Higenamine, a Dual Agonist for ß 1- and ß 2-Adrenergic Receptors Identified by Screening a Traditional Chinese Medicine Library.

Chronic heart failure is the terminal stage of various cardiovascular diseases. Despite the availability of several classes of drugs, there is still an unmet need for effective treatment. Based on bench work during the past two decades, we have proposed that enhancement of ß 2-adrenergic receptor signaling in combination with the presently preferred ß 1-adrenergic receptor blockade would be a promising strategy. Chinese herbal medicines have been shown to be effective in the treatment of heart failure, although the mechanisms largely remain unknown. In the present study, we screened an herbal medicine compound/extract library for ß-adrenergic receptor ligands to determine the target of certain effective botanical remedies and seek a leading compound(s) for chronic heart failure treatment. Using a high-throughput screening assay, we identified higenamine, which has a long history in chronic heart failure treatment in traditional Chinese medicine, to be a potent ß-adrenergic receptor agonist. Further experiments using specific inhibitors showed that higenamine activated both ß 1-adrenergic receptor and ß 2-adrenergic receptor. Inhibition of its action by pertussis toxin (a Gi inhibitor) indicated that it is a ß 2-adrenergic receptor Gs/Gi dual agonist. Contractility experiments demonstrated a positive inotropic effect of higenamine. In conclusion, we found an herbal compound, higenamine, to be a dual agonist for ß 1/ß 2-adrenergic receptors with no preference in stimulating the Gs and Gi pathways in ß 2-adrenergic receptor signaling. Our results elucidated not only the target of higenamine to explain its pharmacological effect in treating chronic heart failure, but also the mechanisms of its cardiac toxicity.

(-)-Epigallocatechin-3-gallate, the major green tea catechin, regulates the desensitization of ß1 adrenoceptor via GRK2 in experimental heart failure.

OBJECTIVE: We investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cardiac function and its mechanism, which focused on the desensitization of ß1-AR and GRK2 in heart failure (HF) rats. METHODS: HF was induced by abdominal aortic coarctation. Four weeks after HF induction, the rats were given EGCG (25, 50, 100 mg/kg/day). Cardiac function was assessed by measuring haemodynamic parameters. Histological changes were analyzed by HE and Masson's trichrome staining. The expression of ß1-AR was detected by immunohistochemistry and immunofluorescence. The membrane expression of ß1-AR and GRK2 was detected by western blot. The expression levels of ß1-AR mRNA and GRK2 mRNA were evaluated by Q-PCR. RESULT: Compared to the control group, the left ventricular end diastolic pressure, mean blood pressure, heart weight/body weight, and posterior wall thickness in the HF group were significantly increased, whereas the left ventricular systolic pressure, maximum rate of left ventricular pressure rise (+ dP/dt max) and maximum rate of left ventricular pressure fall (- dP/dt max) were clearly decreased. EGCG could improve cardiac function by regulating these parameters. Inflammatory cell infiltration, irregularly arranged cardiomyocytes, swelling of cardiomyocytes and myocardial fibrosis were observed in HF rats' myocardial morphology, and EGCG obviously improved the morphological signs. The expression of ß1-AR was significantly decreased in the left ventricle tissue of HF rats by immunohistochemistry and immunofluorescence. The membrane expression of ß1-AR decreased, whereas GRK2 increased in vivo and in vitro by western blot. EGCG could down-regulate the membrane expression of GRK2 and up-regulate the expression of ß1-AR. There were no significant differences in the total expression of ß1-AR mRNA and GRK2 mRNA. CONCLUSIONS: EGCG has therapeutic effects on the heart function of HF rats. The mechanism might be related to the inhibition of the transfer membrane of GRK2 and to the reduction of the desensitization of ß1-AR.

Genetic characterization of physical activity behaviours in university students enrolled in kinesiology degree programs.

