Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Feb 1.
Published in final edited form as: Nitric Oxide. 2024 Nov 7;154:1–7. doi: 10.1016/j.niox.2024.11.001

Downregulation of Neuronal Nitric Oxide Synthase (nNOS) within the Paraventricular Nucleus in Ins2Akita-type-1 Diabetic Mice Contributes to Sympatho-excitation

Tapan A Patel 1, Lie Gao 2, Shane H Boomer 3, Xuefei Liu 3, Kaushik P Patel 1, Hong Zheng 3
PMCID: PMC11729414  NIHMSID: NIHMS2036432  PMID: 39521242

Abstract

Activation of both renin-angiotensin system (RAS) and the sympathetic system is the primary etiologic event in developing cardiovascular complications in diabetes mellitus (DM). However, the precise mechanisms for sympathetic activation in DM have not been elucidated. Here we attempted to investigate diabetes-linked cardiovascular dysregulation due to angiotensin II (Ang II)-mediated reduction in neuronal nitric oxide (NO) synthase (nNOS) within the paraventricular neuleus (PVN). In the present study, we used Ins2+/−Akita (a spontaneous, insulin-dependent genetic diabetic non-obese murine model) and wild-type (WT) littermates mice as controls. At 14 weeks of age, we found the Akita mice had increased renal sympathetic nerve activity and elevated levels of plasma norepinephrine. There was decreased expression of nNOS protein (Akita 0.43 ± 0.11 vs. WT 0.75 ± 0.05, P < 0.05) in the PVN of Akita mice. Akita mice had increased expression of angiotensin-converting enzyme (ACE) (Akita 0.58 ± 0.05 vs. WT 0.34 ± 0.04, P < 0.05) and Ang II type 1 receptor (Akita 0.49 ± 0.03 vs. WT 0.29 ± 0.09, P < 0.05), decreased expressions of ACE2 (Akita 0.17 ± 0.05 vs. WT 0.27 ± 0.03, P < 0.05) and angiotensin (1–7) Mas receptor (Akita 0.46 ± 0.02 vs. WT 0.77 ± 0.07, P < 0.05). Futher, there were increased protein levels of protein inhibitor of nNOS (PIN) (Akita 1.75 ± 0.08 vs. WT 0.71 ± 0.09, P < 0.05) with concomitantly decreased catalytically active dimers of nNOS (Akita 0.11 ± 0.04 vs. WT 0.19 ± 0.02, P < 0.05) in the PVN in Akita mice. Our studies suggest that activation of the excitatory arm of RAS, leads to a decrease NO, causing an over-activation of the sympathetic drive in DM.

Keywords: nNOS, angiotensin II, paraventricular nucleus, neurogenic hypertension

1. Introduction

Diabetes has been established as an independent cause of cardiovascular related morbidity and mortality [1]. Type 1 diabetes, known as insulin dependent diabetes, is characterized by the autoimmune destruction of pancreatic β-cells and is common in children and teenagers [2]. In human and the animal model of type 1 diabetes, there is evidence that the onset of hyperglycemia at the early stages can increase blood pressure while inducing renal hyperfiltration and natriuresis [3], wheras at later stages, it is correlated with urinary albumin excretion [1]. Augmented sympathetic nerve activity is crucial in the pathogenesis of hypertension in diabetes and is characterized by sympathetic dominance and parasympathetic withdrawal [4; 5]. There is accumulating evidence suggesting that excessive activation of the renin angiotensin system (RAS) exaggerates the progression of cardiovascular maladies and sympatho-excitation [1; 6; 7].

The increased sympatho-excitation originates from the central nervous system (CNS), with one specific site of interest being the paraventricular nuclues (PVN) [8; 9]. Neuroanatomical, electrophysiological and functional studies have indicated an important role for the PVN in cardiovascular regulation via innervation to the rostral ventrolateral medulla (RVLM), as well as direct projections to the intermediolateral cell column in the spinal cord, dictating sympathetic outflow [10]. Neuronal nitric oxide (NO) synthase (nNOS) has been identified as a primary source for NO in the PVN [11]. NO within the PVN modulates the release of several neurotransmitters, such as acetylcholine, catecholamine, excitatory and inhibitory amino acids to influence neuronal function [12; 13; 14]. Previously we have shown that the micro-administration of an NO donor into the PVN decreases renal sympathetic nerve activity (RSNA). Conversely, administration of an inhibitor of NOS into the PVN, increases RSNA [15]. Blockade of NOS activity with NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) in the PVN also enhances the angiotensin II (Ang II)-mediated sympatho-excitation, and this enhancement is blunted in heart failure rats known to have exaggerated sympatho-excitation [16]. Interestingly nNOS expression is markedly reduced in streptozotocin (STZ)-induced diabetic model in the PVN, and at the same time there was blunted inhibition of sympathetic activation which correlates with data that shows enhanced sympathetic drive [17; 18]. During diabetes, in the peripheral circulation, NO produced by endothelial NOS (eNOS) is decreased and results in vasoconstriction during diabetes. This vascular disposition is linked to hypertension, atherosclerosis, and other cardiovascualr disease events commonly observed during diabetes [19]. Therefore, the tonicity of NO, produced by either nNOS or eNOS, contributes to cardiovascular dysregulation during diabetes in the brain and periphery, respectively.

