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Changes in vascular structure contribute to vascular events and loss of brain health. We examined the inherent structural plasticity of cerebral arterioles during and after a period of hypertension. Arteriolar wall thickness, diameter, wall-to-lumen ratio, and biological stiffness changed rapidly during hypertension and recovered with blood pressure. In contrast, inward remodeling developed slowly and did not recover. This lack of recovery of arteriolar diameter has implications for the long-term impact of hypertension on vascular determinants of brain health.

The carotid body (CB) is the main peripheral chemoreceptor for arterial respiratory gases O2and CO2and pH, eliciting reflex ventilatory, cardiovascular, and humoral responses to maintain homeostasis. This review examines the fundamental biology underlying CB chemoreceptor function, its contribution to integrated physiological responses, and its role in maintaining health and potentiating disease. Emphasis is placed on 1) transduction mechanisms in chemoreceptor (type I) cells, highlighting the role played by the hypoxic inhibition of O2-dependent K+channels and mitochondrial oxidative metabolism, and their modification by intracellular molecules and other ion channels; 2) synaptic mechanisms linking type I cells and petrosal nerve terminals, focusing on the role played by the main proposed transmitters and modulatory gases, and the participation of glial cells in regulation of the chemosensory process; 3) integrated reflex responses to CB activation, emphasizing that the responses differ dramatically depending on the nature of the physiological, pathological, or environmental challenges, and the interactions of the chemoreceptor reflex with other reflexes in optimizing oxygen delivery to the tissues; and 4) the contribution of enhanced CB chemosensory discharge to autonomic and cardiorespiratory pathophysiology in obstructive sleep apnea, congestive heart failure, resistant hypertension, and metabolic diseases and how modulation of enhanced CB reactivity in disease conditions may attenuate pathophysiology.

Toll-like receptors (TLR) are key components of the innate immune system that elicit inflammatory responses through the adaptor proteins myeloid differentiation protein 88 (MyD88) and Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β (TRIF). Previously, we demonstrated that TRIF mediates the signaling of angiotensin II (ANG II)- induced hypertension and cardiac hypertrophy. Since TRIF is activated selectively by TLR3 and TLR4, our goals in this study were to determine the roles of TLR3 and TLR4 in mediating ANG II-induced hypertension and cardiac hypertrophy, and associated changes in proinflammatory gene expression in heart and kidney. In wild-type (WT) mice, ANG II infusion (1,000 ng·kg−1·min−1for 3 wk) increased systolic blood pressure and caused cardiac hypertrophy. In ANG II-infused TLR4-deficient mice ( Tlr4del), hypertrophy was significantly attenuated despite a preserved or enhanced hypertensive response. In contrast, in TLR3-deficient mice ( Tlr3−/−), both ANG II-induced hypertension and hypertrophy were abrogated. In WT mice, ANG II increased the expression of several proinflammatory genes in hearts and kidneys that were attenuated in both TLR4- and TLR3-deficient mice compared with WT. We conclude that ANG II activates both TLR4-TRIF and TLR3-TRIF pathways in a nonredundant manner whereby hypertension is dependent on activation of the TLR3-TRIF pathway and cardiac hypertrophy is dependent on both TLR3-TRIF and TLR4-TRIF pathways.NEW & NOTEWORTHY Angiotensin II (ANG II)-induced hypertension is dependent on the endosomal Toll-like receptor 3 (TLR3)-Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β (TRIF) pathway of the innate immune system but not on cell membrane localized TLR4. However, ANG II-induced cardiac hypertrophy is regulated by both TLR4-TRIF and TLR3-TRIF pathways. Thus, ANG II-induced rise in systolic blood pressure is independent of TLR4-TRIF effect on cardiac hypertrophy. The TLR3-TRIF pathway may be a potential target of therapeutic intervention.

Calcitonin gene-related peptide (CGRP) can cause migraines, yet it is also a potent vasodilator that protects against hypertension. Given the emerging role of CGRP-targeted antibodies for migraine prevention, an important question is whether the protective actions of CGRP are mediated by vascular or neural CGRP receptors. To address this, we have characterized the cardiovascular phenotype of transgenic nestin/hRAMP1 mice that have selective elevation of a CGRP receptor subunit in the nervous system, human receptor activity-modifying protein 1 (hRAMP1). Nestin/hRAMP1 mice had relatively little hRAMP1 RNA in blood vessels and intravenous injection of CGRP caused a similar blood pressure decrease in transgenic and control mice. At baseline, nestin/hRAMP1 mice exhibited similar mean arterial pressure, heart rate, baroreflex sensitivity, and sympathetic vasomotor tone as control mice. We previously reported that expression of hRAMP1 in all tissues favorably improved autonomic regulation and attenuated hypertension induced by angiotensin II (Ang II). Similarly, in nestin/hRAMP1 mice, hypertension caused by Ang II or phenylephrine was greatly attenuated, and associated autonomic dysregulation and increased sympathetic vasomotor tone were diminished or abolished. We conclude that increased expression of neuronal CGRP receptors is sufficient to induce a protective change in cardiovascular autonomic regulation with implications for migraine therapy.

Rationale: Renal inflammation contributes to the pathophysiology of hypertension. CD161a + immune cells are dominant in the (SHR) spontaneously hypertensive rat and expand in response to nicotinic cholinergic activation. Objective: We aimed to phenotype CD161a + immune cells in prehypertensive SHR after cholinergic activation with nicotine and determine if these cells are involved in renal inflammation and the development of hypertension. Methods and Results: Studies used young SHR and WKY (Wistar–Kyoto) rats. Splenocytes and bone marrow cells were exposed to nicotine ex vivo, and nicotine was infused in vivo. Blood pressures, kidney, serum, and urine were obtained. Flow cytometry, Luminex/ELISA, immunohistochemistry, confocal microscopy, and Western blot were used. Nicotinic cholinergic activation induced proliferation of CD161a + /CD68 + macrophages in SHR-derived splenocytes, their renal infiltration, and premature hypertension in SHR. These changes were associated with increased renal expression of MCP-1 (monocyte chemoattractant protein-1) and VLA-4 (very-late antigen-4). LLT1 (lectin-like transcript 1), the ligand for CD161a, was overexpressed in SHR kidney, whereas vascular cellular and intracellular adhesion molecules were similar to those in WKY. Inflammatory cytokines were elevated in SHR kidney and urine after nicotine infusion. Nicotine-mediated renal macrophage infiltration/inflammation was enhanced in denervated kidneys, not explained by angiotensin II levels or expression of angiotensin type-1/2 receptors. Moreover, expression of the anti-inflammatory α7-nAChR (α7-nicotinic acetylcholine receptor) was similar in young SHR and WKY rats. Conclusions: A novel, inherited nicotinic cholinergic inflammatory effect exists in young SHR, measured by expansion of CD161a + /CD68 + macrophages. This leads to renal inflammation and premature hypertension, which may be partially explained by increased renal expression of LLT-1, MCP-1, and VLA-4.

The satiety effects and metabolic actions of cholecystokinin (CCK) have been recognized as potential therapeutic targets in obesity for decades. We identified a potentially novel Ca(2+)-activated chloride (Cl(–)) current (CaCC) that is induced by CCK in intestinal vagal afferents of nodose neurons. The CaCC subunit Anoctamin 2 (Ano2/TMEM16B) is the dominant contributor to this current. Its expression is reduced, as is CCK current activity in obese mice on a high-fat diet (HFD). Reduced expression of TMEM16B in the heterozygote KO of the channel in sensory neurons results in an obese phenotype with a loss of CCK sensitivity in intestinal nodose neurons, a loss of CCK-induced satiety, and metabolic changes, including decreased energy expenditure. The effect on energy expenditure is further supported by evidence in rats showing that CCK enhances sympathetic nerve activity and thermogenesis in brown adipose tissue, and these effects are abrogated by a HFD and vagotomy. Our findings reveal that Ano2/TMEM16B is a Ca(2+)-activated chloride channel in vagal afferents of nodose neurons and a major determinant of CCK-induced satiety, body weight control, and energy expenditure, making it a potential therapeutic target in obesity.

