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  • MICROVASCULAR DYSFUNCTION IN HEART FAILURE WITH PRESERVED EJECTION FRACTION

    Davila, Alec Christopher; Biomedical Sciences (Augusta University, 2019-05)
    Background. Heart failure with preserved ejection fraction (HFpEF) is often manifested as impaired cardiac and microvascular reserve, for which no current effective therapies are available. We sought to determine if conducted vasodilation, which coordinates microvascular resistance longitudinally becomes compromised in HFpEF. We tested the hypothesis that inhibition of adenosine kinase (ADK), the major adenosine-metabolizing enzyme and novel therapeutic target, augments conducted vasodilation; therefore, improving tissue perfusion and left ventricle (LV) diastolic function. Methods and Results. Conducted vasodilation was assessed ex vivo in coronary arterioles isolated from right atrial appendages of patients with or without HFpEF diagnosis and in skeletal muscle arteries of the rodent model of HFpEF, ZSF1 rats. Obese ZSF1 rats displayed LV diastolic dysfunction over a 20-week lifespan as indicated by reduced E/A ratio and increased deceleration time of mitral flow velocity observed on echocardiogram. Conducted vasodilation in both HFpEF patients and obese ZSF1 rats were significantly reduced, which was associated with increased vascular expression of ADK. Isolated arterioles incubated with ADK inhibitor, ABT-702 (0.1 μM) displayed an improved conducted vasodilation. In vivo treatment of obese ZSF1 rats with ABT-702 (1.5 mg/kg, i.p. for 8-week) prevented LV diastolic dysfunction, and in a crossover design, ADK inhibition improved conducted vasodilation and LV diastolic function. Furthermore, ABT-702 treatment reduced surrogate markers of myocardial hypoxia (carbonic anhydrase 9 expression and fibrosis) in obese ZSF1 rats. Moreover, mice with endothelium-specific deletion of ADK exhibited augmented vasodilation and were protected against the development of transverse aortic constriction-induced LV dysfunction. Conclusion. Collectively, upregulation of microvascular ADK impairs conducted vasodilation in HFpEF. Pharmacological inhibition of ADK improves microvascular vasodilator function and provides beneficial effects on myocardial perfusion and LV diastolic function in HFpEF.
  • NAE1-MEDIATED NEDDYLATION IS REQUIRED FOR POSTNATAL LIVER DEVELOPMENT AND FUNCTION

    Sahay, Khushboo; Department of Physiology (7/26/2018)
    Liver disease is an important health concern and a significant source of morbidity and mortality in the United States and worldwide. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is a novel ubiquitin-like protein modifier. The conjugation of NEDD8 to target proteins, termed neddylation, requires NEDD8 specific E1, E2 and E3 ligases. Neddylation participates in various cellular processes. However, whether neddylation regulates liver development and function is completely unknown. We created mice with hepatocyte specific deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, and identified that they display severe hepatomegaly, hypertriglyceridemia, and hypercholesterolemia from 10 days after birth. By postnatal 14 days, their liver cytoarchitecture is completely disrupted, along with formation of numerous biliary cysts, fibrosis and hypoglycemia, which ultimately result in liver failure and premature death by 6 weeks. Mechanistically, NAE1 deficiency in hepatocytes caused reduced hepatocytespecific gene expression but increased biliary/oval cell gene expression in liver. In vitro, NAE1 inhibition by MLN4924 and CRISPR/Cas9-mediated NAE1 deletion in HepG2 cells recapitulated in vivo findings with repressed expression of hepatocyte specific genes but elevated biliary/oval cell gene expression. Together, these data highlight an essential role for neddylation in regulating hepatocyte lineage commitment and function as well as polycyst formation through trans/de-differentiation of hepatocytes.
  • Assessment of Renal Ischemia Reperfusion Induced Injury in Male and Female Rats

    Crislip, Gene Ryan; Department of Physiology (1/25/2018)
    Acute kidney injury (AKI) is a clinical problem often induced by ischemia reperfusion (IR). Males are reported to have worse outcomes following IR compared to females based on measurements of blood urea nitrogen and creatinine. However, these markers are produced at different levels depending on body mass. The goal of Aims 1 and 2 was to do a complete assessment of the impact of sex on IR to establish a model that displays a sex difference. We measured multiple markers, including inulin clearance which is the gold standard of determining renal function. We determined there is no sex difference in response to IR after 24 hours. However, males had impaired renal function, higher vascular congestion and tubular injury than females 7 days following IR. A consequence of vascular congestion and tubular injury is fluid leakage into interstitial space, which increases renal volume. The goal of Aim 3 was to determine if ultrasound could be used as a tool to detect progressive changes in regional kidney volume following IR. To do this, we compared renal volume measurements with stereological assessment and examined the use of renal volume as an injury marker following IR. We verified the use of ultrasound to monitor renal volume after IR and the changes in volume correlated with the extent of medullary injury. Limiting vascular congestion improves recovery following IR. Pericytes are contractile cells that line the vessels in the renal medulla that are prone to congestion following IR. The goal of Aim 4 was to determine the role of renal pericytes following IR. To do this, we decreased pericytes in rats before IR to determine if this effected injury. We found that lower pericyte density was associated with greater vascular congestion following IR, additionally, males lose more pericytes than females. From these studies, we concluded that there was no sex difference in IR induced injury after 24 hours, however, following 7 days males had poorer recovery than females. We hypothesize that this poorer recovery is attributed to less pericytes in males following IR resulting in the inability to reduce vascular congestion compared to females.
  • The effects of hypertension on neurovascular unit function and structure