Activation of both RAS and the sympathetic system is the primary etiologic event in the development of cardiovascular dysfunctioin including hypertension in diabetes mellitus [3]. Elevated levels of Ang II in diabetic patients [20], STZ-induced diabetic rat [21] and Akita mice [22] are of particular interest since Ang II has central actions in the CNS to increase sympatho-excitation as well as peripheral actions at the nerve terminal to increase norepinephrine (NE) release [23; 24]. Ang II is known to exhibit chronic actions in CNS that are expressed through changes in gene expression, protein expression and enzyme activities [25; 26]. Evidence suggests that local RAS is also activated in the PVN of STZ-induced diabetic model [18]. Consistent with this observation Angiotensin-converting enzyme (ACE) inhibitors [27] and Ang II receptor blockers [23; 28] are effective in reducing sympatho-excitation. Here we used Ins2+/−Akita mice, a spontaneous insulin dependent genetic diabetic murine model, to test the hypothesis that diabetes-linked sympatho-excitation is due to altered Ang II-mediated reduction in nNOS within the PVN.

2. Materials and methods

2.1. Animal model

Male Ins2Akita-type-1 (Akita) mice heterozygous for the insulin-2 spontaneous mutation and controls with identical backgrounds (C57BL/6J, WT) (14 weeks old) obtained from the Jackson Laboratory (Bar Harbor, ME) were fed and housed according to institutional guidelines. The University of Nebraska Medical Center and University of South Dakota Institutional Animal Care and Use Committee approved all protocols which adhere to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, Eighth Edition (National Academic Press; 2011). Blood glucose levels were tested weekly in Akita mice. Insulin was not administered as animals maintained healthy weights and body condition scores throughout the housing period. Blood glucose was consistently above 500 mg/dl in all Akita mice.

2.2. Serum norepinephrine (NE) concentration measurements

Serum NE concentration was measured as a general index of overall sympathetic activation. At 14 weeks old, blood from Akita and WT mice was collected. Serum NE concentration were measured using a commercially available ELISA kit (Labor Diagnostika Nord, Nordhorn, Germany), following the manufacturer’s instructions. The limit of detection of the assay is 1.5 ng/ml NE.

2.3. General surgical procedures

Akita and WT mice were anesthetized by injecting urethane (0.75 g/kg ip) and α-chloralose (70 mg/kg ip). The right femoral artery and femoral vein were cannulated for recording arterial blood pressure and administrating chemicals, respectively. Mean arterial pressure (MAP) and heart rate (HR) were simultaneously recorded on a PowerLab data-acquisition system (8SP, AD Instruments, Colorado Springs, CO).

2.4. Renal sympathetic nerve activity (RSNA) recording

In anesthetized mice a renal nerve bundle near the renal hilus was isolated and gently placed on a bipolar platinum electrode. The electrical signal from the electrode was amplified and recorded with the MacLab. The basal renal nerve discharge recording was obtained. The background noise was determined by cutting the distal end of the nerve. The value of renal nerve activity was calculated by subtracting the background noise from the actual recorded value.

The mean value of the 10-minute baseline RSNA was used to quantify basal RSNA. The basal RSNA was normalized to the peak RSNA response to 13% KCl injection (iv) (RSNAmax), which expressed as a percent of RSNAmax.

2.5. Micro-punch of the PVN area

The brain was removed from the euthanized animal and frozen on dry ice. Six serial coronal sections (100 μm/section) were cut through the hypothalamus at the level of the PVN with a cryostat. The PVN tissue was obtained using the micro-punch method of Palkovit and Brownstein [29] using a diethylpyrocarbonate-treated blunt 18-gauge needle attached to a syringe as documented previously in our laboratory [23; 30].

