Blocking AT(1) receptors prevents the overexpression of CAPON and concomitant decrease in nNOS in the PVN, resulting in attenuation of sympathoexcitation commonly observed in CHF. Taken together, our data highlight the importance of altered expression and subsequent interaction of nNOS and CAPON within the PVN, leading to increased sympathoexcitation in CHF. Identifying this crucial nNOS/CAPON interaction regulated by AT(1) receptors may provide an important potential therapeutic target in CHF.

Angiotensin II is a key neuropeptide that acting within the brain hypothalamic paraventricular nucleus regulates neurohumoral outflow to the circulation. Moreover, an exacerbated angiotensin II action within the paraventricular nucleus contributes to neurohumoral activation in hypertension. While angiotensin II effects involve changes in paraventricular nucleus neuronal activity, the precise underlying mechanisms, cellular targets, and distribution of angiotensin II receptors within the paraventricular nucleus remain largely unknown. Thus, whether angiotensin II 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 angiotensin II actions, which involves astrocytes as major intermediary cellular targets. We found that angiotensin II AT1 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-receptors, increased presympathetic neuronal activity, enhanced sympathoexcitatory outflow, and increased blood pressure. Together, our studies support astrocytes as critical intermediary cell types underlying brain angiotensin II regulation of the circulation, and indicate that angiotensin II-mediated neuronal and sympathoexcitatory effects are dependent on a unique neuro-glial signaling modality involving non-synaptic glutamate transmission.

Our previous studies have shown that the decreased nitric oxide (NO) and increased glutamatergic mechanisms on sympathetic regulation within the paraventricular nucleus (PVN) may contribute to the elevated sympatho-excitation during chronic heart failure (CHF). In the present study, we investigated the effects of neuronal NO synthase (nNOS) gene transfer on N-methyl-D-aspartic acid (NMDA) receptor subunit NR1 in the rats with coronary ligation model of CHF. Adenovirus vectors encoding either nNOS (AdnNOS) or β-galactosidase (AdβGal) were transfected into the PVN in vivo. Five days after application of AdnNOS, the increased expression of nNOS within the PVN was confirmed by NADPH-diaphorase staining, real time PCR and Western blot. In anesthetized rats, AdnNOS treatment significantly enhanced the blunted renal sympathetic nerve activity (RSNA), blood pressure (BP) and heart rate (HR) responses to NOS inhibitor L-NMMA in the rats with CHF compared to CHF-AdβGal group. AdnNOS significantly decreased the enhanced RSNA, BP and HR responses to NMDA in the rats with CHF (RSNA: 44±2% versus 79±6%, P<0.05) compared to CHF-AdβGal group. AdnNOS transfection significantly reduced the increased NR1 receptor mRNA expression (Δ35±5%) and protein levels (Δ24±4%) within the PVN in CHF rats. Furthermore, in neuronal NG-108 cells, NR1 receptor protein expression decreased in a dose-dependent manner after AdnNOS transfection. According to our results nNOS down-regulation enhances glutamate transmission in the PVN by increasing NR1 subunit expression. This mechanism may enhance RSNA in CHF rats.

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.

An increased sympathetic drive is an adverse characteristic in chronic heart failure (CHF). The protein expression of neuronal nitric oxide synthase (nNOS)- and hence nitric oxide (NO)-mediated sympathoinhibition is reduced in the paraventricular nucleus (PVN) of rats with CHF. However, the molecular mechanism(s) of nNOS downregulation remain(s) unclear. The aim of the study was to reveal the underlying molecular mechanism for the downregulation of nNOS in the PVN of CHF rats. Sprague-Dawley rats with CHF (6-8 wk after coronary artery ligation) demonstrated decreased nNOS dimer/monomer ratio (42%), with a concomitant increase in the expression of PIN (a protein inhibitor of nNOS known to dissociate nNOS dimers into monomers) by 47% in the PVN. Similarly, PIN expression is increased in a neuronal cell line (NG108) treated with angiotensin II (ANG II). Furthermore, there is an increased accumulation of high-molecular-weight nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of CHF rats (29%). ANG II treatment in NG108 cells in the presence of a proteasome inhibitor, lactacystin, also leads to a 69% increase in accumulation of nNOS-Ub conjugates immunoprecipitated by an antiubiquitin antibody. There is an ANG II-driven, PIN-mediated decrease in the dimeric catalytically active nNOS in the PVN, due to ubiquitin-dependent proteolytic degradation in CHF. Our results show a novel intermediary mechanism that leads to decreased levels of active nNOS in the PVN, involved in subsequent reduction in sympathoinhibition during CHF, offering a new target for the treatment of CHF and other cardiovascular diseases.

