Publications by authors named "Kaushik P Patel"

115 Publications

Central Ang II (Angiotensin II)-Mediated Sympathoexcitation: Role for HIF-1α (Hypoxia-Inducible Factor-1α) Facilitated Glutamatergic Tone in the Paraventricular Nucleus of the Hypothalamus.

Hypertension 2021 Jan 8;77(1):147-157. Epub 2020 Dec 8.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha (N.M.S., A.S.H., K.K., S.S.N., K.P.P.).

Central infusion of Ang II (angiotensin II) has been associated with increased sympathetic outflow resulting in neurogenic hypertension. In the present study, we appraised whether the chronic increase in central Ang II activates the paraventricular nucleus of the hypothalamus (PVN) resulting in elevated sympathetic tone and altered baro- and chemoreflexes. Further, we evaluated the contribution of HIF-1α (hypoxia-inducible factor-1α), a transcription factor involved in enhancing the expression of N-methyl-D-aspartate receptors and thus glutamatergic-mediated sympathetic tone from the PVN. Ang II infusion (20 ng/minute, intracerebroventricular, 14 days) increased mean arterial pressure (126±9 versus 84±4 mm Hg), cardiac sympathetic tone (96±7 versus 75±6 bpm), and decreased cardiac parasympathetic tone (16±2 versus 36±3 versus bpm) compared with saline-infused controls in conscious rats. The Ang II-infused group also showed an impaired baroreflex control of heart rate (-1.50±0.1 versus -2.50±0.3 bpm/mm Hg), potentiation of the chemoreflex pressor response (53±7 versus 30±7 mm Hg) and increased number of FosB-labeled cells (53±3 versus 19±4) in the PVN. Concomitant with the activation of the PVN, there was an increased expression of HIF-1α and N-Methyl-D-Aspartate-type1 receptors in the PVN. Further, Ang II-infusion showed increased renal sympathetic nerve activity (20.5±2.3% versus 6.4±1.9% of Max) and 3-fold enhanced renal sympathetic nerve activity responses to microinjection of N-methyl-D-aspartate (200 pmol) into the PVN of anesthetized rats. Further, silencing of HIF-1α in NG108 cells abrogated the expression of N-methyl-D-aspartate-N-methyl-D-aspartate-type1 induced by Ang II. Taken together, our studies suggest a novel Ang II-HIF-1α-N-methyl-D-aspartate receptor-mediated activation of preautonomic neurons in the PVN, resulting in increased sympathetic outflow and alterations in baro- and chemoreflexes.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.16002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720881PMC
January 2021

MMP9 inhibition increases autophagic flux in chronic heart failure.

Am J Physiol Heart Circ Physiol 2020 12 16;319(6):H1414-H1437. Epub 2020 Oct 16.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska.

Increased matrix metalloprotease 9 (MMP9) after myocardial infarction (MI) exacerbates ischemia-induced chronic heart failure (CHF). Autophagy is cardioprotective during CHF; however, whether increased MMP9 suppresses autophagic activity in CHF is unknown. This study aimed to determine whether increased MMP9 suppressed autophagic flux and MMP9 inhibition increased autophagic flux in the heart of rats with post-MI CHF. Sprague-Dawley rats underwent either sham surgery or coronary artery ligation 6-8 wk before being treated with MMP9 inhibitor for 7 days, followed by cardiac autophagic flux measurement with lysosomal inhibitor bafilomycin A1. Furthermore, autophagic flux was measured in vitro by treating H9c2 cardiomyocytes with two independent pharmacological MMP9 inhibitors, salvianolic acid B (SalB) and MMP9 inhibitor-I, and CRISPR/cas9-mediated MMP9 genetic ablation. CHF rats showed cardiac infarct, significantly increased left ventricular end-diastolic pressure (LVEDP), and increased MMP9 activity and fibrosis in the peri-infarct areas of left ventricular myocardium. Measurement of the autophagic markers LC3B-II and p62 with lysosomal inhibition showed decreased autophagic flux in the peri-infarct myocardium. Treatment with SalB for 7 days in CHF rats decreased MMP9 activity and cardiac fibrosis but increased autophagic flux in the peri-infarct myocardium. As an in vitro corollary study, measurement of autophagic flux in H9c2 cardiomyocytes and fibroblasts showed that pharmacological inhibition or genetic ablation of MMP9 upregulates autophagic flux. These data are consistent with our observations that MMP9 inhibition upregulates autophagic flux in the heart of rats with CHF. In conclusion, the results in this study suggest that the beneficial outcome of MMP9 inhibition in pathological cardiac remodeling is in part mediated by improved autophagic flux. This study elucidates that the improved cardiac extracellular matrix (ECM) remodeling and cardioprotective effect of matrix metalloprotease 9 (MMP9) inhibition in chronic heart failure (CHF) are via increased autophagic flux. Autophagy is cardioprotective; however, the mechanism of autophagy suppression in CHF is unknown. We for the first time demonstrated here that increased MMP9 suppressed cardiac autophagy and ablation of MMP9 increased cardiac autophagic flux in CHF rats. Restoring the physiological level of autophagy in the failing heart is a challenge, and our study addressed this challenge. The novelty and highlights of this report are as follows: ) MMP9 regulates cardiomyocyte and fibroblast autophagy, ) MMP9 inhibition protects CHF after myocardial infarction (MI) via increased cardiac autophagic flux, ) MMP9 inhibition increased cardiac autophagy via activation of AMP-activated protein kinase (AMPK)α, Beclin-1, Atg7 pathway and suppressed mechanistic target of rapamycin (mTOR) pathway.
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http://dx.doi.org/10.1152/ajpheart.00032.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792705PMC
December 2020

Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication.

J Mol Cell Cardiol 2020 Dec 18;149:27-40. Epub 2020 Sep 18.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address:

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLa cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLa cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.
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http://dx.doi.org/10.1016/j.yjmcc.2020.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736531PMC
December 2020

Nanoformulation of the superoxide dismutase mimic, MnTnBuOE-2-PyP, prevents its acute hypotensive response.