2.6. Immunoblotting

Western blotting was performed on the lysates of PVN punches. Total protein in PVN lysates was extracted with a radioimmunoprecipitation assay buffer (10 mM Tris, 1 mM EDTA, 1% SDS, 0.1% Triton X-100) supplemented with protease inhibitors. The protein lysates (20–30 μg), mixed with SDS-PAGE buffer were fractionated on polyacrylamide gel and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). Non-fat dry milk (5% w/v) in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20) was used to block the membrane at ambient temperature for 30 minutes. Then the membrane was incubated with the primary antibody overnight, followed by the corresponding peroxidase-conjugated secondary antibody for 1 hour. An enhanced chemiluminescence substrate (Pierce Chemical, Rockford, IL) was used to visualize the signals, which were detected by Worklab digital image system. Image J (NIH) was used to quantify the signal.

The following primary antibodies were used: nNOS (sc-5302, 1:500), ACE (sc-23908, 1:250), ACE2 (sc-20998, 1:250), Ang II type 1 receptor (AT1R) (sc-515884, 1:250), Mas receptor (sc-390453, 1:250), protein inhibitor of nNOS (PIN) (sc-13969, 1:500), and β-actin (sc-47778, 1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA).

2.7. Assay of nNOS dimers

Dimeric nNOS (active form) was determined using low-temperature polyacrylamide gel electrophoresis (LT-PAGE) following the Klatt et al. [31] technique and as described previously [32]. The procedure was performed as mentioned above using a 5% separating gel, except that the electrophoresis was done in a cold room at 25 V, and gel and buffers were pre-equilibrated to 4°C. After LT-PAGE, the gels were transferred to the PVDF membrane overnight in a cold room at 30 V and probed with nNOS antibody. Tubulin was used as a housekeeping gene for this experiment.

2.8. Statistical Analysis

Data were expressed as mean ± SEM, and statistical significance was determined at P < 0.05. Statistical comparisons of the groups were made using a Student’s t-test, one-way analysis of variance (ANOVA), followed by Bonferroni’s test for posthoc analysis using Prism 7 (GraphPad Software).

3. Results

3.1. General characteristics

In the present study, we used the Akita mice as insulin-dependent genetic diabetic non-obese murine model. Akita mice had a significant lower body weight at 14 weeks old than WT mice (Akita 19.0 ± 0.1 vs. WT 26.0 ± 0.8 g, P < 0.05). Akita mice also had increased levels of glucose compared to the WT mice (Akita 507.0 ± 33.6 vs. WT 133.0 ± 7.8 mg/dl, P < 0.05) (Figure 1).

Figure 1:

Figure 1:

Open in a new tab

Metabolic parameters (body weight, plasma glucose and serum norepinephrine) of Akita mice and control WT mouse. Values are presented as mean ± SE. *P < 0.05 compared with WT.

Serum concentrations of NE used as an index of overall sympathetic activation was significantly greater in Akita mice compared to WT controls (Akita 2,126 ± 759 vs. WT 548 ± 99 pg/mL, P < 0.05) (Figure 1).

3.2. Basal blood pressure, heart rate and renal sympathetic nerve activity in Akita mice

Examples of recordings showing tracing of basal MAP, HR and RSNA, maximal RSNA after 13% KCl iv injection, from WT and Akita mice are shown in Figure 2A and Figure 2B. The final basal RSNA was expressed as a percentage of peak RSNA (% of Max). Basal RSNA was significantly higher in Akita mice than in WT mice (Akita 54.1 ± 3.1 vs. WT 34.2 ± 4.8 % max, P < 0.05) (Figure 2C). There were no significant differences of basal MAP (Akita 75.3 ± 4.7 vs. WT 77.1 ± 3.6 mmHg, P > 0.05) and HR (Akita 521.8 ± 55.3 vs. WT 506.3 ± 24.6 bpm, P > 0.05) between the Akita and WT groups of mice. The KCL-induced maximal RSNA was not different between the two groups.

Figure 2:

Figure 2:

Open in a new tab

A: Segments of original recordings demonstrating the representative tracing of basal mean arterial pressure (AP), heart rate (HR), raw renal sympathetic nerve activity (RSNA) and integrated RSNA (Int. RSNA) in wild type (WT) and Akita mice. B. Segments of original recordings demonstrating in the end of experiment, the responses of AP, HR, raw RSNA and Int. RSNA to KCL injection in WT and Akita mice. C. Basal RSNA in WT and Akita mice (n = 4/group). Values are presented as mean ± SE. *P < 0.05 compared with WT.

3.3. Decreased nNOS within the PVN in Akita mice

Expression of nNOS protein was significantly reduced (~43%) in the PVN of Akita mice compared to WT controls (Akita 0.43 ± 0.11 vs. WT 0.75 ± 0.05, P < 0.05) (Figure 3).

