Structural basis and mechanism of activation of two different families of G proteins by the same GPCR.

The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).

Roadmap for the use of base editors to decipher drug mechanism of action.

CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The ß2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.

GPCR-ErbB transactivation pathways and clinical implications.

Cell surface receptors including the epidermal growth factor receptor (EGFR) family and G-protein coupled receptors (GPCRs) play quintessential roles in physiology, and in diseases, including cardiovascular diseases. While downstream signaling from these individual receptor families has been well studied, the cross-talk between EGF and GPCR receptor families is still incompletely understood. Including members of both receptor families, the number of receptor and ligand combinations for unique interactions is vast, offering a frontier of pharmacologic targets to explore for preventing and treating disease. This molecular cross-talk, called receptor transactivation, is reviewed here with a focus on the cardiovascular system featuring the well-studied GPCR receptors, but also discussing less-studied receptors from both families for a broad understanding of context of expansile interactions, repertoire of cellular signaling, and disease consequences. Attention is given to cell type, level of chronicity, and disease context given that transactivation and comorbidities, including diabetes, hypertension, coronavirus infection, impact cardiovascular disease and health outcomes.

Cytoprotective Potential of Aged Garlic Extract (AGE) and Its Active Constituent, S-allyl-l-cysteine, in Presence of Carvedilol during Isoproterenol-Induced Myocardial Disturbance and Metabolic Derangements in Rats.

This study was conducted to determine the potential interaction of aged garlic extract (AGE) with carvedilol (CAR), as well as to investigate the role of S-allyl-l-cysteine (SAC), an active constituent of AGE, in rats with isoproterenol (ISO)-induced myocardial dysfunction. At the end of three weeks of treatment with AGE (2 and 5 mL/kg) or SAC (13.1 and 32.76 mg/kg), either alone or along with CAR (10 mg/kg) in the respective groups of animals, ISO was administered subcutaneously to induce myocardial damage. Myocardial infarction (MI) diagnostic predictor enzymes, lactate dehydrogenase (LDH) and creatinine kinase (CK-MB), were measured in both serum and heart tissue homogenates (HTH). Superoxide dismutase (SOD), catalase, and thiobarbituric acid reactive species (TBARS) were estimated in HTH. When compared with other groups, the combined therapy of high doses of AGE and SAC given alone or together with CAR caused a significant decrease in serum LDH and CK-MB activities. Further, significant rise in the LDH and CK-MB activities in HTH was noticed in the combined groups of AGE and SAC with CAR. It was also observed that both doses of AGE and SAC significantly increased endogenous antioxidants in HTH. Furthermore, histopathological observations corroborated the biochemical findings. The cytoprotective potential of SAC and AGE were dose-dependent, and SAC was more potent than AGE. The protection offered by aged garlic may be attributed to SAC. Overall, the results indicated that a high dose of AGE and its constituent SAC, when combined with carvedilol, has a synergistic effect in preventing morphological and physiological changes in the myocardium during ISO-induced myocardial damage.

Comparative Protective Effect of Nigella sativa Oil and Vitis vinifera Seed Oil in an Experimental Model of Isoproterenol-Induced Acute Myocardial Ischemia in Rats.

The study's aim was to characterize the composition of Nigella sativa seed (NSO) and grape seed (GSO) oils, and to evaluate their cardioprotective and anti-inflammatory effect on isoproterenol (ISO)-induced ischemia in rats. Materials and Methods: NSO and GSO supplements were physicochemically characterized. Liquid chromatography-mass spectrometry (HPLC-MS), Fourier-transform infrared spectroscopy (FTIR), and gas chromatography-mass spectrometry (GC-MS) analyses were used to determine the phytochemical composition in the oils. Total polyphenol content (TPC) and in vitro antioxidant activity were also determined. Pretreatment with 4 mL/kg/day NSO or GSO was administered to rats for 14 days. The experimental ischemia was induced by a single administration of ISO 45 mg/kg after 14 days. An electrocardiogram (ECG) was performed initially and 24 h after ISO. Biological evaluation was done at the end of experiment. Results: The HPLC-MS, GC-MS, and FTIR analyses showed that both NSO and GSO are important sources of bioactive compounds, especially catechin and phenolic acids in GSO, while NSO was enriched in flavonoids and thymol derivatives. Pretreatment with GSO and NSO significantly reduced ventricular conduction, prevented the cardiotoxic effect of ISO in ventricular myocardium, and reduced the level of proinflammatory cytokines and CK-Mb. Conclusion: Both NSO and GSO were shown to have an anti-inflammatory and cardioprotective effect in ISO-induced ischemia.