Redox Biol 2020 09 20;36:101610. Epub 2020 Jun 20.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, United States. Electronic address:

Scavenging superoxide (O) via overexpression of superoxide dismutase (SOD) or administration of SOD mimics improves outcomes in multiple experimental models of human disease including cardiovascular disease, neurodegeneration, and cancer. While few SOD mimics have transitioned to clinical trials, MnTnBuOE-2-PyP (BuOE), a manganese porphyrin SOD mimic, is currently in clinical trials as a radioprotector for cancer patients; thus, providing hope for the use of SOD mimics in the clinical setting. However, BuOE transiently alters cardiovascular function including a significant and precipitous decrease in blood pressure. To limit BuOE's acute hypotensive action, we developed a mesoporous silica nanoparticle and lipid bilayer nanoformulation of BuOE (nanoBuOE) that allows for slow and sustained release of the drug. Herein, we tested the hypothesis that unlike native BuOE, nanoBuOE does not induce an acute hypotensive response, as the nanoformulation prevents BuOE from scavenging O while the drug is still encapsulated in the formulation. We report that intact nanoBuOE does not effectively scavenge O, whereas BuOE released from the nanoformulation does retain SOD-like activity. Further, in mice, native BuOE, but not nanoBuOE, rapidly, acutely, and significantly decreases blood pressure, as measured by radiotelemetry. To begin exploring the physiological mechanism by which native BuOE acutely decreases blood pressure, we recorded renal sympathetic nerve activity (RSNA) in rats. RSNA significantly decreased immediately following intravenous injection of BuOE, but not nanoBuOE. These data indicate that nanoformulation of BuOE, a SOD mimic currently in clinical trials in cancer patients, prevents BuOE's negative side effects on blood pressure homeostasis.
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http://dx.doi.org/10.1016/j.redox.2020.101610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327277PMC
September 2020

Angiotensin-converting enzyme 2 activator, DIZE in the basolateral amygdala attenuates the tachycardic response to acute stress by modulating glutamatergic tone.

Neuropeptides 2020 Oct 4;83:102076. Epub 2020 Aug 4.

Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.. Electronic address:

The basolateral amygdala (BLA) is critical in the control of the sympathetic output during stress. Studies demonstrated the involvement of the renin-angiotensin system components in the BLA. Angiotensin-(1-7) [Ang-(1-7)], acting through Mas receptors, reduces stress effects. Considering that angiotensin-converting enzyme 2 (ACE2) is the principal enzyme for the production of Ang-(1-7), here we evaluate the cardiovascular reactivity to acute stress after administration of the ACE2 activator, diminazene aceturate (DIZE) into the BLA. We also tested whether systemic treatment with DIZE could modify synaptic activity in the BLA and its effect directly on the expression of the N-methyl-d-aspartate receptors (NMDARs) in NG108 neurons in-vitro. Administration of DIZE into the BLA (200 pmol/100 nL) attenuated the tachycardia to stress (ΔHR, bpm: vehicle = 103 ± 17 vs DIZE = 49 ± 7 p = 0.018); this effect was inhibited by Ang-(1-7) antagonist, A-779 (ΔHR, bpm: DIZE = 49 ± 7 vs A-779 + DIZE = 100 ± 15 p = 0.04). Systemic treatment with DIZE attenuated the excitatory synaptic activity in the BLA (Frequency (Hz): vehicle = 2.9 ± 0.4 vs. DIZE =1.8 ± 0.3 p < 0.04). NG108 cells treated with DIZE demonstrated decreased expression of l subunit NMDAR-NR1 (NR1 expression (a.u): control = 0.534 ± 0.0593 vs. DIZE = 0.254 ± 0.0260) of NMDAR and increases of Mas receptors expression. These data demonstrate that DIZE attenuates the tachycardia evoked by acute stress. This effect results from a central action in the BLA involving activation of Mas receptors. The ACE2 activation via DIZE treatment attenuated the frequency of excitatory synaptic activity in the basolateral amygdala and this effect can be related with the decreases of the NMDAR-NR1 receptor expression.
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http://dx.doi.org/10.1016/j.npep.2020.102076DOI Listing
October 2020

GLP-1 mediated diuresis and natriuresis are blunted in heart failure and restored by selective afferent renal denervation.

Cardiovasc Diabetol 2020 05 8;19(1):57. Epub 2020 May 8.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE, 68198-5850, USA.

Background: Glucagon-like peptide-1 (GLP-1) induces diuresis and natriuresis. Previously we have shown that GLP-1 activates afferent renal nerve to increase efferent renal sympathetic nerve activity that negates the diuresis and natriuresis as a negative feedback mechanism in normal rats. However, renal effects of GLP-1 in heart failure (HF) has not been elucidated. The present study was designed to assess GLP-1-induced diuresis and natriuresis in rats with HF and its interactions with renal nerve activity.

Methods: HF was induced in rats by coronary artery ligation. The direct recording of afferent renal nerve activity (ARNA) with intrapelvic injection of GLP-1 and total renal sympathetic nerve activity (RSNA) with intravenous infusion of GLP-1 were performed. GLP-1 receptor expression in renal pelvis, densely innervated by afferent renal nerve, was assessed by real-time PCR and western blot analysis. In separate group of rats after coronary artery ligation selective afferent renal denervation (A-RDN) was performed by periaxonal application of capsaicin, then intravenous infusion of GLP-1-induced diuresis and natriuresis were evaluated.

Results: In HF, compared to sham-operated control; (1) response of increase in ARNA to intrapelvic injection of GLP-1 was enhanced (3.7 ± 0.4 vs. 2.0 ± 0.4 µV s), (2) GLP-1 receptor expression was increased in renal pelvis, (3) response of increase in RSNA to intravenous infusion of GLP-1 was enhanced (132 ± 30% vs. 70 ± 16% of the baseline level), and (4) diuretic and natriuretic responses to intravenous infusion of GLP-1 were blunted (urine flow 53.4 ± 4.3 vs. 78.6 ± 4.4 µl/min/gkw, sodium excretion 7.4 ± 0.8 vs. 10.9 ± 1.0 µEq/min/gkw). A-RDN induced significant increases in diuretic and natriuretic responses to GLP-1 in HF (urine flow 96.0 ± 1.9 vs. 53.4 ± 4.3 µl/min/gkw, sodium excretion 13.6 ± 1.4 vs. 7.4 ± 0.8 µEq/min/gkw).

Conclusions: The excessive activation of neural circuitry involving afferent and efferent renal nerves suppresses diuretic and natriuretic responses to GLP-1 in HF. These pathophysiological responses to GLP-1 might be involved in the interaction between incretin-based medicines and established HF condition. RDN restores diuretic and natriuretic effects of GLP-1 and thus has potential beneficial therapeutic implication for diabetic HF patients.
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http://dx.doi.org/10.1186/s12933-020-01029-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206815PMC
May 2020

Central angiotensin II-Protein inhibitor of neuronal nitric oxide synthase (PIN) axis contribute to neurogenic hypertension.