Figure 3:

Figure 3:

Open in a new tab

Representative western blots gel image and mean values of nNOS protein expression within the PVN in WT and Akita mice (n = 6/group). Values are presented as mean ± SE. *P < 0.05 compared with WT.

3.4. Increased activation of RAS in the PVN in Akita mice

We found that Akita mice had increased expression of ACE (Akita 0.58 ± 0.05 vs. WT 0.34 ± 0.04, P < 0.05) (Figure 4A) and decreased expression of ACE2 (Akita 0.17 ± 0.05 vs. WT 0.27 ± 0.03, P < 0.05) in the PVN (Figure 4B). Akita mice also had increased AT1R (Akita 0.49 ± 0.03 vs. WT 0.29 ± 0.09, P < 0.05) (Figure 5A) and decreased Mas receptor (WT 0.77 ± 0.07 vs. Akita 0.46 ± 0.02, P < 0.05) in the PVN (Figure 5B).

Figure 4:

Figure 4:

Open in a new tab

Representative western blots gel image and mean values of ACE (A) and ACE2 (B) protein expressions within the PVN in WT and Akita mice (n = 7/group). Values are presented as mean ± SE. *P < 0.05 compared with WT.

Figure 5:

Figure 5:

Open in a new tab

Representative western blots gel image and mean values of AT1R (A) and Mas receptor (B) protein expressions within the PVN in WT and Akita mice (n = 6–7/group). Values are presented as mean ± SE. *P < 0.05 compared with WT.

3.5. Increased PIN expression and decreased nNOS dimer/monomer within the PVN in Akita mice

Expression of PIN protein was significantly increased (~146%) in the PVN of Akita (Akita 1.75 ± 0.08 vs. WT 0.71 ±0.09, P < 0.05) (Figure 6A). We also examined the level of monomeric and dimeric forms of nNOS in PVN lysates using LT-PAGE so that the SDS-resistant dimeric form of nNOS could be measured. We observed a significant decrease in dimer/monomer ratio of nNOS in the PVN of Akita mice compared to WT controls (Akita 0.11 ± 0.04 vs. WT 0.19 ± 0.01, P < 0.05) (Figure 6B).

Figure 6:

Figure 6:

Open in a new tab

A. Representative western blots gel image and mean values of PIN protein expression within the PVN in WT and Akita mice (n = 6/group). B. nNOS dimers in the PVN in WT and Akita mice (n = 4/group). Top panel: Representative LT-PAGE blot showing dimer and monomer of nNOS, bottom panel: densitometry analyses of monomer and dimer nNOS levels represented as a ratio of the dimer to the monomer. Values are mean ± SEM. *P < 0.05 compared with WT.

4. Discussion

The salient findings of this study are; 1) Akita mice had elevated levels of plasma NE (index of general sympatho-excitation) as well as an increase in RSNA; 2) There was decreased expression of nNOS protein with a concomitant increase in levels of PIN in the PVN of Akita mice; 3) Consistent with the levels of PIN, there were decreased catalytically active dimers of nNOS in the PVN of Akita mice; 4) Further, Akita mice had increased expressions of ACE and AT1R, while there were decreased expressions of ACE2 and angiotensin Mas receptor. Overall these studies indicate that an activated RAS causes a reduction of the inhibitory nNOS-NO mechanisms within the PVN resulting in an over-activation of the sympathetic drive in DM.

In the present study, we used the Akita mice as insulin-dependent diabetic model, which serves as a well-established model for investigating type 1 diabetes [33]. The autosomal dominant point mutation Mody (Cys/Tyr) in the Ins2 insulin gene causes a primary defect in protein processing that renders pancreatic β cells incapable of insulin secretion resulting in chronic hypoinsulinemia and hyperglycemia. The diabetic phenotype is apparent as early as 4 weeks after birth resulting in significant elevations in blood glucose by 7 weeks of age and increased systolic blood pressure at 14 weeks of age [34; 35]. Our 14 weeks Akita mice have higher glucose levels (> 500 mg/dl) than the WT mice.

Sympathetic over-activation is crucial in the pathogenesis of cardiovascular complications in diabetes [4; 5; 36]. Many studies have identified the PVN of the hypothalamus as being an essential integration site for regulating sympathetic nerve activity [10; 37; 38]. The PVN plays an important role in integrating signals/inputs from circumventricular organs and other brain areas involved in cardiovascular regulation and generating an output to the RVLM and other downstream areas to influence overall sympathetic activity. NO in the PVN is involved in regulating sympathetic outflow [15; 39]. Previously, we have shown nNOS expression is markedly reduced in STZ-induced diabetic model in the PVN, which correlates with data that shows enhanced sympathetic drive [17; 18]. The Akita mice in the present study showed increased basal sympathetic nerve activity and reduced nNOS expression in the PVN, which was consistent with previous diabetic studies. Overall, NO synthesized by nNOS is downregulated in the PVN under diabetic conditions. However, the mechanism/s remained elusive.

