adrenergic receptors in the liver of neonatal calves.

Glucocorticoids increase plasma glucose concentrations in neonatal calves, but not hepatic gluconeogenic enzyme mRNA levels and activities. Catecholamines, too, enhance plasma glucose levels and regulate hepatic glucose metabolism. We have measured hepatic mRNA levels of beta-adrenergic receptors and beta-adrenergic receptor binding in neonatal calves on day 5 of life. Calves were fed either colostrums (C) or an isoenergetic milk-based formula (F), and in each feeding group, half of the calves were treated with dexamethasone (DEXA; 30 microg/(kg body weightday)). Abundance of mRNA was highest (P < 0.01) for beta2-adrenergic receptors and was higher (P < 0.01) for beta1- than for beta3-adrenergic receptors. DEXA treatment decreased (P < 0.05) beta1- and beta2-adrenergic receptor mRNA levels. Beta3-adrenergic receptor mRNA levels were higher (P < 0.05) in colostrum- than in formula-fed calves. Competitive binding revealed highest affinities for alprenolol, propranolol (both beta1- and beta2-antagonists), and ICI-188,551 (beta2-antagonist), which did not significantly differ from each other. Atenolol (beta1-antagonist) up to 10(-5) M did not displace (3H)-CGP-12177 from receptors. Competitive binding for adrenaline was best fitted by a two-receptor model. DEXA decreased (P < 0.05) (3H)-CGP-12177 binding capacities, whereas binding affinity of (3H)-CGP-12177 was not affected by DEXA or different feeding. Binding sites correlated positively with mRNA levels of beta2-adrenergic receptors (r = 0.56; P < 0.01). In conclusion, beta2-adrenergic receptors were the dominant subtype in the hepatic tissue. Feeding did not significantly affect beta2-adrenergic binding sites. However, DEXA decreased beta2-adrenergic binding sites and this was regulated at the transcriptional level.

Determination of dissociation constants by competitive binding in partial filling capillary electrophoresis.

The determination of dissociation constands (K(d)) by competitive ligand binding in partial filling capillary electrophoresis is demonstrated. Two different strategies were applied, one of which only uses a single reporter ligand and a more elaborated one which suppresses systemic disturbances by using a racemic mixture as reporter. The dissociation constants obtained by both alternatives were virtually identical and in good agreement with those previously reported.

Metabolite characterization in drug discovery utilizing robotic liquid-handling, quadruple time-of-flight mass spectrometry and in-silico prediction.

An assay method for identification of metabolites from in vitro microsomal incubations was developed for use in the early stage of drug discovery. We have developed a practical approach which involves integrated sample generation, sample preparation, bioanalysis, and data handling to maximize sample throughput and speed up the process for identification of metabolites. The assay system consisted of a robotic liquid handler (Genesis workstation) to generate and process samples, PALLAS MetabolExpert software to predict possible metabolites, exact mass measurement via a tandem quadrupole time-of-flight mass spectrometer (QTOF-MS) coupled with liquid chromatography to analyze samples, MetaboLynx software to find potential metabolites and Advanced Chemistry Development/MS (ACD/MS) software to provide guidance to the most likely hypothetical metabolite chemical structures. For purposes of evaluating this new method, dextromethorphan, alprenolol, and propranolol were incubated separately for up to 60 minutes with rat and human hepatic microsomes. The incubation and sample preparation were carried out in 96-well plates using the Genesis workstation. The bioanalysis was performed by LC-MS/MS using QTOF with MetaboLynx software to find metabolites. Metabolic products formed in vitro by rat and human microsomes were separated using an analytical column C18 with gradient elution at flow rate of 250 micro l/min. The internal mass calibration was performed by continuous postcolumn infusion of Haloperidol. The mass spectra from incubations containing NADPH were compared to those without NADPH (control) using the MetaboLynx software to find potential metabolites. Finally, the MS/MS spectra were processed by the ACD/MS software to predict the chemical structure. MetaboLynx software successfully identified metabolites for each of the drugs studied by automatically discerning expected metabolites. Exact differences in masses between each metabolite and parent drug were measured from five replicate sample injections. All measured values are accurate to less than 0.001Da or 3.8 ppm with the standard deviation within 0.0015 Da, which allowed good prediction/confirmation of empirical formulae. Hypothetical chemical structures were achieved by the ACD/MS software and provided a useful tool to assist in prediction of the metabolic pathways of the drugs. The metabolites identified were in good agreement with previously published results for all three compounds. This new method will greatly enhance throughput, which in turn will facilitate our ability to rapidly provide this guidance to the synthetic chemist.