Parasympathetic activity is often reduced in hypertension and can elicit anti-inflammatory mechanisms. Thus we hypothesized that chronic vagal nerve stimulation (VNS) may alleviate cardiovascular end-organ damage in stroke-prone spontaneously hypertensive rats. Vagal nerve stimulators were implanted, a high-salt diet initiated, and the stimulators turned on (VNS, n = 10) or left off (sham, n = 14) for 4 wk. Arterial pressure increased equally in both groups. After 4 wk, endothelial function, assessed by in vivo imaging of the long posterior ciliary artery (LPCA) after stimulation (pilocarpine) and inhibition ( Nω-nitro-l-arginine methyl ester) of endothelial nitric oxide synthase (eNOS), had significantly declined (−2.3 ± 1.2 μm, P < 0.05) in sham, but was maintained (−0.7 ± 0.8 μm, nonsignificant) in VNS. Furthermore, aortic eNOS activation (phosphorylated to total eNOS protein content ratio) was greater in VNS (0.83 ± 0.07) than in sham (0.47 ± 0.08, P < 0.05). After only 3 wk, ultrasound imaging of the aorta demonstrated decreased aortic strain (−9.7 ± 2.2%, P < 0.05) and distensibility (−2.39 ± 0.49 1,000/mmHg, P < 0.05) and increased pulse-wave velocity (+2.4 ± 0.7 m/s, P < 0.05) in sham but not in VNS (−3.8 ± 3.8%, −0.70 ± 1.4 1,000/mmHg, and +0.1 ± 0.7 m/s, all nonsignificant). Interleukin (IL)-6 serum concentrations tended to be higher in VNS than in sham (34.3 ± 8.3 vs. 16.1 ± 4.6 pg/ml, P = 0.06), and positive correlations were found between NO-dependent relaxation of the LPCA and serum levels of IL-6 ( r = +0.70, P < 0.05) and IL-10 (r = +0.56, P < 0.05) and between aortic eNOS activation and IL-10 ( r = +0.48, P < 0.05). In conclusion, chronic VNS prevents hypertension-induced endothelial dysfunction and aortic stiffening in an animal model of severe hypertension. We speculate that anti-inflammatory mechanisms may contribute to these effects. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/chronic-vagal-nerve-stimulation-in-stroke-prone-shr/ .

The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo-conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox-mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate-induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.

Muscular dystrophies are a group of heterogeneous genetic disorders that cause progressive muscle weakness and wasting, dilated cardiomyopathy and early mortality. There are different types of muscular dystrophies with varying aetiologies but they all have a common hallmark of myofibre degeneration, atrophy and decreased mobility. Mutation in Sgcd (sarcoglycan-δ), a subunit of dystrophin glycoprotein complex, causes LGMD2F (limb girdle muscular dystrophy 2F). Previously, we have reported that Sgcd-deficient (Sgcd−/−) mice exhibit AngII (angiotensin II)-induced autonomic and skeletal muscle dysfunction at a young age, which contributes to onset of dilated cardiomyopathy and mortality at older ages. Two counter-regulatory RAS (renin–angiotensin system) pathways have been identified: deleterious actions of AngII acting on the AT1R (AngII type 1 receptor) compared with the protective actions of Ang-(1–7) [angiotensin-(1–7)] acting on the receptor Mas. We propose that the balance between the AngII/AT1R and Ang-(1–7)/Mas axes is disturbed in Sgcd−/− mice. Control C57BL/6J and Sgcd−/− mice were treated with Ang-(1–7) included in hydroxypropyl β-cyclodextrin (in drinking water) for 8–9 weeks beginning at 3 weeks of age. Ang-(1–7) treatment restored the AngII/AT1R compared with Ang-(1–7)/Mas balance, decreased oxidative stress and fibrosis in skeletal muscle, increased locomotor activity, and prevented autonomic dysfunction without lowering blood pressure in Sgcd−/− mice. Our results suggest that correcting the early autonomic dysregulation by administering Ang-(1–7) or enhancing its endogenous production may provide a novel therapeutic approach in muscular dystrophy.

While the importance of regulating arterial blood pressure within a 'normal' range is widely appreciated, the definition of 'normal' and the means by which humans and other species regulate blood pressure under various conditions remain hotly debated. The effects of diverse physiological, pathological and environmental challenges on blood pressure and the mechanisms that attempt to maintain it at an optimal level are reviewed and critically analyzed in a series of articles published in this themed issue of the European Journal of Applied Physiology. We summarize here the major points made in these reviews, with emphasis on unifying concepts of regulatory mechanisms and future directions for research.

Hypertension is considered an immunologic disorder. However, the role of the IL-17 family in genetic hypertension in the spontaneously hypertensive rat (SHR) has not been investigated.We tested the hypothesis that enhanced TWe measured expression of the CD161 surface marker on splenocytes in SHRs and normotensive control Wistar-Kyoto (WKY) rats from birth to adulthood. We compared expression of IL-17A and IL-17F in splenic cells under different conditions. We then determined the functional effect of these cytokines on vascular reactivity. Finally, we tested whether pharmacologic inhibition of RORγt can attenuate hypertension in SHRs.SHRs exhibited an abnormally large population of CD161SHRs have a markedly enhanced potential for RORγt-driven expression of proinflammatory and prohypertensive IL-17F in response to innate immune activation. Increased RORγt and IL-17F levels contribute to SHR hypertension and might be therapeutic targets.

The autonomic and the immune systems play major roles in the pathogenesis of cardiovascular disease and hypertension. To date, those 2 systems have been studied extensively but independently by cardiovascular biologists and by immunologists. The notion that the autonomic system can modulate the immune system and thereby influence the pathogenesis of cardiovascular disease and hypertension and their clinical outcome is novel and critical. In this brief review we focus on that interaction and an integrated understanding of the neuro-immune axis. We also highlight recent progress and future research directions. The main theme is that dysregulation of the autonomic system enhances the inflammatory response of the innate and adaptive immune systems leading to the initiation or acceleration of pathological processes and worsening of cardiovascular risks. The therapeutic potential of restoring an optimal autonomic control of the immune system is very promising. Both components of the neuro-immune axis may be involved in its disruption. One is the autonomic nervous system, which may be dysregulated or imbalanced with increased sympathetic and decreased parasympathetic activation. The other is the immune system itself, which may be abnormally sensitive to the modulatory influence of the autonomic system. These 2 components are briefly described below. #### Autonomic Dysregulation in Chronic Hypertension The complexity of cardiovascular mechanisms that contribute to hypertension has been challenging. The roles of vascular and renal abnormalities are undeniable. Changes in vasomotor tone, sodium retention, and renal renin release are all well established. For decades the contribution of the sympathetic nervous system to chronic hypertension was not fully appreciated. Its importance is now well recognized.1–5 In a 1982 review, we described the neural sites and mechanisms involved in exaggerated sympathetic nerve activity (SNA) in several animal models, as well as in human hypertension.1 Moreover, there has been a surge of data highlighting the damaging cardiovascular effects …

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium- or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K(+) channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)α activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.

Objective— Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. Approach and Results— Control, hypertensive, hypercholesterolemic ( Apoe −/− ), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. Conclusions— Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.

The methods used to assess cardiac parasympathetic (cardiovagal) activity and its effects on the heart in both humans and animal models are reviewed. Heart rate (HR)-based methods include measurements of the HR response to blockade of muscarinic cholinergic receptors (parasympathetic tone), beat-to-beat HR variability (HRV) (parasympathetic modulation), rate of post-exercise HR recovery (parasympathetic reactivation), and reflex-mediated changes in HR evoked by activation or inhibition of sensory (afferent) nerves. Sources of excitatory afferent input that increase cardiovagal activity and decrease HR include baroreceptors, chemoreceptors, trigeminal receptors, and subsets of cardiopulmonary receptors with vagal afferents. Sources of inhibitory afferent input include pulmonary stretch receptors with vagal afferents and subsets of visceral and somatic receptors with spinal afferents. The different methods used to assess cardiovagal control of the heart engage different mechanisms, and therefore provide unique and complementary insights into underlying physiology and pathophysiology. In addition, techniques for direct recording of cardiovagal nerve activity in animals; the use of decerebrate and in vitro preparations that avoid confounding effects of anesthesia; cardiovagal control of cardiac conduction, contractility, and refractoriness; and noncholinergic mechanisms are described. Advantages and limitations of the various methods are addressed, and future directions are proposed.

