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The Molecular Mechanism of Angiotensin II on Cardiovascular Regulation in the Nucleus Tractus Solitarii of RatsCheng, Wen-han 06 August 2008 (has links)
Angiotensin II (Ang II) exerts diverse physiological actions in both peripheral and central nervous system. It has been demonstrated to implicate in central mechanisms leading to hypertension in the nucleus tractus solitarii (NTS) of rats, and mediated by the type-1 receptors (AT1R). Our previous studies already suggested that inhibition of NO synthesis in the NTS causes sustained hypertension. It was reported that the activity of Ang II was higher in the NTS of spontaneously hypertensive rat (SHR) and AT1R are colocalized in the neurons of the NTS, providing the local reactive oxygen species (ROS) production by Ang II. However, the signaling mechanisms of Ang II that induce hypertension remain uncertain. In the present study, we investigated the possible signal pathways involved in the cardiovascular regulation of Ang II in the NTS. Male SHR was treated with AT1R blocker, losartan (30 mg/kg/day) or superoxide dismutase (SOD) mimetic, tempol (1 mM/kg/day) for two weeks, systolic blood pressure was decreased significantly in losartan- or tempol-treated SHR. The NTS was excised for dihydroethidium (DHE) staining, NO analysis, immunoblotting and immunohistochemistry. Our results demonstrated that DHE staining revealed of ROS was much more in the NTS of SHR than in the NTS of wistar-Kyoto (WKY) rat. The ROS in the NTS of SHR was reduced by losartan. The NO content in the NTS of SHR was lower than WKY, while losartan and tempol could increase NO in the NTS of SHR. Immunoblotting and immunohistochemistry studies demonstrated that Ang II-induced hypertension inhibited neuronal NO synthase (nNOS), ERK and RSK phosphorylation levels in the NTS of SHR. These results suggest that Ang II induces ROS production in the NTS of SHR. In addition, the cardiovascular modulatory effects of Ang II in the NTS are accomplished by downregulation of ERK1/2-RSK phosphorylation levels and then nNOS level.
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The Adaptive Role of Neuronal Nitric Oxide Synthase in Maintaining Oxygen Homeostasis during Acute AnemiaTsui, Albert King-Yeung 31 August 2012 (has links)
Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.
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The Adaptive Role of Neuronal Nitric Oxide Synthase in Maintaining Oxygen Homeostasis during Acute AnemiaTsui, Albert King-Yeung 31 August 2012 (has links)
Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.
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Localization of Multiple Neurotransmitters in Surgically Derived Specimens of Human Atrial GangliaHoover, D. B., Isaacs, E. R., Jacques, F., Hoard, J. L., Pagé, P., Armour, J. A. 15 December 2009 (has links)
Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.
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A novel role of Lipin1 in the regulation of expression and function of nNOS.Azzam, Ayat 16 May 2023 (has links)
No description available.