Studies of physical activity behaviours have increasingly shown the importance of heritable factors such as genetic variation. Nonsynonymous polymorphisms of alpha-actinin 3 (ACTN3) and the ß-adrenergic receptors 1 and 3 (ADRB1 and ADRB3) have been previously associated with exercise capacity and cardiometabolic health. We thus hypothesized that these polymorphisms are also related to physical activity behaviours in young adults. To test this hypothesis we examined relationships between ACTN3 (R577X), ARDB1 (Arg389Gly), ADRB3 (Trp64Arg), and physical activity behaviours in university students. We stratified for student enrollment in kinesiology degree programs compared with nonmajors as we previously found this to be a predictor of physical activity. We did not identify novel associations between physical activity and ACTN3. However, the minor alleles of ADRB1 and ADRB3 were significantly underrepresented in kinesiology students compared with nonmajors. Furthermore, carriers of the ADRB1 minor allele reported reduced participation in moderate physical activity and increased afternoon fatigue compared with ancestral allele homozygotes. Together, these findings suggest that the heritability of physical activity behaviours in young adults may be linked to nonsynonymous polymorphisms within ß-adrenergic receptors.

ß1/2 or M2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints.

Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of ß1/ß2 receptors and M2/M3 receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in ß1/2 receptor-knockout mice and M2/3 receptor-knockout mice. We found that knockout of the M2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the ß1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in ß1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in ß1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M2/3 receptor-knockout mice without altering gastrointestinal transition in wild-type littermates of either. Our study revealed that M2/3 receptors are required for the gastrointestinal motility associated with whole gastrointestinal transition enhanced by acupuncture at heterotopic acupoints, whereas ß1/2 receptors are required for the same gastrointestinal motility processes inhibited by acupuncture at homotopic acupoints. Therefore, our findings reveal important biological mechanisms underlying acupuncture treatment of disorders involving gastrointestinal motility dysfunction.

Zinc Methionine Supplementation Impacts Gene and Protein Expression in Calf-Fed Holstein Steers with Minimal Impact on Feedlot Performance.

Providing cattle a more bioavailable zinc (Zn) source prior to administering a beta adrenergic agonist (ßAA) may enhance the metabolic pool of primary nutrients that will influence the magnitude of the ßAA response. Calf-fed Holstein steers were supplemented with a Zn methionine supplement (ZnMet; ZINPRO(®); Zinpro Corporation, Eden Prairie, MN) for 115 ± 5 days prior to harvest along with zilpaterol hydrochloride (ZH; Zilmax(®); Merck Animal Health, Summit, NJ) for the last 20 days with a 3-day withdrawal to evaluate the effects on growth and carcass performance together with gene and protein expression of skeletal muscle, adipose tissue, and fatty acid composition of polar and neutral lipid depots. Steers (n = 1296; initial weight = 468.5 ± 0.5 kg) were sorted by weight, blocked by harvest date, and randomly assigned to pens (n = 12) and treatments: control (90 ppm Zn from ZnSO4) and ZnMet (Control plus 720 mg Zn from ZnMet/hd/d). There were no differences (P > 0.05) in growth performance or carcass characteristics. The ZnMet-fed cattle had reduced (P < 0.05) abundance of myosin heavy chain (MHC)-IIX, ß1-adrenergic receptor (ßAR), peroxisome proliferator-activated receptor gamma, and stearoyl-CoA desaturase mRNA in skeletal muscle tissue. The ZnMet cattle had greater (P < 0.05) abundance of MHC-II protein, increased MHC-IIA and IIX cross-sectional areas (P < 0.05), an increased percentage of MHC-I fibers (P < 0.05), and a decreased percentage of MHC-IIX fibers (P < 0.05). The combination of ZnMet and ZH had positive biological effects on musculoskeletal tissue; however, these molecular effects were not significant enough to impact overall feedlot and carcass performance.

Screening ß1AR inhibitors by cell membrane chromatography and offline UPLC/MS method for protecting myocardial ischemia.

A high expression ß1AR/cell membrane chromatography (ß1AR-CMC) and offline UPLC/MS method has been developed for screening active ingredients from Coptis chinensis. In this study, the fractions retained by CMC column were separated and identified by UPLC/MS system. Using metoprolol as a positive control drug, coptisine from C. chinensis was identified as the active component which could inhibit ß1AR. Compared with the control group: coptisine could attenuate the infarct size and release malondialdehyde (MDA) while increasing superoxide dismutase (SOD) activity, suggesting a role in reducing myocardial injury. In vitro, coptisine could decrease apoptosis, showing their protective effects upon cardiomyocytes. This ß1AR-CMC-offline-UPLC/MS method can be applied for screening active components acting on ß1AR from traditional Chinese medicines.