    Iddings, Jennifer Ann; Department of Physiology (2015)
    Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. The goal of this dissertation project was to test the central hypothesis that hypertension-induced impairments in functional hyperemia are mediated by a specific disruption of communication within the neurovascular unit at the parenchymal arteriole level of the cerebrovascular tree. To test our hypothesis, we measured parenchymal arteriole reactivity, vascular smooth muscle cell Ca2+ dynamics, parenchymal arteriole remodeling and cerebral vascular density in cortical brain slices from normotensive (WKY) and hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A2 receptor agonist U46619 and basal vascular smooth muscle cell Ca2+ oscillation frequency were increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was increased in SHR. While K+-induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Parenchymal arteriole wall to lumen ratio and wall thickness were increased in SHR. Vascular density was also increased in deeper cortical layers in SHR. Our data indicate that although SHR parenchymal arterioles display vascular remodeling, neurovascular coupling is not impaired in SHR, at least at the parenchymal arteriole level.
  • The role of ceramide in the regulation of ciliogenesis

    He, Qian; Department of Physiology (2015)
    The primary cilium is a single, antenna-like protrusion of mammalian cells, involved in many signaling pathways important for cellular processes. In Madin-Darby Canine Kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) was observed at the base of the primary cilia. Ceramide and Rab11a vesicles showed similar protein and lipid profiles. The lipid and protein composition suggested the presence of a ceramide associated lipid-protein complex containing atypical protein kinase C (aPKC), Cdc42, Sec8, Rab11a, and Rab8 in MDCK cells. Ceramide vesicles and Rab11a vesicles were highly enriched with C16 and C18 ceramides. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppressed ciliogenesis, indicating that not only the association of ceramide with aPKC, but the activation of aPKC is critical for ciliogenesis in MDCK cells. In neural progenitors (NPs) differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), the ceramide-protein interaction underlying ciliogenesis showed parallels to that with MDCK cells, but also significant differences. Ceramide was enriched in the apical region of the cell as well as primary cilia. In addition, the ceramide level was elevated by 3-fold after neural differentiation, especially C16 and C24:1 ceramide. Immunostaining showed that C16 ceramide was mainly distributed in the apical region and primary cilium, while the C24:1 antibody showed signals at the apicolateral cell membrane in addition to the apical area and the primary cilium. Immunostaining of aPKC also showed a signal at the apicolateral membrane as well as the primary cilium. Decreasing ceramide levels led to not only reduced ciliogenesis, but also translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora A kinase (AurA). Incubation of ceramide-depleted cells with C24:1 ceramide restored ciliogenesis as well as membrane distribution of aPKC and accelerated neuronal process formation. The histone deacetylase (HDAC) inhibitor trichostatin A rescued ciliogenesis in ceramide-depleted MDCK cells and NPs, indicating that ceramide promotes tubulin acetylation in cilia. In summary, we concluded that ceramide promotes ciliogenesis by inhibiting HDAC6 activity in both of these two models, but via different molecular signaling pathways.
  • Toll-like receptor 9 contributes to vascular dysfunction in hypertension

    McCarthy, Cameron; Department of Physiology (2016-03)
    Inappropriate immune system activation is common in hypertension; however, the exact mechanisms by which this occurs are not well understood. Innate immune system recognition and response to damage-associated molecular patterns (DAMPs) is becoming an increasingly accepted mechanism. Mitochondrial DNA (mtDNA) is a DAMP that is recognized by Toll-like receptor (TLR)9, and it is elevated in the circulation of spontaneously hypertensive rats (SHR). Therefore, we hypothesized that (1) inhibition of TLR9 in SHR with a TLR9 antagonist (ODN2088) or TLR9 inhibitor (chloroquine) would lower blood pressure and improve vascular function and that (2) treatment of normotensive rats with a TLR9 agonist (ODN2395) would cause vascular dysfunction and increase blood pressure. Both ODN2088 and chloroquine lowered high blood pressure in SHR and treatment with chloroquine also improved cyclooxygenase-dependent endothelial function and prevented the full recruitment of the adaptive immune system in SHR. On the other hand, treatment of normotensive rats with ODN2395 increased blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to cyclooxygenase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability. In conclusion, these findings support the involvement of the innate immune system pattern recognition receptor TLR9 in the pathogenesis and maintenance of hypertension. Specifically, circulating mtDNA may activate TLR9 and contribute to high blood pressure and endothelial dysfunction in SHR.
  • Toll-like receptor 2 contributes to cerebrovascular dysfunction and cognitive impairment in diabetes