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Modifying function and fibrosis of cardiac and skeletal muscle from mdx micevan Erp, Christel January 2005 (has links)
Duchenne Muscular Dystrophy (DMD) is a fatal condition occurring in approximately 1 in 3500 male births and is due to the lack of a protein called dystrophin. Initially DMD was considered a skeletal myopathy, but the pathology and consequences of cardiomyopathy are being increasingly recognised. Fibrosis, resulting from continual cycles of degeneration of the muscle tissues followed by inadequate regeneration of the muscles, is progressive in both cardiac and skeletal dystrophic muscle. In the heart fibrosis interferes with contractility and rhythm whereas it affects contractile function and causes contractures in skeletal muscles. This study utilised the mdx mouse which exhibits a pathological loss of muscle fibres and fibrosis characteristic of DMD, to examine a range of mechanisms that can influence muscle function and fibrosis. Ageing and workload both appear to contribute to the development of dystrophic features in cardiac and skeletal muscle of the mdx mouse. Therefore the effect of eccentric exercise on cardiac and skeletal muscle was examined in older mdx mice. Mice ran in 30 minute sessions for five months, 5 days per week. Downhill treadmill running did not exacerbate the contractile function or fibrosis of the mdx heart or the EDL, SOL or diaphragm muscles suggesting that cytokines influence function and fibrosis to a greater extent than workload alone. The role of the cytokine TGF-beta was examined by treating mdx mice with the TGF-beta antagonist pirfenidone at 0.4, 0.8 or 1.2 per cent in drinking water for six months. Pirfenidone improved cardiac contractility (P<0.01) and coronary flow (P<0.05), to levels comparable to control mice, despite no reduction in cardiac fibrosis. Pirfenidone did not reduce fibrosis or improve function in skeletal muscle. A deficiency of neuronal nitric oxide synthase (nNOS) in DMD and mdx mice causes a lowered production of nitric oxide indicating that the substrate of nNOS, l-arginine, may be beneficial to cardiac and skeletal muscle function in mdx mice. Oral l-arginine (5 mg/g bw) improved cardiac contractility, coronary flow and reduced cardiac fibrosis (P<0.05) without improving skeletal muscle function or fibrosis. In contrast, 10 mg/g bw l-arginine improved cardiac function and coronary flow (P<0.01), despite also elevating cardiac collagen. This increment in collagen was prevented by co-administration of prednisone. The experiments described in this dissertation reveal for the first time that pharmacological treatments in mdx mice can improve cardiac structure and function. Further elucidation of the optimum time and doses of such treatments may result in future pharmacological treatments to improve cardiac function and fibrosis in DMD.
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Regional neurochemical characterization of the flinders sensitive line rat with regard to glutamate-nitric oxide and cGMP signalling pathways / Estella Lily Minnaar.Minnaar, Estella Lily January 2008 (has links)
The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may
realize great importance as a fundamental substrate underlying the pathophysiology
of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway.
The aim was to determine whether the depressive-like behaviour of the
hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP
pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL)
rats differ neurochemically with regard to basal hippocampal and frontal cortical
NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined.
Additionally, select behavioural assessments were performed to confirm the
anxiogenic phenotype of the FSL strain.
For neurochemical determinations a sensitive fluorometric reversed phase highperformance
liquid chromatographic (HPLC) assay was developed to analyze total
nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before
derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent
product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the
amino acid L-citrulline was measured by HPLC with electrochemical detection after
o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index
for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to
determine the downstream activity of the NO-pathway in brain tissue.
FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A .
receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT)
behavioural assessment was performed to compare FSL with FRL groups under
baseline conditions according to their level of inherent anxiety. The parameters used
to measure anxiety were number of line crosses (locomotor activity), time spent in
middle blocks and social interaction time between pairs of rats. As an additional
behavioural assessment, the Forced Swim Test (FST) was performed to assess
behavioural restraint measured as time of immobility.
Basal cGMP levels in the frontal cortex were found to be significantly less in FSL
than in FRL rats, whereas the levels in the hippocampus did not differ significantly.
No other significant differences with respect to NO and nNOS activity were apparent
in either of the brain areas. The hypothermia test confirmed a significantly greater
decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm
any differences in immobility time between the two rat strains. In the OFT, FSL rat
groups exhibited behaviour that indicated significantly more anxiety than FRL rats.
Under basal conditions, FSL rats do not present with significant changes in markers
of the NO cascade in the hippocampus and frontal cortex compared to FRL controls,
including NOS activity as well as NO accumUlation. However, cGMP levels were
found to be significantly lower in the frontal cortex of FSL rats versus FRL rats,
although not in the hippocampus. Since the FSL rat is known to be hypercholinergic,
these data support an interaction between the NO/cGMP pathway and the
cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The
mechanisms and implications of such a mutual involvement need further clarification.