Nitric Oxide 2020 01 22;94:54-62. Epub 2019 Oct 22.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, 68198-5850, USA.

Activation of renin-angiotensin- system, nitric oxide (NO•) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases, including hypertension. Previously we have shown increased protein expression of PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with heart failure. To elucidate the molecular mechanism by which Angiotensin II (Ang II) increases PIN expression, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 0.5 μl/h) or saline as vehicle (Veh) for 14 days through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline renal sympathetic nerve activity and mean arterial pressure. Ang II infusion increased the expression of PIN (1.24 ± 0.04* Ang II vs. 0.65 ± 0.07 Veh) with a concomitant 50% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1.00 ± 0.03 Veh) in the PVN. Substrate-dependent ligase assay in cells transfected with pCMV-(HA-Ub)8 vector revealed a reduction of HA-Ub-PIN conjugates after Ang II and a proteasome inhibitor, Lactacystin (LC), treatment (4.5 ± 0.7* LC Ang II vs. 9.2 ± 2.5 LC). TUBE (Tandem Ubiquitin-Binding Entities) assay showed decrease PIN-Ub conjugates in Ang II-treated cells (0.82 ± 0.12* LC Ang II vs. 1.21 ± 0.06 LC) while ATR blocker, Losartan (Los) treatment diminished the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.16 ± 0.04* LC Ang II Los). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO• on pre-autonomic neurons in the PVN resulting in increased sympathetic outflow.
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http://dx.doi.org/10.1016/j.niox.2019.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911640PMC
January 2020

Central Glucagon-like Peptide-1 Receptor Signaling via Brainstem Catecholamine Neurons Counteracts Hypertension in Spontaneously Hypertensive Rats.

Sci Rep 2019 09 19;9(1):12986. Epub 2019 Sep 19.

Division of Integrative Physiology, Department of Physiology, Jichi Medical University School of Medicine, Shimotsuke, Tochigi, 329-0498, Japan.

Glucagon-like peptide-1 receptor (GLP-1R) agonists, widely used to treat type 2 diabetes, reduce blood pressure (BP) in hypertensive patients. Whether this action involves central mechanisms is unknown. We here report that repeated lateral ventricular (LV) injection of GLP-1R agonist, liraglutide, once daily for 15 days counteracted the development of hypertension in spontaneously hypertensive rats (SHR). In parallel, it suppressed urinary norepinephrine excretion, and induced c-Fos expressions in the area postrema (AP) and nucleus tractus solitarius (NTS) of brainstem including the NTS neurons immunoreactive to dopamine beta-hydroxylase (DBH). Acute administration of liraglutide into fourth ventricle, the area with easy access to the AP and NTS, transiently decreased BP in SHR and this effect was attenuated after lesion of NTS DBH neurons with anti-DBH conjugated to saporin (anti-DBH-SAP). In anti-DBH-SAP injected SHR, the antihypertensive effect of repeated LV injection of liraglutide for 14 days was also attenuated. These findings demonstrate that the central GLP-1R signaling via NTS DBH neurons counteracts the development of hypertension in SHR, accompanied by attenuated sympathetic nerve activity.
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http://dx.doi.org/10.1038/s41598-019-49364-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753091PMC
September 2019

Renal denervation improves sodium excretion in rats with chronic heart failure: effects on expression of renal ENaC and AQP2.

Am J Physiol Heart Circ Physiol 2019 11 6;317(5):H958-H968. Epub 2019 Sep 6.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska.

Previously we have shown that increased expression of renal epithelial sodium channels (ENaC) may contribute to the renal sodium and water retention observed during chronic heart failure (CHF). The goal of this study was to examine whether renal denervation (RDN) changed the expressions of renal sodium transporters ENaC, sodium-hydrogen exchanger-3 proteins (NHE3), and water channel aquaporin 2 (AQP2) in rats with CHF. CHF was produced by left coronary artery ligation in rats. Four weeks after ligation surgery, surgical bilateral RDN was performed. The expression of ENaC, NHE3, and AQP2 in both renal cortex and medulla were measured. As a functional test for ENaC activation, diuretic and natriuretic responses to ENaC inhibitor benzamil were monitored in four groups of rats (Sham, Sham+RDN, CHF, CHF+RDN). Western blot analysis indicated that RDN (1 wk later) significantly reduced protein levels of α-ENaC, β-ENaC, γ-ENaC, and AQP2 in the renal cortex of CHF rats. RDN had no significant effects on the protein expression of kidney NHE3 in both Sham and CHF rats. Immunofluorescence studies of kidney sections confirmed the reduced signaling of ENaC and AQP2 in the CHF+RDN rats compared with the CHF rats. There were increases in diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. RDN reduced the diuretic and natriuretic responses to benzamil in CHF rats. These findings suggest a critical role for renal nerves in the enhanced expression of ENaC and AQP2 and subsequent pathophysiology of renal sodium and water retention associated with CHF. This is the first study to show in a comprehensive way that renal denervation initiated after a period of chronic heart failure reduces the expression of epithelial sodium channels and aquaporin 2 leading to reduced epithelial sodium channel function and sodium retention.
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http://dx.doi.org/10.1152/ajpheart.00299.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879918PMC
November 2019

Does glucagon-like peptide-1 induce diuresis and natriuresis by modulating afferent renal nerve activity?

