We have recently shown that the octapeptide angiotensin II is a potent stimulus of protein synthesis and growth in cultured cardiomyocytes. The present study was performed to determine if the renin-angiotensin system was involved in regulating cardiac cell growth in vivo. The pressure-overload cardiac hypertrophy model that develops in abdominal aorta-constricted rats was studied. At 7 and 15 days after abdominal aorta constriction, rats developed significant left ventricular hypertrophy. The increase in left ventricular mass was completely prevented in animals fed the angiotensin-converting enzyme inhibitor, enalapril maleate (0.2 mg/ml) in their drinking water. Cardiac afterload was the same in both groups of animals in that carotid artery pressures were not different in conscious awake aortic-constricted animals receiving and not receiving enalapril. These data suggest a direct growth effect of angiotensin II on the left ventricle and indicate a role for the renin-angiotensin system in the cardiac hypertrophy that develops in response to pressure overload. The presence and chamber localization of angiotensinogen mRNA was determined using Northern hybridization and S1 nuclease mapping analysis. Angiotensinogen mRNA, as determined by dot-blot hybridization analysis, was significantly increased in hypertrophied left ventricles at both 7 and 15 days after the surgery, when compared with sham-operated controls. The activity of the circulating renin-angiotensin system, as indexed by plasma renin activity was increased at 1 day following surgery [6.0 +/- 2.0 ng.ml-1.h-1 angiotensin I (control) vs. 41.8 +/- 10.9 ng.ml-1.h-1 angiotensin I (experimental)], but returned to control values by day 3 postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

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There is increasing evidence that the renin-angiotensin system (RAS) modulates cardiovascular function through both blood-borne and tissue-derived components. The existence of a local RAS has been proposed in the heart based on biochemical and molecular biological studies that identify angiotensinogen and renin. We conducted the present study to determine the chamber localization of angiotensinogen and renin mRNA in neonatal rat heart and whether these components could be identified in cultured cardiomyocytes and fibroblasts obtained from neonatal rat heart. Experiments using polymerase chain reaction (PCR) indicated that whole hearts obtained from neonatal rats contained both angiotensinogen and renin mRNA. With the use of radiolabeled cDNA probes and in situ hybridization, angiotensinogen and renin transcripts were localized both in the atria and ventricles of neonatal rat hearts. Relative signal strengths for angiotensinogen were highest in the left and right ventricles. In contrast, renin signal strength was overall much lower and preferentially localized in the left ventricle. To investigate the cellular source of angiotensinogen and renin, cultured neonatal heart cardiomyocytes and ventricular fibroblasts were screened for angiotensinogen and renin messenger RNA and protein using PCR and indirect immunofluorescent staining, respectively. These experiments demonstrated that both cell types produce transcripts and the respective translation products for angiotensinogen and renin. These data suggest that the site of angiotensin II synthesis can occur at the level of the individual cardiomyocyte and fibroblast, where it may serve to directly and/or indirectly regulate cardiac rate, force, growth, and development in the neonate.

Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4–20 spots while some of the OTR diffuses throughout the nucleoplasm.The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

Muscle invasive bladder cancer (MIBC) is a common malignancy and major cause of morbidity worldwide. Over the last decade mortality rates for MIBC have not decreased as compared to other cancers indicating a need for novel strategies. The molecular chaperones HSP70 and HSP90 fold and maintain the 3-dimensional structures of numerous client proteins that signal for cancer cell growth and survival. Inhibition of HSP70 or HSP90 results in client protein degradation and associated oncogenic signaling. Here we targeted HSP70 and HSP90 with small molecule inhibitors that trap or block each chaperone in a low client-affinity “open” conformation. HSP70 inhibitors, VER155008 (VER) and MAL3-101 (MAL), along with HSP90 inhibitor, STA-9090 (STA), were tested alone and in combination for their ability to reduce cell viability and alter protein levels in 4 MIBC cell lines. When combined, VER+MAL synergistically reduced cell viability in each MIBC cell line while not inducing expression of heat shock proteins (HSPs). STA+MAL also synergistically reduced cell viability in each cell line but induced expression of cytoprotective HSPs indicating the merits of targeting HSP70 with VER+MAL. Additionally, we observed that STA induced the expression of the stress-related transcription factor HSF2 while reducing levels of the co-chaperone TTI1.