Further, this anatomical differentiation may have important implications for
understanding the role of NO in the depressive-like behaviour of the FSL rat and,
indeed, may reveal more on the neurobiology and treatment of depression. Through
the performed behavioural assessments, the FSL and FRL rats were successfully
separated with respect to their anxiety phenotype as well as their heightened
response to serotonergic challenge, thus confirming a contribution of both the
serotonergic and cholinergic systems to the depressogenic nature of these animals.
As concluding remark can be said that under normal basal conditions markers of the
NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP
levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent
mechanism of cGMP regulation, possibly involving ACh. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.
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Regional neurochemical characterization of the flinders sensitive line rat with regard to glutamate-nitric oxide and cGMP signalling pathways / Estella Lily Minnaar.Minnaar, Estella Lily January 2008 (has links)
The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may
realize great importance as a fundamental substrate underlying the pathophysiology
of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway.
The aim was to determine whether the depressive-like behaviour of the
hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP
pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL)
rats differ neurochemically with regard to basal hippocampal and frontal cortical
NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined.
Additionally, select behavioural assessments were performed to confirm the
anxiogenic phenotype of the FSL strain.
For neurochemical determinations a sensitive fluorometric reversed phase highperformance
liquid chromatographic (HPLC) assay was developed to analyze total
nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before
derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent
product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the
amino acid L-citrulline was measured by HPLC with electrochemical detection after
o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index
for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to
determine the downstream activity of the NO-pathway in brain tissue.
FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A .
receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT)
behavioural assessment was performed to compare FSL with FRL groups under
baseline conditions according to their level of inherent anxiety. The parameters used
to measure anxiety were number of line crosses (locomotor activity), time spent in
middle blocks and social interaction time between pairs of rats. As an additional
behavioural assessment, the Forced Swim Test (FST) was performed to assess
behavioural restraint measured as time of immobility.
Basal cGMP levels in the frontal cortex were found to be significantly less in FSL
than in FRL rats, whereas the levels in the hippocampus did not differ significantly.
No other significant differences with respect to NO and nNOS activity were apparent
in either of the brain areas. The hypothermia test confirmed a significantly greater
decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm
any differences in immobility time between the two rat strains. In the OFT, FSL rat
groups exhibited behaviour that indicated significantly more anxiety than FRL rats.
Under basal conditions, FSL rats do not present with significant changes in markers
of the NO cascade in the hippocampus and frontal cortex compared to FRL controls,
including NOS activity as well as NO accumUlation. However, cGMP levels were
found to be significantly lower in the frontal cortex of FSL rats versus FRL rats,
although not in the hippocampus. Since the FSL rat is known to be hypercholinergic,
these data support an interaction between the NO/cGMP pathway and the
cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The
mechanisms and implications of such a mutual involvement need further clarification.
Further, this anatomical differentiation may have important implications for
understanding the role of NO in the depressive-like behaviour of the FSL rat and,
indeed, may reveal more on the neurobiology and treatment of depression. Through
the performed behavioural assessments, the FSL and FRL rats were successfully
separated with respect to their anxiety phenotype as well as their heightened
response to serotonergic challenge, thus confirming a contribution of both the
serotonergic and cholinergic systems to the depressogenic nature of these animals.
As concluding remark can be said that under normal basal conditions markers of the
NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP
levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent
mechanism of cGMP regulation, possibly involving ACh. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.
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Elevated Levels of NR2A and PSD-95 in the Lateral Amygdala in DepressionKarolewicz, Beata, Szebeni, Katalin, Gilmore, Tempestt, MacIag, Dorota, Stockmeier, Craig A., Ordway, Gregory A. 01 March 2009 (has links)
Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 1314 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.
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Glutamate Signaling Proteins and Tyrosine Hydroxylase in the Locus Coeruleus of AlcoholicsKarolewicz, Beata, Johnson, Laurel, Szebeni, Katalin, Stockmeier, Craig A., Ordway, Gregory A. 01 January 2008 (has links)
It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.
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