• 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.
    • Needle Bore Size and The Degree of Hemolysis During Blood Specimen Acquisition

      Carr, Rebecca Lamb; Department of Physiological and Technological Nursing (1983-05)
      This study examined the degree of detectable hemolysis in- blood specimens _as-pirated with needles'p~ :small and 1.arge bore_ size. A quasiexperimental design was used to test the resea;ch hypothesis that blood . specimens. aspira~ed_~tth a 22~gauge_needle will have a higher degree of -detectable-hemolysis than blood specimens asp:irated .with a 20-gauge needle. Data collectionconsisted of aspirating from each subject - (n=31) one blood-·speci~en--wi-th a 22-gauge-needle and-one-:.blood specimen·: _ _, . ,.,. : . . . . ' . . with a 20~gauge ·needle~- .. Th~- -plasma -hemoglobin df each specimen was . . . . . . '• . mea~ured and data were 'analyzed using the independent t~test~ The - results- indicated- that the mean difference in the plasma hemogl_obin· . ' . . . . . - ·between the two blood specimens w·as not stattstically · si'gnif1cant', -t-hus ·· .. the research hypothesis was not supported .. Thefindi~gs sug\Jested that . . '·· heme lysis was not influenced by the bore si z~ of the needle us.ed during blood specimen acq-uisition.
    • Neonatal Nurses' Knowledge of Their Standards of Practice: A Reflection of Accountability

      Chadwich, Jean; Department of Nursing (1984-02)
      The purpose of this study was to examine the neonatal nurses'- knowledge of standards of nursing practice. It was post~lated the degree to which a nurse understands and accepts her professional . accou~ta.bility depend·s :upon her own understanding of her status~ The level of knowledge was obtained from the investigator's selfdesigned tool. Additionally, the study .examined··whether or not a ' . ' ' . difference existed between the knowledge of nu'rses who care for . . . infants who require varying degree~ of 'nursing care, as evidenced by the t:y-pe of hospital ·they are employed in· und~r :regionalization guide~ lines. It further examined the relationship be~ween ·the personal -· variable of type of basic nursing education with the t·otal and subscale scores on the tool. A descriptive--correlational design was ·used to examine the study data generated from· 61 registered nurses, distri~uted _among 15 hospitals in one Southeastern stat_e. Analysis of the study .data using descriptive stati.stics indicated that the neonatal nurses wereknowledgeable of their standards of practice. Pe~rson correlation coefficients revealed no significant difference exis~ed amongst the nurses in Level I,. II _and III neonatal care facilities, nor was there any correlation between the nurses' educational preparation and their level of knowledge of the standards of practice. Additionally~ Pearson correlation coefficients· and Cronhach' s alpha ·were. used to determine the statistic:al validity and r~liabi.li ty es-timates of the tool •..
    • Neuregulin1 promotes excitatory synapse development specifically in GABAergic interneurons

      Tin, Kin Lai; Department of Neuroscience and Regenerative Medicine (2010-03)
      Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD- 95 puncta in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no consistent effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in postsynaptic differentiation in interneurons. Taken together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.
    • Neuregulin3 Regulation of Glutamatergic Transmission

      Figueiredo, Dwight; Department of Neuroscience and Regenerative Medicine (2015-12)
      Synapses are fundamental communication units in the brain, essential for meaningful response to stimuli received from the environment. Abnormal synaptic communication leads to mental disorders. My studies focus on Neuregulin3, a member of the Neuregulin family. Single Nucleotide Polymorphisms (SNPs) within the NRG3 gene are associated with schizophrenia in different populations. Analysis of postmortem human brain samples of schizophrenia patients revealed abnormal levels of NRG3. However, unlike its well-studied family member NRG1, NRG3’s role in synaptic transmission is not understood. I studied how depletion of Nrg3 protein in the brain could affect synaptic transmission. I measured the amplitude and the frequency of spontaneous as well as miniature Excitatory Post Synaptic Currents (sEPSC and mEPSC, respectively) at hippocampal CA1 neurons of GFAP
    • 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.
    • Neuron-derived estrogen and neural function