Am J Physiol Renal Physiol 2019 10 7;317(4):F1010-F1021. Epub 2019 Aug 7.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, has diuretic and natriuretic effects. The present study was designed to explore the possible underlying mechanisms for the diuretic and natriuretic effects of GLP-1 via renal nerves in rats. Immunohistochemistry revealed that GLP-1 receptors were avidly expressed in the pelvic wall, the wall being adjacent to afferent renal nerves immunoreactive to calcitonin gene-related peptide, which is the dominant neurotransmitter for renal afferents. GLP-1 (3 μM) infused into the left renal pelvis increased ipsilateral afferent renal nerve activity (110.0 ± 15.6% of basal value). Intravenous infusion of GLP-1 (1 µg·kg·min) for 30 min increased renal sympathetic nerve activity (RSNA). After the distal end of the renal nerve was cut to eliminate the afferent signal, the increase in efferent renal nerve activity during intravenous infusion of GLP-1 was diminished compared with the increase in total RSNA (17.0 ± 9.0% vs. 68.1 ± 20.0% of the basal value). Diuretic and natriuretic responses to intravenous infusion of GLP-1 were enhanced by total renal denervation (T-RDN) with acute surgical cutting of the renal nerves. Selective afferent renal nerve denervation (A-RDN) was performed by bilateral perivascular application of capsaicin on the renal nerves. Similar to T-RDN, A-RDN enhanced diuretic and natriuretic responses to GLP-1. Urine flow and Na excretion responses to GLP-1 were not significantly different between T-RDN and A-RDN groups. These results indicate that the diuretic and natriuretic effects of GLP-1 are partly governed via activation of afferent renal nerves by GLP-1 acting on sensory nerve fibers within the pelvis of the kidney.
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http://dx.doi.org/10.1152/ajprenal.00028.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843041PMC
October 2019

Exercise training augments neuronal nitric oxide synthase dimerization in the paraventricular nucleus of rats with chronic heart failure.

Nitric Oxide 2019 06 13;87:73-82. Epub 2019 Mar 13.

Department of Cellular and Integrative Physiology, UNMC, Omaha, NE 68198-5850, USA.

Exercise training (ExT) is an established non-pharmacological therapy that improves the health and quality of life in patients with chronic heart failure (CHF). Exaggerated sympathetic drive characterizes CHF due to an imbalance of the autonomic nervous system. Neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) produce nitric oxide (NO•), which is known to regulate the sympathetic tone. Previously we have shown that during CHF, the catalytically active dimeric form of nNOS is significantly decreased with a concurrent increase in protein inhibitor of nNOS (PIN) expression, a protein that dissociates dimeric nNOS to monomers and facilitates its degradation. Dimerization of nNOS also requires (6R)-5,6,7,8-tetrahydrobiopterin (BH4) for stability and activity. Previously, we have shown that ExT improves NO-mediated sympathetic inhibition in the PVN; however, the molecular mechanism remains elusive. We hypothesized; ExT restores the sympathetic drive by increasing the levels and catalytically active form of nNOS by abrogating changes in the PIN in the PVN of CHF rats. CHF was induced in adult male Sprague-Dawley rats by coronary artery ligation, which reliably mimics CHF in patients with myocardial infarction. After 4 weeks of surgery, Sham and CHF rats were subjected to 3 weeks of progressive treadmill exercise. ExT significantly (p < 0.05) decreased PIN expression and increased dimer/monomer ratio of nNOS in the PVN of rats with CHF. Moreover, we found decreased GTP cyclohydrolase 1(GCH1) expression: a rate-limiting enzyme for BH4 biosynthesis in the PVN of CHF rats suggesting that perhaps reduced BH4 availability may also contribute to decreased nNOS dimers. Interestingly, CHF induced decrease in GCH1 expression was increased with ExT. Our findings revealed that ExT rectified decreased PIN and GCH1 expression and increased dimer/monomer ratio of nNOS in the PVN, which may lead to increase NO• bioavailability resulting in amelioration of activated sympathetic drive during CHF.
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http://dx.doi.org/10.1016/j.niox.2019.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527363PMC
June 2019

Specific Afferent Renal Denervation Prevents Reduction in Neuronal Nitric Oxide Synthase Within the Paraventricular Nucleus in Rats With Chronic Heart Failure.

Hypertension 2018 09;72(3):667-675

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha (K.K., K.P.P.).

Renal denervation (RDN) has been shown to restore endogenous neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) and reduce sympathetic drive during chronic heart failure (CHF). The purpose of the present study was to assess the contribution of afferent renal nerves to the nNOS-mediated sympathetic outflow within the PVN in rats with CHF. CHF was induced in rats by ligation of the left coronary artery. Four weeks after surgery, selective afferent RDN (A-RDN) was performed by bilateral perivascular application of capsaicin on the renal arteries. Seven days after intervention, nNOS protein expression, nNOS immunostaining signaling, and diaphorase-positive stained cells were significantly decreased in the PVN of CHF rats, changes that were reversed by A-RDN. A-RDN reduced basal lumbar sympathetic nerve activity in rats with CHF (8.5%±0.5% versus 17.0%±1.2% of max). Microinjection of nNOS inhibitor L-NMMA (L-N-monomethyl arginine citrate) into the PVN produced a blunted increase in lumbar sympathetic nerve activity in rats with CHF. This response was significantly improved after A-RDN (Δ lumbar sympathetic nerve activity: 25.7%±2.4% versus 11.2%±0.9%). Resting afferent renal nerves activity was substantially increased in CHF compared with sham rats (56.3%±2.4% versus 33.0%±4.7%). These results suggest that intact afferent renal nerves contribute to the reduction of nNOS in the PVN. A-RDN restores nNOS and thus attenuates the sympathoexcitation. Also, resting afferent renal nerves activity is elevated in CHF rats, which may highlight a crucial neural mechanism arising from the kidney in the maintenance of enhanced sympathetic drive in CHF.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.11071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202134PMC
September 2018

GABA-containing liposomes: neuroscience applications and translational perspectives for targeting neurological diseases.

Nanomedicine 2018 04 24;14(3):781-788. Epub 2017 Dec 24.

Department of Physiology & Biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil. Electronic address:

There are multiple challenges for neuropharmacology in the future. Undoubtedly, one of the greatest challenges is the development of strategies for pharmacological targeting of specific brain regions for treatment of diseases. GABA is the main inhibitory neurotransmitter in the central nervous system, and dysfunction of GABAergic mechanisms is associated with different neurological conditions. Liposomes are lipid vesicles that are able to encapsulate chemical compounds and are used for chronic drug delivery. This short review reports our experience with the development of liposomes for encapsulation and chronic delivery of GABA to sites within the brain. Directions for future research regarding the efficacy and practical use of GABA-containing liposomes for extended periods of time as well as understanding and targeting neurological conditions are discussed.
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http://dx.doi.org/10.1016/j.nano.2017.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415746PMC
April 2018

A Hypothalamic Leptin-Glutamate Interaction in the Regulation of Sympathetic Nerve Activity.

Neural Plast 2017 3;2017:2361675. Epub 2017 Aug 3.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850, USA.

