Xiuying Ma

The Ion Channel ASIC2 Is Required for Baroreceptor and Autonomic Control of the Circulation

Arterial baroreceptors provide a neural sensory input that reflexly regulates the autonomic drive of circulation. Our goal was to test the hypothesis that a member of the acid-sensing ion channel (ASIC) subfamily of the DEG/ENaC superfamily is an important determinant of the arterial baroreceptor reflex. We found that aortic baroreceptor neurons in the nodose ganglia and their terminals express ASIC2. Conscious ASIC2 null mice developed hypertension, had exaggerated sympathetic and depressed parasympathetic control of the circulation, and a decreased gain of the baroreflex, all indicative of an impaired baroreceptor reflex. Multiple measures of baroreceptor activity each suggest that mechanosensitivity is diminished in ASIC2 null mice. The results define ASIC2 as an important determinant of autonomic circulatory control and of baroreceptor sensitivity. The genetic disruption of ASIC2 recapitulates the pathological dysautonomia seen in heart failure and hypertension and defines a molecular defect that may be relevant to its development.

Mechanisms determining sensitivity of baroreceptor afferents in health and disease.

Abstract: Baroreceptors sense and signal the central nervous system of changes in arterial pressure through a series of sensory processes. An increase in arterial pressure causes vascular distension and baroreceptor deformation, the magnitude of which depends on the mechanical viscoelastic properties of the vessel wall. Classic methods (e.g., isolated carotid sinus preparation) and new approaches, including studies of isolated baroreceptor neurons in culture, gene transfer using viral vectors, and genetically modified mice have been used to define the cellular and molecular mechanisms that determine baroreceptor sensitivity. Deformation depolarizes the nerve endings by opening a new class of mechanosensitive ion channel. This depolarization triggers action potential discharge through opening of voltage‐dependent sodium (Na+) and potassium (K+) channels at the “spike initiating zone” (SIZ) near the sensory terminals. The resulting baroreceptor activity and its sensitivity to changes in pressure are modulated through a variety of mechanisms that influence these sensory processes. Modulation of voltage‐dependent Na+ and K+ channels and the Na+ pump at the SIZ by membrance potential, action potential discharge, and chemical autocrine and paracrine factors are important mechanisms contributing to changes in baroreceptor sensitivity during sustained increases in arterial pressure and in pathological states associated with endothelial dysfunction, oxidative stress, and platelet activation.

A novel effect of angiotensin on renal sympathetic nerve activity in mice.

Objective The goals of this study were to characterize the effects of angiotensin II (Ang II) on renal sympathetic nerve activity (RSNA) and to define mechanisms of its actions in mice. Design The experiments were performed in sodium pentobarbital anesthetized C57BL/6J mice to investigate the effects of intravenous administration of Ang II on RSNA recorded from renal sympathetic post-ganglionic nerve fibers. Results Intravenous (i.v.) administration of Ang II (4 ng/g) increased arterial pressure and evoked a biphasic change in RSNA: inhibition of high-amplitude phasic bursts of RSNA secondary to the initial rise of arterial pressure followed by activation of low-amplitude continuously discharging RSNA that exceeded baseline activity (255 ± 72% baseline, n = 8). The peak change of mean arterial pressure (MAP) was +60 ± 4 mmHg (n = 8). In the same group of animals, norepinephrine (40 ng/g) caused an equivalent increase in MAP (+57 ± 5 mmHg) and essentially abolished RSNA. The Ang II-induced activation of RSNA was dose-dependent (0.5–4 ng/g, n = 7) and was abolished by the Ang II type 1 (AT1) receptor blocker, losartan (10 μg/g, i.v.) (301 ± 61 versus 117 ± 22% baseline , before versus after losartan, n = 5). The ganglionic blocker, hexamethonium (30 μg/g, i.v.), eliminated baseline high-amplitude bursts of RSNA but did not blunt the Ang II-induced RSNA (n = 6). In baroreceptor denervated and vagotomized mice, Ang II failed to inhibit high-amplitude bursts of RSNA but continued to trigger low-amplitude continuous RSNA. Conclusion We conclude that Ang II activates renal sympathetic nerves that discharge in a continuous pattern, distinctly different than the normal baseline high-amplitude bursts of RSNA. The mechanism may involve direct activation of post-ganglionic sympathetic neurons mediated through AT1 receptors.

