Characterization of cAMP accumulation mediated by three α1—adrenoceptor subtypes in HEK293 cells

AIM:To investigate the characterization of cAMP response mediated by α1-adrenoceptor (α1-AR) subtypes in HEK293 cells. METHODS:(1) Full-length cDNA encoding three α1-AR subtypes were transfected into HEK293 cells by the calcium phosphate precipitation method, respectively. (2) The densities of α1-AR subtypes expressed in HEK293 cells were measured by radioligand binding assay. (3)cAMP accumulation was measured by [^3H] adenine prelabeling method. RESULTS: (1)Activation of each of three subtypes resulted in an increase of cAMP accumulation in HEK293 cells in a dose-dependent manner, which was inhibited by selective α1-AR antagonist prazosin. (2) Comparing the pharmacological property, the maximal responses of α1A-AR to agonists were the most potent, while the sensitivity of α1-AR subtypes to norepinephrine(NE) was the highest. CONCLUSION: Each of three α1-AR subtypes can mediate cAMP accumulation in HEK293 cell line, and there are differences in pharmacological property.     

Receptor subtype involved in α1-adrenergic receptor-mediated Ca^2＋ signaling in cardiomyocytes

Aim： The enhancement of intracellular Ca^＋signaling in response to α1-adrenergic receptor （α1-AR） stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role（s） of the α1-AR subtype（s） in mediating this response. Methods： We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca^2＋ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results： After being cultured for 48 h, the myocytes exhibited spontaneous local Ca^2＋ release, sparks, and global Ca^2＋ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca^2＋ transients with an EC5o value of 2.3 larnol/L. Blocking the α1A-AR subtype with 5-methylurapidil （5-Mu） inhi- bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca^2＋ spark numbers were also increased by the activation of the α1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency （EC50 value =6.9 nmol/L） in the potentiation of Ca^2＋ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion： These results indicate that α1-adrenergic stimulation of intracellular Ca^2＋ activity is mediated selectively by the α1-AR.     

Real-time detection of α1A-AR movement stimulated by phenylephrine in single living cells

Aim： To investigate the movement of α1A-adrenergic receptors（α1A-AR） stimulated by agonist, phenylephrine （PE）, and the dynamics of receptor movement in real time in single living cells with millisecond resolution. Methods： We labeled α1A-AR using the monoclonal, anti-FLAG （a kind of tag） antibody and Cy3-conju- gated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. Results： The specific detection of α1A-AR on the surface of living HEK293A-α1A cells was achieved, α1A-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20min, apparent colocalization was found between α1A-AR and F-actins. After 40 rain stimulation of PE, trajectories of approximate linear motion in HEK293A-α1A cells were recorded, and their velocity was calculated. Conclusion： The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect α1A-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.     

Crosstalk between different adrenoceptors in rat hearts and HEK293 cells

There exist α_(1A), α_(1B), α_(1D), β_1, and β_2 subtypes ofadrenoceptors (AR) in rat hearts. Its physiological significancehas not been clear. We investigated the crosstalks between thosecoexisted subtypes in the rat heart and HEK 293 cells. Ourresults indicated: (1) In electric driven isolated rat left atria,either α_(1A~-) or α_(1B~(-AR)) per se induced positive inotropic response(PIR). But when they were artivated together with β-AR, theα_(1A)-AR inihibited, while the α_(1B)-AR potentiated the β-AR mediatedPIR and cAMP accumulation. The α_(1D)-AR did not show aboveeffects. When all the subtypes of α_1-AR were acticated with β-AR, the α_(1A)-AR played a dominated effect. Activation of α_1-ARshowed no effect on the foskolin-induced cAMP accumulation. Italso did not change the binding characteristiics of β-AR. Those     

β-Adrenoceptors potentiate α1-adrenoceptor-mediated inotropic response in rat left atria

AIM: To investigate whether stimulation of β-adrenoceptor (AR) and its subtypes augment α1-AR-evoked positive inotropic response and inositol phosphate (InsP) accumulation in isolated rat left atria. METHODS: Inotropic response was determined by contractile function experiment in isolated electrically driven rat left atria. ^3H-InsP accumulations were measured by ^3H-inositol incorporation and column chromatography. RESULTS: (1) Stimula-tion of α1-AR by phenylephrine (PE) or norepinephrine (NE) in the presence of propranolol (Prop) evoked positive inotropic response and ^3H-InsP accumulations, while stimulation of β-AR by isoprenaline (ISO) or NE in the presence of phentolamine (Phen) only evoked positive inotropic response, but not ^3H-InsP accumulations. (2) Simultaneous stimulation of α1- and β-AR by NE or ISO plus PE significantly shifted the concentration-dependent inotropic response curves and ^3H-InsP accumulation curves to the left and upward compared with individual α1-AR stimulation by PE or NE in the presence of Prop. (3) In the presence of ICI118551 (selective β2-AR antagonist) or CGP12177 (selective β1-AR antagonist), stimulation of either β1- or β2-AR did not change α1-AR-evoked inotropic response and ^3H-InsP accumulations. CONCLUSION: Stimulation of β1-AR and β2-AR potentiates α1-AR-mediated positive inotropic response and InsP accumulation in isolated rat left atria.     