Accumulated evidence indicates that obesity-induced type 2 diabetes (T2D) is associated with enhanced sympathetic activation. The present study was conducted to investigate the role for leptin-glutamate signaling within the hypothalamus in regulating sympathetic nerve activity. In anesthetized rats, microinjections of leptin (5 ng ~ 100 ng) into the arcuate nucleus (ARCN) and paraventricular nucleus (PVN) induced increases in renal sympathetic nerve activity (RSNA), blood pressure (BP), and heart rate (HR). Prior microinjections of NMDA receptor antagonist AP5 (16 pmol) into the ARCN or PVN reduced leptin-induced increases in RSNA, BP, and HR in both ARCN and PVN. Knockdown of a leptin receptor with siRNA inhibited NMDA-induced increases in RSNA, BP, and HR in the ARCN but not in the PVN. Confocal calcium imaging in the neuronal NG108 and astrocytic C6 cells demonstrated that preincubation with leptin induced an increase in intracellular calcium green fluorescence when the cells were challenged with glutamate. In high-fat diet and low-dose streptozotocin-induced T2D rats, we found that leptin receptor and NMDA NR receptor expressions in the ARCN and PVN were significantly increased. In conclusion, these studies provide evidence that within the hypothalamic nuclei, leptin-glutamate signaling regulates the sympathetic activation. This may contribute to the sympathoexcitation commonly observed in obesity-related T2D.
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http://dx.doi.org/10.1155/2017/2361675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560058PMC
May 2018

A novel role for miR-133a in centrally mediated activation of the renin-angiotensin system in congestive heart failure.

Am J Physiol Heart Circ Physiol 2017 May 10;312(5):H968-H979. Epub 2017 Mar 10.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska; and.

An activated renin-angiotensin system (RAS) within the central nervous system has been implicated in sympathoexcitation during various disease conditions including congestive heart failure (CHF). In particular, activation of the RAS in the paraventricular nucleus (PVN) of the hypothalamus has been recognized to augment sympathoexcitation in CHF. We observed a 2.6-fold increase in angiotensinogen (AGT) in the PVN of CHF. To elucidate the molecular mechanism for increased expression of AGT, we performed in silico analysis of the 3'-untranslated region (3'-UTR) of AGT and found a potential binding site for microRNA (miR)-133a. We hypothesized that decreased miR-133a might contribute to increased AGT in the PVN of CHF rats. Overexpression of miR-133a in NG108 cells resulted in 1.4- and 1.5-fold decreases in AGT and angiotensin type II (ANG II) type 1 receptor (ATR) mRNA levels, respectively. A luciferase reporter assay performed on NG108 cells confirmed miR-133a binding to the 3'-UTR of AGT. Consistent with these in vitro data, we observed a 1.9-fold decrease in miR-133a expression with a concomitant increase in AGT and ATR expression within the PVN of CHF rats. Furthermore, restoring the levels of miR-133a within the PVN of CHF rats with viral transduction resulted in a significant reduction of AGT (1.4-fold) and ATR (1.5-fold) levels with a concomitant decrease in basal renal sympathetic nerve activity (RSNA). Restoration of miR-133a also abrogated the enhanced RSNA responses to microinjected ANG II within the PVN of CHF rats. These results reveal a novel and potentially unique role for miR-133a in the regulation of ANG II within the PVN of CHF rats, which may potentially contribute to the commonly observed sympathoexcitation in CHF. Angiotensinogen (AGT) expression is upregulated in the paraventricular nucleus of the hypothalamus through posttranscriptional mechanism interceded by microRNA-133a in heart failure. Understanding the mechanism of increased expression of AGT in pathological conditions leading to increased sympathoexcitation may provide the basis for the possible development of new therapeutic agents with enhanced specificity.
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http://dx.doi.org/10.1152/ajpheart.00721.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146304PMC
May 2017

Post-translational regulation of neuronal nitric oxide synthase: implications for sympathoexcitatory states.

Expert Opin Ther Targets 2017 Jan 2;21(1):11-22. Epub 2016 Dec 2.

a Department of Cellular & Integrative Physiology , University of Nebraska Medical Center , Omaha , NE , USA.

Introduction: Nitric oxide (NO) synthesized via neuronal nitric oxide synthase (nNOS) plays a significant role in regulation/modulation of autonomic control of circulation. Various pathological states are associated with diminished nNOS expression and blunted autonomic effects of NO in the central nervous system (CNS) including heart failure, hypertension, diabetes mellitus, chronic renal failure etc. Therefore, elucidation of the molecular mechanism/s involved in dysregulation of nNOS is essential to understand the pathogenesis of increased sympathoexcitation in these diseased states. Areas covered: nNOS is a highly regulated enzyme, being regulated at transcriptional and posttranslational levels via protein-protein interactions and modifications viz. phosphorylation, ubiquitination, and sumoylation. The enzyme activity of nNOS also depends on the optimal concentration of substrate, cofactors and association with regulatory proteins. This review focuses on the posttranslational regulation of nNOS in the context of normal and diseased states within the CNS. Expert opinion: Gaining insight into the mechanism/s involved in the regulation of nNOS would provide novel strategies for manipulating nNOS directed therapeutic modalities in the future, including catalytically active dimer stabilization and protein-protein interactions with intracellular protein effectors. Ultimately, this is expected to provide tools to improve autonomic dysregulation in various diseases such as heart failure, hypertension, and diabetes.
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http://dx.doi.org/10.1080/14728222.2017.1265505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488701PMC
January 2017

Hypoxia-Inducible Factor-1α Mediates Increased Sympathoexcitation via Glutamatergic N-Methyl-d-Aspartate Receptors in the Paraventricular Nucleus of Rats With Chronic Heart Failure.

Circ Heart Fail 2016 Nov;9(11)

From the Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha.

Background: Increased sympathetic outflow is a major contributor to the progression of chronic heart failure (CHF). Potentiation of glutamatergic tone has been causally related to the sympathoexcitation in CHF. Specifically, an increase in the N-methyl-d-aspartate-type 1 receptor (NMDA-NR) expression within the paraventricular nucleus (PVN) is critically linked to the increased sympathoexcitation during CHF. However, the molecular mechanism(s) for the upregulation of NMDA-NR remains unexplored. We hypothesized that hypoxia via hypoxia-inducible factor 1α (HIF-1α) might contribute to the augmentation of the NMDA-NR-mediated sympathoexcitatory responses from the PVN in CHF.