      Lu, Yujiao; Department of Neuroscience and Regenerative Medicine (Augusta University, 2020-05)
      17β-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. We created a forebrain neuron-specific aromatase knockout (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice. Under normal conditions, FBN-ARO-KO mice showed a 70-80% decrease in aromatase and forebrain E2 levels. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent cognitive functions. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, electrophysiological study suggested normal long-term potentiation (LTP) induction, but significantly decreased amplitude in FBN-ARO-KO mice which could be fully rescued by acute E2 treatment in vitro. Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. After global cerebral ischemia (GCI), ovariectomized female FBN-ARO-KO mice had significantly attenuated aromatase and hippocampal E2 levels. Intriguingly, FBN-ARO-KO mice exhibited a robust reduction in astrocyte activation, as well as exacerbated neuronal damage and worse cognitive dysfunction after GCI. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. In vivo E2 replacement rescued the compromised reactive astrogliosis and cognitive deficits. Moreover, neuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was dramatically increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling in astrocytes by central injection of an FGFR3 antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Collectively, our data provides novel genetic evidence for the roles of neuron-derived E2 in regulating synaptic plasticity, cognitive function in the non-injured brain, and astrocyte activation and neuroprotection in the injured brain.
    • Neuron-Glia interaction and role of Nrf2 in hyperhomocysteinemic retina

      Navneet, Soumya; Biomedical Sciences (Augusta University, 2019-05)
      Elevated level of the excitatory amino acid homocysteine (Hcy) or hyperhomocysteinemia (Hhcy) has been reported in patients with glaucoma, a disease characterized by increased oxidative stress and retinal ganglion cell (RGC) degeneration. Whether Hhcy is causative or merely a biomarker for glaucoma is not known. Primary RGCs exhibit acute sensitivity to Hcy exposure, while in vivo murine models of Hhcy demonstrate a more modest RGC loss (∼20%) over a period of several months. This differential response to Hhcy in isolated cells and the intact retina suggests the presence a buffering mechanism invoked by the retinal milieu. Oxidative stress has been implicated as a mechanism of Hcy-induced neuronal loss. Owing to the key role of Müller glial cells (MCs) in retinal antioxidant defense we hypothesized that MCs protect RGCs under conditions of Hhcy via the NRF2 antioxidant pathway. Compared to RGCs, MCs were less sensitive to Hcy. Hcy exposure increased oxidative stress and induced apoptosis in RGCs, whereas in MCs Hcy evoked several cytoprotective responses including reduced oxidative stress, increased antioxidant levels and improved mitochondrial function. Hcy upregulated the expression of Nrf2 and several downstream antioxidant targets including glutathione in MCs. To investigate the role of NRF2 in Hcy-induced RGC degeneration, we crossed Nrf2-/- mice with two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice) and generated Cbs+/-Nrf2-/- and Mthfr+/-Nrf2-/- mouse models and analyzed their retinas. Absence of NRF2 reduced inner retinal thickness and visual acuity, accelerated RGC loss and increased gliosis in Hhcy mice. To understand the role of Müller glial specific NRF2 in RGC survival, we established an ex-vivo indirect co-culture system using primary RGCs and MCs. Apoptosis induced by Hcy exposure in primary RGCs were abrogated when the RGCs were co-cultured with wild type (WT) MCs but not with Nrf2-/- MCs. Hhcy induced robust mitochondrial and glycolytic response in WT MCs, but not in Nrf2-/- MCs. Altogether, the in vivo and in vitro data here suggest that the deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of retinal antioxidant response mechanism.
    • Neuronal and Astroglial Injury and Recovery from Stroke-Induced Depolarizations

      Risher, William Christopher; Department of Neurosurgery (2010-12)
    • Neurotransmitter Gases as Modulators of GnRH and the Preovulatory LH Surge

