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.

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.

Our previous studies have shown that the decreased NO and increased glutamatergic mechanisms on sympathetic regulation within the paraventricular nucleus (PVN) may contribute to the elevated sympathoexcitation 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 receptor subunit NR ₁ in the rats with a coronary ligation model of CHF. Adenovirus vectors encoding nNOS (AdnNOS) or adenovirus vectors encoding β-galactosidase 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, blood pressure, and heart rate responses to NO synthase inhibitor N ^G -monomethyl- l -arginine in the rats with CHF compared with CHF-adenovirus vectors encoding β-galactosidase group. AdnNOS significantly decreased the enhanced renal sympathetic nerve activity, blood pressure, and heart rate responses to N -methyl- d -aspartic acid in the rats with CHF (renal sympathetic nerve activity: 44±2% versus 79±6%; P <0.05) compared with CHF-adenovirus vectors encoding the β-galactosidase group. AdnNOS transfection significantly reduced the increased NR ₁ receptor mRNA expression (Δ35±5%) and protein levels (Δ24±4%) within the PVN in CHF rats. Furthermore, in neuronal NG-108 cells, NR ₁ receptor protein expression decreased in a dose-dependent manner after AdnNOS transfection. According to our results, nNOS downregulation enhances glutamate transmission in the PVN by increasing NR ₁ subunit expression. This mechanism may enhance renal sympathetic nerve activity in CHF rats.

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 NG-monomethyl l-arginine into the PVN resulted in a blunted increase in lumbar sympathetic nerve activity (11±2% versus 24±2%) in H...

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.

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 (AT₁) 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. AT₁ receptor protein expression was increased 50% in HF-Sed group compared with sham-Sed group. In the HF-ExT group, AT₁ 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.