    Hardigan, Trevor; Department of Physiology (2016-03)
    The risk of cognitive decline in diabetes (Type 1 and Type 2) is significantly greater compared to normoglycemic patients, and the risk of developing dementia in diabetic patients is doubled. The etiology for this is likely multifactorial, but one mechanism that has gained increasing attention is decreased cerebral blood flow (CBF) as a result of cerebrovascular dysfunction. The innate immune system has been shown to play a role in diabetic vascular complications, notably through Toll-like receptor (TLR) stimulated release of proinflammatory cytokines and chemokines that leads to vascular damage. TLR2 has been implicated in the development of diabetic microvascular complications such as nephropathy, and thus we hypothesized that TLR2-mediated cerebrovascular dysfunction leads to decreased CBF and cognitive impairment in diabetes. Vascular TLR2 expression was increased and local TLR2 antagonism improved cerebrovascular function in diabetes. While the anti-hyperglycemic dipeptidylpeptidase-IV (DPP-IV) inhibitor linagliptin prevented TLR2 expression in brain microvascular endothelial cells (BMVEC) when applied locally, chronic in vivo treatment did not decrease vascular smooth muscle TLR2 expression. Treatment with linagliptin restored CBF in diabetes independent of effects on blood glucose levels, and this increase in CBF was correlated with decreased endothelin-1 (ET-1)-mediated vasoconstriction, decreased pathological remodeling, and increased endothelium-dependent relaxation. Knockout of TLR2 conferred protection from impaired CBF in early-stage diabetes and from hyperperfusion in long-term diabetes, prevented the development of endothelium dependent vascular dysfunction in diabetes, created a hyperactive and anxiolytic phenotype, and protected against diabetes induced impairment of long term hippocampal- and prefrontal cortex- mediated fear learning. In conclusion, these findings support the involvement of TLR2 in the pathogenesis of diabetic vascular disease and cognitive impairment.
  • Protein Kinase D Restrains Angiotensin II-Induced Aldosterone Secretion in Primary Adrenal Glomerulosa Cells

    Shapiro, Brian A.; Department of Physiology (2007-07)
    Misregulation of the renin-angiotensin II (Angll)-aldosterone (Aldo) system is a key feature of cardiovascular disease. A focus of study in this system is the Angll-elicited secretion of Aldo from the adrenocortical zona glomerulosa. An excellent model in which to study this phenomenon is primary cultures of bovine adrenal glomerulosa (AG) cells. These cells secrete detectable quantities of Aldo in response to secretagogues, such as Angll, elevated potassium (K+), adrenocorticotrophic hormone (ACTH) and phorbol 12-myristate 13-acetate (PMA), within 30 minutes. The serine (Ser)/threonine kinase protein kinase D (PKD) is reported to be activated by Angll in several systems, including the adrenocortical carcinoma cell line NCI H295R, and is thought to have a positive role in chronic (24 hours) Angll-evoked Aldo secretion. Because the role of PKD in acute Angll-elicited Aldo secretion has never been examined in a primary culture system, we undertook to study the role of PKD in acute (minutes to one hour) Aldo secretion. Thus, Angll (10 nM) and PMA (100 nM), but not elevated K+ (15 mM) and ACTH (10 nM), induced phosphorylation of PKD on Ser910, a marker of PKD activation, in primary bovine AG cells. This finding was confirmed by an in vitro kinase activity assay. Angll and PMA were also able to induce PKD activation in H295R cells. Furthermore, this activation was concentration dependent, and was rapidly induced (by 5 min). PKD activation was dependent on Angll type 1 (AT-1), but not AT-2 receptor, signaling, and was independent of tyrosine kinase signaling. Finally, we introduced, via adenovirus transduction, wild-type PKDwt and dominant negative PKDS738/742A constructs into primary AG cells and monitored Angll-evoked Aldo secretion. PKDwt -transduced AG cells exhibited decreased Angll-stimulated Aldo secretion, while in the PKDS738A742A - infected AG cells Angll-stimulated Aldo was enhanced. Thus, we hypothesize that PKD has an anti-secretory role in Angll-induced acute Aldo secretion.
  • Involvement of calpain in angiotensin II-induced aldosterone production in adrenal glomerulosa cells

    Seremwe, Mutsa P.; Department of Physiology (2014-03)
    Aldosterone is a steroid hormone important in the regulation of blood pressure. Aberrant production of aldosterone results in the development and progression of diseases such as hypertension, cardiofibrosis and congestive heart failure; therefore, a complete understanding of this process is important for developing more effective treatment strategies. Angiotensin II (Angll) regulates aldosterone production, in part through its ability to increase intracellular calcium levels. Calcium can activate calpains, proteases classified as typical or atypical based on the presence or absence of penta- EF-hands. Calpains are involved in various cellular responses which include actin cytoskeletal remodeling and Angll/AT1R signaling. We hypothesized that calpain, in particular calpain 10, is activated by angiotensin II in adrenal glomerulosa cells and underlies increased aldosterone production. We conducted our experiments in two different adrenal glomerulosa cell models: primary bovine zona glomerulosa (zG) cells and human adrenocortical carcinoma cells (HAC15). Our results showed that the pan-calpain inhibitors, calpeptin and MDL 28170, inhibited Angll-induced aldosterone production and CYP11B2 expression in these cells, in addition, Angll induced calpain activation in HAC15 cells. The typical (classical) calpain inhibitors PD-150606 and calpastatin peptide had no effect on Angll-elicited aldosterone production, suggesting a lack of involvement of a classical calpain in this process. Atypical calpains expressed by HAC15 cells include calpain 5, 7, 10 and 15. The calpain-10 inhibitor, CYGAK inhibited both Angll-induced aldosterone production and CYP11B2 expression. Consistent with this result, knockdown of calpain 10 by an RNA interference technique inhibited aldosterone production and CYP11B2 expression. On the contrary overexpression of calpain-10 using adenoviral infection induced an increase in aldosterone production in the presence and absence of Angll. Our results indicate that Angll-induced activation of calpain 10 in adrenal glomerulosa cells underlies aldosterone production. Our results identify calpain-10 as a potential target for the development of drug therapies to inhibit aldosterone production for the treatment of hypertension.
  • The Implementation of Controlled Physical Training and the Physiological Effects of Exercise in Women with Sickle Cell Disease