Sympathoexcitation, increased arterial blood pressure and enhanced central Ang II signaling have been documented in various cardiovascular disease states such as hypertension, diabetes and chronic heart failure [16; 18; 40; 41]. Multiple previous studies from our laboratory have explored the association between Ang II and decreased NO bioavailability within the PVN, which may eventually contribute to enhanced sympatho-excitation [16; 23]. Our present data confirmed the local RAS (increased ACE and AT1R, decreased ACE2 and Mas receptor) was activated in the PVN of Akita mice, which led to the hypothesis Ang II-dependent mechanisms are involved in the downregulation of nNOS within the PVN in diabetic mice. Consistent with our results increased levels of AT1R and downregulation of Mas receptors were notoed in the brain cortexes of diabetic, hypertensive, and diabetic-hypertensive rats [42]. Enhanced sympathetic activity as well as upregulation of angiotensinogen and AT1R was also observed in the subfornical organ, supraoptic nucleus and PVN of diabetic rat model indicating upregulation of central RAS [43].

nNOS is regulated by various mechanisms, including selective proteolytic degradation involving ubiquitination [44]. Honodimerzation of nNOS and its association with Ca2+-calmodulin is necessary for the production of NO [45; 46]. A dimer/monomer ratio decrease is thought to decrease NO production and bioavailability. Dimer instability is reported to trigger ubiquitination and proteasomal degradation [47]. PIN, a small molecular weight protein, was so named because it was thought to regulate nNOS activity and hence NO generation negatively [48; 49]. PIN binding destabilizes the dimeric structure of nNOS [50] and acts as a molecular trigger for ubiquitination and proteasomal degradation of nNOS [32]. We have shown that Ang II treatment of neurons results in increased accumulation of PIN and increased PIN-nNOS binding in vitro, suggesting a relevant functional interaction between these two proteins [32]. This effect is associated with the decreased expression and active catalytic dimers of nNOS, which would be expected to result in reduced NO bioavailability. In this study, we have observed approximately 1.5-fold higher levels of PIN and a 50% decrease in the nNOS dimer to monomer ratio. The possible mechanism of reduced nNOS in Akita mice may be via central Ang II increasing the levels of PIN within the PVN, reducing the active dimeric form of nNOS, which consequently results in an increased sympathetic outflow and a robust hypertensive response.

The role of PIN to inhibit the activity of nNOS, blood pressure and consequentlyregulating sympathetic activity has been examined in spontaneously hypertensive rats [51]. This study demonstrated that inhibition of PIN expression by siRNA attenuates the increased blood pressure and development of hypertension in spontaneously hypertensive rats at 12 weeks of age. Increased PIN expression and decreased nNOS dimers are also reported in the RVLM of high-fructose diet-fed rats contributing to increased sympatho-excitation and hypertension [52]. The results of the present studies provide a potential explanation that PIN levels within the PVN may be post-translationally regulated by Ang II-mediated ubiquitination. Therefore, the increased levels of Ang II enhance the expression of PIN post-translationally in specific areas of the brain, specifically, the PVN. Reduced levels of active nNOS dimers due to increased levels of PIN lead to the reduction of NO centrally within the PVN, resulting in reduced inhibitory influences to neurons, and thereby enhanced sympatho-excitation observed in DM (Figure 7).

Figure 7.

Figure 7.

Open in a new tab

Proposed model for the up-regulation of the PIN by post-translational regulation in the PVN. Elevated central Ang II levels via AT1R in the PVN increase the expression of PIN via decreasing the ubiquitination. Increased expression of PIN destabilizes nNOS dimers, which renders nNOS catalytically inactive, either by interfering with the assembly or dimer stability. A reduced level of functional nNOS reduces NO production in the PVN, causing an increase in sympatho-excitation and associated blood pressure.

5. Conclusion

In summary, the observations in this study provide a unique insight into the RAS mediated upregulation of PIN resulting in decreased inhibitory nNOS-NO mechanism(s) within the PVN that contribute to the over-activation of sympathetic drive, and offer possible new target/s for treating sympatho-excitation that leads to cardiovascular complications in DM.

Acknowledgements

This work was supported by National Institutes of Health grants [R01-DK-114663, R01-DK-129311, and endowed McIntyre Professorship fund to K.P.P].