[Involvement of beta-adrenoceptors in cardioprotective effect of electroacupuncture intervention in mice with swimming fatigue-induced myocardial ischemia].

OBJECTIVE: To observe the cardioprotective effect of electroacupuncture (EA) stimulation at "Neiguan" (PC 6) in mice with myocardial ischemia (MI) and to explore the involvement of cardiac beta-adrenergic receptors (beta-AR) in the cardioprotective effect of EA intervention. METHODS: Adult male C 57 BL/6 mice of wild type and beta1/beta2-AR double-knockout (beta1/beta2-AR(-/-)) mice were randomly and respectively divided into control group and EA group, with 8 mice in each group. These mice were subject to procedures of basic control, fatigue swimming and fatigue swimming + EA. The model was established by fatigue swimming for inducing acute MI. EA (2 Hz, 0.5 mA) was applied to bilateral "Neiguan" (PC 6) for 30 min, once daily for 7 days in both EA groups. The ST-segment of electrocardiogram (ECG) of standard limb lead II, heart rate were recorded by using a biophysical amplifier and the arrhythmia score was assessed according to Curtis and Walker's methods (1988). RESULTS: Self-comparison showed that, compared with the baseline, the amplitude of ECG-ST II segment was obviously increased in swimming fatigue C 57 BL/6 mice (P < 0.01) and further obviously increased in beta1/beta2-AR(-/-) mice (P < 0.01), suggesting an acute MI in C 57 BL/6 mice and a worsened MI in beta1/beta2-AR(-/-) mice. Simultaneously, the heart rate was markedly decreased (P < 0.05), and the score of arrhythmia obviously increased in both C 57 BL/6 and beta1/beta2-AR(-/-) mice (P < 0.05). Compared with the modeling procedures, ECG-ST II amplitude and arrhythmia score were significantly decreased in C 57 BL/6 mice of the EA group (P < 0.01, P < 0.05), rather than in the beta1/beta2-AR(-/-) mice of the EA group (P > 0.05). In addition, heart rate levels of both C 57 BL/6 mice and beta1/beta2-AR(-/-) mice had no significant differences between control and EA groups (P > 0.05). CONCLUSION: EA at "Neiguan" (PC 6) can protect the heart from swimming fatigue-induced MI in C 57 BL/6 mice not in beta1/beta2-AR(-/-) mice, suggesting an involvement of beta1/beta2-AR in the protective effect of EA.

Heart-protective effect of n-3 PUFA demonstrated in a rat model of diabetic cardiomyopathy.

This study was designed to examine in vivo functional changes of the heart in the early stages of streptozotocin (STZ)-induced diabetic cardiomyopathy and to evaluate the effects of n-3 PUFA intake. Moreover, we investigated whether modulation of diabetes-related abnormalities of myocardial connexin-43 (Cx43), ß-myosin heavy chain (ß-MHC), and ß1-adrenergic receptors (ß1-AR) might be implicated in the cardioprotective mechanism of n-3 PUFA. Our results showed significantly reduced cardiac output and ejection fraction (using the microtip pressure-volume catheter technique) as well as stroke volume and stroke work, 4 weeks after STZ-induced diabetes, with improvement of these parameters due to n-3 PUFA consumption. Myocardial expression of Cx43 mRNA estimated by real-time polymerase chain reaction did not change in diabetic rats regardless of n-3 PUFA consumption (100 mg/100 g b.w./day). In contrast, the total and functional phosphorylated form of Cx43 protein increased significantly, and its cardiomyocyte-related distribution was disordered in the diabetic heart, but these changes normalized because of n-3 PUFA intake. Furthermore, acute diabetes was accompanied by decrease of myocardial ß1-AR mRNA expression and mild yet nonsignificant increase of ß-MHC mRNA. These alterations were not significantly affected by n-3 PUFA. In conclusion, the results point out that STZ-diabetic rats benefit from n-3 PUFA consumption particularly because of the attenuation of myocardial Cx43 abnormalities that most likely contributes to improvement of cardiac function.