      Lamar, Charisee; Institute of Molecular Medicine and Genetics (1998-04)
      Until recently nitric oxide (NO) and carbon monoxide (CO) were viewed only as toxic substances. However, there has been a substantial amount of evidence in the past decade that has redefined these gaseous molecules as physiological messengers. Along these lines, NO and CO are now recognized as modulators of immunological defense, vasodilation, endocrine signaling, and neurotransmission (1-8). As neurotransmitters, NO and CO are unique when compared to classical neurotransmitters. For instance, unlike all other classical neurotransmitters NO and CO are, 1) lipophilic gases with short half lives, 2) not stored in synaptic vesicles, 3) their effects are not mediated through classic receptor proteins - rather their effects result from NO and CO binding to the heme moiety of heme-proteins such as guanylate cyclase and cyclooxygenase, and 4) their effects are terminated by diffusion from target tissues ( 1,2,5,7,9). Production of NO and CO relies on the activity of the enzymes nitric oxide synthase (NOS) and heme oxygenase (HO), respectively. NOS uses the substrate Larginine to generate NO (2, 10), while HO uses the substrate heme to generate CO (9,11-16). NOS exists as three isoforms, macrophage NOS (mNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (17-21). HO also exists as three isoforms, heme oxygenase-1 (HO-1), heme oxygenase-2 (HO-2), and heme oxygenase-3 (HO-3) HO-1 is inducible, while HO-2 and HO-3 are constitutive enzymes; however, HO-3 is currently viewed as a poor heme catalyst (12,13,22). There is a growing body of evidence that suggests that NO and CO regulate hypothalamic function. For example, recent studies have shown that the hypothalamus produces significant quantities of NO, primarily through the actions of nNOS (23,24). Likewise, the hypothalamus possesses one of the largest production rates of CO in the brain (25,26). That NO and CO can regulate neurohormone release from the hypothalamus is evidenced by findings demonstrating that NO and CO regulate corticotropin-releasing hormone (CRH) (27-33), vasopressin (29-31,34,35), and oxytocin secretion (29,36). With respect to reproductive function, numerous studies (23,37-45) have provided evidence for a significant role of NO in the control of the hypothalamic releasing factor, gonadotropin-releasing hormone (GnRH). For instance, it has been demonstrated (37,46,47) that NO neurons are located in close proximity to GnRH generating cells bodies in the hypothalamus and thus strategically located to exert regulatory effects over GnRH neurons. That NO can exert regulatory effects on GnRH neurons was demonstrated in studies where exogenously applied NO markedly stimulated GnRH release from male hypothalamic fragments (1,48,49) and immortalized GnRH (GT1-7) cells in vitro (1,48). A physiological role for NO in the steroid-induced luteinizing hormone (LH) surge has been suggested based on studies in which the LH surge was attenuated by the administration of NOS inhibitors (43,44) or NOS antisense oligonucleotides (38) No studies, however, have reported nNOS mRNA, protein, or NOS activity levels in the hypothalamus during the LH surge so as to verify that an increased NO tone actually occurs during this critical time. To address this deficit, Aim 1 of this study was designed to examine NO tone on proestrus in the cycling female rat-the day the natural preovulatory LH surge occurs. Since heme oxygenase, the enzyme that produces CO from heme molecule metabolism, is located in the hypothalamus (2,11,12,14,15), and CO production in the hypothalamus is one of the highest in the brain (25,26), it is conceivable that CO could play a role in regulating GnRH secretion. This possibility has not been investigated and thus studies on this issue appear warranted. Therefore, Aims 2-4 of this study were designed to assess the possible role of CO in the control of GnRH and LH secretion.
    • Neurotransmitter gases as modulators of GnRH and the preovulatory LH surge

      Lamar, Charisee; School of Graduate Studies (1998-04)
    • Neurotransmitter receptor binding and protein phosphorylation in the rabbit iris smooth muscle

      Taft, William C; Department of Cell and Molecular Biology (1982-06)
    • Neurotransmitters and the phospholipid effect in rabbit iris muscle

      Owen, Mary Pruitt; Department of Cell and Molecular Biology (1976-06)
    • NEUROVASCULAR DEGENERATION FOLLOWING RETINAL ISCHEMIA REPERFUSION INJURY: ROLE OF ARGINASE 2