Methods And Results: Immunohistochemistry staining, mRNA, and protein for hypoxia-inducible factor 1α were upregulated within the PVN of left coronary artery-ligated CHF rats. In neuronal cell line (NG108-15) in vitro, hypoxia caused a significant increase in mRNA and protein for HIF-1α (2-fold) with the concomitant increase in NMDA-NR mRNA, protein levels, and glutamate-induced Ca influx. Chromatin immunoprecipitation assay identified HIF-1α binding to NMDA-NR promoter during hypoxia. Silencing of HIF-1α in NG108 cells leads to a significant decrease in expression of NMDA-NR, suggesting that expression of HIF-1α is necessary for the upregulation of NMDA-NR. Consistent with these observations, HIF-1α silencing within the PVN abrogated the increased basal sympathetic tone and sympathoexcitatory responses to microinjection of NMDA in the PVN of rats with CHF.

Conclusions: These results uncover a critical role for HIF-1 in the upregulation of NMDA-NR to mediate sympathoexcitation in CHF. We conclude that subtle hypoxia within the PVN may act as a metabolic cue to modulate sympathoexcitation during CHF.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.116.003423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123857PMC
November 2016

Liposome-entrapped GABA modulates the expression of nNOS in NG108-15 cells.

J Neurosci Methods 2016 11 11;273:55-63. Epub 2016 Aug 11.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850, United States. Electronic address:

Background: Liposomes are concentric lipid vesicles that allow a sustained release of entrapped substances. GABA (γ-aminobutyric acid) is the most prevalent inhibitory neurotransmitter in the central nervous system.

New Method: Using GABA-containing liposomes (GL) prepared by the freeze-thawing method, we determined the effect of sustained release of GABA on expression of neuronal nitric oxide synthase (nNOS) and GABA receptor (GABAR) in an in vitro neuronal model.

Results: Neuronal cell line NG108-15 treated with different doses of GL during 24h showed an increase in expression of GABAR (54 and 50% with 10 and 20ng doses, respectively) and nNOS (138, 157 and 165% with 20, 50 and 100ng doses, respectively) compared with cells treated with empty liposomes (EL). Additionally, cells treated with 50ng of GL showed an increase in GABAR (23%) after 1h followed by an increase in nNOS (55, 46 and 55%) at 8, 12 and 24h time points, respectively. Immunofluorescence experiments confirmed an increase in nNOS (134%) and basal intracellular levels of nitric oxide (84%) after GL treatment. Further, treatment of cells with GL showed a decrease in expression of a protein inhibitor of nNOS (PIN) (26, 66 and 57% with 20, 50 and 100ng doses respectively) compared with control.

Comparison With Existing Methods: This is first demonstration for the development of GL that allows sustained slow release of this neurotransmitter.

Conclusion: These results suggest that a slow release of GABA can change the expression of nNOS possibly via alteration in PIN levels in neuronal cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075514PMC
http://dx.doi.org/10.1016/j.jneumeth.2016.08.004DOI Listing
November 2016

Astrocytes Contribute to Angiotensin II Stimulation of Hypothalamic Neuronal Activity and Sympathetic Outflow.

Hypertension 2016 12 3;68(6):1483-1493. Epub 2016 Oct 3.

From the Department of Physiology, Augusta University, GA (J.E.S., S.S., V.C.B.); and Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha (H.Z., N.S., K.P.P.).

Angiotensin II (AngII) is a key neuropeptide that acting within the brain hypothalamic paraventricular nucleus regulates neurohumoral outflow to the circulation. Moreover, an exacerbated AngII action within the paraventricular nucleus contributes to neurohumoral activation in hypertension. Although AngII effects involve changes in paraventricular nucleus neuronal activity, the precise underlying mechanisms, cellular targets, and distribution of AngII receptors within the paraventricular nucleus remain largely unknown. Thus, whether AngII effects involve direct actions on paraventricular neurons, or whether it acts via intermediary cells, such as astrocytes, is still controversial. To address this important gap in our knowledge, we used a multidisciplinary approach combining patch-clamp electrophysiology in presympathetic paraventricular neurons and astrocytes, along with in vivo sympathetic nerve recordings and astrocyte-targeted gene manipulations. We present evidence for a novel mechanism underlying central AngII actions, which involves astrocytes as major intermediary cellular targets. We found that AngII type 1 receptor mRNA is expressed in paraventricular astrocytes. Moreover, we report that AngII inhibited glutamate transporter function, increasing in turn extracellular glutamate levels. This resulted in the activation of neuronal extrasynaptic NMDA (N-methyl-d-aspartate) receptors, increased presympathetic neuronal activity, enhanced sympathoexcitatory outflow, and increased blood pressure. Together, our studies support astrocytes as critical intermediary cell types mediating brain AngII regulation of the circulation and indicate that AngII-mediated neuronal and sympathoexcitatory effects are dependent on a unique neuroglial signaling modality involving nonsynaptic glutamate transmission.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.116.07747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159229PMC
December 2016

Integration of renal sensory afferents at the level of the paraventricular nucleus dictating sympathetic outflow.

Auton Neurosci 2017 05 6;204:57-64. Epub 2016 Aug 6.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850, United States. Electronic address:

The sympathetic nervous system has been identified as a major contributor to the pathophysiology of chronic heart failure (CHF) and other diseases such as hypertension and diabetes, both in experimental animal models and patients. The kidneys have a dense afferent sensory innervation positioning it to be the origin of multimodal input to the central nervous system. Afferent renal nerve (ARN) signals are centrally integrated, and their activation results in a general increase in sympathetic tone, which is directed toward the kidneys as well as other peripheral organs innervated by the sympathetic nerves. In the central nervous system, stimulation of ARN increases the neuronal discharge frequency and neuronal activity in the paraventricular nucleus (PVN) of the hypothalamus. The activity of the neurons in the PVN is attenuated during iontophoretic application of glutamate receptor blocker, AP5. An enhanced afferent renal input to the PVN may be critically involved in dictating sympathoexcitation in CHF. Furthermore, renal denervation abrogates the enhanced neuronal activity within the PVN in rats with CHF, thereby possibly contributing to the reduction in sympathetic tone. Renal denervation also restores the decreased endogenous levels of neuronal nitric oxide synthase (nNOS) in the PVN of rats with CHF. Overall, these data demonstrate that sensory information originating in the kidney excites pre-autonomic sympathetic neurons within the PVN and this "renal-PVN afferent pathway" may contribute to elevated sympathetic nerve activity in hyper-sympathetic disease conditions such as CHF and hypertension.
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http://dx.doi.org/10.1016/j.autneu.2016.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293680PMC
May 2017

Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase.