High-expression ß(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

ß-Adrenergic receptors are important targets for drug discovery. We have developed a new ß1 -adrenergic receptor cell membrane chromatography (ß1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high ß1 AR expression levels were established and used to prepare a cell membrane stationary phase in a ß1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on ß1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of ß1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource.

Downregulation of ß1 -adrenergic receptors in rat C6 glioblastoma cells by hyperforin and hyperoside from St John's wort.

OBJECTIVES: While the use of St John's wort extracts as treatment for mild to moderate depression is well established the mode of action is still under investigation. Individual constituents of St John's wort extract were tested for possible effects on the ß1 AR density and a subsequent change in downstream signalling in rat C6 glioblastoma cells. METHODS: The effect of compounds from St John's wort extract on the downregulation of ß1 -adrenergic receptor-GFP fusion proteins (ß1 AR-green fluorescent protein (GFP)) of transfected rat C6 gliobastoma cells (C6-ß1 AR-GFP) was investigated by means of confocal laser scanning microscopy (LSM). The influence on the lateral mobility of ß1 AR-GFP in C6-ß1 AR-GFP was investigated by fluorescence correlation spectroscopy. The formation of second messenger was determined by c-AMP-assay. KEY FINDINGS: Confocal LSM revealed that pretreatment of cells with 1 µm of hyperforin and hyperoside for 6 days, respectively, led to an internalization of ß1 AR-GFP under non-stimulating conditions. Observation by fluorescence correlation spectroscopy showed two diffusion time constants for control cells, with τdiff1 = 0.78 ± 0.18 ms and τdiff2 = 122.53 ± 69.41 ms, similarly distributed. Pretreatment with 1 µm hyperforin or 1 µm hyperoside for 3 days did not alter the τdiff values but decreased the fraction of τdiff1 whereas the fraction of τdiff2 increased significantly. An elevated level of ß1 AR-GFP with hindered lateral mobility was in line with ß1 AR-GFP internalization induced by hyperforin and hyperoside, respectively. A reduced ß1 -adrenergic responsiveness was assumed for C6 gliobastoma cells after pretreatment for 6 days with 1 µm of both hyperforin and hyperoside, which was confirmed by decreased cAMP formation of about 10% and 5% under non-stimulating conditions. Decrease in cAMP formation by 23% for hyperforin and 15% for hyperoside was more pronounced after stimulation with 10 µm dobutamine for 30 min. CONCLUSIONS: The treatment of C6 gliobastoma cells with hyperforin and hyperoside results in a reduced ß1 AR density in the plasma membrane and a subsequent reduced downstream signalling.

[Effects of electroacupuncture stimulation of scalp-point on cardiac sympathetic discharges, myocardial beta1-adrenoceptor protein expression and plasma norepinephrine concentration in myocardial ischemia-reperfusion injury rats].

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) of scalp-point in the management of myocardial ischemia-reperfusion injury (MI/RI) by examining its effects on left cardiac sympathetic nerve activity, myocardial beta1-adrenaline receptor (AR) protein expression and plasma norepinephrine (NE) concentration in MI/RI rats. METHODS: Eighteen Wistar rats were randomly divided into sham, model and EA groups (n = 6). MI/RI model was induced by ligation of the left anterior descending coronary artery for 30 min, followed by release of the ligation for 15 min. EA was applied to bilateral Epangxian I (MS 2) for 15 min. The left cardiac sympathetic nerve activity was recorded with BL-420 E+ biological signal acquisition system. Myocardial beta1-AR protein expression was examined by western blot and plasma NE level detected by enzyme-labeled immunosorbent assay. RESULTS: Compared with the sham group, the left cardiac sympathetic discharges and plasma NE levels and myocardial beta1-AR protein expression were markedly increased in the model group (P < 0.01), whereas in comparison with the model group, the sympathetic discharges, plasma NE level and myocardial beta1-AR protein expression in the EA group were down-regulated significantly (P < 0.05). CONCLUSION: Scalp-point EA intervention can suppress MI/RI induced increase of sympathetic nerve activity and plasma NE level, and beta1-AR protein expression, which may contribute to its effect in relieving myocardial ischemia.