Qualitative and quantitative morphology of renal nerves in C57BL/6J mice

The detailed morphology of the renal nerves in mice has not been reported previously. The aims of this study were to describe the general morphology of the extrinsic renal nerve in C57BL/6 mice, and determine its morphometric parameters. The major renal nerve innervating the left kidney was isolated in five mice. Thin sections of the nerve segments were then examined by transmission electron microscopy. The renal nerve averaged 35.4 ± 3.6 (S.E.M.) μm in diameter and 741 ± 104 μm in area. The renal nerve contained an average of 830 ± 169 unmyelinated fibers and only 4.6 ± 1.7 myelinated fibers. The axon diameter of myelinated and unmyelinated fibers averaged 2.2 ± 0.3 μm and 0.76 ± 0.02 μm, respectively. The diameter of the unmyelinated fibers ranged from 0.3 to 2.0 μm, and the distribution histogram was unimodal. The majority of fibers (85%) had diameters of 0.6–1.0 μm. These results are similar to those obtained for renal nerves of rats with respect to the predominance of unmyelinated fibers. However, the diameter of unmyelinated fibers is larger in rats and the distribution histogram of rat unmyelinated fibers is bimodal, in contrast to the unimodal distribution in mice. The morphological description of the renal nerves in mice provides baseline data for further investigations of the structural basis of altered autonomic reflexes. The results will be useful in analyses of genes that influence the development and structure of sympathetic and sensory innervation of the kidney in genetically manipulated mice. Anat Rec 268:399–404, 2002.

Ganglionic Action of Angiotensin Contributes to Sympathetic Activity in Renin-Angiotensinogen Transgenic Mice

Abstract—In addition to central nervous system actions, angiotensin (Ang) II may increase sympathetic nerve activity (SNA) via a direct action on sympathetic ganglia. We hypothesized that sympathetic ganglionic actions of endogenous Ang II contribute to SNA in transgenic mice that overexpress renin and angiotensinogen (R+A+ mice). Renal SNA and arterial pressure were recorded in anesthetized R+A+ and littermate control mice before and after ganglionic blockade, and after additional blockade of angiotensin type 1 (AT1) receptors with losartan. Ganglionic blockade essentially abolished SNA in control mice, but only reduced SNA to 47±18% of baseline in R+A+ mice. The residual SNA remaining after ganglionic blockade in R+A+ mice was reduced from 47±18% to 8±6% of baseline by losartan (P <0.05). The sympathoinhibitory response to losartan was accompanied by an enhanced decrease in arterial pressure in R+A+ mice compared with that observed in control mice. AT1 receptor expression in sympathetic ganglia, as measured by real-time reverse transcription–polymerase chain reaction, was increased ≈3-fold in R+A+ versus control mice. The results demonstrate that, as anticipated, essentially all of the renal postganglionic SNA in control mice is driven by preganglionic input. The major new finding is that Ang II–evoked ganglionic activity accounts for ≈40% of total SNA in R+A+ mice. The significant contribution of the direct ganglionic action of Ang II in R+A+ mice likely reflects both increased levels of Ang II and upregulation of AT1 receptors in sympathetic ganglia.

Dual mechanisms of angiotensin‐induced activation of mouse sympathetic neurones

Ang II directly activates neurones in sympathetic ganglia. Our goal was to define the electrophysiological basis of this activation. Neurones from mouse aortic–renal and coeliac ganglia were identified as either ‘tonic’ or ‘phasic’. With injections of depolarizing currents, action potentials (APs) were abundant and sustained in tonic neurones (TNs) and scarce or absent in phasic neurones (PNs). Resting membrane potentials were equivalent in TNs (−48 ± 2 mV, n= 18) and PNs (−48 ± 1 mV, n= 23) while membrane resistance was significantly higher in TNs. Ang II depolarized and increased membrane resistance equally in both TNs (n= 8) and PNs (n= 8) but it induced APs only in TNs, and enhanced current‐evoked APs much more markedly in TNs (P < 0.05). The AT1 receptor antagonist losartan (2 μm, n= 6) abolished all responses to Ang II, whereas the AT2 receptor blocker PD123,319 had no effect. The transient K+ current (IA), which was more than twice as large in TNs as in PNs, was significantly inhibited by Ang II in TNs only whereas the delayed sustained K+ current (IK), which was comparable in both TNs and PNs, was not inhibited. M currents were more prominent in PNs and were inhibited by Ang II. The IA channel blocker 4‐aminopyridine triggered AP generation in TNs and prevented the Ang II‐induced APs but not the depolarization. Blockade of M currents by oxotremorine M or linopirdine prevented the depolarizing action of Ang II. The protein kinase C (PKC) inhibitor H7 (10 μm, n= 9) also prevented the Ang II‐induced inhibition of IA and the generation APs but not the depolarization nor the inhibition of M currents. Conversely, the PKC agonist phorbol 12‐myristate 13‐acetate mimicked the Ang II effects by triggering APs. The results indicate that Ang II may increase AP generation in sympathetic neurones by inducing a PKC‐dependent inhibition of IA currents, and a PKC‐independent depolarization through inhibition of M currents. The differential expression of various K+ channels and their sensitivity to phosphorylation by PKC may determine the degree of activation of sympathetic neurones and hence may influence the severity of the hypertensive response.