    Ramsey, Leigh-Taylor; Department of Physiology (1999-12)
    Sickle cell disease (SCD) comprises a group of genetic disorders of hemoglobin that affect more than 70,000 Americans and is associated with increased morbidity and health care cost (1). W ith improved medical management, individuals with SCD are now surviving well into adulthood. However, adults with SCD are often sedentary and because of medical conservatism are frequently discouraged from exercise fo r fear of precipitating acute vasoocclusive or painful episodes. A review of the literature revealed little baseline data on perceived health status and well-being, physical fitness (cardiovascular and muscular), and body composition in adults with SCD. It is currently unknown whether exercise or physical training (PT) will have beneficial, adverse, or no effect in SCD patients. As a result, exercise recommendations for adults with SCD are vague, and implementation and effectiveness of PT have not been reported. Moreover, there are no reports on the effects of controlled PT on perceived health status, physical fitness, and body composition parameters in patients with SCD. The effect of exercise on inflammatory mediators (i.e., tum or necrosis factor-alpha [TNF-a], interleukin - 6 [IL-6], and C-reactive protein [CRP]) and vasoactive mediators (i.e., nitric oxide metabolites [NOx] and endothelin-1 [ET-1]) has not been reported in patients with sickle cell anemia (SCA). Since changes in these parameters may influence the occurrence of vasoocclusive episodes by increasing adhesion of erythrocytes to the endothelium and by other mechanisms, such as vasoconstriction, it is important to clarify changes that may occur with exercise. Physical training holds the promise of allowing patients with SCD to preserve functional capacity and improve their quality of life, as has been reported for other chronic diseases. Yet, there are no repotted studies assessing the implementation and effectiveness of PT in adults with SCD. The study reported in this thesis provides new information concerning the impact of controlled PT on fitness and well-being in women with SCD. It also provides new information on the effects of three consecutive days of exercise, which might be used in a standard PT program. This project is a first step in the assessment of the role of regular exercise for SCD patients.
  • Neuro-vascular Communication in the Hypothalamic Supraoptic Nucleus in Rats. Do nitric oxide and vasopressin play a role?

    Du, Wenting; Department of Physiology (2015-03)
    The classical model of neurovascular coupling (NVC) proposes that activity-dependent synaptically released glutamate dilates arterioles. However, whether this model is also applicable to brain areas that use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritically released vasopressin (VP) can be found. Bath-applied VP significantly constricted SON arterioles via activation of the V ia receptor subtype. Vasoconstriction was also observed in response to single VP neuronal stimulation, an effect prevented by V ia receptor blockade (V2255). Conversely, osmotically-driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide (NO)- mediated vasodilation. Activity-dependent vasodilation was followed by a VP-mediated vasoconstriction, which acted to reset vascular tone. Taken together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between activity-dependent dendritic released VP and NO, in the generation of proper NVC responses.
  • Adrenal Zona Glomerulosa Targeting in Transgenic Mice

    Parmar, Jeniel; Department of Physiology (2009-12)
    The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11B2). CYP11B2 is primarily expressed in the zona glomerulosa (ZG) of the adrenal cortex. Adrenocortical expression of CYP11B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Considerable in vitro analyses have been performed towards defining the mechanisms that control CYP11B2 expression. Previous studies from our laboratory and others have identified several c/s-regulatory elements on the 5' flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11B2 gene transcription. Moreover, key transcription factors that bind these c/s-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified. Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP11B2 gene. However, thus far, all evidence is based on in vitro analyses of transcriptional regulation, which does not always depict in vivo occurrences. To initiate our in vivo assessment of CYP11B2 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP11B2 genes, which interestingly revealed high sequence similarity in the 5' flanking promoter region of the CYP11B2 gene. This result suggested that the cisregulatory regions identified by in vitro analyses likely plays an important role in CYP11B2 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11b2 promoter driving expression of a LacZ reporter gene. Importantly, 4 founder Tg mouse lines revealed LacZ expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed LacZ mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein (the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of LacZ exclusively in the adrenal ZG of Tg mice. Moreover, with the expression of LacZ properly restricted to adrenal ZG, we concluded that regions required for Cyp11b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation. While developing Tg mice, we also focused on characterization and development of novel adrenocortical cell lines. As aforementioned, in vitro culture models have allowed a multitude of studies that have broadened our understanding of normal adrenocortical endocrine function. Primary cultures of adrenocortical cells have been an excellent source for in vitro studies. However, the eventual onset of senescence in primary cultures of cells creates a recurring need for the costly and difficult isolations of fresh adrenocortical cells. Hence, the use of primary cultures has been increasingly supplemented by immortalized cell lines. We utilized an adrenocortical carcinoma to develop a human adrenocortical cell line. We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15). HAC15 represents only the second human adrenocortical cell line available that exhibits physiological hormonal responses, steroidogenesis, and expression of steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and ACTH makes it the first adrenal cell line capable of responding to the three main physiologic regulators of the adrenal cortex.
  • Angiotensin II Regulation of Aldosterone Synthase