Hypothalamic gene expression in ω-3 PUFA-deficient male rats before, and following, development of hypertension.

Dietary deficiency of ω-3 fatty acids (ω-3 DEF) produces hypertension in later life. This study examined the effect of ω-3 DEF on blood pressure and hypothalamic gene expression in young rats, before the development of hypertension, and in older rats following the onset of hypertension. Animals were fed experimental diets that were deficient in ω-3 fatty acids, sufficient in short-chain ω-3 fatty acids or sufficient in short- and long-chain ω-3 fatty acids, from the prenatal period until 10 or 36 weeks-of-age. There was no difference in blood pressure between groups at 10 weeks-of-age; however, at 36 weeks-of-age ω-3 DEF animals were hypertensive in relation to sufficient groups. At 10 weeks, expression of angiotensin-II(1A) receptors and dopamine D(3) receptors were significantly increased in the hypothalamic tissue of ω-3 DEF animals. In contrast, at 36 weeks, α(2a) and ß(1) adrenergic receptor expression was significantly reduced in the ω-3 DEF group. Brain docosahexaenoic acid was significantly lower in ω-3 DEF group compared with sufficient groups. This study demonstrates that dietary ω-3 DEF causes changes both in the expression of key genes involved in central blood pressure regulation and in blood pressure. The data may indicate that hypertension resulting from ω-3 DEF is mediated by the central adrenergic system.

[Effects of acupuncture on expression of norepinephrine transporter mRNA in the cervical sympathetic ganglion and beta1-AR mRNA in the heart in cerebral-cardiac syndrome rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on the expression of norepinephrine transporter (NET) mRNA in the middle cervical-stellate ganglion complex (MC-SGC) and beta1-adrenergic receptor (beta1-AR) mRNA in the left ventricular myocardium in cerebral-cardiac syndrome (CCS) rats, so as to study its underlying mechanism in the prevention and treatment of CCS. METHODS: Eighteen Wistar rats were randomized into sham operation, model and EA groups (n=6). CCS model was duplicated by intravenous injection of 1 microL of saline containing collagenase (1 U/microL) + heparin (7 U/microL) into the caudate nucleus. EA (2 Hz, 5 V) was applied to "Shuigou" (GV 26)-"Fengfu" (GV 16), and "Neiguan" (PC 6)-"Xinshu" (BL 15) for 20 min, once daily for 3 days. Seventy-two hours after modeling, pathological changes of the brain and cardiac tissues were observed after sectioning and stained with HE method. The expression of MC-SGC NET mRNA and the myocardial p1-AR mRNA of the left ventricle were detected by fluorescence quantitative PCR. RESULTS: (1) In comparison with the model group, the number of the exuded red blood cells in the brain tissue around the caudate Nucleus region was fewer, the severity of the tissue edema, cell necrosis, and inflammatory cell infiltration lighter in the EA group. Compared with the model group, the severity of degeneration and necrosis of myocytes was lighter and the number of the broken cardiac smooth muscle fewer in the EA group. (2) The expression levels of both MC-SGC NET mRNA and myocardial beta1-AR mRNA were significantly lower in the model group than in the sham operation group (P < 0.01). Compared with the model group, the expression levels of both MC-SGC NET mRNA and myocardial beta1-AR mRNA were upregulated considerably in the EA group (P < 0.01). CONCLUSION: EA of GV 26-GV 16 and PC 6-BL 15 can upregulate the expression of both MC-SGC NET mRNA and myocardial beta1-AR mRNA in CCS rats which may contribute to its effect in the prevention and treatment of CCS syndrome.

The effects of acute triiodothyronine therapy on myocardial gene expression in brain stem dead cardiac donors.