Spectroelectrochemical Determination of Isoprenaline in a Pharmaceutical Sample.

UV/Vis absorption spectroelectrochemistry (SEC) is a multi-response technique that has been commonly used for the characterization of materials and the study of reaction mechanisms. However, it has been scarcely used for quantitative purposes. SEC allows us to obtain two analytical signals simultaneously, yielding a dual sensor in just one experiment. In the last years, our group has developed new devices useful for analysis. In this work, a SEC device in parallel configuration, based on optical fibers fixed on screen-printed electrodes, was used to determine isoprenaline in a commercial drug, using both, the electrochemical and the spectroscopic signals. In this commercial drug, isoprenaline is accompanied in solution by other compounds. Among them is sodium metabisulfite, an antioxidant that strongly interferes in the isoprenaline determination. A simple pretreatment of the drug sample by bubbling wet-air allows us to avoid the interference of metabisulfite. Here, we demonstrate again the capabilities of UV/Vis absorption SEC as double sensor for analysis and we propose a simple pretreatment to remove interfering compounds.

Brain pharmacokinetics and biodistribution of 11C-labeled isoproterenol in rodents.

INTRODUCTION: Isoproterenol is a non-selective ß receptor agonist, which is a drug approved for bradycardia and bronchial asthma in many countries. Recently, isoproterenol has been reported to have the potential as a drug for the treatment of Alzheimer's disease by inhibiting the aggregation of tau protein. Isoproterenol is a highly potent drug causing increases in heart rates even when its plasma concentration is very low. Thus, it is critical to know if potentially effective therapeutic levels of isoproterenol can be achieved, maintaining safe plasma levels without any untoward pharmacological effects. The purpose of the study is to investigate the brain pharmacokinetics and biodistribution of 11C-labeled isoproterenol in rodents. METHODS: We performed positron emission tomography (PET) brain imaging and biodistribution studies of [11C]isoproterenol. 120-min scans with arterial blood sampling were performed in rats. Additionally, plasma and brain homogenates were analyzed with radio-HPLC to characterize its metabolite profiles. As a measure of [11C]isoproterenol brain uptake, total distribution volumes were determined by a pharmacokinetic compartment model. Biodistribution of [11C]isoproterenol was investigated in mice at six-time points from 1-min to 90-min after injection. RESULTS: We found a modest brain uptake of [11C]isoproterenol. Its brain pharmacokinetics showed that the concentration of isoproterenol in the brain at equilibrium was about two-fold higher than in the plasma (total distribution volumes 2.0 ± 0.2 cm3/mL). Only unmetabolized isoproterenol was detected in the brain at 30 min after injection, although isoproterenol was rapidly metabolized in plasma. The biodistribution study showed that isoproterenol and its metabolite are excreted mainly via the urinary system. CONCLUSIONS, ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: In this study, we have shown that rat brain concentrations of isoproterenol are only two-fold of that in plasma at equilibrium. If the brain pharmacokinetics are similar in the human brain, it may be difficult to achieve potentially therapeutic levels of this drug safely in humans. Further studies appear warranted to investigate the brain pharmacokinetics in humans with PET using [11C]isoproterenol.

Micro-patterned SU-8 cantilever integrated with metal electrode for enhanced electromechanical stimulation of cardiac cells.

Over the past few years, cardiac tissue engineering has undergone tremendous progress. Various in vitro methods have been developed to improve the accuracy in the result of drug-induced cardiac toxicity screening. Herein, we propose a novel SU-8 cantilever integrated with an electromechanical-stimulator to enhance the maturation of cultured cardiac cells. The simultaneous electromechanical stimulation significantly enhances the contraction force of the cardiomyocytes, thereby increasing cantilever displacement. Fluorescence microscopy analysis was performed to confirm the improved maturation of the cardiomyocytes. After the initial experiments, the contractile behaviors of the cultured cardiomyocytes were investigated by measuring the mechanical deformation of the SU-8 cantilever. Finally, the proposed electromechanical-stimulator-integrated SU-8 cantilever was used to evaluate the adverse effects of different cardiac vascular drugs, i.e., verapamil, lidocaine, and isoproterenol, on the cultured cardiomyocytes. The physiology of the cardiac-drug-treated cardiomyocytes was examined with and without electrical stimulation of the cardiomyocytes. The experimental results indicate that the proposed cantilever platform can be used as a predictive assay system for preliminary cardiac drug toxicity screening applications.

Cyanobacteria as Nanogold Factories II: Chemical Reactivity and anti-Myocardial Infraction Properties of Customized Gold Nanoparticles Biosynthesized by Cyanothece sp.