    Nogueira, Edson da F.; Department of Physiology (2009-07)
    Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. The major goal of our study was to define the Ang II-induced mechanisms regulating the expression of aldosterone synthase (CYP11B2) in adrenocortical cells. We approached the analysis of the protein synthesis-dependent regulation of this enzyme by defining, through microarray and real time PCR analysis, the transcription factors that are rapidly induced by Ang II incubation of adrenocortical cell models from three species (human, bovine, and rat). The gene list generated by this comparison included: ATF3, BTG2, NR4A1, NR4A2, NR4A3, EGR1, FOS, FOSB, and JUNB. Importantly, pretreatment of H295R cells with cycloheximide had no effect on Ang II induction of these genes, suggesting that they are direct targets of Ang II signaling. Co-transfection studies, used to investigate the role of these transcription factors in the regulation of CYP11B2, determined that out of the nine transcription factors listed above, only the NGFI-B family members (NGFI-B, NURR1, and NOR1) increased expression of CYP11B2. The importance of NGFI-B in the regulation of CYP11B2 was confirmed by the decrease in CYP11B2 expression in the presence of a dominant-negative (DN)- NGFI-B. A pharmacological approach used to characterize the Ang II pathways regulating transcription of NGFI-B family genes suggested that Ang II binding to the AT1R increases activity of protein kinase C (PKC), Ca -dependent calmodulin kinases (CaMK), and SRC kinase (SRC), which act to regulate the expression of the family of NGFI-B genes as well as CYP11B2. In the current study we also analyzed protein synthesis-independent mechanisms regulating CYP11B2 expression. We studied the role of the ATF/CREB family of transcription factors (ATF1, ATF2, CREB, and CREM), which may bind the cAMP response element (CRE) in the promoter region of the CYP11B2 gene. Importantly, analysis of these transcription factors in the human H295R adrenocortical cell line revealed very low expression of CREB in comparison to the other CRE-binding proteins herein studied. We investigated Ang II-induced phosphorylation of these transcription factors, their binding to the promoter region of CYP11B2, and their effect on CYP11B2 expression. Ang II time-dependently induced phosphorylation of ATF1, ATF2, and CREM in H295R cells. The association of these transcription factors with the CYP11B2 promoter region was induced by Ang II and K+. Transfection of siRNA for ATF1, ATF2, and CREM significantly reduced CYP11B2 expression in Ang II-stimulated conditions. Expression of NURR-1 alone or with constitutively active ATF1, ATF2, CREB, and CREM increased the promoter activity of CYP11B2 in H295R cells. In summary, Ang II rapidly induces expression of newly synthesized transcription factors as well as the phosphorylation of transcription factors already present in the adrenocortical cell. These events are followed by increased CYP11B2 expression and, therefore, represent important mechanisms to increase the adrenal capacity to produce aldosterone.
  • Tetrahydrobiopterin-Dependent Vasodilation is Impaired in Experimental Hypertension

    Mitchell, Brett M.; Department of Physiology (2003-05)
    Decreased nitric oxide (NO) bioavailability leads to decreased vasodilation and increased blood presssure. Adequate amounts of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH4), optimizes NO production. Reduced BH4 results in decreased NO and increased superoxide production. Therefore, we hypothesized that decreased GTP cyclohydrolase (GTPCH), the rate-limiting enzyme in BH4 production, decreases NO leading to impaired vasodilation and increased blood pressure. To examine the effect of in vivo GTPCH inhibition on vasodilation and blood pressure, we administered DAHP in the drinking water of rats. Systolic blood pressure increased significantly in DAHP-treated rats. Endothelium-dependent relaxation was decreased in aortas from DAHP-treated rats, but restored with superoxide dismutase or sepiapterin, which produces BH4 via a salvage pathway. In conclusion, in vivo GTPCH inhibition leads to decreased NO production resulting in decreased BH4-dependent vasodilation and increased blood pressure. Excess glucocorticoids (GC) cause hypertension. To assess the effect of GCs on BH4 biosynthesis and vasodilation, we implanted dexamethasone (DEX), a synthetic GC, in rats. Aortas were isolated after 12 hours, 4 days, or 15 days of DEX-treatment to examine the role of GTPCH in the onset, development, and maintenance of GC-induced hypertension, respectively. Aortic relaxation and GTPCH and eNOS mRNA levels were decreased significantly in aortas from 4- and 15-day DEX-treated rats and restored with sepiapterin. In conclusion, excess GCs down-regulate GTPCH leading to decreased BH4-dependent vasodilation, which contributes to GC-induced hypertension. To assess if GCs act directly on the blood vessel to down-regulate GTPCH and decrease vasodilation, we incubated rat aortic rings with various compounds and found endothelium-dependent relaxation and GTPCH mRNA decreased significantly following incubation with DEX for 6 hours. This effect was blocked by a GC receptor antagonist and reversed by sepiapterin. In conclusion, GCs act through the GC receptor to down-regulate GTPCH leading to decreased BH4- dependent vasodilation. The results support the overall hypothesis that decreased BH4 biosynthesis, either by GCs or pharmacological inhibition, results in decreased NO production leading to reduced vasodilation and increased blood pressure.
  • The Role of Iron Induced Oxidative Stress in Acute Ischemic Stroke and the Potential Role for Fasciculations in their Therapy