Rev1 and DNA polymerase (Pol) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Pol lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Pol with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pulldown and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Pol-dependent TLS pathway. Taken together, the data suggest that Pol and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.Cellular DNA is continuously attacked by endogenous and exogenous agents that damage the bases and the DNA backbone. Damage that is not repaired prior to the S phase of cell cycle can block the replication machinery, because most lesions cannot be accommodated in the highly selective active site of replicative DNA polymerases (1, 2). Replication stalling activates several damage tolerance mechanisms that help bypass the damage in either an accurate or mutagenic manner. Translesion DNA synthesis (TLS) 3 is an important damage tolerance pathway, in which specialized DNA polymerases are recruited to synthesize DNA through template lesions (3). Structural studies showed that TLS polymerases have a more open active site that allows them to accommodate a variety of DNA lesions and catalyze polymerization on damaged templates (4). The accuracy of TLS can vary depending on the particular lesion and the DNA polymerases involved. It is, however, an inherently mutagenic process, because the damage can alter the coding properties of the bases, and because TLS polymerases generally have low fidelity (2).In human cells, TLS polymerases include Y family enzymes Pol, Pol, Pol, and REV1, and the B family enzyme Pol. Although the Y family polymerases share little amino acid sequence similarity with the classical DNA polymerases, they have a similar overall "right-hand" architecture with the "palm," "thumb," and "fingers" domains. The thumb and fingers domains of the Y family enzymes, however, are short and do not make as many contacts wi...

Kleiber AC, Zheng H, Sharma NM, Patel KP. Chronic AT1 receptor blockade normalizes NMDA-mediated changes in renal sympathetic nerve activity and NR1 expression within the PVN in rats with heart failure. Am J Physiol Heart Circ Physiol 298: H1546 -H1555, 2010. First published February 19, 2010 doi:10.1152/ajpheart.01006.2009.-Exercise training normalizes enhanced glutamatergic mechanisms within the paraventricular nucleus (PVN) concomitant with the normalization of increased plasma ANG II levels in rats with heart failure (HF). We tested whether ANG II type 1 (AT1) receptors are involved in the normalization of PVN glutamatergic mechanisms using chronic AT1 receptor blockade with losartan (Los; 50 mg · kg Ϫ1 · day Ϫ1 in drinking water for 3 wk). Left ventricular end-diastolic pressure was increased in both HF ϩ vehicle (Veh) and HF ϩ Los groups compared with sham-operated animals (Sham group), although it was significantly attenuated in the HF ϩ Los group compared with the HF ϩ Veh group. The effect of Los on cardiac function was similar to exercise training. At the highest dose of N-methyl-D-aspartate (NMDA; 200 pmol) injected into the PVN, the increase in renal sympathetic nerve activity was 93 Ϯ 13% in the HF ϩ Veh group, which was significantly higher (P Ͻ 0.05) than the increase in the Sham ϩ Veh (45 Ϯ 2%) and HF ϩ Los (47 Ϯ 2%) groups. Relative NMDA receptor subunit NR1 mRNA expression within the PVN was increased 120% in the HF ϩ Veh group compared with the Sham ϩ Veh group (P Ͻ 0.05) but was significantly attenuated in the HF ϩ Los group compared with the HF ϩ Veh group (P Ͻ 0.05). NR1 protein expression increased 87% in the HF ϩ Veh group compared with the Sham ϩ Veh group but was significantly attenuated in the HF ϩ Los group compared with the HF ϩ Veh group (P Ͻ 0.05). Furthermore, in in vitro experiments using neuronal NG-108 cells, we found that ANG II treatment stimulated NR1 protein expression and that Los significantly ameliorated the NR1 expression induced by ANG II. These data are consistent with our hypothesis that chronic AT1 receptor blockade normalizes glutamatergic mechanisms within the PVN in rats with HF. paraventricular nucleus; N-methyl-D-aspartate receptor; angiotensin type 1 receptor; sympathetic nerve activity INCREASED ACTIVATION of the sympathetic nervous system is associated with heart failure (HF) (46). This increased sympathoexcitation is due, in part, to increased glutamatergic mechanisms within the paraventricular nucleus (PVN) of the hypothalamus (16). Specifically, the increase in renal sympathetic nerve activity (RSNA) in response to N-methyl-D-aspartate (NMDA) microinjected into the PVN is higher in rats with HF than in sham-operated rats (16). Additionally, expression of a subunit of the NMDA receptor, NR 1 , is increased within the PVN in rats with HF (16). We (15) have recently shown that exercise training normalizes the increased RSNA response to NMDA microinjected into the PVN as well as the increased NR 1 expression within the PVN.A functional NMDA receptor comprise...

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the underlying mechanisms are not known. We hypothesized ExT would normalize the augmented activation of the paraventricular nucleus (PVN) via an angiotensinergic mechanism during HF. Four groups of rats used were the following: 1) sham-sedentary (Sed); 2) sham-ExT; 3) HF-Sed, and 4) HF-ExT. HF was induced by left coronary artery ligation. Four weeks after surgery, 3 wk of treadmill running was performed in ExT groups. The number of FosB-positive cells in the PVN was significantly increased in HF-Sed group compared with the sham-Sed group. ExT normalized (negated) this increase in the rats with HF. In anesthetized condition, the increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in response to microinjection of angiotensin (ANG) II (50∼200 pmol) in the PVN of HF-Sed group were significantly greater than of the sham-Sed group. In the HF-ExT group the responses to microinjection of ANG II were not different from sham-Sed or sham-ExT groups. Blockade of ANG II type 1 (AT(1)) receptors with losartan in the PVN produced a significantly greater decrease in RSNA, MAP, and HR in HF-Sed group compared with sham-Sed group. ExT prevented the difference between HF and sham groups. AT(1) receptor protein expression was increased 50% in HF-Sed group compared with sham-Sed group. In the HF-ExT group, AT(1) receptor protein expression was not significantly different from sham-Sed or sham-ExT groups. In conclusion, one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of angiotensinergic mechanisms within the PVN.