Footnotes

Declaration of competing interest

The authors declare no potential conflicts of interest.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Refeferences

  • [1].Perin PC, Maule S, Quadri R, Sympathetic nervous system, diabetes, and hypertension, Clin Exp Hypertens 23 (2001) 45–55. [DOI] [PubMed] [Google Scholar]
  • [2].Kloppel G, Clemens A, [Insulin-dependent diabetes mellitus. Current aspects of morphology, etiology and pathogenesis], Pathologe 17 (1996) 269–75. [DOI] [PubMed] [Google Scholar]
  • [3].Brands MW, Fitzgerald SM, Blood pressure control early in diabetes: a balance between angiotensin II and nitric oxide, Clin Exp Pharmacol Physiol 29 (2002) 127–31. [DOI] [PubMed] [Google Scholar]
  • [4].Jermendy G, Clinical consequences of cardiovascular autonomic neuropathy in diabetic patients, Acta Diabetol 40 Suppl 2 (2003) S370–4. [DOI] [PubMed] [Google Scholar]
  • [5].Rosengard-Barlund M, Bernardi L, Fagerudd J, Mantysaari M, Af Bjorkesten CG, Lindholm H, Forsblom C, Waden J, Groop PH, FinnDiane Study G, Early autonomic dysfunction in type 1 diabetes: a reversible disorder?, Diabetologia 52 (2009) 1164–72. [DOI] [PubMed] [Google Scholar]
  • [6].Zucker IH, Wang W, Pliquett RU, Liu JL, Patel KP, The regulation of sympathetic outflow in heart failure. The roles of angiotensin II, nitric oxide, and exercise training, Ann N Y Acad Sci 940 (2001) 431–43. [PubMed] [Google Scholar]
  • [7].Mehta JK, Kaur G, Buttar HS, Bagabir HA, Bagabir RA, Bagabir SA, Haque S, Tuli HS, Telessy IG, Role of the renin-angiotensin system in the pathophysiology of coronary heart disease and heart failure: Diagnostic biomarkers and therapy with drugs and natural products, Front Physiol 14 (2023) 1034170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Patel KP, Schmid PG, Role of paraventricular nucleus (PVH) in baroreflex-mediated changes in lumbar sympathetic nerve activity and heart rate, J Auton Nerv Syst 22 (1988) 211–9. [DOI] [PubMed] [Google Scholar]
  • [9].Patel KP, Role of paraventricular nucleus in mediating sympathetic outflow in heart failure, Heart Fail Rev 5 (2000) 73–86. [DOI] [PubMed] [Google Scholar]
  • [10].Swanson LW, Sawchenko PE, Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms, Neuroendocrinology 31 (1980) 410–7. [DOI] [PubMed] [Google Scholar]
  • [11].Guix FX, Uribesalgo I, Coma M, Munoz FJ, The physiology and pathophysiology of nitric oxide in the brain, Prog Neurobiol 76 (2005) 126–52. [DOI] [PubMed] [Google Scholar]
  • [12].Schuman EM, Madison DV, Nitric oxide and synaptic function, Annu Rev Neurosci 17 (1994) 153–83. [DOI] [PubMed] [Google Scholar]
  • [13].Patel KP, Li YF, Hirooka Y, Role of nitric oxide in central sympathetic outflow, Exp Biol Med (Maywood) 226 (2001) 814–24. [DOI] [PubMed] [Google Scholar]
  • [14].Horn T, Smith PM, McLaughlin BE, Bauce L, Marks GS, Pittman QJ, Ferguson AV, Nitric oxide actions in paraventricular nucleus: cardiovascular and neurochemical implications, Am J Physiol 266 (1994) R306–13. [DOI] [PubMed] [Google Scholar]
  • [15].Zhang K, Mayhan WG, Patel KP, Nitric oxide within the paraventricular nucleus mediates changes in renal sympathetic nerve activity, Am J Physiol 273 (1997) R864–72. [DOI] [PubMed] [Google Scholar]
  • [16].Li YF, Patel KP, Paraventricular nucleus of the hypothalamus and elevated sympathetic activity in heart failure: the altered inhibitory mechanisms, Acta Physiol Scand 177 (2003) 17–26. [DOI] [PubMed] [Google Scholar]
  • [17].Zheng H, Mayhan WG, Bidasee KR, Patel KP, Blunted nitric oxide-mediated inhibition of sympathetic nerve activity within the paraventricular nucleus in diabetic rats, Am J Physiol Regul Integr Comp Physiol 290 (2006) R992–R1002. [DOI] [PubMed] [Google Scholar]