Characterization of [3H]CGP 12177 binding to beta-adrenergic receptors in intact eel hepatocytes.

The aim of this study was to characterize [3H]CGP 12177 (CGP) binding to beta-adrenergic receptors in isolated hepatocytes of the European eel (Anguilla anguilla), in which the involvement of cAMP in epinephrine-induced glucose release has been previously observed. Specific binding of CGP was saturable, reversible, and linear as a function of cell number. Analysis of binding data suggested a single class of binding sites, with a Kd of 1.31 nM and a number of approximately 7000 beta-adrenergic receptors per cell. The potency order of specific inhibition of [3H]CGP binding was CGP > propranolol > or = alprenolol >> butoxamine > or = atenolol, while phentolamine and prazosin failed to significantly displace the tracer at concentrations up to 100 microM. The binding kinetics of CGP were closely related to its biological effect. In fact, the drug dose-dependently counteracted the enhancement of intracellular cAMP levels induced by epinephrine in isolated hepatocytes with a Kd of 1.06 nM. Moreover, it antagonized the hormone-induced stimulation of adenylyl cyclase activity in hepatic membranes as well as of glucose release from cells. These data clearly show that beta-adrenergic receptors are coupled to the adenylyl cyclase/cAMP transduction pathway in eel liver.

In vitro permeability of eight beta-blockers through Caco-2 monolayers utilizing liquid chromatography/electrospray ionization mass spectrometry.

It is demonstrated that the apparent permeability (P(app)) coefficients of beta-adrenoceptor antagonist drugs can easily be determined for Caco-2 cell culture intestinal models utilizing liquid chromatography/mass spectrometry (LC/MS). The LC/MS method with electrospray ionization in the single ion monitoring mode showed an increased sensitivity of 1000-fold compared with LC/UV detection and enhanced selectivity with respect to both LC/UV and radioactivity assays. The P(app) coefficients of beta-adrenoceptor antagonists determined by LC/MS have the same ranking order as those determined by LC/UV and radioactivity assays. However, the P(app) coefficients determined in this study showed significant discrepancies from those determined in other laboratories. There are several experimental factors that directly affect the absolute value of the P(app) coefficients, including pH gradients, additional diffusion barriers (i.e. unstirred water layer and type of filter support), analyte concentration, detection method and possibly cell culture variations. These parameters should be controlled when generating Caco-2 P(app) coefficients for different compounds.

Study of the interaction between aryloxypropanolamines and Asn386 in helix VII of the human 5-hydroxytryptamine1A receptor.