Cyanothece sp., a coccoid, unicellular, nitrogen-fixing and hydrogen-producing cyanobacterium, has been used in this study to biosynthesize customized gold nanoparticles under certain chemical conditions. The produced gold nanoparticles had a characteristic absorption band at 525-535 nm. Two types of gold nanoparticle, the purple and blue, were formed according to the chemical environment in which the cyanobacterium was grown. Dynamic light scattering was implemented to estimate the size of the purple and blue nanoparticles, which ranged from 80 ± 30 nm and 129 ± 40 nm in diameter, respectively. The highest scattering of laser light was recorded for the blue gold nanoparticles, which was possibly due to their larger size and higher concentration. The appearance of anodic and cathodic peaks in cyclic voltammetric scans of the blue gold nanoparticles reflected the oxidation into gold oxide, followed by the subsequent reduction into the nano metal state. The two produced forms of gold nanoparticles were used to treat isoproterenol-induced myocardial infarction in experimental rats. Both forms of nanoparticles ameliorated myocardial infarction injury, with a slight difference in their curative activity with the purple being more effective. Mechanisms that might explain the curative effect of these nanoparticles on the myocardial infarction were proposed. The morphological, physiological, and biochemical attributes of the Cyanothece sp. cyanobacterium were fundamental for the successful production of "tailored" nanoparticles, and complemented the chemical conditions for the differential biosynthesis process. The present research represents a novel approach to manipulate cyanobacterial cells towards the production of different-sized gold nanoparticles whose curative impacts vary accordingly. This is the first report on that type of manipulated gold nanoparticles biosynthesis which will hopefully open doors for further investigations and biotechnological applications.

In situ immobilization of sulfated-ß-cyclodextrin as stationary phase for capillary electrochromatography enantioseparation.

In this work, a novel sulfated-ß-cyclodextrin (S-ß-CD) coated stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by attaching polydopamine/sulfated-ß-cyclodextrin (PDA/S-ß-CD) onto the gold nanoparticles (AuNPs) coated capillary which was pretreated with polydopamine. The results of scanning electron microscopy (SEM) and energy dispersive X-ray analysis spectroscopy (EDS) indicated that polydopamine/sulfated-ß-cyclodextrin was successfully fixed on the gold nanoparticles coated capillary. To evaluate the performance of the prepared open tubular (OT) column, the enantioseparation was carried out by using ten chiral drugs as model analytes. Under the optimal conditions, salbutamol, terbutaline, trantinterol, tulobuterol, clorprenaline, pheniramine, chlorpheniramine, brompheniramine, isoprenaline and tolterodine were baseline separated with the resolution (Rs) values of 3.25, 1.76, 2.51, 1.89, 3.17, 2.17, 1.99, 1.72, 2.01 and 3.20, respectively. Repeatability of the column was studied, with the relative standard deviations for run-to-run, day-to-day and column-to-column lower than 5.7%.

Hybridization of ß-Adrenergic Agonists and Antagonists Confers G Protein Bias.

Starting from the ß-adrenoceptor agonist isoprenaline and beta-blocker carvedilol, we designed and synthesized three different chemotypes of agonist/antagonist hybrids. Investigations of ligand-mediated receptor activation using bioluminescence resonance energy transfer biosensors revealed a predominant effect of the aromatic head group on the intrinsic activity of our ligands, as ligands with a carvedilol head group were devoid of agonistic activity. Ligands composed of a catechol head group and an antagonist-like oxypropylene spacer possess significant intrinsic activity for the activation of Gαs, while they only show weak or even no ß-arrestin-2 recruitment at both ß1- and ß2-AR. Molecular dynamics simulations suggest that the difference in G protein efficacy and ß-arrestin recruitment of the hybrid ( S)-22, the full agonist epinephrine, and the ß2-selective, G protein-biased partial agonist salmeterol depends on specific hydrogen bonding between Ser5.46 and Asn6.55, and the aromatic head group of the ligands.

Sensitive enantioseparation and determination of isoprenaline in human plasma and pharmaceutical formulations.

A simple, sensitive and fast RPHPLC method was developed and validated for the enantioselective determination of (RS)-isoprenaline (Ipn) in human plasma. The enantiomers were converted to diastereomeric derivatives using s-triazine (cyanuric chloride) based chiral derivatizing reagents. l-isoleucine and l-methionine were introduced as chiral auxiliary in s-triazine and two new monochloro-s-triazine reagents were synthesized. These reagents were characterized and used for synthesis of diastereomeric derivatives of (RS)-Ipn spiked in human plasma. (RS)-Ipn was isolated (purified and characterized) from a commercial pharmaceutical formulation and was used as the standard racemic sample. Structures of the two diastereomeric derivatives were optimized for lowest energy using the Gaussian 09 Rev A. 02 program and hybrid density functional B3LYP with 6-31G* basis set which showed the spatial orientation of hydrophobic groups on stereogenic centers in the diastereomeric derivatives. The results were correlated with the mechanism of separation and elution order. Limit of detection values were found to be 24.6 and 26.8 ng mL-1 for the first and second eluting diastereomeric derivatives, respectively.

