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

Abstract

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 AT 1 R 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.

Central Ang II (Angiotensin II)-Mediated Sympathoexcitation

“…We have standardized all procedures overtime to specifically punch areas within the PVN as evident from our previous publications. 15 , 21 , 29 , 36 , 37 It is also recognized that the PVN has a myriad of different neuronal phenotypes but the specific focus of our interest was on the preautonomic neurons activated by glutamate. Future studies, targeting glutamatergic neurons specifically in the PVN using optogenetics technique would strengthen our conclusions.…”

“…Rats with intracerebroventricular infusion of Ang II (20 ng/minute, 0.5 µL/hour for 14 days) were used as a model of neurogenic hypertension as described. 29 The dose of Ang II was based on previous studies showing the central action of Ang II. 30 – 32 The baroreflex control of heart rate (HR) was evaluated by measuring the reflex changes in HR in response to transient increases or decreases in MAP (mm Hg) induced by bolus injections of phenylephrine (2.5–25 µg/mL in 0.1 mL, IV) or sodium nitroprusside (5–50 µg/mL in 0.1 mL, IV), respectively, as described previously.…”

“…Six serial coronal sections (100 µm) of the PVN were cut following the Palkovit and Brownstein 39 and bilaterally punched as described previously. 36 , 37 Methods for cell culture and molecular biology experiments were extensively used previously, 15 , 21 , 29 and described in detail in the Data Supplement .…”

Lactacystin-induced kidney fibrosis: Protection by melatonin and captopril

“…In line with this, surprisingly, lactacystin did not reduce but increased blood pressure and fibrotic rebuilding in the left ventricle ( Simko et al, 2017 ). Thus, lactacystin administration-induced BP elevation was recently characterized as a novel model of experimental hypertension ( Vrankova et al, 2010 ; Simko et al, 2017 ; Sharma et al, 2020 ). Proteasome inhibition may cause hypertension either because of an increased endogenous protein inhibitor of neuronal nitric oxide synthase, leading to decreased NO bioavailability in the paraventricular nucleus ( Sharma et al, 2020 ), or from tyrosine hydroxylase upregulation and activation in the hypothalamus and brainstem ( Congo Carbajosa et al, 2015 ), both resulting in increased sympathetic outflow.…”

“…Chronic lactacystin administration induces a mild but significant rise in systolic blood pressure (BP) along with fibrotic remodeling of the left ventricle (LV) in Wistar rats ( Simko et al, 2017 ), and hypertrophy of the aorta in L-NAME (L-NG-nitro arginine methyl ester)-induced hypertension ( Vrankova et al, 2010 ). Several hypothetical mechanisms for hemodynamic alterations and heart and vessel damage by lactacystin have been proposed, such as enhanced oxidative stress ( Vrankova et al, 2010 ; Huseby et al, 2016 ; Parajuli, 2019 ), decreased NO bioavailability ( Simko et al, 2017 ; Sharma et al, 2020 ), sympathetic nervous system activation ( Congo Carbajosa et al, 2015 ) and modification of various cytosolic, nuclear, and myofibrillar protein turnovers ( Mearini et al, 2008 ). However, data on lactacystin’s renal effects are not available.…”