Browsing by Subject "osmoregulation"
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Item Molecular Cloning, Expression, and Regulation of the Na+/Myo-Inosiotl Cotransporter Gene(1996-08-01) Zhou, Cheng; Chaitin, Michael; Easom, Richard; Garner, MargaretZhou, Cheng, Molecular Cloning, Expression, and Regulation of the NA+/Myo-Inositol Cotransporter Gene. Doctor of Philosophy (Biomedical Sciences), August 1996. Mammalian cells respond to osmotic stress by accumulation of high concentrations of intracellular osmolytes. Osmotic-induced accumulation of the osmolyte, myo-inositol (MI), is achieved through activation of the NA+/MI cotransporter. Hypertonic stress results in elevated NA+/MI cotransporter mRNA abundance and transcription rate, and increased transporter activity. The goals of this dissertation are to establish the osmoregulation of the NA+/MI cotransporter gene expression in lens cells, and to investigate the transcriptional regulation of the NA+/MI cotransporter gene. Expression of the Na+/MI cotransporter in cultured bovine lens epithelial cells (BLECs) was demonstrated by RT-PCR amplification and Northern blot analysis. Hypertonic stress resulted in induction of the NA+/MI contransporter mRNA abundance in cultured BLECs. The induction patterns of the NA+/MI cotransporter and aldose reductase mRNA abundance by hypertonic stress indicated that osmoregulation of MI and sorbitol accumulations were regulated in concert. Accumulation of MI is an early-onset protective system, which is suppressed by the elevated sorbitol, the late-onset protective system. 5’-RACE analysis indicated that multiple transcription start sites were utilized in controlling of the expression of the NA+/MI cotransporter. Osmotic stress resulted in preferential utilization of a hypertonic promoter a. The bovine NA+/MI cotransporter gene was cloned and analyzed. The regulation of the Na+/MI cotransporter expression was investigated by transient transfection assays using promoter-luciferase constructs. Although multiple promoters were functional in cultured BLECs, only the hypertonic promoter a was osmotically responsive. Characterization of this osmotic-responsive element(s) between -536 to -300 bp upstream of the hypertonic transcription start site a. The studies presented in this dissertation refined the osmoregulation of the Na+/MI cotransporter gene expression. Hypertonicity induces MI accumulation by activation of an osmotic-responsive promoter. The consequences of the activation of this promoter lead to more cotransporter mRNA, more cotransporter protein, and higher transporter activity, resulting in accumulation of a higher concentration of intracellular Mi.Item The role of Angiotensin II in central autonomic and endocrine regulation(2014-08-01) Saxena, Ashwini; Cunningham, J. Thomas; Mifflin, Steve W.; Schreihofer, Ann M.Renin-Angiotensin system (RAS) is a peptidergic hormonal system that is known to regulate hemodynamic and fluid balance. Clinical success of RAS inhibitors in several cardiovascular and renal diseases such as hypertension (HTN), chronic heart failure, and diabetic nephropathy underscore its involvement in their pathophysiology. Recent research efforts are not only helping us understand the mechanisms through which RAS orchestrate the pathophysiology of cardiovascular diseases but are also identifying its involvement in other pathological conditions such as mood disorders and cancer. Although, initially identified as an endocrine system in peripheral circulation, the discovery of renin in the brain ushered in the concept of ‘local’ or ‘tissue’ RAS. Now, local RAS components are increasingly identified to be present in nearly all organ systems. The In the first project we investigated the role played by the communication between circulating Ang II and subfornical organ (SFO), a circumventricular organ lacking blood brain barrier, in chronic intermittent hypoxia (CIH) associated sustained increase in MAP even during period of normoxic breathing. Using viral mediated delivery of shRNA against Ang II type 1a receptors (AT1aR), it was found that the rats that received AT1aRshRNA in their SFO, exhibited increased MAP responses during CIH but their MAP recovered to levels of normoxic control during room air breathing. Also, disruption of this communication led to decreased FosB/ΔFosB staining in the autonomic regions of forebrain. FosB/ΔFosB staining identifies the expression of transcription factor FosB and its splice variant ΔFosB. These transcription factors are known to orchestrate transcriptional adaptations that lead to lasting neuroplastic adaptations. These data suggest that Ang II-SFO communication is essential in neuroplastic adaptations of forebrain autonomic nuclei, which may sustain the CIH associated increase in MAP even during periods of room air breathing. Second project was initiated to study the mechanisms through which synaptically released Ang II could affect post-synaptic neuronal sensitivity and function. It is known that bile duct ligated rats, an experimental model of cirrhosis, exhibit impaired osmoregulation. We previously reported that bile duct ligated rats show increased presence of a non-specific cation channel (TRPV4) in the hypothalamic membrane extracts. In addition, an activated RAS is also associated with fluid-electrolyte imbalance, a hallmark feature of cirrhosis. In this project it was investigated if Ang II could translocate TRPV4 to neuronal surface in vitro, in a hypothalamic neuronal cell line, 4B. In 4B cells, Ang II incubation was associated with increased TRPV4 localization to the cell membrane and was also associated with increased calcium influx in response to specific TRPV4 agonist, GSK 1016790A. In addition, these effects were completely blocked in the presence of AT1R receptor antagonist (Losartan) and Src kinase inhibitor, PP2. Taken together, these data suggest that Ang II could translocate TRPV4 to neuronal membrane via Src kinase pathway. These observations could explain one of the mechanisms through which Ang II could contribute to the pathophysiological adaptations that lead to increased water retention and dilutional hyponatremia associated with chronic liver failure.