SummaryArterial 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.

The baroreflex control of heart rate (HR) is reduced following chronic intermittent hypoxia (CIH). Since the nucleus ambiguus (NA) plays a key role in baroreflex control of HR, we examined whether CIH remodels vagal efferent projections to cardiac ganglia. C57BL/6J mice (3-4 months of age) were exposed to either room air (RA) or CIH for 3 months. Confocal microscopy was used to examine NA axons and terminals in cardiac ganglia following Fluoro-Gold (FG) injections to label cardiac ganglia, and microinjections of tracer DiI into the left NA to anterogradely label vagal efferents. We found that: 1) Cardiac ganglia were widely distributed on the dorsal surface of the atria. Although the total number of cardiac ganglia did not differ between RA and CIH mice, the size of ganglia and the somatic area of cardiac principal neurons (PNs) were significantly decreased (P0.01), and the size of the PN nuclei was increased following CIH (P0.01). 2) NA axons entered cardiac ganglia and innervated PNs with dense basket endings in both RA and CIH mice, and the percentage of innervated PNs was similar (RA: 50 +/- 1.0%; CIH: 49 +/- 1.0%; P0.10). In CIH mice, however, swollen cardiac axons and terminals without close contacts to PNs were found. Furthermore, varicose endings around PNs appeared swollen and the axonal varicose area around PNs was almost doubled in size (CIH: 163.1 +/- 6.4 microm(2); RA: 88 +/- 3.9 microm(2), P0.01). Thus, CIH significantly altered the structure of cardiac ganglia and resulted in reorganized vagal efferent projections to cardiac ganglia. Such remodeling of cardiac ganglia and vagal efferent projections provides new insight into the effects of CIH on the brain-heart circuitry of C57BL/6J mice.

Baroreflex control of heart rate (HR) is impaired after chronic intermittent hypoxia (CIH). However, the location and nature of this response remain unclear. We examined baroreceptor afferent, vagal efferent, and central components of the baroreflex circuitry. Fischer 344 (F344) rats were exposed to room air (RA) or CIH for 35–50 days and were then anesthetized with isoflurane, ventilated, and catheterized for measurement of mean arterial blood pressure (MAP) and HR. Baroreceptor function was characterized by measuring percent changes of integrated aortic depressor nerve (ADN) activity (Int ADNA) relative to the baseline value in response to sodium nitroprusside- and phenylephrine-induced changes in MAP. Data were fitted to a sigmoid logistic function curve. HR responses to electrical stimulation of the left ADN and the right vagus nerve were assessed under ketamine-acepromazine anesthesia. Compared with RA controls, CIH significantly increased maximum baroreceptor gain or maximum slope, maximum Int ADNA, and Int ADNA range (maximum − minimum Int ADNA), whereas other parameters of the logistic function were unchanged. In addition, CIH increased the maximum amplitude of bradycardic response to vagal efferent stimulation and decreased the time from stimulus onset to peak response. In contrast, CIH significantly reduced the maximum amplitude of bradycardic response to left ADN stimulation and increased the time from stimulus onset to peak response. Therefore, CIH decreased central mediation of the baroreflex but augmented baroreceptor afferent function and vagal efferent control of HR.

Chronic intermittent hypoxia (CIH) leads to increased sympathetic nerve activity and arterial hypertension. In this study, we tested the hypothesis that CIH impairs baroreflex (BR) control of heart rate (HR) in mice, and that decreased cardiac chronotropic responsiveness to vagal efferent activity contributes to such impairment. C57BL/6J mice were exposed to either room air (RA) or CIH (6-min alternations of 21% O2and 5.7% O2, 12 h/day) for 90 days. After the treatment period, mice were anesthetized (Avertin) and arterial blood pressure (ABP) was measured from the femoral artery. Mean ABP (MABP) was significantly increased in mice exposed to CIH (98.7 ± 2.5 vs. RA: 78.9 ± 1.4 mmHg, P < 0.001). CIH increased HR significantly (584.7 ± 8.9 beats/min; RA: 518.2 ± 17.9 beats/min, P < 0.05). Sustained infusion of phenylephrine (PE) at different doses (0.1–0.4 μg/min) significantly increased MABP in both CIH and RA mice, but the ABP-mediated decreases in HR were significantly attenuated in mice exposed to CIH ( P < 0.001). In contrast, decreases in HR in response to electrical stimulation of the left vagus nerve (30 μA, 2-ms pulses) were significantly enhanced in mice exposed to CIH compared with RA mice at low frequencies. We conclude that CIH elicits a sustained impairment of baroreflex control of HR in mice. The blunted BR-mediated bradycardia occurs despite enhanced cardiac chronotropic responsiveness to vagal efferent stimulation. This suggests that an afferent and/or a central defect is responsible for the baroreflex impairment following CIH.

Baroreflex responses to changes in arterial pressure are impaired in spontaneously hypertensive rats (SHR). Mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances were measured before and during electrical stimulation (5-90 Hz) of the left aortic depressor nerve (ADN) in conscious SHR and normotensive control rats (NCR). The protocol was repeated after beta-adrenergic-receptor blockade with atenolol. SHR exhibited higher basal MAP (150 +/- 5 vs. 103 +/- 2 mmHg) and HR (393 +/- 9 vs. 360 +/- 5 beats/min). The frequency-dependent hypotensive response to ADN stimulation was preserved or enhanced in SHR. The greater absolute fall in MAP at higher frequencies (-68 +/- 5 vs. -38 +/- 3 mmHg at 90-Hz stimulation) in SHR was associated with a preferential decrease in hindquarter (-43 +/- 5%) vs. mesenteric (-27 +/- 3%) resistance. In contrast, ADN stimulation decreased hindquarter and mesenteric resistances equivalently in NCR (-33 +/- 7% and -30 +/- 7%). Reflex bradycardia was also preserved in SHR, although its mechanism differed. Atenolol attenuated the bradycardia in SHR (-88 +/- 14 vs. -129 +/- 18 beats/min at 90-Hz stimulation) but did not alter the bradycardia in NCR (-116 +/- 16 vs. -133 +/- 13 beats/min). The residual bradycardia under atenolol (parasympathetic component) was reduced in SHR. MAP and HR responses to ADN stimulation were also preserved or enhanced in SHR vs. NCR after deafferentation of carotid sinuses and contralateral right ADN. The results demonstrate distinct differences in central baroreflex control in conscious SHR vs. NCR. Inhibition of cardiac sympathetic tone maintains reflex bradycardia during ADN stimulation in SHR despite impaired parasympathetic activation, and depressor responses to ADN stimulation are equivalent or even greater in SHR due to augmented hindquarter vasodilation.

Chronic musculoskeletal pain (CMP) conditions, like fibromyalgia, are associated with widespread pain and alterations in autonomic functions. Regular physical activity prevents the development of CMP and can reduce autonomic dysfunction. We tested if there were alterations in autonomic function of sedentary mice with CMP, and whether exercise reduced the autonomic dysfunction and pain induced by CMP. Chronic musculoskeletal pain was induced by 2 intramuscular injections of pH 5.0 in combination with a single fatiguing exercise task. A running wheel was placed into cages so that the mouse had free access to it for either 5 days or 8 weeks (exercise groups) and these animals were compared to sedentary mice without running wheels. Autonomic function and nociceptive withdrawal thresholds of the paw and muscle were assessed before and after induction of CMP in exercised and sedentary mice. In sedentary mice, we show decreased baroreflex sensitivity, increased blood pressure variability, decreased heart rate variability, and decreased withdrawal thresholds of the paw and muscle 24 hours after induction of CMP. There were no sex differences after induction of the CMP in any outcome measure. We further show that both 5 days and 8 weeks of physical activity prevent the development of autonomic dysfunction and decreases in withdrawal threshold induced by CMP. Thus, this study uniquely shows the development of autonomic dysfunction in animals with chronic muscle hyperalgesia, which can be prevented with as little as 5 days of physical activity, and suggest that physical activity may prevent the development of pain and autonomic dysfunction in people with CMP.

Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors are key determinants of the immunologic outcome through their proinflammatory response. Toll-like receptor–activated signaling pathways use several adaptor proteins of which adaptor proteins myeloid differentiation protein 88 (MyD88) and toll-interleukin receptor domain–containing adaptor protein–inducing interferon-β (TRIF) define 2 major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)–induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression ( Tnfa, Nox4 , and Agtr1a ) was significantly greater in the heart and kidney of MyD88-deficient mice when compared with wild-type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. However, in mice with nonfunctional TRIF ( Trif mut mice), Ang II–induced hypertension and cardiac hypertrophy were abrogated, and proinflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a proinflammatory innate immune response, causing hypertension and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88-dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses.

Background Multifunctional calcium/calmodulin‐dependent kinase II (Ca MKII ) is activated by angiotensin II (Ang II ) in cultured vascular smooth muscle cells ( VSMCs ), but its function in experimental hypertension has not been explored. The aim of this study was to determine the impact of Ca MKII inhibition selectively in VSMCs on Ang II hypertension. Methods and Results Transgenic expression of a Ca MKII peptide inhibitor in VSMCs ( TG SM ‐Ca MKIIN model ) reduced the blood pressure response to chronic Ang II infusion. The aortic depressor nerve activity was reset in hypertensive versus normotensive wild‐type animals but not in TG SM ‐Ca MKIIN mice, suggesting that changes in baroreceptor activity account for the blood pressure difference between genotypes. Accordingly, aortic pulse wave velocity, a measure of arterial wall stiffness and a determinant of baroreceptor activity, increased in hypertensive versus normotensive wild‐type animals but did not change in TG SM ‐Ca MKIIN mice. Moreover, examination of blood pressure and heart rate under ganglionic blockade revealed that VSMC Ca MKII inhibition abolished the augmented efferent sympathetic outflow and renal and splanchnic nerve activity in Ang II hypertension. Consequently, we hypothesized that VSMC Ca MKII controls baroreceptor activity by modifying arterial wall remodeling in Ang II hypertension. Gene expression analysis in aortas from normotensive and Ang II –infused mice revealed that TG SM ‐Ca MKIIN aortas were protected from Ang II –induced upregulation of genes that control extracellular matrix production, including collagen. VSMC Ca MKII inhibition also strongly altered the expression of muscle contractile genes under Ang II . Conclusions CaMKII in VSMCs regulates blood pressure under Ang II hypertension by controlling structural gene expression, wall stiffness, and baroreceptor activity.

What is the central question of this study? Is autonomic dysregulation in a mouse model of muscular dystrophy dependent on left ventricular systolic dysfunction and/or activation of the renin-angiotensin system (RAS) and does it predict development of dilated cardiomyopathy (DCM)? What is the main finding and its importance? The results demonstrate that autonomic dysregulation precedes and predicts left ventricular dysfunction and DCM in sarcoglycan-δ-deficient (Sgcd-/-) mice. The autonomic dysregulation is prevented by treatment of young Sgcd-/- mice with the angiotensin II type 1 receptor blocker losartan. Measurements of RAS activation and autonomic dysregulation may predict risk of DCM, and therapies targeting the RAS and autonomic dysregulation at a young age may slow disease progression in patients. Sarcoglycan mutations cause muscular dystrophy. Patients with muscular dystrophy develop autonomic dysregulation and dilated cardiomyopathy (DCM), but the temporal relationship and mechanism of autonomic dysregulation are not well understood. We hypothesized that activation of the renin-angiotensin system (RAS) causes autonomic dysregulation prior to development of DCM in sarcoglycan-δ-deficient (Sgcd-/-) mice and that the severity of autonomic dysfunction at a young age predicts the severity of DCM at older ages. At 10-12 weeks of age, when left ventricular function assessed by echocardiography remained normal, Sgcd-/- mice exhibited decreases in arterial pressure, locomotor activity, baroreflex sensitivity and cardiovagal tone and increased sympathetic tone compared with age-matched C57BL/6 control mice (P0.05). Systemic and skeletal muscle RAS were activated, and angiotensin II type 1 receptor (AT1 R) expression, superoxide and fibrosis were increased in dystrophic skeletal muscle (P0.05). Treatment with the AT1 R blocker losartan for 7-9 weeks beginning at 3 weeks of age prevented or strongly attenuated the abnormalities in Sgcd-/- mice (P0.05). Repeated assessment of phenotypes between 10 and 75 weeks of age demonstrated worsening of autonomic function, progressive cardiac dysfunction and DCM and increased mortality in Sgcd-/- mice. High sympathetic tone predicted subsequent left ventricular dysfunction. We conclude that activation of the RAS causes severe autonomic dysregulation in young Sgcd-/- mice, which portends a worse long-term prognosis. Therapeutic targeting of the RAS at a young age may improve autonomic function and slow disease progression in muscular dystrophy.

We developed transgenic mice with targeted expression of human renin (hREN) and human angiotensinogen (hAGT) to either neurons (N-AII mice) or glia (G-AII mice) to test the hypothesis that neuronal and glial ANG II may have differential function. Since baseline blood pressure (BP) did not differ between the models (109 ± 3 vs. 114 ± 4 mmHg), we stressed the BP regulatory pathway by measuring the heart rate (HR) (baroreflex) response to phenylephrine- and nitroprusside-induced changes in arterial BP. The midpoint of the baroreflex curve (BP50) was reset to a significantly higher BP in N-AII mice (131 ± 5 mmHg) compared with littermate controls (115 ± 3 mmHg). Baroreflex gain (slope of BP-HR relation) was similar in N-AII and control mice (12 ± 1 vs. 14 ± 2 beats·min−1·mmHg−1). In contrast, G-AII mice exhibited less of an increase in BP50 (125 ± 5 mmHg) but a larger decrease in baroreflex gain (8 ± 1 beats·min−1·mmHg−1) compared with both control and N-AII mice. Differences in BP50 and gain between N-AII, G-AII, and control mice persisted after parasympathetic blockade with atropine but were eliminated after sympathetic blockade with propranolol, indicating the effects of ANG II were selective for cardiosympathetic arm of the reflex. ANG II-like immunoreactivity was observed more prominently around the paraventricular nucleus and nucleus tractus solitarii in G-AII mice but more prominently in the ventrolateral medulla in N-AII mice. We conclude that ANG II differentially modulates baroreflex control of HR in mice producing ANG II in neurons vs. glia, and its differential function may reflect regional differences in the production of ANG II in cardiovascular control nuclei of the brain.

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. 2. We provide here a brief overview of: (i) experimental approaches used to assess autonomic and reflex control of the circulation in mice; (ii) novel mechanisms of neurocardiovascular regulation revealed using these approaches; and (iii) findings from recent studies involving mouse models of cardiovascular disease.