NAD(P)H oxidase-induced oxidative stress in sympathetic ganglia of apolipoprotein E deficient mice.

Superoxide anion (O2*-) is increased throughout the arterial wall in atherosclerosis. The oxidative stress contributes to lesion formation and vascular dysfunction. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived O2*- is increased in nodose sensory ganglia and sympathetic ganglia of apolipoprotein E deficient (apoE-/-) mice, an established animal model of atherosclerosis. O2*- measured ex vivo by L-012-enhanced chemiluminescence was increased by 79+/-17% in whole sympathetic ganglia from apoE-/- mice (n=5) compared with sympathetic ganglia from control mice (n=5) (P<0.05). In contrast, O2*- was not elevated in nodose ganglia from apoE-/- mice. Dihydroethidium staining confirmed the selective increase in O2*- in sympathetic ganglia of apoE-/- mice, and revealed the contribution of both neurons and non-neuronal cells to the O2*- generation. We investigated the enzymatic source of increased O2*- in sympathetic ganglia of apoE-/- mice. The mRNA expression of gp91phox, p22phox, p67phox, and p47phox subunits of NAD(P)H oxidase measured by real time RT-PCR was increased approximately 3-4 fold in sympathetic ganglia of apoE-/- mice (n=5) compared with control ganglia (n=5). NADPH oxidase activity measured by lucigenin chemiluminescence was increased by 68+/-12% in homogenates of sympathetic ganglia from apoE-/- mice (n=7) compared with control ganglia (n=7) (P<0.05). The results identify sympathetic ganglia as a novel site of oxidative stress in atherosclerosis, and suggest that upregulation of NAD(P)H oxidase is the source of increased O2*- generation. We speculate that oxidative stress in sympathetic ganglia may contribute to impaired baroreflex control of sympathetic nerve activity.

Neurocardiovascular regulation in mice: Experimental approaches and novel findings

1. Neural mechanisms are of major importance in the regulation of arterial blood pressure, blood volume and other aspects of cardiovascular function. The recent explosion in gene discovery and advances in molecular technologies now provide the opportunity to define the molecular and cellular mechanisms essential to integrative neurocardiovascular regulation. The unique susceptibility of mice to genetic manipulation makes this species an attractive model for such investigation.

Autocrine/paracrine modulation of baroreceptor activity after antidromic stimulation of aortic depressor nerve in vivo

Activation of the sensory nerve endings of non-myelinated C-fiber afferents evokes release of autocrine/paracrine factors that cause localized vasodilation, neurogenic inflammation, and modulation of sensory nerve activity. The aims of this study were to determine the effect of antidromic electrical stimulation on afferent baroreceptor activity in vivo, and investigate the role of endogenous prostanoids and hydrogen peroxide (H2O2) in mediating changes in nerve activity. Baroreceptor activity was recorded from the left aortic depressor nerve (ADN) in anesthetized rats before and after stimulating the ADN for brief (5–20 s) periods. The rostral end of the ADN was crushed or sectioned beforehand to prevent reflex changes in blood pressure. Antidromic stimulation of ADN using parameters that activate both myelinated A-fibers and non-myelinated C-fibers caused pronounced and long-lasting (> 1 min) inhibition of baroreceptor activity (n = 9, P < 0.05), with the magnitude and duration of inhibition dependent on the duration of the stimulation period (n = 5). Baroreceptor activity was only transiently inhibited after selective stimulation of A-fibers. The inhibition of activity after antidromic stimulation of A and C fibers was prolonged after administration of the cyclooxygenase inhibitor indomethacin (5 mg/kg, IV, n = 7) and abolished after administration of PEG-catalase (104 units/kg, IV, n = 7), an enzyme that catalyzes the decomposition of H2O2 to water and oxygen. The results demonstrate a long-lasting inhibition of baroreceptor activity after antidromic stimulation of ADN and suggest that endogenous prostanoids and H2O2 oppose and mediate the inhibition, respectively. These mechanisms may contribute to rapid baroreceptor resetting during acute hypertension and be engaged during chronic baroreceptor activation therapy in patients with hypertension.