    Mehta, Shyamal H.; Department of Physiology (2003-07)
    (Introductory Paragraphs) Stroke accounts for about one of every 15 deaths in the United States. It is the third leading cause of death behind heart disease and cancer in the United States and the second most common cause of death worldwide, according to the National Center for Health Statistics (1,2). Stroke is also the leading cause of serious disability in the United States; four million people are coping with the debilitating consequences of surviving a stroke which adds to the significant public financial burden (3). Based on the Framingham Heart Study 500,000 people suffer a new or recurrent stroke each year, of whom one third die over the next year, one-third remain permanently disabled and the remaining one-third make a reasonable recovery (1,4). Stroke is a sudden loss of brain function resulting from a disruption in the supply of blood and oxygen to the central nervous system (CNS) giving rise to hypoxic-ischemic conditions within the tissue. Acute stroke can be classified either as: 1. Ischemic stroke involves an interruption in blood supply to the CNS secondary to a vaso-occlusive phenomenon, accounting for 80% of the stroke cases. On basis of its etiology it can be further arbitrarily classified to extra-cranial or intracranial thrombosis and embolism (5). 2. Hemorrhagic stroke involves an interference in blood supply secondary to vascular disruption, accounting for 20% of the cases, which can be further classified to intracranial hemorrhage and subarachnoid hemorrhage (5). A progressing stroke or a stroke in evolution is an extremely complex event whose etiopathogenesis is poorly understood. Its multifactorial etiology makes it difficult to predict and treat by means of clinical, imaging and laboratory data currently available in clinical practice. The hemodynamic changes in the cerebral milieu and the biochemical mechanisms that hasten the progression of neurological injury are crucial to understand in order to reduce neurological morbidity and to design clinically effective interventions. In cerebral ischemia there is an ischemic gradient which can be divided into the core, which is the central ischemic zone and the penumbra, which is the area peripheral to the core. In the penumbra, functional impairment occurs in the neurons and the glia, with the neurons being more susceptible to ischemic injury due their dependence on oxidative metabolism (5). A better understanding of the pathologic mechanisms in ischemic injury would help limit the neurological injury in the penumbra through therapeutic intervention. The major pathogenic mechanisms include energy failure and excitotoxicity, loss of protein translation in the susceptible neurons, apoptotic mechanisms, inflammation and lastly, injury mediated by oxidative stress through the generation of reactive oxygen species (ROS) (6). Many of the above mentioned mechanisms are influenced by the generation of ROS. It has been directly demonstrated in numerous studies that ROS are involved in oxidative damage through peroxidation of lipids, proteins and nucleic acids in ischemic tissues (7). In addition, ROS also function as signaling molecules in cellular ischemia and reperfusion. In this dissertation we tried to elucidate the role of ROS in exacerbation of neurological injury in acute ischemic stroke. In order to gain a better understanding of the pathophysiological mechanisms underlying oxidative stress, we studied iron induced oxidative stress, as iron generates ROS through the Fenton reaction. We believe that ROS exacerbate ischemic injury, hence we wanted to demonstrate the neuroprotective ability of various antioxidants. In the end, we present a model of neuronal behavior in vitro that may have possible implications in post-injury remodeling and repair. Chapter 1 will review the literature in the field of antioxidants and ROS in stroke. In addition, the prevailing theories on the role of iron-induced oxidative stress and the various antioxidant agents used in stroke will be critically reviewed.
  • Genomic approaches towards understanding primary aldosteronism