Rapid and mild fabrication of protein membrane coated capillary based on supramolecular assemble for chiral separation in capillary electrochromatography.

Exploration of the simple and stable coating methods is of great significance in capillary electrochromatography (CEC). In this work, a lysozyme assemble supramolecular membrane coated capillary column was developed for CEC chiral separation. Taking advantage of phase transformation of lysozyme, the coating process was achieved within 1.0 h thus to a large extent reduced the capillary preparation time and simplified the coating procedure. The successful fabrication of the supramolecular membrane coated capillary was verified by scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), fluorescence imaging, and electroosmotic flow (EOF). The separation capacity of the coated capillary was evaluated by analysis of different chiral analytes, including chiral amine drug and neurotransmitters, and good enantioseparation efficiency was achieved for the three pairs of enantiomers. For three consecutive runs, the relative standard deviations (RSD) for the migration time of the analytes for intra-day (n = 3), inter-day (n = 3) and column-to-column (n = 3) were in the range of 0.7-1.5%, 2.7-3.6%, and 4.5-5.8%, respectively. Additionally, the supramolecular membrane coated capillary column could run consecutively 100 times without observable change in the separation efficiency, proving the feasibility of the coating method based on the adhesion of the protein-based supramolecular membrane.

Design and optimization of a luminescent Samarium complex of isoprenaline: A chemometric approach based on Factorial design and Box-Behnken response surface methodology.

A chemometrically optimized procedure has been developed for the determination of isoprenaline (ISO) in the parent substance as well as in its respective pharmaceutical preparation. It is worth mentioning that although spectroscopic determination of Isoprenaline metal complexes has been described in literature, yet, no methods for the quantification of Isoprenaline with Samarium nor any other lanthanide metal have been reported. Fractional factorial design (FFD) was implemented in the initial screening procedure of the four designated factors, namely, reaction time (RT), metal volume (MV), pH and temperature (T) followed by Response Surface Methodology (RSM) optimization tool performed by the aid of Box Behnken design (BBD).The proposed techniques are based on a multivariate approach where a complexation reaction between Isoprenaline (ISO) and Samarium III (Sm3+) metal was exploited for the first time to synthesize novel fluorescence and absorbance probes of ISO-Sm. Maximum fluorescence intensity (Y1) as well as maximum absorbance (Y2) of the produced complex were attained at λex/λem = 315/450 and λ 295 nm for spectrofluorimetric and spectrophotometric determinations, respectively, against blank solutions. Using assessment quality tools such as, Pareto charts, normal probability plots and statistical analysis of variance testing (ANOVA), significant factors were successfully indicated (p < 0.05). Furthermore, the proposed methods verified specificity and accuracy for the determination of Isoprenaline in its pure and pharmaceutical preparation using spectrofluorimetric (Technique A) and spectrophotometric (Technique B) techniques, respectively. Linearity was obtained in the range of (0.02-0.50 µg/mL) and (2-12 µg/mL) upon employing both techniques A and B, respectively. Furthermore, limit of detection (LOD) and limit of quantification (LOQ), were found to be 5.1877 ∗ 10-3 µg/mL, 0.01572 µg/mL and 0.5593 µg/mL, 1.6949 µg/mL, upon employing techniques A and B, respectively. Standard addition method was applied for both techniques. The analysis was successfully applied to the assay of pure powder and pharmaceutical dosage forms after which the corresponding mean recoveries were computed and were found to be in the range of 99.546%-100.257% (Technique A) and 99.872%-99.887% (Technique B) with RSD (<1).

Binding of anti-cardiovascular drug to serum albumin: an insight in the light of spectroscopic and computational approaches.