CONTEXT: After brain stem death (BSD), a low T(3) state is common, and T(3) supplementation has been advocated to improve heart function and yield for transplantation. OBJECTIVES: The aim of the study was to assess the effects of T(3) on expression of mRNAs encoding T(3)-responsive genes in the post-BSD human heart. DESIGN: Within a prospective double-blind trial, potential BSD cardiac donors undergoing hemodynamic optimization were randomized to T(3) (0.8 microg . kg(-1) bolus; infusion 0.113 microg . kg(-1) . h(-1)) or placebo (5% dextrose) for up to 6 h. Left ventricular biopsies were obtained at end-assessment from 30 donors (T(3); n=16). TaqMan real-time PCR was performed to investigate mRNA expression of the voltage-gated potassium channel Kv1.5, beta-1 adrenergic receptor (ADRB1), sarcoplasmic reticulum calcium ATPase type 2a (SERCA2a), and phospholamban (PLB). RESULTS: Time between diagnosis of BSD and donor management was 13.2 h (range, 9.7-16.8 h). T(3) donors were managed for 7.6 (6.9-8.3) h. Median serum free T(3) (fT3) at baseline was 2.9 (2.3-3.8) pmol . liter(-1) (reference range, 3.3-7.5 pmol . liter(-1)). At baseline, 19 of 30 (56.7%) had low serum fT3, and T(3) treatment increased fT3 to supraphysiological levels (P < 0.001). Expression of mRNAs encoding Kv1.5 and SERCA2a was increased 1.99-fold and 1.51-fold (P = 0.015 and 0.043). There was no significant change in the expression of mRNAs encoding ADRB1 and PLB. Treatment with T(3) did not improve hemodynamic function compared with placebo. CONCLUSIONS: Acute administration of T(3) in the BSD cardiac donor reverses the low T(3) state and increases expression of the mRNAs encoding Kv1.5 and SERCA2a, but not ADRB1 or PLB and is not associated with any improvement in hemodynamic performance.

Mutagenesis of important amino acid reveals unconventional homologous internalization of beta(1)-adrenergic receptor.

AIMS: The study was designed to examine the internalization of Asp104Lys mutant of beta(1)-adrenergic receptor (beta(1)-AR) and compared to other mutant (Asp104Ala) and wild type receptors. Moreover, this study needs to perform the role of GRK2 (betaARK1) and beta-arrestin1 on this internalization of Asp104Lys mutant of beta(1)-AR. MAIN METHODS: Binding affinity, functional potency of agonist and agonist-induced internalization were determined for wild type and both mutants of beta(1)-ARs stably expressed in HEK 293 cells as assessed by [(3)H] CGP12177 radioligand. We have performed GRK2 and beta-arrestin1 expression levels by western blot analysis and also performed internalization of this mutant receptor after over expression and deletion of beta-arrestin1 gene. KEY FINDINGS: In the present study, the binding affinity of (-)-isoproterenol for both mutants were significantly decreased compared to wild type. Though the mutant Asp104Ala showed agonist-induced receptor activation, interestingly this mutant was not internalized. However, the mutant Asp104Lys, which showed uncoupling with G protein, was internalized 31.77+/-3.13% from cell surface. Asp104Lys mutant produced the same level of GRK2 expression in (-)-isoproterenol induced stimulation of wild type receptor and addition of (-)-isoproterenol further increased GRK2 expression in mutant receptors. In addition, overexpression of beta-arrestin1 in mutant Asp104Lys promoted (39.75+/-2.19%) and knockdown of beta-arrestin1 by siRNA decreased (3.55+/-1.75%) internalization compared to Asp104Lys mutant of beta(1)-ARs. SIGNIFICANCE: The present studies suggest that Asp104Lys mutant beta(1)-ARs triggers unconventional homologous internalization induced by G protein independent signals, where GRK2 and beta-arrestin1 play an important role for beta(1)-AR internalization.

Oxidative stress triggers cardiac fibrosis in the heart of diabetic rats.