A powerful interaction between the autonomic and the immune systems plays a prominent role in the initiation and maintenance of hypertension and significantly contributes to cardiovascular pathology, end-organ damage and mortality. Studies have shown consistent association between hypertension, proinflammatory cytokines and the cells of the innate and adaptive immune systems. The sympathetic nervous system, a major determinant of hypertension, innervates the bone marrow, spleen and peripheral lymphatic system and is proinflammatory, whereas the parasympathetic nerve activity dampens the inflammatory response through α7-nicotinic acetylcholine receptors. The neuro-immune synapse is bidirectional as cytokines may enhance the sympathetic activity through their central nervous system action that in turn increases the mobilization, migration and infiltration of immune cells in the end organs. Kidneys may be infiltrated by immune cells and mesangial cells that may originate in the bone marrow and release inflammatory cytokines that cause renal damage. Hypertension is also accompanied by infiltration of the adventitia and perivascular adipose tissue by inflammatory immune cells including macrophages. Increased cytokine production induces myogenic and structural changes in the resistance vessels, causing elevated blood pressure. Cardiac hypertrophy in hypertension may result from the mechanical afterload and the inflammatory response to resident or migratory immune cells. Toll-like receptors on innate immune cells function as sterile injury detectors and initiate the inflammatory pathway. Finally, abnormalities of innate immune cells and the molecular determinants of their activation that include toll-like receptor, adrenergic, cholinergic and AT1 receptors can define the severity of inflammation in hypertension. These receptors are putative therapeutic targets.

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.

Abstract —Angiotensin II (Ang II) increases renal sympathetic nerve activity in anesthetized mice before and after ganglionic blockade, suggesting that Ang II may directly activate postganglionic sympathetic neurons. The present study directly tested this hypothesis in vitro. Neurons were dissociated from aortic-renal and celiac ganglia of C57BL/6J mice. Cytosolic Ca 2+ concentration ([Ca 2+ ] i ) was measured with ratio imaging using fura 2. Ang II increased [Ca 2+ ] i in a subpopulation of sympathetic neurons. At a concentration of 200 nmol/L, 14 (67%) of 21 neurons responded with a rise in [Ca 2+ ] i . The Ang II type 1 (AT 1 ) receptor blocker (losartan, 2 μmol/L) but not the Ang II type 2 (AT 2 ) receptor blocker (PD123,319, 4 μmol/L) blocked this effect. The Ang II–induced [Ca 2+ ] i increase was abolished by removal of extracellular Ca 2+ but not altered by depletion of intracellular Ca 2+ stores with thapsigargin. Ang II no longer elicited a [Ca 2+ ] i increase in the presence of lanthanum (25 μmol/L). The specific N-type and L-type Ca 2+ channel blockers, ω-conotoxin GVIA and nifedipine, respectively, significantly inhibited the Ang II–induced [Ca 2+ ] i increase. The protein kinase C inhibitor H7 but not the protein kinase A inhibitor H89 blocked the response to Ang II. These results demonstrate that Ang II selectively activates a subpopulation of postganglionic sympathetic neurons in aortic-renal and celiac ganglia, triggering Ca 2+ influx through voltage-gated Ca 2+ channels. This effect is mediated through AT 1 receptors and requires the activation of protein kinase C. The activation of a subgroup of sympathetic neurons by Ang II may exert unique effects on kidney function in pathological states associated with elevated Ang II.

New Findings What is the topic of this review? This symposium report summarizes autonomic, cardiac and skeletal muscle abnormalities in sarcoglycan-δ-deficient mice (Sgcd-/-), a mouse model of limb girdle muscular dystrophy, with emphasis on the roles of autonomic dysregulation and activation of the renin-angiotensin system at a young age. What advances does it highlight? The contributions of the autonomic nervous system and the renin-angiotensin system to the pathogenesis of muscular dystrophy are highlighted. Results demonstrate that autonomic dysregulation precedes and predicts later development of cardiac dysfunction in Sgcd-/- mice and that treatment of young Sgcd-/- mice with the angiotensin type 1 receptor antagonist losartan or with angiotensin-(1-7) abrogates the autonomic dysregulation, attenuates skeletal muscle pathology and increases spontaneous locomotor activity. Muscular dystrophies are a heterogeneous group of genetic muscle diseases characterized by muscle weakness and atrophy. Mutations in sarcoglycans and other subunits of the dystrophin-glycoprotein complex cause muscular dystrophy and dilated cardiomyopathy in animals and humans. Aberrant autonomic signalling is recognized in a variety of neuromuscular disorders. We hypothesized that activation of the renin-angiotensin system contributes to skeletal muscle and autonomic dysfunction in mice deficient in the sarcoglycan-δ (Sgcd) gene at a young age and that this early autonomic dysfunction contributes to the later development of left ventricular (LV) dysfunction and increased mortality. We demonstrated that young Sgcd-/- mice exhibit histopathological features of skeletal muscle dystrophy, decreased locomotor activity and severe autonomic dysregulation, but normal LV function. Autonomic regulation continued to deteriorate in Sgcd-/- mice with age and was accompanied by LV dysfunction and dilated cardiomyopathy at older ages. Autonomic dysregulation at a young age predicted later development of LV dysfunction and higher mortality in Sgcd-/- mice. Treatment of Sgcd-/- mice with the angiotensin type 1 receptor blocker losartan for 8-9 weeks, beginning at 3 weeks of age, decreased fibrosis and oxidative stress in skeletal muscle, increased locomotor activity and prevented autonomic dysfunction. Chronic infusion of the counter-regulatory peptide angiotensin-(1-7) resulted in similar protection. We conclude that activation of the renin-angiotensin system, at a young age, contributes to skeletal muscle and autonomic dysfunction in muscular dystrophy. We speculate that the latter is mediated via abnormal sensory nerve and/or cytokine signalling from dystrophic skeletal muscle to the brain and contributes to age-related LV dysfunction, dilated cardiomyopathy, arrhythmias and premature death. Therefore, correcting the early autonomic dysregulation and renin-angiotensin system activation may provide a novel therapeutic approach in muscular dystrophy.

Cell-attached patch-clamp experiments were performed on dissociated neurons from nodose ganglia of adult rats. Putative aortic baroreceptor neurons were identified by labeling nerve endings in the adventitia of the aortic arch with the carbocyanine dye DiI. Whereas previous experiments demonstrated the presence of mechanosensitive (MS) whole cell currents, these experiments studied single MS ion channels and examined the influence of culture conditions on their expression. Single MS channels were activated by applying negative pressure through the recording pipette. Channel openings became more frequent as the negative pressure was increased, with open probability increasing significantly above 30 mmHg. MS channels had a slope conductance of 114 pS and a reversal potential of ∼0 mV, consistent with a nonspecific cation conductance. Channels were not affected by antagonists of voltage-gated conductances but were blocked by 20 μM gadolinium, a known blocker of MS ion channels. When nodose neurons were cocultured with aortic endothelial cells, but not aortic smooth muscle cells, the percentage of patches exhibiting MS ion channels increased significantly, suggesting that aortic endothelial cells secrete a diffusible factor that increases channel expression.

Recent advances have enabled transfer of genes to various types of cells and tissues. The goals of the present study were to transfer genes to nodose sensory neurons using replication-deficient adenovirus vectors and to define the conditions needed to optimize the gene transfer. Neurons were dissociated from rat nodose ganglia and maintained in culture. Cultures were exposed for 30 min to vectors containing the beta-galactosidase gene lacZ driven by either the Rous sarcoma virus (RSV) or the cytomegalovirus (CMV) promoter. Cultures were fixed and treated with X-gal to evaluate lacZ expression 1-7 days after exposure to virus. Increasing concentrations of virus led to dose-related increases in the number of neurons expressing lacZ. LacZ was expressed in 8 +/- 2, 39 +/- 6, and 82 +/- 3% of neurons 1 day after exposure to 10(7), 10(8), and 10(9) pfu/ml of AdRSVlacZ, respectively (P0.05). The same doses of AdCMVlacZ led to expression in 41 +/- 9, 60 +/- 10, and 86 +/- 4% of neurons. Expression driven by the CMV promoter was essentially maximal within 1 day and remained stable for at least 7 days. In contrast, expression driven by the RSV promoter was less on day 1 but increased over time (1-7 days). There was no lacZ expression in vehicle-treated cultures and exposure to the adenovirus vectors did not adversely influence cell viability. Exposure of the neuronal cultures to an adenovirus vector containing the gene for green fluorescent protein (AdRSVgfp, 10(9) pfu/ml) enabled visualization of successful gene transfer in living neurons. The results indicate that gene transfer to cultured nodose neurons can be accomplished using adenovirus vectors. The expression of the transferred gene persists for at least 7 days, occurs more rapidly when expression is driven by the CMV compared with the RSV promoter, and occurs without adversely affecting cell viability.