We studied the stereoselective interaction between aryloxypropanolamines and the human 5-hydroxytryptamine1A (5-HT1A) receptor. R- and S-enantiomers of propranolol, penbutolol, and alprenolol were investigated for their ability to bind to human 5-HT1A wild-type and Asn386Val mutant receptors. Asn386 seemed to act as a chiral discriminator. Although both aryloxypropanol enantiomers displayed lower affinity for the mutant receptors, the affinities for the S-enantiomers were more affected. Receptor affinities of other structurally unrelated 5-HT1A ligands were not decreased by the mutation of Asn386 to valine. In addition, a series of analogues of propranolol with structural variation in the oxypropanolamine moiety was synthesized, and affinities for wild-type and Asn386Val mutant 5-HT1A receptors were determined. Both the hydroxyl and the ether oxygen atoms of the oxypropanol moiety seem to be required for binding at wild-type 5-HT1A receptors. The hydroxyl group of propranolol probably directly interacts with Asn386. The ether oxygen atom may be important for steric reasons but can also be involved in a direct interaction with Asn386. These findings are in agreement with the interactions of aryloxypropanolamines with Asn386 in rat 5-HT1A receptors that we previously proposed. The loss of affinity for propranolol by the Asn386Val mutation could be regained by replacement of the hydroxyl group of the ligand by a methoxy group. This modification of the propranolol structure has no effect on the affinity of both enantiomers for the wild-type 5-HT1A receptor, which provides an alternative hypothesis for the interaction of Asn386 with the oxypropanol oxygen atoms. According to this novel hypothesis, the oxypropanol oxygen atoms may both act as hydrogen bond acceptors from the NH2 group of Asn386.

Relationship between amount of beta-blockers permeating through the stratum corneum and skin irritation after application of beta-blocker adhesive patches to guinea pig skin.

We evaluated the relationship between the cumulative amounts of 5 kinds of beta-blockers (alprenolol, oxprenolol, timolol, acebutolol and atenolol) permeating through the stratum corneum and a* values obtained by measuring the formation of erythema, a skin irritation reaction, with a chromameter after transdermal application of adhesive patches containing 2 beta-blocker to the skin of guinea pigs. The cumulative amount of beta-blocker released from each adhesive patch to the skin increased with the increase in application time. The contents of alprenolol, oxprenolol and timolol in the stratum corneum and in the stripped skin increased markedly up to 4 h after application and thereafter were maintained at high levels up to 24 h. The contents of acebutolol and atenolol, on the other hand, increased up to 24 h, but these values were low. a* values of all adhesive patches 24 h after application were higher than those before application. The correlation coefficients between the cumulative amounts of alprenolol, oxprenolol, timolol, acebutolol or atenolol permeating through the stratum corneum and (delta a* -delta a*Placebo) values were 0.739, 0.717, 0.722, 0.551 and 0.633, respectively. The correlation coefficient calculated by averaging the cumulative amounts of 6 kinds of beta-blockers permeating through the stratum corneum [including propranolol which was reported previously (Kobayashi I., et al., Biol. Pharm. Bull., 19, 839-844 (1996))] was 0.731, higher than the correlation coefficient between contents of these beta-blockers in the stripped skin and (delta a* -delta a*Placebo) values (r = 0.552). This suggests that there was a high correlation between the cumulative amounts of beta-blockers permeating through the stratum corneum and (delta a* -delta a*Placebo) values.

Effects of active immunization against clenbuterol on the growth-promoting effect of clenbuterol in rats.

We examined the effect of active immunization against clenbuterol on the growth-promoting effect of clenbuterol in rats in two experiments. Six-week-old female Sprague-Dawley rats were immunized against clenbuterol conjugated to histone by diazotization and then received clenbuterol approximately 4 wk after the initiation of immunization. Antibody titers were determined using indirect ELISA with diazotized clenbuterol-BSA conjugate as an antigen in coating the microwells. Antibody titer increased during booster injections. No significant difference in titer value was observed between two doses of immunogen (.1 vs .5 mg). Competitive ELISA showed that terbutaline cross-reacted with anti-clenbuterol antibodies, and the cross-reactivity was 12%. Alprenolol, propranolol, phentolamine, epinephrine, norepinephrine, and L644,969 showed no affinity for anti-clenbuterol antibodies. The rats immunized against clenbuterol-histone conjugate had 11% lower body weight gain during the 23-d immunization period than the rats immunized against histone only. When clenbuterol was administered after the immunization, no significant difference in growth rate was observed between the rats immunized against clenbuterol-histone conjugate and rats immunized against histone only. No significant difference in muscle weight was observed between the two groups at the termination of the experiment. Results indicate that active immunization against clenbuterol before clenbuterol administration did not modify the growth-promoting effects of clenbuterol in rats.