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Regulation of beta-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2.

ABSTRACT: The regulation of the expression of beta-adrenoceptor (beta-ARs) is not thoroughly understood. We demonstrate that the rat heart cell-line H9c2 expresses both beta 1- and beta 2-ARs. In radioligand-binding experiments, the maximal binding capacity of (-)-[125I]-iodocyanopindolol was determined as 18 +/- 0.6 fmol/mg of protein with a KD of 35.4 +/- 4.1 pM. Competitive radioligand-binding experiments with subtype-specific beta-antagonists reveal a subtype ratio of beta 1- to beta 2-ARs of 29%: 71%. With competitive reverse-transcriptase PCR we found beta 2-mRNA to be up to 1600 times more frequent than beta 1-mRNA. Treatment of the H9c2 cell-line with the beta-adrenergic agonist (-)-isoproterenol (10(-6) M), the antagonist (-)-propranolol (10(-6) M) and the glucocorticoid dexamethasone (500 nM) induces regulatory effects on both the beta-AR protein and mRNA level. Isoproterenol treatment leads to down-regulation of the total receptor number by 56 +/- 4%, due to a decrease in beta 2-ARs, while maintaining the beta 1-AR number constant. On the transcription level, both beta 1-and beta 2-mRNAs are decreased by 30% and 42% respectively. mRNA stability measurements reveal a reduced half-life of beta 2-mRNA from 9.3 h to 6.5 h after isoproterenol treatment. Incubation of cells with (-)-propranolol does not affect the amounts of beta-ARs and their mRNAs. Dexamethasone induces a 1.8 +/- 0.2-fold increase in beta-AR number over the basal level as well as a 1.9 +/- 0.2-fold increase in the amount of beta 2-mRNA. Because the half-life of beta 2-mRNA was unaffected by dexamethasone, the increased beta 2-mRNA level must be due to an enhanced transcription rate. The beta 1-mRNA levels are unchanged during dexamethasone-incubation of the cells. Our data clearly demonstrate that treatment of H9c2 rat heart cells with isoproterenol and dexamethasone induces alterations in the level of RNA stability as well as gene transcription, leading to altered receptor numbers.

SUBMITTER: Dangel V

PROVIDER: S-EPMC1217574 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Project description:Chinese hamster ovary (CHO) cells transfected to express human beta 2- or beta 3-adrenoceptors (beta 2-CHO and beta 3-CHO cells) were exposed to the beta-adrenoceptor agonist isoprenaline at various concentrations and for differing times. Sustained exposure of the beta 2-CHO but not beta 3-CHO cells to isoprenaline resulted in a time- and concentration-dependent down-regulation of the receptor as measured by a reduction in specific binding of [125I]cyanopindolol. Such maintained exposure of cells expressing either receptor to the agonist produced a marked down-regulation of immunologically detectable levels of the alpha subunit of the stimulatory guanine-nucleotide-binding protein Gs. This effect was specific for Gs because levels of both G12 alpha and Gq alpha/G11 alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on Gs alpha down-regulation was some 30-fold more potent in the beta 2-CHO than in the beta 3-CHO cells. Time courses of isoprenaline-induced down-regulation of Gs alpha were not different, however, in the two cell lines. Isoprenaline treatment of the beta 3-CHO cells produced a desensitization of agonist-mediated regulation of adenylyl cyclase, manifested by a 4-fold reduction in the potency and a 30% reduction in maximal effect of the agonist, whereas desensitization of the beta 2-CHO cells was considerably greater (25-fold reduction in potency and 70% reduction in maximal effect). These results demonstrate that agonist-induced down-regulation of the G-protein which interacts with a receptor can be produced by both beta 2- and beta 3-adrenoceptors. Despite apparent concurrence of down-regulation of receptors and G-proteins in other systems [e.g. Adie, Mullaney, McKenzie and Milligan (1992) Biochem. J. 285, 529-536], agonist-induced receptor down-regulation does not appear to be a prerequisite for down-regulation of the G-protein. Furthermore, the results suggest that agonist-induced down-regulation of a G-protein may be sufficient, in the absence of receptor regulation, to induce some agonist desensitization of effector function.

Project description:Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (?AR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific ?AR-cAMP signaling within cardiomyocyte membrane microdomains. Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and ?AR-cAMP signaling. Results: ?2AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM ? p < 0.05). There was no effect of PMU on ?2AR-cAMP signaling in RII_Protein Kinase A domains. ?1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that ?2AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM ?? p < 0.01, ??? p < 0.001 vs. loaded TT). By comparison ?1AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (Po) were reduced by PMU (loaded TT Po 0.073 ± 0.011% vs. loaded crest Po 0.027 ± 0.006% N,n 5/18-26 mean ± SEM ? p < 0.05) (unloaded TT 0.0350 ± 0.003% vs. unloaded crest Po 0.025 N,n 5/20-30 mean ± SEM NS # p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2. Discussion: PMU suppresses' ?2AR-cAMP and LTCC activity. When activated, the signaling of ?2AR-cAMP and LTCC become more far-reaching after PMU. We suggest that a situation of 'suppression/decompartmentation' is elicited by the loss of refined cardiomyocyte structure following PMU. As PMU is a component of modern device therapy for heart failure this study has clinical ramifications and raises important questions for regenerative medicine.

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Regulation of beta-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2.

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