Abstract Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch. The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia. Replication-deficient adenovirus containing the gene for Escherichia coli β-galactosidase (β-Gal) was applied topically to the carotid sinuses of anesthetized rabbits. Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light). Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg. β-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days. Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses. Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses. In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation. The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.

1. Previous studies indicate that prostacyclin (PGI2) increases the activity of baroreceptor afferent fibres. The purpose of this study was to test the hypothesis that PGI2 inhibits Ca(2+)-activated K+ current (IK(Ca))in isolated baroreceptor neurones in culture. 2. Rat aortic baroreceptor neurones in the nodose ganglia were labelled in vivo by applying a fluorescent dye (DiI) to the aortic arch 1-2 weeks before dissociation of the neurones. Outward K+ currents in baroreceptor neurones evoked by depolarizing voltage steps from a holding potential of -40 mV were recorded using the whole-cell patch-clamp technique. 3. Exposure of baroreceptor neurones to the stable PGI2 analogue carbacyclin significantly inhibited the steady-state K+ current in a dose-dependent and reversible manner. The inhibition of K+ current was not caused indirectly by changes in cytosolic Ca2+ concentration. The Ca(2+)-activated K+ channel blocker charybdotoxin (ChTX, 10(-7) M) also inhibited the K+ current. In the presence of ChTX or in the absence of Ca2+, carbacyclin failed to inhibit the residual K+ current. Furthermore, in the presence of high concentrations of carbacyclin, ChTX did not cause further reduction of K+ current. 4. Carbacyclin-induced inhibition of IK(Ca) was mimicked by 8-bromo-cAMP and by activation of G-protein with GTP gamma S. The inhibitory effect of carbacyclin on IK(Ca) was abolished by GDP beta S, which blocks G-protein activation, and by a selective inhibitor of cAMP-dependent protein kinase, PKI5-24. 5. The results demonstrate that carbacyclin inhibits ChTX-sensitive IK(Ca) in isolated aortic baroreceptor neurones by a G-protein-coupled activation of cAMP-dependent protein kinase. This mechanism may contribute to the PGI2-induced increase in baroreceptor activity demonstrated previously.

Autonomic testing is used to define the role of the autonomic nervous system in diverse clinical and research settings. Because most of the autonomic nervous system is inaccessible to direct physiological testing, in the clinical setting the most widely used techniques entail the assessment of an end-organ response to a physiological provocation. The noninvasive measures of cardiovascular parasympathetic function involve the assessment of heart rate variability while the measures of cardiovascular sympathetic function assess the blood pressure response to physiological stimuli. Tilt-table testing, with or without pharmacological provocation, has become an important tool in the assessment of a predisposition to neurally mediated (vasovagal) syncope, the postural tachycardia syndrome, and orthostatic hypotension. Distal, postganglionic, sympathetic cholinergic (sudomotor) function may be evaluated by provoking axon reflex mediated sweating, e.g., the quantitative sudomotor axon reflex (QSART) or the quantitative direct and indirect axon reflex (QDIRT). The thermoregulatory sweat test provides a nonlocalizing measure of global pre- and postganglionic sudomotor function. Frequency domain analyses of heart rate and blood pressure variability, microneurography, and baroreflex assessment are currently research tools but may find a place in the clinical assessment of autonomic function in the future.

We appreciate the opportunity to clarify the queries regarding the analysis of our data raised in the Letter to the Editor in this issue from Buckler (2013). The reciprocal sensitivity to hypoxia (cyanide – CN) vs. low pH (acidic solution) that we have reported is represented in the regression analyses (Y=aX+b) of responses of individual glomus cells to CN (Y) vs. acid (X). Changes in the slopes (a) were inversely related to increased or decreased sensitivity to acid. These regression analyses are portrayed in Fig. 6 and in the Supplemental Table 1. The data in Fig. 6 are adjusted to go through zero, and the data in the Supplemental Table 1 include both the adjusted as well as the unadjusted regressions and their Y-intercepts. Both sets of analyses support our interpretation. The data on rat cells do define three distinct populations of cells. Of a total of 109 cells, one group responded only to hypoxia (19% of cells), another responded only to acid (13%), and a third responded to both (68%; Figs 3B–D). Figure 3E portrays the range of responses to acid vs. hypoxia by decadal increments in Δ[Ca2+]i. The description and analyses of those results as reported reveal two points: One is that the responses to hypoxia were significantly larger (Δ40 ± 2 nm, n= 95) than those to acid (Δ28 ± 3 nm, n= 88) with a P value of 0.015. The second point is evident in Fig. 3E. In the group of low responding cells (Δ10 to 20 nm), there were 4 times as many cells responding to acid as to hypoxia (n= 50 vs. 13); and conversely, in the high responding cells (Δ30–160 nm) there were 4 times as many cells responding to hypoxia as to acidosis (n= 57 vs. 14). The choice of Δ30 nm[Ca2+]i was not arbitrary. It seemed to uncouple most of the more intensively cells responsive to hypoxia from most of the responsive cells to acid. A χ2 test analysis of the distribution of responsive cells to hypoxia or to acid, based on the intensity of the Ca2+ signal being higher or lower than Δ30 nm, gave a very significant P value of 5.21 × 10−9. We had not reported this statistical analysis in the legend of Fig. 3 and should have done so. With respect to the data in mice, in Fig. 4 we first show individual cells in the same cluster responding with greater intensity to either CN or pH 6.0. In Fig. 5, we show progressive increases in responses to pH 6.0, which are small in ASIC3-KO (n= 205), higher in WT (n= 207), and still higher in ASIC3-Tg (n= 32). The responses to CN were reciprocally reduced significantly in the ASIC-Tg. In Fig. 6, responses of each cell to CN vs. acid were plotted and regression analyses were carried out in each of the three genotypes. By genetically increasing or decreasing the sensitivity to acid, we saw inverse (reciprocal) responses to CN. These changes are reflected in the plotted regression lines and the changes in the slopes of these regressions. Our main finding is that in both populations of responsive cells, the CN > acid (gradient >1 – red cells) and the CN acid groups (gradient >1), the slopes of the regressions were at a high of 2.8 in ASIC3−/− and decreased to 2.1 in WT and to 1.2 in ASIC3-Tg, notwithstanding the admittedly underpowered Tg group. The corresponding ‘unadjusted’ regressions also showed the inverse relation. These results, published in the Supplemental Table 1, were 2.07, 1.56, and 1.1, respectively. In the CN < acid groups (gradient

Abstract Structural changes in large arteries are often considered the predominant mechanism responsible for decreased baroreflex sensitivity and baroreceptor resetting in hypertension, atherosclerosis, and aging. Recent work has demonstrated that “functional” mechanisms, both at the level of the peripheral sensory endings and within the central nervous system, contribute significantly to altered baroreflex responses. We have conducted both reductive studies of mechanoelectrical transduction in cultured baroreceptor neurons and integrative studies with in vivo recordings of the activity of baroreceptor afferent fibers and efferent sympathetic nerves. Results suggest that the primary mechanism of mechanical activation of baroreceptor neurons involves opening of stretch-activated ion channels susceptible to blockade by gadolinium. Baroreceptor nerve activity is modulated by the activity of potassium channels and the sodium-potassium pump and by paracrine factors, including prostacyclin, oxygen free radicals, and factors released from aggregating platelets. Endothelial dysfunction and altered release of these paracrine factors contribute significantly to the decreased baroreceptor sensitivity in hypertension and atherosclerosis. The central mediation of the baroreflex depends on the pulse phasic pattern of afferent baroreceptor discharge. Baroreflex-mediated inhibition of sympathetic nerve activity is well maintained during pulse phasic afferent activity. Continuous, nonphasic baroreceptor discharge or a rapid (>1.5 Hz) pulse phasic discharge results in disinhibition of sympathetic activity. This disinhibition during continuous baroreceptor input is exaggerated with aging. Thus, a defect in central mediation of the baroreflex may be a major cause of the impaired baroreflex and sympathoexcitation in the elderly. In summary, functional neural mechanisms, in addition to structural vascular changes, contribute importantly to altered baroreflex responses in normal and pathophysiological states. Therefore, it may be possible to implement therapies that reverse functional changes and restore baroreflex sensitivity long before the reversal of structural vascular changes.