Isoprenaline hydrochloride is a potential cardiovascular drug helps in the smooth functioning of the heart muscles. So, we have performed the binding study of ISO with BSA. This study was investigated by UV absorption, fluorescence, synchronous fluorescence, circular dichroism, etc. The analysis of intrinsic fluorescence data showed the low binding affinity of ISO. The binding constant Kb was 2.8 × 103 M-1 and binding stoichiometry (n) was approximately one and the Gibb's free energy change at 310 K was determined to be -8.69 kcal mol-1. Negative Gibb's free energy change shows the spontaneity of the BSA and ISO interaction. We have found ISO-induced alternation in the UV absorption, synchronous fluorescence and CD spectra in the absence and presence of the quencher indicates the complex formation. In synchronous fluorescence, red shift was obtained because of the complex formation of BSA and ISO. The distance (r) between the BSA (donor) and ISO (acceptor) was 2.89 nm, determined by FRET. DLS measurements interpreted complex formation due to the reduction in hydrodynamic radii of the protein in the presence of the drug. The binding site of ISO was found to be nearer to Trp 134 with the help of molecular docking and the ΔG° was found to be -10.2 kcal mol-1. The esterase activity result suggests that ISO acts as competitive inhibitor. Thus, this study would help to determine the binding capacity of the drug to the protein which may indicate the efficiency of diffusion of ISO into the blood for the treatment of heart diseases.

Altered myocyte contractility and calcium homeostasis in alpha-myosin heavy chain point mutations linked to familial dilated cardiomyopathy.

Mutations in the human cardiac motor protein beta-myosin heavy chain (ßMHC) have been long recognized as a cause of familial hypertrophic cardiomyopathy. Recently, mutations (P830L and A1004S) in the less abundant but faster isoform alpha-myosin heavy chain (αMHC) have been linked to dilated cardiomyopathy (DCM). In this study, we sought to determine the cellular contractile phenotype associated with these point mutations. Ventricular myocytes were isolated from 2 month male Sprague Dawley rats. Cells were cultured in M199 media and infected with recombinant adenovirus containing the P830L or the A1004S mutant human αMHC at a MOI of 500 for 18 h. Uninfected cells (UI), human ßMHC (MOI 500, 18 h), and human αMHC (MOI 500, 18 h) were used as controls. Cells were loaded with fura-2 (1 µM, 15 min) after 48 h. Sarcomere shortening and calcium transients were recorded in CO2 buffered M199 media (36°±1 C) with and without 10 nM isoproterenol (Iso). The A1004S mutation resulted in decreased peak sarcomere shortening while P830L demonstrated near normal shortening kinetics at baseline. In the presence of Iso, the A1004S sarcomere shortening was identical to the ßMHC shortening while the P830L was identical to the αMHC control. All experimental groups had identical calcium transients. Despite a shared association with DCM, the P830L and A1004S αMHC mutations alter myocyte contractility in completely different ways while at the same preserving peak intracellular calcium.

Nanobodies to Study G Protein-Coupled Receptor Structure and Function.

Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools.

Lumican-null mice are susceptible to aging and isoproterenol-induced myocardial fibrosis.

With aging and stress, the myocardium undergoes structural remodeling, often leading to fibrosis. The purpose of this study is to examine whether lumican, one of the class II small leucine-rich proteoglycans, has a protective role in cardiac remodeling and fibrosis. In attempts to elucidate the hypothesis that lumican may have a protective role in cardiac remodeling and fibrosis, we compared the cardiac phenotypes of young (3-month-old) and elder (6-month- and 12-month-old) lumican-null (Lum-/-) mice. Extra-cellular matrix remodeling and apoptosis are examined to determine the roles of lumican on age-dependent cardiac fibrosis induced by isoproterenol. Compared to wild type littermates, Lum-/- mice exhibited higher mortality due to significantly impaired systolic function, which was associated with an increase of atrial natriuretic peptide (ANP) secreted by the ventricles in response to excessive stretching of myocytes. Masson's Trichrome and silver stains showed significantly more severe ventricle fibrosis in Lum-/- mice. Interestingly, rate of cell death mediated via apoptosis illustrated by the expression of caspase 3 and TUNEL assay was lower in Lum-/- mice after isoproterenol infusion. In addition, Lum-/- mice exhibited higher levels of TGF-ß, collagen I/III, and membrane-type matrix metalloproteinase-1 (MT1-MMP/MMP-14) during cardiac remodeling. This study shows that alternations of lumican might be implicated in the pathogenesis of cardiac fibrosis and suggests lumican as novel targets for cardiac fibrosis therapy. Further studies are required to define the mechanism by which lumican modulates cardiac remodeling.

Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

Single-molecule imaging reveals the stoichiometry change of ß2-adrenergic receptors by a pharmacological biased ligand.

The stoichiometry of the ß2-adrenergic receptor (ß2AR) was determined using single-molecule fluorescence imaging in living cells. The results showed that ß2AR mainly existed as monomers under physiological conditions and exhibited ß-arrestin-dependent dimerization upon stimulation with the pharmacological biased ligand carvedilol. The association of ß2AR dimerization with biased signalling is revealed.