Diabetic cardiomyopathy is characterized by myocyte loss and myocardial fibrosis, leading to decreased elasticity and impaired contractile function. The study examines the downstream signaling whereby oxidative stress, induced by hyperglycemia, leads to myocardial fibrosis and impaired contractile function in the left ventricle of diabetic rats. It also examines the effects of dehydroepiandrosterone (DHEA), which prevents the oxidative damage induced by hyperglycemia in experimental models. DHEA was administered for 6 wk in the diet [0.02%, wt/wt)] to rats with streptozotocin-induced diabetes. Oxidative balance, advanced glycated end products (AGEs) and AGE receptors, transcription factors nuclear factor-kappaB and activator protein-1, and profibrogenic growth factors (connective tissue growth factor and TGFbeta1) were determined in the left ventricle of treated and untreated streptozotocin-diabetic rats. Structural and ultrastructural changes, and the contractile force developed by electrically driven papillary muscles, under basal conditions and after stimulation with isoproterenol, were also evaluated. Oxidative stress induced by hyperglycemia increased AGEs and AGE receptors and triggered a cascade of signaling, eventually leading to interstitial fibrosis. DHEA treatment, by improving oxidative balance, counteracted the enhanced AGE receptor activation and increase of profibrogenic factors and restored tissue levels of collagen I, collagen IV, and fibronectin to those of control animals. Moreover, DHEA completely restored the contractility of isolated papillary muscle. Oxidative stress led to cardiac fibrosis, the most important pathogenetic factor of the heart's impaired functional integrity in diabetes. Structural and ultrastructural changes and impairment of muscle function induced by experimental diabetes were minimized by DHEA treatment.

Down-regulation of beta1-adrenoceptors gene expression by short interfering RNA impairs the memory retrieval in the basolateral amygdala of rats.

The influence of basolateral amygdala (BLA) on memory is known to depend critically on adrenergic neurotransmission. However, the roles of noradrenergic receptors on memory retrieval have been elusive and controversial. Here, we investigated the effect of beta(1)-adrenoceptor (beta(1)-AR) on auditory fear memory in the rat BLA. We attenuated the expression of beta(1)-AR by RNA interference, a popular means to specific suppress gene expression. Bilaterally microinjection of beta(1)-AR short interfering RNA (siRNA) could reach a satisfying transfection in the BLA: beta(1)-AR protein expression was reduced transiently by siRNA in vivo at day 3. The behavioral tests indicated that memory retrieval was impaired as beta(1)-AR protein expression was prevented, and the memory was restored when the beta(1)-AR protein got back to normal level. The results suggested that beta(1)-AR might be critical for the retrieval of auditory fear memory.

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.

Ser49Gly of beta1-adrenergic receptor is associated with effective beta-blocker dose in dilated cardiomyopathy.

OBJECTIVE: Our objective was to evaluate the influence of polymorphisms at codons 49 and 389 of the beta1-adrenergic receptor (beta1-AR) on the response to beta-blockers and outcome in patients with dilated cardiomyopathy. METHODS: We genotyped both codons of the beta1-AR in 375 patients with dilated cardiomyopathy and 492 control subjects. RESULTS: Neither of the polymorphisms was associated with susceptibility for dilated cardiomyopathy. In a retrospective analysis of patients receiving beta-blockers, there was a significant association between long-term survival rate and codon 49 (P = .014) but not codon 389 (P = .08). Despite a similar mean heart rate (69 beats/min), patients with the Ser49 genotype tended to have higher doses of beta-blockade compared with Gly49 carriers (P = .065). In patients receiving a low dose of beta-blockade (< or = 50% of targeted full dose), the 5-year mortality rate was lower among Gly49 carriers than Ser49 patients (risk ratio [RR], 0.24; 95% confidence interval [CI], 0.07-0.80; P = .020). In patients receiving high doses of beta-blockers, there was no significant difference in outcome between genotypes (P = .20), which was attributable to a better outcome for Ser49 patients treated with a high dose of beta-blockade as compared with a low dose. Gly49 carriers had a similar survival rate with different doses of beta-blockers. With low-dose beta-blockers, both codon 49 (RR, 0.26; 95% CI, 0.08-0.89; P = .029) and codon 389 (RR, 2.42; 95% CI, 1.04-5.63, P = .039) were related to 5-year mortality rate. CONCLUSION: In patients with heart failure, the influence of codon 49 on the outcome and effect of beta-blockers appeared to be more pronounced than that of codon 389. The more common Ser49Ser genotype responded less beneficially to beta-blockade and would motivate genotyping to promote higher doses for the best outcome effect.