Diabetes 2016 10 13;65(10):3075-90. Epub 2016 Jul 13.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE

MicroRNAs (miRNAs) have a fundamental role in diabetic heart failure. The cardioprotective miRNA-133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts. Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating β-adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme, tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT). However, the cross talk/link between TAT and TH in the heart is unclear. To determine whether miR-133a plays a role in the cross talk between TH and TAT and regulates contractility by influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes. The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results revealed that miR-133a mimic treatment improved the contractility of the diabetic rat's heart concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and plasma levels of NE. In miR-133aTg mice, cardiac-specific miR-133a overexpression prevented upregulation of TAT and suppression of TH in the heart after streptozotocin was administered. Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating NE biosynthesis, and consequently, β-AR and cardiac function.
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http://dx.doi.org/10.2337/db16-0023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033264PMC
October 2016

Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells.

J Endocrinol 2016 09 8;230(3):309-23. Epub 2016 Jul 8.

Department of Cellular and Integrative PhysiologyUniversity of Nebraska Medical Center, Omaha, NE, USA

One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic-adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition.
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http://dx.doi.org/10.1530/JOE-16-0146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980258PMC
September 2016

Renal denervation improves cardiac function in rats with chronic heart failure: Effects on expression of β-adrenoceptors.

Am J Physiol Heart Circ Physiol 2016 08 10;311(2):H337-46. Epub 2016 Jun 10.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska

Chronic activation of the sympathetic drive contributes to cardiac remodeling and dysfunction during chronic heart failure (HF). The present study was undertaken to assess whether renal denervation (RDN) would abrogate the sympathoexcitation in HF and ameliorate the adrenergic dysfunction and cardiac damage. Ligation of the left coronary artery was used to induce HF in Sprague-Dawley rats. Four weeks after surgery, RDN was performed, 1 wk before the final measurements. At the end of the protocol, cardiac function was assessed by measuring ventricular hemodynamics. Rats with HF had an average infarct area >30% of the left ventricle and left ventricular end-diastolic pressure (LVEDP) >20 mmHg. β1- and β2-adrenoceptor proteins in the left ventricle were reduced by 37 and 49%, respectively, in the rats with HF. RDN lowered elevated levels of urinary excretion of norepinephrine and brain natriuretic peptide levels in the hearts of rats with HF. RDN also decreased LVEDP to 10 mmHg and improved basal dP/dt to within the normal range in rats with HF. RDN blunted loss of β1-adrenoceptor (by 47%) and β2-adrenoceptor (by 100%) protein expression and improved isoproterenol (0.5 μg/kg)-induced increase in +dP/dt (by 71%) and -dP/dt (by 62%) in rats with HF. RDN also attenuated the increase in collagen 1 expression in the left ventricles of rats with HF. These findings demonstrate that RDN initiated in chronic HF condition improves cardiac function mediated by adrenergic agonist and blunts β-adrenoceptor expression loss, providing mechanistic insights for RDN-induced improvements in cardiac function in the HF condition.
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http://dx.doi.org/10.1152/ajpheart.00999.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504438PMC
August 2016

Renal Denervation Improves Exaggerated Sympathoexcitation in Rats With Heart Failure: A Role for Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus.

Hypertension 2016 07 16;68(1):175-84. Epub 2016 May 16.

From the Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha.

Renal denervation (RDN) has been postulated to reduce sympathetic drive during heart failure (HF), but the central mechanisms are not completely understood. The purpose of the present study was to assess the contribution of neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN) in modulating sympathetic outflow in rats with HF that underwent RDN. HF was induced in rats by ligation of the left coronary artery. Four weeks after surgery, bilateral RDN was performed. Rats with HF had an increase in FosB-positive cells in the PVN with a concomitant increase in urinary excretion of norepinephrine, and both of these parameters were ameliorated after RDN. nNOS-positive cells immunostaining, diaphorase staining, and nNOS protein expression were significantly decreased in the PVN of HF rats, findings that were ameliorated by RDN. Microinjection of nNOS inhibitor N(G)-monomethyl l-arginine into the PVN resulted in a blunted increase in lumbar sympathetic nerve activity (11±2% versus 24±2%) in HF than in sham group. This response was normalized after RDN. Stimulation of afferent renal nerves produced a greater activation of PVN neurons in rats with HF. Afferent renal nerve stimulation elicited a greater increase in lumbar sympathetic nerve activity in rats with HF than in sham rats (45±5% versus 22±2%). These results suggest that intact renal nerves contribute to the reduction of nNOS in the PVN, resulting in the activation of the neurons in the PVN of rats with HF. RDN restores nNOS and thus attenuates the sympathoexcitation commonly observed in HF.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.115.06794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900899PMC
July 2016

Exercise Training Attenuates Upregulation of p47(phox) and p67(phox) in Hearts of Diabetic Rats.

Oxid Med Cell Longev 2016 16;2016:5868913. Epub 2016 Feb 16.

Department of Cellular & Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

Exercise training (ExT) is currently being used as a nonpharmacological strategy to improve cardiac function in diabetic patients. However, the molecular mechanism(s) underlying its beneficial effects remains poorly understood. Oxidative stress is known to play a key role in the pathogenesis of diabetic cardiomyopathy and one of the enzyme systems that produce reactive oxygen species is NADH/NADPH oxidase. The goal of this study was to investigate the effect of streptozotocin- (STZ-) induced diabetes on expression of p47(phox) and p67(phox), key regulatory subunits of NADPH oxidase, in cardiac tissues and determine whether ExT can attenuate these changes. Four weeks after STZ treatment, expression of p47(phox) and p67(phox) increased 2.3-fold and 1.6-fold, respectively, in left ventricles of diabetic rats and these increases were attenuated with three weeks of ExT, initiated 1 week after onset of diabetes. In atrial tissues, there was increased expression of p47(phox) (74%), which was decreased by ExT in diabetic rats. Furthermore, increased collagen III levels in diabetic hearts (52%) were significantly reduced by ExT. Taken together, ExT attenuates the increased expression of p47(phox) and p67(phox) in the hearts of diabetic rats which could be an underlying mechanism for improving intracardiac matrix and thus cardiac function and prevent cardiac remodeling in diabetic cardiomyopathy.
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http://dx.doi.org/10.1155/2016/5868913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771908PMC
December 2016

Electrical stimulation of the aortic depressor nerve in conscious rats overcomes the attenuation of the baroreflex in chronic heart failure.