  • [18].Patel KP, Mayhan WG, Bidasee KR, Zheng H, Enhanced angiotensin II-mediated central sympathoexcitation in streptozotocin-induced diabetes: role of superoxide anion, Am J Physiol Regul Integr Comp Physiol 300 (2011) R311–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Oak JH, Cai H, Attenuation of angiotensin II signaling recouples eNOS and inhibits nonendothelial NOX activity in diabetic mice, Diabetes 56 (2007) 118–26. [DOI] [PubMed] [Google Scholar]
  • [20].Chu KY, Leung PS, Angiotensin II in type 2 diabetes mellitus, Curr Protein Pept Sci 10 (2009) 75–84. [DOI] [PubMed] [Google Scholar]
  • [21].Zimpelmann J, Kumar D, Levine DZ, Wehbi G, Imig JD, Navar LG, Burns KD, Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat, Kidney Int 58 (2000) 2320–30. [DOI] [PubMed] [Google Scholar]
  • [22].Zhang L, Xiong XQ, Fan ZD, Gan XB, Gao XY, Zhu GQ, Involvement of enhanced cardiac sympathetic afferent reflex in sympathetic activation in early stage of diabetes, J Appl Physiol (1985) 113 (2012) 47–55. [DOI] [PubMed] [Google Scholar]
  • [23].Li YF, Wang W, Mayhan WG, Patel KP, Angiotensin-mediated increase in renal sympathetic nerve discharge within the PVN: role of nitric oxide, Am J Physiol Regul Integr Comp Physiol 290 (2006) R1035–43. [DOI] [PubMed] [Google Scholar]
  • [24].Buttler L, Ribeiro IM, Ferreira-Neto HC, Antunes VR, Angiotensin II acting on PVN induces sympathoexcitation and pressor responses via the PI3K-dependent pathway, Auton Neurosci 198 (2016) 54–8. [DOI] [PubMed] [Google Scholar]
  • [25].Sumners C, Fleegal MA, Zhu M, Angiotensin AT1 receptor signalling pathways in neurons, Clin Exp Pharmacol Physiol 29 (2002) 483–90. [DOI] [PubMed] [Google Scholar]
  • [26].Park HS, You MJ, Yang B, Jang KB, Yoo J, Choi HJ, Lee SH, Bang M, Kwon MS, Chronically infused angiotensin II induces depressive-like behavior via microglia activation, Sci Rep 10 (2020) 22082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Kinugawa T, Ogino K, Kitamura H, Saitoh M, Omodani H, Osaki S, Hisatome I, Miyakoda H, Catecholamines, renin-angiotensin-aldosterone system, and atrial natriuretic peptide at rest and during submaximal exercise in patients with congestive heart failure, Am J Med Sci 312 (1996) 110–7. [DOI] [PubMed] [Google Scholar]
  • [28].Rezacova L, Hojna S, Kopkan L, Rauchova H, Kadlecova M, Zicha J, Vaneckova I, Role of angiotensin II in chronic blood pressure control of heterozygous Ren-2 transgenic rats: Peripheral vasoconstriction versus central sympathoexcitation, Biomed Pharmacother 116 (2019) 108996. [DOI] [PubMed] [Google Scholar]
  • [29].Palkovits M, Brownstein M, Brain microdissection techniques. in: Cuello AC (Ed.), John Wiley & Sons, Inc., Chichester, 1983. [Google Scholar]
  • [30].Li YF, Cornish KG, Patel KP, Alteration of NMDA NR1 receptors within the paraventricular nucleus of hypothalamus in rats with heart failure, Circ Res 93 (2003) 990–7. [DOI] [PubMed] [Google Scholar]
  • [31].Klatt P, Schmidt K, Lehner D, Glatter O, Bachinger HP, Mayer B, Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer, EMBO J 14 (1995) 3687–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Sharma NM, Llewellyn TL, Zheng H, Patel KP, Angiotensin II-mediated posttranslational modification of nNOS in the PVN of rats with CHF: role for PIN, Am J Physiol Heart Circ Physiol 305 (2013) H843–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Yoshioka M, Kayo T, Ikeda T, Koizumi A, A novel locus, Mody4, distal to D7Mit189 on chromosome 7 determines early-onset NIDDM in nonobese C57BL/6 (Akita) mutant mice, Diabetes 46 (1997) 887–94. [DOI] [PubMed] [Google Scholar]
  • [34].Abdo S, Lo CS, Chenier I, Shamsuyarova A, Filep JG, Ingelfinger JR, Zhang SL, Chan JS, Heterogeneous nuclear ribonucleoproteins F and K mediate insulin inhibition of renal angiotensinogen gene expression and prevention of hypertension and kidney injury in diabetic mice, Diabetologia 56 (2013) 1649–60. [DOI] [PubMed] [Google Scholar]