    Hattangady, Namita G; Department of Physiology (2014-10)
    The human adrenal glands are complex endocrine organs that are physiologically located above the kidney. The cortex of the adrenal gland may be considered as a combination of three different steroidogenic tissue-types which form concentric zones within each adrenal. The three cortical zones include zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Each zone, under independent regulation, produces unique steroid(s) which exhibit specific functions. The outermost ZG layer secretes the steroid, aldosterone due to ZG specific expression of aldosterone synthase (CYP11B2). Aldosterone regulates sodium reabsorption, and therefore, blood pressure. Aldosterone production is tightly regulated by the renin-angiotensin-aldosterone system. Thus, aldosterone levels are in direct proportion with renin levels. Other known physiological regulators of aldosterone production include serum K+ and adrenocorticotrophic hormone. A type of endocrine hypertension termed ‘Primary Aldosteronism’ (PA), is characterized by aldosterone secretion under suppressed renin levels. PA accounts for almost 10 % of hypertension. More recently, genetic mutations in an inward rectifying K+ channel (KCNJ5) that occur as both, somatic and germline cases, have been implicated in the pathology of PA. The goal of this dissertation is to define the role of KCNJ5 mutations in PA. In this dissertation, I will summarize my studies that describe the acute and chronic events involved in mutated KCNJ5 mediated aldosterone excess. In addition, I will define a novel mutation in KCNJ5 of germline nature identified at Georgia Regents University. Finally, I will also describe some interesting lessons we learnt from the expression of mutated KCNJ5 in primary cultures of human adrenals. The prevalence of a hereditary form of PA termed as Familial Hyperaldosteronism type III (FH III) is very rare. Thus far, only a few mutations in the KCNJ5 gene, including T158A, G151R, G151E and I157S, are confirmed as causing FH III, following Mendelian genetics. Perhaps the most interesting feature of this disease is the varied phenotype between the different mutations. T158A-affected patients present with massive hyperplasia and require bilateral adrenalectomy. In contrast, patients affected by the G151E mutation have more severe hypertension, although their adrenals are near normal in appearance. In this study we identify a new germline mutation (Y152C). The index case was a 61 year old woman who underwent unilateral adrenalectomy. The patient with the Y152C mutation exhibited a milder hypertension phenotype (like the G151E-affected patient) with extensive hyperplasia (as seen in the T158A-affected patient). In vitro analyses of the Y152C mutation indicated a pathology similar to other known mutations in KCNJ5, including change in conductance to Na+ ions and elevated calcium levels, and increase in CYP11B2 mRNA and aldosterone production. The inherent challenge presented by current studies utilizing constitutive expression of KCNJ5 mutations is the limitation in studying acute temporal events such as post translational modifications of steroidogenic enzymes and transcription factors. To address this issue, we generated a doxycycline inducible cell model system for the T158A harboring KCNJ5 transgene. Herein, we demonstrate a useful system that was amenable to the study of acute and chronic events involved in mutant-KCNJ5 mediated aldosterone excess. Our findings suggest that mutant KNCJ5 increases CYP11B2 expression through the activation of transcriptional activators of CYP11B2. Additionally, this is the first study to demonstrate that mutant KCNJ5 also activates steroidogenic acute regulatory protein (StAR) at the levels of translation and post translational phosphorylation. We also demonstrate calcium channel blocker, verapamil as an efficient blocker of mKCNJ5 mediated aldosterone production. Finally, one of the sharp advantages of our study was the use of primary cultures of human adrenal cells to confirm the effects of mutated KCNJ5. Interestingly, transduction of cells with constitutive viruses for mutant KCNJ5, confirmed an increase in KCNJ5 mRNA, although no change in CYP11B2 expression levels was observed. Pilot data including treatment of primary cells with calcium ionophores indicated that ZF/ZR cells may have a phenotype that is ‘muted’ for calcium mediated pathways. We could also speculate that this may disprove some current hypotheses that APA harboring KCNJ5 mutations may originate from the ZF. Overall, this study has improved our knowledge regarding the pathogenesis of PA caused by KCNJ5 mutations and has identified verapamil as a potentially effective therapeutic strategy in the inhibition of aldosterone excess in this type of PA.
  • Integrated Effects of Leptin in the Forebrain and Hindbrain

    Desai, Bhavna N; Department of Physiology (2014-11)
    Obesity develops because of a sustained positive shift in energy balance. The hormone leptin was identified as a key negative feedback signal in energy balance regulation, yet it has been ineffective in reversing human obesity. Leptin injection studies in experimental animals have identified leptin receptors (ObRb) in the forebrain and hindbrain as critical and independent mediators of leptin responses. We hypothesized that under near physiological conditions; activation of ObRb in both these areas is required to reduce body fat. We used a male Sprague Dawley double cannulation rat model (3rd and 4th ventricle) and infused either saline (S) or sub-threshold doses of leptin (L) for 12 days (0.1μg leptin/24h in 3rd, 0.6μg leptin/24h in 4th) in different combinations SS, SL, LS, LL (3rd-4th), to test for integration of forebrain and hindbrain responses. There was no effect of leptin in single ventricle infused groups (LS, SL) compared to controls (SS). Rats with sub-threshold leptin infusions into both ventricles (LL) showed a 60% reduction in energy intake that reversed after day 6 and a 20% weight loss which stabilized at day 6. Body fat of LL rats was decreased by 30% in 6 days, and 50% after 12 days despite correction of energy intake. LL rats displayed normal activity and maintained normal energy expenditure despite weight loss. We further investigated which brain nuclei are involved in this integrated response using phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) as a marker of ObRb activation and delta FosB (ΔFosB) as a marker of chronic neuronal activation. The weight loss in LL rats was associated with a significant increase in pSTAT3 and ΔFosB within multiple hypothalamic nuclei, including the arcuate, ventromedial and dorsomedial nuclei, with no changes in activation of brainstem nuclei. Our results suggest that under near physiologial conditions, the simultaneous activation of both forebain and hindbrain ObRb is required for leptin to reduce body fat and this is facilitated by leptin in the hindbrain promoting activation of pSTAT3 in the hypothalamus. This provides a new perspective on the physiological role of leptin and could lead to new strategies to treat obesity.
  • Protein Kinase D In Keratinocyte Maturation