Am J Physiol Regul Integr Comp Physiol 2016 Apr 3;310(7):R612-8. Epub 2016 Feb 3.

Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil;

Chronic heart failure (CHF) is characterized by autonomic dysfunction combined with baroreflex attenuation. The hypotensive and bradycardic responses produced by electrical stimulation of the aortic depressor nerve (ADN) were examined in conscious CHF and control male Wistar rats (12-13 wk old). Furthermore, the role of parasympathetic and sympathetic nervous system in mediating the cardiovascular responses to baroreflex activation was evaluated by selective β1-adrenergic and muscarinic receptor antagonists. CHF was induced by myocardial infarction. After 6 wk, the subjects were implanted with electrodes for ADN stimulation. Twenty-four hours later, electrical stimulation of the ADN was applied for 20 s using five different frequencies (5, 15, 30, 60, and 90 Hz), while the arterial pressure was recorded by a catheter implanted into the femoral artery. Electrical stimulation of the ADN elicited progressive and similar hypotensive and bradycardic responses in control (n = 12) and CHF (n = 11) rats, while the hypotensive response was not affected by methylatropine. Nevertheless, the reflex bradycardia was attenuated by methylatropine in control, but not in CHF rats. Atenolol did not affect the hypotensive or bradycardic response in either group. The ADN function was examined under anesthesia through electroneurographic recordings. The arterial pressure-ADN activity relationship was attenuated in CHF rats. In conclusion, despite the attenuation of baroreceptor function in CHF rats, the electrical stimulation of the ADN elicited a stimulus-dependent hypotension and bradycardia of similar magnitude as observed in control rats. Therefore, electrical activation of the aortic baroreflex overcomes both the attenuation of parasympathetic function and the sympathetic overdrive.
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http://dx.doi.org/10.1152/ajpregu.00392.2015DOI Listing
April 2016

Urinary Proteolytic Activation of Renal Epithelial Na+ Channels in Chronic Heart Failure.

Hypertension 2016 Jan 30;67(1):197-205. Epub 2015 Nov 30.

From the Department of Cellular and Integrative Physiology (H.Z., X.L., N.M.S., K.P.P.) and Department of Emergency Medicine (Y.L.), University of Nebraska Medical Center, Omaha; and Department of Internal Medicine II, University Hospital Halle, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany (R.U.P.).

One of the key mechanisms involved in renal Na(+) retention in chronic heart failure (CHF) is activation of epithelial Na(+) channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC, resulting in renal Na(+) retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared with sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06, and plasmin 3.57 versus sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch clamp was conducted in cultured renal collecting duct M-1 cells to record Na(+) currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na(+) inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ≈2-folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid, which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na(+) retention commonly observed in CHF.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.115.05838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679598PMC
January 2016

Role of Chemoreceptor Activation in Hemodynamic Responses to Electrical Stimulation of the Carotid Sinus in Conscious Rats.

Hypertension 2015 Sep 20;66(3):598-603. Epub 2015 Jul 20.

From the Department of Physiology, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil (P.L.K., J.A.C., D.P.M.D., R.F., H.C.S.); and Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha (K.P.P.).

Electric carotid baroreflex activation has been used to treat patients with resistant hypertension. It is hypothesized that, in conscious rats, combined activation of carotid baro- and chemoreceptors afferences attenuates the reflex hypotension. Rats were divided into 4 groups: (1) control group, with unilateral denervation of the right carotid chemoreceptors; (2) chemoreceptor denervation group, with bilateral ligation of the carotid body artery; (3) baroreceptor denervation group, with unilateral denervation of the left carotid baroreceptors and right carotid chemoreceptors; and (4) carotid bifurcation denervation group, with denervation of the left carotid baroreceptors and chemoreceptors, plus denervation of the right carotid chemoreceptors. Animals were subjected to 4 rounds of electric stimulation (5 V, 1 ms), with 15, 30, 60, and 90 Hz applied randomly for 20 s. Electric stimulation caused greater hypotensive responses in the chemoreceptor denervation group than in the control group, at 60 Hz (-37 versus -19 mm Hg) and 90 Hz (-33 versus -19 mm Hg). The baroreceptor denervation group showed hypertensive responses at all frequencies of stimulation. In contrast, the carotid sinus denervation group showed no hemodynamic responses. The control group presented no changes in heart rate, whereas the chemoreceptor denervation group and the baroreceptor denervation group showed bradycardic responses. These data demonstrate that carotid chemoreceptor activation attenuates the reflex hypotension caused by combined electric stimulation of the carotid sinus and the carotid sinus nerve in conscious rats. These findings may provide useful insight for clinical studies using baroreflex activation therapy in resistant hypertension and heart failure.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.115.05316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537377PMC
September 2015

Modulation of angiotensin II signaling following exercise training in heart failure.

Am J Physiol Heart Circ Physiol 2015 Apr 13;308(8):H781-91. Epub 2015 Feb 13.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska.

Sympathetic activation is a consistent finding in the chronic heart failure (CHF) state. Current therapy for CHF targets the renin-angiotensin II (ANG II) and adrenergic systems. Angiotensin converting enzyme (ACE) inhibitors and ANG II receptor blockers are standard treatments along with β-adrenergic blockade. However, the mortality and morbidity of this disease is still extremely high, even with good medical management. Exercise training (ExT) is currently being used in many centers as an adjunctive therapy for CHF. Clinical studies have shown that ExT is a safe, effective, and inexpensive way to improve quality of life, work capacity, and longevity in patients with CHF. This review discusses the potential neural interactions between ANG II and sympatho-excitation in CHF and the modulation of this interaction by ExT. We briefly review the current understanding of the modulation of the angiotensin type 1 receptor in sympatho-excitatory areas of the brain and in the periphery (i.e., in the carotid body and skeletal muscle). We discuss possible cellular mechanisms by which ExT may impact the sympatho-excitatory process by reducing oxidative stress, increasing nitric oxide. and reducing ANG II. We also discuss the potential role of ACE2 and Ang 1-7 in the sympathetic response to ExT. Fruitful areas of further investigation are the role and mechanisms by which pre-sympathetic neuronal metabolic activity in response to individual bouts of exercise regulate redox mechanisms and discharge at rest in CHF and other sympatho-excitatory states.
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http://dx.doi.org/10.1152/ajpheart.00026.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398865PMC
April 2015