Trafficking of α1B-adrenergic receptor mediated by inverse agonist in living cells

AIM The project is aimed at understanding the action of inverse agonist at single molecule level and capturing the real time picture of molecular behavior of α1B-adrenergic receptor (AR) mediated by inverse agonist in living cells by single molecule detection (SMD). METHODS The location and distribution of α1B-AR was detected by laser confocal and whole cell ^3H-prazosin binding assay. Dynamic imaging of BODIPY-FL-labeled prazosin (Praz), specific antagonist of (1-AR, was observed in α1B-AR stably expressed human embryonic kidney 293 (HEK293) living cells. The detection of real-time dynamic behaviors of AR was achieved by using fluorescence-labeled AR and its ligand combined with SMD techniques. RESULTS α1B-AR was predominantly distributed on the cell surface and 8.2% of the total receptors were located in cytosol.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Competitive binding of postsynaptic density 95 and Ca^2＋-calmodulin dependent protein kinase Ⅱ to N-methyl-D-aspartate receptor subunit 2B in rat brain

AIM: To investigate the interactions among postsynaptic density 95 (PSD-95), Ca^2 -calmodulin dependent protein kinase Ⅱα (CaMKⅡα), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats. METHODS: Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKⅡα to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKⅡ) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins. RESULTS: The alternations of the binding level of PSD-95 and CaMKⅡα to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKⅡα to NR2B. CONCLUSION: PSD-95 competes with CaMKⅡ to bind to NR2B during ischemia and reperfusion in rat hippocampus.     

Identification of glutamate transporters and receptors in mouse testis

INTRODUCTION Glutamate is the predominant excitatory neu-rotransmitter in the central nervous system (CNS). Itsneurotransmission can be mediated by various ligand-gated ion channels, of which there are three subtypes.These subtypes, which are classified on the basis ofsequence homologies and agonist affinities, are N-methyl-D-aspartate (NMDA) receptors (NR1 andNR2A-D), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (GluR1-4), and kainate(KA) receptors (GluR5-7…     

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim： Several nicotinic acetylcholine receptor （nAChR） subunits have been engineered as fluorescent protein （FP） fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors, Methods： A gene construct was generated to introduce yellow fluorescent protein （YFP）, in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit （α7Y）. SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits （α7） or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin （I-Bgt） binding, laser scanning confocal microscopy, and total internal reflectance fluorescence （TIRF） microscopy. Results： Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion： nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs, α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.     

Evaluation of hepatitis B virus replication and proteomic analysis of HepG2.2.15 cell line after cyclosporine A treatment

Aim： The effect of cyclosporine A （CsA） on hepatitis B virus （HBV） replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication. Methods： Methyl thiazolyl tetrazolium （MTT） assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens （HBsAg） and Hepatitis B e antigens （HBeAg） in culture supernatant, while the intracellular HBV DNA replication level was analyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search. Results： CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed significantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors （eIF） 3k, otubain 1, 14.3.3 protein, eIF2-1α, eIF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The downregulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa （ECP-51）, stathmin 1/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally- controlled 1, was shifted, suggesting it had undergone posttranslational modifications. Conclusion： Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.     

Novel derivative of Danshensu acts as anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57

OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase(PDI)family proteins including ERp57.To further investigate the underlying mechanism of ADTM to modulate ERp57 to regulate platelet function with direct interaction withαⅡbβ3 integrin.METHODS To isolate the protein targets that bound to ADTM,a biotin-conjugated ADTM analogue(BAA)was designed and synthesized.BAA(300μmol·L-1)was incubated with rat blood platelet lysates and the BAA-protein complexes were pulled down with NeutrAvidin-agarose followed by protein profiling using LC-MS/MS.To determine platelet aggregation in vitro,rabbit platelets were incubated with the indicated concentrations of compounds and aggregation was induced by ADP(10μmol·L-1)or AA(200μmol·L-1)and measured using a platelet aggregometer.To determine platelet aggregation-induced by ADP in rat in vivo,ADTM(5-20mg·kg-1)in comparison with DSS(10mg·kg-1)and clopidogrel(18mg·kg-1)were administered daily by i.v.injection for 5d,respectively.To determine the action of ADTM on the ERp57/αⅡbβ3 interaction,it was examined by immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting.RESULTS BAA could bind to various proteins involved in platelet function.In particular,platelet aggregation-associated proteins were identified with>95% protein identification probability including ERp72,ERp57ERp5 and PDI,which are members of the protein disulfide isomerase(PDI)family related to platelet function and redox homeostasis.ADTM exhibited potent inhibition on the redox activity of ERp57 in a concentration-dependent manner(IC50=100 300μmol·L-1).In in vitro studies,ADTM exhibited concentration-dependent inhibition on ADP-induced and AA-induced platelet aggregation with comparable effects to aspirin and clopidogrel.In vivo study showed that ADP-induced platelet aggregation was significantly compromised(>40%reduction)in rats treated with ADTM(20mg·kg-1).Similarly,ADTM also exhibited significant anti-thrombotic effect in vivo as shown in the ferric chloride(FeCl3)-induced venous thrombosis.Immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting showed that ADTM disrupted the interaction of ERp57 with αⅡ bβ3.CONCLUSION These results demonstrated that ADTM exhibited broad-spectrum anti-platelet activities and ERp57 is a potential therapeutic target for anti-platelet therapy.     

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

Aim： Studies of the α7-type neuronal nicotinic acetylcholine receptor （AChR）, one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs. The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods： Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [125Ⅰ]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results： The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [125Ⅰ]aBTX saturable binding with an apparent KD of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-PI-hα7- Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-PI-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion： The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardio- myocytes

β-adrenergic receptor （β-AR） agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 （Cx43）, the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication （GJIC） function.
Methods： The level of Cx43 expression in neonatal rat cardiomyocytes （NRCM） was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.
Results： Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β2-AR inhibitor ICI 118551, but not β1-AR inhibitor CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and Gi protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.
Conclusion： Taken together, β2-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A-and G1-dependent manner.