      Shosha, Esraa; Department of Cellular Biology and Anatomy (2017)
      Ischemic retinopathies such as retinopathy of prematurity, central retinal artery occlusion and diabetic retinopathy are leading causes of visual impairment and blindness. These pathologies share common features of oxidative stress, activation of inflammatory pathways and neurovascular damage. There is no clinically effective treatment for these conditions because the underlying mechanisms are still not fully understood. In the current study, we used a mouse model of retinal ischemia reperfusion (I/R) insult to explore the underlying mechanisms of neurovascular degeneration in ischemic retinopathies. The arginase enzyme utilizes the L-arginine amino acid for the production of L-ornithine and urea. Here, we investigated the role of the mitochondrial arginase isoform, arginase 2 (A2) in retinal I/R induced neurovascular injury. We found that retinal I/R induced neurovascular degeneration, superoxide and nitrotyrosine formation, glial activation, cell death by necroptosis and impairment of inner retinal function in wild type (WT) mice. A2 homozygous deletion (A2-/-) significantly protected against the neurovascular degeneration after retinal I/R. That was attributed to decreased oxidative stress and glial activation. A2 deletion protected against I/R induced retinal function impairment. Using Optical coherence tomography (OCT), we evaluated the retinal structure in live animals and found that A2-/- retinas showed a more preserved structure and less retinal detachment. To investigate the underlying mechanisms of A2 induced vascular damage after I/R, we used an in vitro model of oxygen glucose deprivation/ reperfusion (OGD/R) in bovine retinal endothelial cells (BRECs). Analysis of oxidative metabolism showed impaired mitochondrial function. We also found an increase in dynamin elated protein 1 (Drp1), a mitochondrial fission marker. Mitochondria labeling studies showed fragmented mitochondria after OGD/R. Arginase inhibition reduced mitochondrial fragmentation in OGD/R insult. This dissertation presents A2 as a new therapeutic target in reducing neurovascular damage in ischemic retinopathies.
    • Neutrophil function studies with respect to antibiotic tolerant staphylococcus aureus and patients with recurring infection

      Raynor, Robert H; Depatment of Cell and Molecular Biology (1980-08)
      Polymorphonuclear neutrophils (PMNs) from s~ven patients who experien<;: ed recurring staphylococcal infections, were tested in order to establish if measurements of phagocytic and bactericidal capacity would reveal a functional cause for the repeated episodes of disease. Using a microprocedure developed for this 'purpose, the results for six of these patients were found to be comparable to normal values. PMNs from one patient, however, showed a deficiency in their ability to both phagocytize and kill s. aureus. This defect could not be attributed to serum deficiencies or ascribed to any previously defined class of nel.ltrophil function. In other studies, six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol+). The other st;r-ains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol-). Lysis curves of these strains revealed that the Tal+ strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. Cells of each phenotype, previously labeled with [ 14C]Glycerol, secreted radioactivity when inhibited by oxacillin. However,. the Tal+ strains rele.ased over twice as much labe 1 as the Talstrains. The behavior of 60 to 65%_ of the labeled material released by. inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol- strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid. Several investigators have recently reported an increase in the severity of infections due to Tol+ strains. Since the enhanced'excretion of LTA by Tol+ staphylococci is the only knownphenotypJ.c difference between these two strains, the effect of this molecule on neutrophil function was measured using the microprocedure. The addition of LTA to the incubation mix·ture res1..1lted in a dose dependent inhibition of phagocytosis •. The decrease~ uptake of. S. aureus in the presence of LTA was accompanied by a corresponding increase in the number of organisms surviving in the presence of the phagocytes. Additional studies suggested that LTA interferes with the process' of opsonization, prior to engulfment.
    • Neutrophil-mediated endothelial dysfunction

      Chen, Xilin; Department of Pharmacology and Toxicology (1992-08)
    • A New Method For Analyzing 1:N Matched Case Control Studies With Incomplete Data