Abstract The goal of this study was to determine whether nitric oxide (NO) and the NO donor, S -nitrosocysteine (cysNO), modulate the activity of carotid sinus baroreceptors. Baroreceptor activity was recorded from the vascularly isolated carotid sinus in anesthetized rabbits. Baroreceptor activity decreased in a dose-dependent manner after injection of either NO or cysNO as constant pressure was maintained, and activity recovered spontaneously over time, within seconds to minutes. The baroreceptor pressure-activity relation was shifted significantly to the right by cysNO, with a profound suppression of activity at high pressure. Baroreceptor activity at 160 mm Hg averaged 76±8%, 60±6%, and 36±5% of the control maximum during exposure to 10 −4 , 2 to 3×10 −4 , and 10 −3 mol/L cysNO, respectively. The inhibition of activity by the l and d isomers of cysNO was equivalent and was blocked by reduced hemoglobin, suggesting that the effect was mediated by NO. The suppression of baroreceptor activity by cysNO was not related to vascular relaxation as measured by videomicrometer. Inhibition of soluble guanylate cyclase with methylene blue or 6-anilinoquinoline-5,8-quinone (LY83583, 10 −5 mol/L) did not attenuate and dibutyryl cGMP (10 −3 mol/L) did not mimic the suppression of baroreceptor activity by cysNO, suggesting a cGMP-independent mechanism. Activation of endogenous NO formation with thimerosal (10 −5 to 10 −4 mol/L) reduced maximum baroreceptor activity in five of eight experiments to 59±7% of the control maximum. The NO synthase inhibitor nitro- l -arginine methyl ester (L-NAME, 10 −4 mol/L) by itself failed to influence baroreceptor activity but prevented thimerosal-induced suppression of activity. Addition of l -arginine (10 −3 mol/L) after L-NAME restored the inhibitory influence of thimerosal. The results indicate that NO and cysNO suppress baroreceptor activity through a mechanism independent of guanylate cyclase activation and vascular relaxation and that endogenous NO released by chemical activation suppresses baroreceptor activity.

We have demonstrated recently that platelets aggregating in the carotid sinus decrease baroreceptor sensitivity. The goals of the present study were to determine whether platelet-induced suppression of baroreceptor activity is mediated by a diffusible, transferable factor and, if true, whether the factor is short-lived or stable. Baroreceptor activity was recorded from the isolated carotid sinus during slow ramp increases in nonpulsatile pressure in rabbits anesthetized with sodium pentobarbital. Intraluminal exposure of the carotid sinus to washed rabbit platelets resuspended in Krebs buffer (3-5 x 10(8) cells/ml) and activated by thrombin decreased baroreceptor activity significantly (n = 7, P0.05). Maximum baroreceptor activity recorded at a pressure of 140 mmHg was reduced to 81 +/- 7% of the control maximum. Injection of cell-free supernatant obtained from filtered thrombin-activated platelets also suppressed baroreceptor activity to a similar extent after 10 min (n = 7) and after 2 h (n = 5) of incubation when maximum baroreceptor activity was reduced to 84 +/- 5 and 82 +/- 5% of the control maximum, respectively. The inhibitory influence of activated platelets and platelet supernatant on baroreceptor activity was still apparent after 10-60 min of heating (95 degrees C) (n = 5) and was reversible upon removal of platelets and supernatant from the sinus. The results indicate that activated platelets release a stable diffusible factor that suppresses baroreceptor activity. We speculate that this 'inhibitory factor' may contribute to impairment of the baroreceptor reflex and neurally-mediated increases in arterial pressure in atherosclerotic and thrombotic states.

The determinants of the nerve activity generated at the baroreceptor endings have been examined. 1) In the isolated carotid sinus, the placement of activated bovine aortic endothelial cells decreased baroreceptor activity (BRA) in a reversible manner. Both endothelin and nitric oxide (NO) suppress BRA, whereas prostacyclin (PGI2) increases activity. 2) The BRA in single units declines and often ceases during non-pulsatile increases in carotid sinus pressure sustained over several minutes. This "adaptation" is attenuated by the transient potassium channel (IA) blocker 4-aminopyridine (4-AP) and not by inhibition of the Na+/K+ pump. 3) In preliminary studies, mechano-electrical transduction was examined in isolated and cultured nodose ganglion neurons. Opening of stretch-activated (SA) channels by suction on the cell-attached patch was seen infrequently; however, probing the neurons consistently increased their intracellular calcium [Ca++]i measured with fura-2. This increase in [Ca++]i is blocked by gadolinium (Gd3+), a trivalent lanthanide reported to block SA channels. Gd3+ also blocks the BRA in the carotid sinus. We conclude that paracrine factors significantly modulate BR sensitivity, that selective ionic mechanisms (the 4-AP sensitive K+ channels) determine the degree of "adaptation" of BR to elevated pressure, and that SA channels sensitive to Gd3+ may be the mechano-electrical transducers in BR neurons.

Rationale: Inflammation and autonomic dysfunction contribute to the pathophysiology of hypertension. Cholinergic stimulation suppresses innate immune responses. Angiotensin II (Ang II) induces hypertension and is associated with proinflammatory immune responses. Objective: Our goal was to define the innate immune response in a model of genetic hypertension and the influences of cholinergic stimulation and Ang II. Methods and Results: Studies were conducted on 4- to 5-week-old prehypertensive spontaneously hypertensive rats (SHRs) and age-matched normotensive control, Wistar Kyoto (WKY) rats. Isolated splenocytes were preexposed to nicotine or Ang II before Toll-like receptor (TLR) activation. Culture supernatants were tested for cytokines (tumor necrosis factor-α, interleukin [IL]-10, and IL-6). TLR-mediated cytokine responses were most pronounced with TLR7/8 and TLR9 activation and similar between WKY rats and SHRs. Nicotine and Ang II enhanced this TLR-mediated IL-6 response in prehypertensive SHR splenocytes. In contrast, nicotine suppressed the TLR-mediated IL-6 response in WKY rats, whereas Ang II had no effect. In vivo, nicotine enhanced plasma levels of TLR7/8-mediated IL-6 and IL-1β responses in prehypertensive SHRs but suppressed these responses in WKY rats. Flow cytometry revealed an increase in a CD161+ innate immune cell population, which was enhanced by nicotine in the prehypertensive SHR spleen but not in WKY. Conclusions: There is a pronounced anti-inflammatory nicotinic/cholinergic modulation of the innate immune system in WKY rats, which is reversed in prehypertensive SHRs. The results support the novel concept that neurohormonal regulation of the innate immune system plays a role in the pathogenesis of genetic hypertension and provide putative molecular targets for treatment of hypertension.

Endotracheal suctioning of intubated infants produces profound changes in cardiovascular and cerebral hemodynamics, but the mechanisms regulating these changes are not fully understood. To determine the role of the autonomic nervous system in regulating these physiologic changes, we investigated the effects of endotracheal suctioning on heart rate (HR), mean arterial blood pressure (MABP), and renal sympathetic nerve activity (RSNA) in nine ventilated newborn lambs. In the first part of the study (n = 6), ventilation was interrupted for suctioning. With suctioning (15 s), HR decreased by 39 +/- 6% (p0.05), whereas MABP and RSNA increased significantly (p0.05) by 36 +/- 5% and 68 +/- 8%, respectively. These changes were significantly (p0.05) larger than changes observed during disconnection from the ventilator (15 s) without suctioning. Administration of atropine (0.02 mg/kg) blocked the HR response to suctioning without altering MABP or RSNA changes. After bilateral vagotomy, suctioning produced no changes in any parameter. When a closed tracheal suction system was used and ventilation was maintained, suctioning again resulted in significant (p0.05) increases in MABP (+10 +/- 3%) and RSNA (+34 +/- 5%) and a decrease in HR (-15 +/- 4%). These data suggest that suctioning stimulates sympathoexcitatory receptors localized in large airways whose afferent fibers course within the vagus, resulting in increased sympathetic activity, which induces peripheral vasoconstriction and elevates MABP. In contrast, the HR response appears to be mediated by increased parasympathetic activity as this is abolished by atropine.