  • [35].Lo CS, Liu F, Shi Y, Maachi H, Chenier I, Godin N, Filep JG, Ingelfinger JR, Zhang SL, Chan JS, Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice, Am J Physiol Renal Physiol 302 (2012) F840–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Kanagala A, Harsoda JM, Sympathetic Overactivity and Parasympathetic Impairment in Type 2 Diabetes: An Analysis of Cardiovascular Autonomic Functions, Cureus 16 (2024) e59561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Pyner S, Coote JH, Identification of branching paraventricular neurons of the hypothalamus that project to the rostroventrolateral medulla and spinal cord, Neuroscience 100 (2000) 549–56. [DOI] [PubMed] [Google Scholar]
  • [38].Li T, Chen Y, Gua C, Wu B, Elevated Oxidative Stress and Inflammation in Hypothalamic Paraventricular Nucleus Are Associated With Sympathetic Excitation and Hypertension in Rats Exposed to Chronic Intermittent Hypoxia, Front Physiol 9 (2018) 840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Krukoff TL, Central regulation of autonomic function: no brakes?, Clin Exp Pharmacol Physiol 25 (1998) 474–8. [DOI] [PubMed] [Google Scholar]
  • [40].Young BE, Holwerda SW, Vranish JR, Keller DM, Fadel PJ, Sympathetic Transduction in Type 2 Diabetes Mellitus, Hypertension 74 (2019) 201–207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Heusser K, Tank J, Diedrich A, Fischer A, Heise T, Jordan J, Limited evidence for sympathetic neural overactivation in older patients with type 2 diabetes mellitus, Front Neurosci 16 (2022) 1107752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Oyesiji Abiodun A, AlDosari DI, Alghamdi A, Aziz Al-Amri A, Ahmad S, Ola MS, Diabetes-induced stimulation of the renin-angiotensin system in the rat brain cortex, Saudi J Biol Sci 30 (2023) 103779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Liu Y, Li L, Qiu M, Tan L, Zhang M, Li J, Zhu H, Jiang S, Su X, Li A, Renal and cerebral RAS interaction contributes to diabetic kidney disease, Am J Transl Res 11 (2019) 2925–2939. [PMC free article] [PubMed] [Google Scholar]
  • [44].Clapp KM, Peng HM, Jenkins GJ, Ford MJ, Morishima Y, Lau M, Osawa Y, Ubiquitination of neuronal nitric-oxide synthase in the calmodulin-binding site triggers proteasomal degradation of the protein, J Biol Chem 287 (2012) 42601–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Alderton WK, Cooper CE, Knowles RG, Nitric oxide synthases: structure, function and inhibition, Biochem J 357 (2001) 593–615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Chachlaki K, Prevot V, Nitric oxide signalling in the brain and its control of bodily functions, Br J Pharmacol 177 (2020) 5437–5458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Dunbar AY, Kamada Y, Jenkins GJ, Lowe ER, Billecke SS, Osawa Y, Ubiquitination and degradation of neuronal nitric-oxide synthase in vitro: dimer stabilization protects the enzyme from proteolysis, Mol Pharmacol 66 (2004) 964–9. [DOI] [PubMed] [Google Scholar]
  • [48].Fan JS, Zhang Q, Li M, Tochio H, Yamazaki T, Shimizu M, Zhang M, Protein inhibitor of neuronal nitric-oxide synthase, PIN, binds to a 17-amino acid residue fragment of the enzyme, J Biol Chem 273 (1998) 33472–81. [DOI] [PubMed] [Google Scholar]
  • [49].Xia Y, Berlowitz CO, Zweier JL, PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase, Biochim Biophys Acta 1760 (2006) 1445–9. [DOI] [PubMed] [Google Scholar]
  • [50].Jaffrey SR, Snyder SH, PIN: an associated protein inhibitor of neuronal nitric oxide synthase, Science 274 (1996) 774–7. [DOI] [PubMed] [Google Scholar]
  • [51].Wang SC, Lin KM, Chien SJ, Huang LT, Hsu CN, Tain YL, RNA silencing targeting PIN (protein inhibitor of neuronal nitric oxide synthase) attenuates the development of hypertension in young spontaneously hypertensive rats, J Am Soc Hypertens 8 (2014) 5–13. [DOI] [PubMed] [Google Scholar]
  • [52].Wu KL, Chao YM, Tsay SJ, Chen CH, Chan SH, Dovinova I, Chan JY, Role of nitric oxide synthase uncoupling at rostral ventrolateral medulla in redox-sensitive hypertension associated with metabolic syndrome, Hypertension 64 (2014) 815–24. [DOI] [PubMed] [Google Scholar]

RESOURCES