      Jin, Chan; Department of Biostatisctics and Epidemiology (5/8/2017)
      1:n matched case-control studies are commonly used to evaluate the association between the exposure to a risk factor and a disease, where one case is matched to up till n controls. The odds ratio is typically used to quantify such association. Difficulties in estimating the true odds ratio arise, when the exposure status is unknown for at least one individual in a group. In the case where the exposure status is known for all individuals in a group, the true odds ratio is estimated as the ratio of the counts in the discordant cells of the observed two-by-two table. In the case where all data are independent, the odds ratio is estimated using the cross-product ratio from the observed table. Conditional logistic regression estimates are used for incomplete matching data. In this dissertation we suggest a simple method for estimating the odds ratio when the sample consists of a combination of paired and unpaired observations, with 1:n matching. This method uses a weighted average of the odds ratio calculations described above. This dissertation compares the new method to existing methods via simulation.
    • Nitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cells

      Cai, Zheqing; Department of Internal Medicine (2001-06)
      Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that renal tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin-1 (ET-1), express caveolin-1 and protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoform(s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOS 1 was significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET-1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOS 1; furthermore, the ETA receptor antagonist, A 127722, rather than the ETB receptor antagonist, A 192621. inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. express caveolin-1, but none of the NOS isoforms appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS I is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 pM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of NOS 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cnr for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD cells. Therefore, in IMCD cells, NOS1 expression is up-regulated by ET-1 through activation of the ETA receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
    • Nitric oxide synthase regulation in inner medullary collecting duct cells

      Cai, Zheqing; School of Graduate Studies (2001-06)
      Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that ren_al tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin:-1 (ET-1), express caveolin-1 artd protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoforrri.( s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary-collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOSl was . significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET .;.1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOSl; furthermore, the ETA receptor antagonist, A127722, rather than the ETB receptor antagonist, A1926_21,- · inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also express caveolin-1, but none of the NOS isofonns appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting ·that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS 1 is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 μM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of Nos·· 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cm2 for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD . cells. Therefore, in IMCD cells, NOS 1 expression is up-regulated by ET-1 through activation of the ET A receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
    • Nitroxyl anion as a novel relaxant molecule in the rat pudendal artery and metformin as treatment for angiotensin II-induced erectile dysfunction

      Labazi, Hicham; Department of Physiology (2012)
      Cardiovascular diseases, which are the leading cause of illness and death in the United States, frequently share similar risk factors (hypertension, smoking, aging, etc…) as erectile dysfunction (ED). Hypertension is an important risk factor for both heart failure and ED; hypertension and ED are closely intertwined diseases, which have vascular and endothelial dysfunction as a common cause. Recently it has been shown that ED is an independent predictor of cardiovascular diseases (CVD). Thus, studying mechanisms of erectile function and ED will be of great importance for developing treatments for ED, as well as reducing the burden of CVD. In this dissertation, the mechanisms of angiotensin II (AngII)-induced ED was investigated. In addition, we also investigated the effect of metformin on erectile function in an AngII-hypertensive model of ED. We hypothesized that AngII infusion for 4 weeks results in ED and treatment with metformin improves erectile function in the AngII-infused rats. We observed that AngII infusion resulted in ED, which was accompanied by an increased contraction and decreased relaxation response of the corpus cavernosum and pudendal arteries. Furthermore, it was observed that ERK1/2 activation contributes to ED in the AngIIhypertensive model of ED. Treatment with metformin restored erectile function in AngIIhypertensive rats, with a reversal of the increased contractility and decreased relaxation seen in both the corpus cavernosum and pudendal arteries of the hypertensive rats. Our data suggest that metformin may have potential therapeutic effects in ED, independent of its anti-diabetic effects. Additionally we investigated the contribution of nitroxyl anion (HNO), a NO congener, to erectile function. We observed that the endothelium mediated relaxation in pudendal arteries was partially mediated through HNO, and that this relaxation was soluble guanylyl cyclase-dependent (sGC), resulting in activation of voltage-dependent potassium channels (KV +) and large conductance calcium-activated potassium channel (BKCa). The identification of this novel pathway will enhance our understanding of erectile function and possibly allow for development of therapeutic agents for the treatment of ED.