Microtubule-Mediated Regulation of ß2AR Translation and Function in Failing Hearts.

BACKGROUND: ß1AR (beta-1 adrenergic receptor) and ß2AR (beta-2 adrenergic receptor)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac ß-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that ß-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. METHODS: The localization pattern of ß-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on ß-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible ß-AR translation sites in cardiomyocytes. The mechanism by which ß-AR mRNA is redistributed post-heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post-myocardial infarction and detubulated cardiomyocytes. RESULTS: ß1AR and ß2AR mRNAs show differential localization in cardiomyocytes, with ß1AR found in the perinuclear region and ß2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of ß2AR transcripts toward the perinuclear region. The close proximity between ß2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of ß2AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both ß1AR and ß2AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to ß-AR mRNA redistribution and impaired ß2AR function in failing hearts. CONCLUSIONS: Asymmetrical microtubule-dependent trafficking dictates differential ß1AR and ß2AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 ß-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted ß2AR-mediated cyclic adenosine monophosphate signaling.

Responses of bone marrow gamma-glutamyltranspeptidase and alkaline phosphatase in vitro to tumor-elaborated granulocytopoietic factors.

Evidence has been obtained for the humoral mediation of the recently noted tumor-induced rise of the host bone marrow gamma-glutamyltranspeptidase (gamma GT) and alkaline phosphatase (AP) content in vivo: normal rat bone marrow suspensions, if incubated for 18 hr to 3 days with serum from mammary carcinoma hosts, show 2- to 8-fold elevations (per cell) of the same 2 enzymes. The active substance(s) is in the acid-stable, HCI-ethanol-soluble polypeptide fraction of the mammary carcinoma extract, and of the hosts' blood serum. The larger the size of the neoplasm, and the faster its growth rate, the greater the effect of the host serum on the gamma GT and AP of the normal bone marrow cells. In host rats in vivo, this response is followed by increases in the number (as well as the gamma GT and AP content) of circulating granulocytes. Therefore, a positive response on the part of these enzymes in the bone marrow suspension was also sought, and found, upon incubation with preparations which enhance granulocyte colony formation in agar cultures (i.e., colony-stimulating factor and serum from endotoxin-treated rats). The results indicate: (a) that the increase in gamma GT and AP is a necessary prelude to stimulation of granulocyte multiplication by appropriate growth factors; and (b) that measurement of these enzymes in the short-term liquid culture offers a biochemical test for such factors elaborated by cancers or in nonneoplastic conditions.

Regulator of G protein signaling 20 enhances cancer cell aggregation, migration, invasion and adhesion.

Several RGS (regulator of G protein signaling) proteins are known to be upregulated in a variety of tumors but their roles in modulating tumorigenesis remain undefined. Since the expression of RGS20 is elevated in metastatic melanoma and breast tumors, we examined the effects of RGS20 overexpression and knockdown on the cell mobility and adhesive properties of different human cancer cell lines, including cervical cancer HeLa, breast adenocarcinoma MDA-MB-231, and non-small cell lung carcinoma H1299 and A549 cells. Expression of RGS20 enhanced cell aggregation, migration, invasion and adhesion as determined by hanging drop aggregation, wound healing, transwell chamber migration and invasion assays. Conversely, shRNA-mediated knockdown of endogenous RGS20 impaired these responses. In addition, RGS20 elevated the expression of vimentin (a mesenchymal cell marker) but down-regulated the expression of E-cadherin, two indicators commonly associated with metastasis. These results suggest that the expression of RGS20 may promote metastasis of tumor cells.

beta-Adrenergic receptors in the developing rabbit lung.

beta-Adrenergic receptors and catecholamine-sensitive adenylate cyclase were identified and partially characterized in membrane fractions of rabbit lungs from day 25 of gestation to adulthood with the beta-adrenergic antagonists (--)-[3H]dihydroalprenolol [(--)-[3H]DHA] and (--)-[125I]iodohydroxybenzylpindolol [(--)-[125I]HYP]. beta-Adrenergic receptor number (Bmax) increased 11.5-fold during this time period, increasing progressively during the latter days of gestation and the early neonatal period, from 37 +/- 10 fmol/mg protein at 25 days gestation to 425 +/- 51 fmol/mg in the adult rabbit lung (mean +/- SD). Receptor affinity for (--)-[3H]DHA (KD = 1.8 nM) or (--)-[125I]HYP (KD - 0.104 nM) and the proportion of beta 1- and beta 2-adrenergic receptor subtypes (60% beta 1 and 40% beta 2) did not change with advancing age. Basal adenylate cyclase activity in lung homogenates decreased significantly with increasing age, whereas the activity in the presence of catecholamine or NaF remained nearly constant. Catecholamines stimulated adenylate cyclase activity at all ages studied supporting a role of the maturation of beta-adrenergic receptors in the regulation of pulmonary function.