Calcitonin gene-related peptide (CGRP) is a powerful vasodilator that interacts with the autonomic nervous system. A subunit of the CGRP receptor complex, receptor activity-modifying protein 1 (RAMP1), is required for trafficking of the receptor to the cell surface and high-affinity binding to CGRP. We hypothesized that upregulation of RAMP1 would favorably enhance autonomic regulation and attenuate hypertension. Blood pressure, heart rate, and locomotor activity were measured by radiotelemetry in transgenic mice with ubiquitous expression of human RAMP1 (hRAMP1) and littermate controls. Compared with control mice, hRAMP1 mice exhibited similar mean arterial pressure, a lower mean heart rate, increased heart rate variability, reduced blood pressure variability, and increased baroreflex sensitivity (2.83±0.20 versus 1.49±0.10 ms/mm Hg in controls;PPP8-37was significantly greater (P

Aggregating platelets release factors that act in a local paracrine manner to alter vascular tone. The purpose of the present study was to explore the possibility that factors released from aggregating platelets may alter the sensitivity of arterial baroreceptors. Baroreceptor activity was recorded from the vascularly isolated carotid sinus of rabbits anesthetized with sodium pentobarbital. The carotid sinus was filled with oxygenated Krebs-Henseleit buffer and distended with slow ramp increases in nonpulsatile pressure. Sensitivity of baroreceptors to increased pressure was determined before and during intraluminal exposure of the sinus to washed human platelets suspended in Krebs' buffer. Platelets activated with thrombin (0.4 units/ml) decreased baroreceptor activity and the slope of the pressure-activity curve significantly (n = 6). The platelet-induced decrease in baroreceptor sensitivity was related to the duration of exposure to platelets with no change in baroreceptor activity after 4 minutes and a progressive decrease in activity over the next 12 minutes. The slope of the pressure-nerve activity relation averaged 1.26 +/- 0.08 %/mm Hg during control and decreased to 0.97 +/- 0.22, 0.80 +/- 0.19, and 0.53 +/- 0.15 %/mm Hg after 12-16 minutes of exposure to 10(7), 10(8), and 3-6 x 10(8) activated platelets/ml, respectively (p less than 0.05). Baroreceptor sensitivity was restored after removal of platelets from the carotid sinus. Thrombin alone had no effect on baroreceptor sensitivity. Activated platelets did not alter the carotid pressure-diameter relation, suggesting a direct inhibitory effect on baroreceptors. The slope of the pressure-activity curve and maximum baroreceptor activity were not decreased by the stable thromboxane analogue U46619, serotonin, or ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Rationale : Increased sympathetic nerve activity has been linked to the pathogenesis of hypertension in humans and animal models. Enhanced peripheral chemoreceptor sensitivity which increases sympathetic nerve activity has been observed in established hypertension but has not been identified as a possible mechanism for initiating an increase in sympathetic nerve activity before the onset of hypertension. Objective : We tested this hypothesis by measuring the pH sensitivity of isolated carotid body glomus cells from young spontaneously hypertensive rats (SHR) before the onset of hypertension and their control normotensive Wistar–Kyoto (WKY) rats. Methods and Results : We found a significant increase in the depolarizing effect of low pH in SHR versus WKY glomus cells which was caused by overexpression of 2 acid-sensing non–voltage-gated channels. One is the amiloride-sensitive acid-sensing sodium channel (ASIC3), which is activated by low pH and the other is the 2-pore domain acid-sensing K + channel (TASK1), which is inhibited by low pH and blocked by quinidine. Moreover, we found that the increase in sympathetic nerve activity in response to stimulation of chemoreceptors with sodium cyanide was markedly enhanced in the still normotensive young SHR compared to control WKY rats. Conclusions : Our results establish a novel molecular basis for increased chemotransduction that contributes to excessive sympathetic activity before the onset of hypertension.

Intrauterine environmental pertubations have been linked to the development of adult hypertension. We sought to evaluate the interrelated roles of sex, nitric oxide, and reactive oxygen species (ROS) in programmed cardiovascular disease. Programming was induced in mice by maternal dietary intervention (DI; partial substitution of protein with carbohydrates and fat) or carbenoxolone administration (CX, to increase fetal glucocorticoid exposure). Adult blood pressure and locomotor activity were recorded by radiotelemetry at baseline, after a week of high salt, and after a week of high salt plus nitric oxide synthase inhibition (by l-NAME). In male offspring, DI or CX programmed an elevation in blood pressure that was exacerbated by Nω-nitro-l-arginine methyl ester administration, but not high salt alone. Mesenteric resistance vessels from DI male offspring displayed impaired vasorelaxation to ACh and nitroprusside, which was blocked by catalase and superoxide dismutase. CX-exposed females were normotensive, while DI females had nitric oxide synthase-dependent hypotension and enhanced mesenteric dilation. Despite the disparate cardiovascular phenotypes, both male and female DI offspring displayed increases in locomotor activity and aortic superoxide production. Despite dissimilar blood pressures, DI and CX-exposed females had reductions in cardiac baroreflex sensitivity. In conclusion, both maternal malnutrition and fetal glucocorticoid exposure program increases in arterial pressure in male but not female offspring. While maternal DI increased both superoxide-mediated vasoconstriction and nitric oxide mediated vasodilation, the balance of these factors favored the development of hypertension in males and hypotension in females.

The purpose of this study was to test the hypothesis that prostaglandins released from vascular endothelial cells contribute to activation of baroreceptors during increases in arterial pressure. Baroreceptor activity was recorded from the vascularly isolated carotid sinus in rabbits anesthetized with chloralose. Baroreceptor activity was measured during ramp or step increases in nonpulsatile carotid sinus pressure over a range of 0-175 mm Hg. Exposure of the isolated carotid sinus to inhibitors of prostaglandin formation (indomethacin [n = 10] or aspirin [n = 6]) decreased baroreceptor activity significantly (p less than 0.05). The slope of the pressure-activity relation averaged 0.80 +/- 0.07 %/mm Hg (mean +/- SEM) during control measurements and 0.72 +/- 0.06 and 0.63 +/- 0.05 %/mm Hg during exposure to 10 and 20 microM indomethacin, respectively. Exposure of the carotid sinus to exogenous prostacyclin (PGI2 [n = 11]) increased baroreceptor activity significantly. The slope of the pressure-activity relation averaged 0.89 +/- 0.10, 1.09 +/- 0.09, and 1.26 +/- 0.16 %/mm Hg during control and during exposure to 10 and 20 microM PGI2, respectively. Activity returned to control after removal of PGI2 (0.89 +/- 0.12 %/mm Hg). Removal of endothelium with either a balloon catheter (n = 4) or a jet of a 95% O2-5% CO2 gas mixture (n = 6) decreased the slope of the pressure-activity relation from 0.92 +/- 0.09 to 0.56 +/- 0.08 %/mm Hg (p less than 0.05). Exposure of the denuded sinus to exogenous PGI2 (20 microM [n = 4]) restored activity (slope = 1.09 +/- 0.24 %/mm Hg). Neither indomethacin (n = 5) nor PGI2 (n = 5) nor denudation (n = 5) significantly altered the pressure-diameter relation of the carotid sinus (sonomicrometers), suggesting that the effects on baroreceptor discharge are not caused by altered stretch of the carotid sinus at a given pressure. The results suggest that prostaglandins (e.g., PGI2) released from endothelium contribute in a paracrine manner to activation of baroreceptors during increases in arterial pressure.