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

Highlights

• Intracerebroventricular (icv) infusion of Ang II increases the expression of PIN in the PVN.

• Ubiquitination of PIN is decreased in NG108 cells treated with Ang II and also in the PVN of rats infused with AngII (icv).

• Ang II via the Ubiquitin-Proteasomal pathway may regulate PIN expression in the PVN at the post-translational level.

• The increased PIN would be expected to decrease active nNOS dimers and hence reduce the production of nitric oxide.

• The reduced NO• levels would lead to abrogated inhibition of sympathetic tone mediated from the PVN.

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 AT₁R 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.

Introduction

Reduced bioavailability of nitric oxide (NO•) concomitant with increased sympathetic outflow is implicated in the pathogenesis of many cardiovascular diseases, including hypertension, heart failure, and diabetes [[1], [2], [3], [4], [5]]. Further, hypertensive patients and animal models of hypertension have demonstrated increased sympathetic nerve activity (SNA), accompanied by alterations in body fluid homeostasis [6]. The NO•-pathway contributes to the blood pressure-lowering effect of many commonly used therapeutic agents and emerging basic science research on this pathway is elucidating potential new antihypertensive drug targets. NO• is a gaseous paracrine molecule produced by a family of NADPH-dependent enzymes named nitric oxide synthases (NOS) [[7], [8], [9]]. NO• acts as a pivotal signaling molecule that regulates blood flow and tissue oxygenation by activating soluble guanylate cyclase in the vascular smooth muscle [10]. NO• produced by the neuronal isoform of NOS (nNOS) plays an essential role in the paraventricular nucleus (PVN) via acting as a retrograde inhibitory neurotransmitter that regulates synaptic efficacy and modulates neuronal activity leading to changes in SNA and consequently neurogenic control of blood pressure [[11], [12], [13]]. Of the three NOS isoforms, nNOS is the largest, due to a 300-amino-acid PDZ (PSD-95/Discs large/zona occludens-1) domain insertion at the N-terminus targeting nNOS to the postsynaptic density (PSD) protein in the synaptic spines of neurons [14]. The catalytic activity of nNOS is regulated by several other scaffolding proteins such as syntrophin, PSD93, PSD95 and CAPON (carboxy-terminal PDZ ligand of nNOS) which have been identified to interact with nNOS in neurons and modify its activity. In neurons, another nNOS-interacting protein the dynein light chain, LC8 (DYNLL1) or PIN (protein inhibitor of nNOS) physically interacts with nNOS and inhibits its activity [15]. PIN is a highly conserved, small homodimer cytosolic protein that is reported to bind to a 17-residue peptide fragment from Met-228 to His-244 of nNOS [16] and destabilizes the nNOS homodimer [15], a conformation necessary for the catalytic activity of the enzyme. Monomerization of nNOS promotes ubiquitination and degradation of nNOS by the proteasomal pathway [17,18]. Previously, we have shown that there is a decrease in nNOS expression [19] and enhanced expression of CAPON [20] and PIN [4] in the PVN of rats with heart failure. Moreover, Losartan (Los) an AT₁R blocker treatment restores the elevated SNA and normalizes the changes in expression of nNOS, CAPON, and PIN suggesting that the AT1 receptor activation may be linked to the reciprocal changes in these regulators(CAPON and PIN) and nNOS in the PVN [4,20]. Furthermore, we described the in vitro molecular mechanism of downregulation of nNOS involving interactions between nNOS and PIN through an Ang II-mediated signaling pathway(s) and suggested that post-translational processes, such as protein degradation/stabilization are involved in Ang II-dependent downregulation of nNOS [4]. To further understand how Ang II to PIN link, regulates nNOS, we dissect the molecular mechanism of increased PIN expression in a well-established model of neurogenic hypertension produced by intracerebroventricular infusion of Ang II for two weeks.