    Dodd, M. Ernest; Department of Physiology (2004-08)
    The epidermis is important for the body's maintenance of water homeostasis and resistance to environmental stress, and the m ajor cell type of the epidermis is the keratinocyte. Keratinocyte maturation requires proliferation, followed by terminal differentiation, and diseases of the skin often exhibit deregulated epidermal maturation. Protein kinase D (PKD) expression correlates with proliferation in keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. W e have hypothesized that PKD functions as a pro-proliferative and/or anti-differentiative signal in primary mouse keratinocytes and have predicted that agents that stimulate differentiation might also initiate a reduction in PKD expression and/or activation to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation and activity were analyzed upon treatment with differentiating agents and with 1 2 -0 - tetradecanoylphorbol-13-acetate, TPA, which stimulates differentiation acutely and proliferation chronically. 1,25-dihydroxyvitamin D3 -, elevated extracellular calcium-, and acute TPA-induced differentiation down-modulated PKD levels and autophosphorylation at serine 916. In addition, elevated extracellular calcium- and acute TPA-induced differentiation down-modulated PKD activity. Chronic TPA treatment stimulated proliferation and caused a recovery o f PKD levels, autophosphorylation and activity. In co-transfection experiments in keratinocytes, co-expression of PKD increased and decreased the promoter activities of keratin 5, a marker of proliferation, and involucrin, a marker of differentiation, respectively, and opposed the effects of elevated extracellular calcium on the expression of these markers. W hile cloning PKD for expression studies, we identified a splice variant of PKD, PKD{3, which is differentially spliced in a region important in activation and subcellular localization. Therefore, we hypothesized that this splice variant may have dissimilar activation properties and/or alternate roles in keratinocyte maturation. However, in vitro activation studies demonstrated equal activation of PK D a (full length) and PKDj3 by TPA and DAG. Co-transfection experiments showed that P K D a and PKDp affected marker expression to the same degree and similarly opposed the effects of elevated extracellular calcium-induced differentiation on marker expression. Our work represents the first demonstration of: 1) down-modulation o f PKD during differentiation, 2) pro-proliferative/anti-differentiative effects of PKD on keratinocyte marker expression and 3) existence of a splice variant of PKD.
  • Increased S-nitrosylation Impairs Contraction and Relaxation in Mouse Aorta

    Choi, Hyehun; Department of Physiology (2011-06)
    S-Nitrosylation is a ubiquitous protein modification in redox-based signaling. This modification uses nitric oxide (NO) to forms S-nitrosothiol (SNO) on cysteine residues. Thioredoxin (Trx) and Trx reductase (TrxR) play a role in limiting Snitrosylation. We hypothesized overall that S-nitrosylation of intracellular signaling molecules impairs contraction and relaxation of vascular smooth muscle cells. Aortic rings from C57BL/6 mice were used to measure vascular contraction and relaxation. The rings were treated with TrxR inhibitors, auranofin or 1-chloro-2,4-dinitrobenzene (DNCB), and/or NO donors, propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO), to increase Snitrosylation. Contractile responses of aortic rings to phorbol-12,13-dibutyrate (PDBu), a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. PKCa S-nitrosylation was increased by a TrxR inhibitor and CysNO; concomitantly, PKCa activity and downstream signaling were inhibited as compared to control protein. Vascular relaxation in aortic rings from normotensive (Sham) and angiotensin II (Angll)-induced hypertensive mice was measured after contraction with phenylephrine in the presence or absence of DNCB. DNCB reduced relaxation to acetylcholine (ACh) compared to vehicle, but the antioxidants, apocynin and tempol, normalized DNCB-induced impaired relaxation to ACh in sham aorta. Soluble guanylyl cyclase (sGC) S-nitrosylation was increased by DNCB, and sGC activity (cyclic GMP assay) was reduced in sham aorta. In aortic rings from Angll-treated mice, DNCB did not change relaxation to ACh compared to vehicle. DNCB decreased relaxation to sodium nitroprusside (SNP) in aortic rings from both sham and Angll mice. Total protein S-nitrosylation was enhanced in Angll aorta compared to sham, and TrxR activity was inhibited in Angll aorta compared to sham. These data suggest that PKC is inactivated by S-nitrosylation and this modification inhibits contractile responses to PDBu. TrxR inhibition reduces vascular relaxation via increasing oxidative stress and sGC S-nitrosylation. In Angll-induced hypertensive mice, augmented S-nitrosylation is associated with impaired vasodilation. Thus, TrxR and Snitrosylation may provide a critical mechanism in hypertension associated with abnormal vascular reactivity.

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