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SUMMARY Arterial baroreceptors provide a neural sensory input that reflexly regulates the autonomic drive of the 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 suggests 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.

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 SNA prior to 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) prior to 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 two 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 two-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 prior to the onset of hypertension.

Abstract-Carotid body chemoreceptors sense hypoxemia, hypercapnia, and acidosis and play an important role in cardiorespiratory regulation. The molecular mechanism of pH sensing by chemoreceptors is not clear, although it has been proposed to be mediated by a drop in intracellular pH of carotid body glomus cells, which inhibits a K ϩ current. Recently, pH-sensitive ion channels have been described in glomus cells that respond directly to extracellular acidosis. In this study, we investigated the possible molecular mechanisms of carotid body pH sensing by recording the responses of glomus cells isolated from rat carotid body to rapid changes in extracellular pH using the whole-cell patch-clamping technique. Extracellular acidosis evoked transient inward current in glomus cells that was inhibited by the acid-sensing ion channel (ASIC) blocker amiloride, absent in Na ϩ -free bathing solution, and enhanced by either Ca 2ϩ -free buffer or addition of lactate. In addition, ASIC1 and ASIC3 were shown to be expressed in rat carotid body by quantitative PCR and immunohistochemistry. In the current-clamp mode, extracellular acidosis evoked both a transient and sustained depolarizations. The initial transient component of depolarization was blocked by amiloride, whereas the sustained component was eliminated by removal of K ϩ from the pipette solution and partially blocked by the TASK (tandem-p-domain, acid-sensitive K ϩ channel) blockers anandamide and quinidine. The results provide the first evidence that ASICs may contribute to chemotransduction of low pH by carotid body chemoreceptors and that extracellular acidosis directly activates carotid body chemoreceptors through both ASIC and TASK channels. (Circ Res. 2007;101:1009-1019.)Key Words: carotid body Ⅲ ASIC Ⅲ glomus cell Ⅲ chemoreceptors Ⅲ pH sensitivity T he carotid bodies function as major peripheral chemoreceptors sensing changes in arterial blood oxygen, carbon dioxide, and pH in mammals. [1][2][3] Glomus type I cells are generally accepted as the chemosensitive elements activated by hypoxemia, hypercapnia, and acidosis. 3 The common cellular mechanism for glomus cell transduction is the release of neurotransmitters such as ATP, acetylcholine, and dopamine as a result of an increase in intracellular Ca 2ϩ caused by stimulus-induced membrane depolarization. 4 -7 This leads to synaptic activation of adjacent carotid nerve endings that elicits both hyperventilation and sympathetic activation. 1,8 The molecular identity of the P O2 , P CO2 , and pH sensors is still an important question. The depolarization induced by hypoxemia has been ascribed to a closure of K ϩ channels including large-conductance, Ca 2ϩ -activated potassium (BK) channels. 3,9 -12 The transduction of acidosis to membrane depolarization was proposed to be mediated by a drop in intracellular pH which would inhibit 1 or more types of K ϩ channels, leading to membrane depolarization. 3,13,14 The findings of extracellular pH-sensitive ion conductances, including pH-sensitive Cl Ϫ currents 15 and...

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. Key Words: calcium imaging Ⅲ calcium influx Ⅲ calcium channel blockers Ⅲ protein kinase activation T he renin-angiotensin system plays an important role in the regulation of arterial blood pressure and body fluid and electrolyte homeostasis. Angiotensin II (Ang II) directly contracts vascular smooth muscle cells and enhances the renal tubular reabsorption of sodium. 1,2 In addition, Ang II stimulates the sympathetic nervous system, thereby enhancing vasoconstriction and sodium reabsorption. 3,4 Mounting evidence supports a role of the sympathetic nervous system in the long-term regulation of arterial pressure. [5][6][7][8] Ang II has been shown to modulate the sympathetic nervous system function through actions within the central nervous system (eg, at the area postrema and rostral ventrolateral medulla) and through facilitation of sympathetic ganglionic neurotransmission and neurotransmitter release from efferent sympathetic terminals. 9 We recently described biphasic effects of Ang II on renal sympathetic nerve activity (RSNA) in intact mice. 10 Intravenous administration of Ang II increased arterial pressure and evoked a biphasic change in RSNA: inhibition of highamplitude phasic bursts of RSNA secondary to the initial rise of arterial pressure followed by the activation of lowamplitude continuously discharging RSNA. The initial inhibition of RSNA was prevented by baroreceptor denervation, indicating that it was indirectly mediated by the baroreflex response to the rise in arterial pressure. Consistent with this interpretation, the ganglionic blocker hexamethonium eliminated the baseline high-amplitude phasic bursts of RSNA. However, neither denervation nor hexamethonium prevented the Ang II-induced activation of low-amplitude continuously discharging RSNA, suggesting a direct action of Ang II on postganglionic sympathetic neurons. The goal of the present study was to test this hypothesis in vitro by directly measuring Ang II-induced changes in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in cultured sympathetic neurons isolated from mouse aortic-renal (ARG) and celiac (CG) ganglia. Materials and Methods AnimalsAll experiments were performed on cells obtained from C57BL/6J mice (Harlan; Madison, Wis). The project had been approved by the University of Iowa Animal Care and Use Committee. Cell CulturePrimary cultures of sympathetic neurons were prepared from the ARG and CG of mice. Adult C57BL/6J mice (25 to 30 g) were Original

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Key points• Carotid body glomus cells are activated by hypoxia and acidosis, but their capacity to differentiate between the two has been undefined.• This is the first work to quantify a differential sensory transduction of hypoxia and acidosis with reciprocal responses in individual glomus cells.• Cytoplasmic [Ca 2+ ] in clusters of glomus cells indicates 68% of glomus cells respond to both hypoxia and acidosis but are selectively more sensitive to one or the other; the rest respond to either hypoxia (19%) or acidosis (13%).• This uncoupling/reciprocal response was recapitulated in a mouse model by genetically altering the expression of ASIC3, an acid-sensing ion channel that we had identified in earlier studies as a mediator of pH sensitivity in carotid body.• We speculate that selective sensory transduction of glomus cells to either hypoxia or acidosis may result in activation of afferents preferentially more sensitive to hypoxia or acidosis, perhaps evoking more specific autonomic adjustments to each stimulus.Abstract Carotid body glomus cells are the primary sites of chemotransduction of hypoxaemia and acidosis in peripheral arterial chemoreceptors. They exhibit pronounced morphological heterogeneity. A quantitative assessment of their functional capacity to differentiate between these two major chemical signals has remained undefined. We tested the hypothesis that there is a differential sensory transduction of hypoxia and acidosis at the level of glomus cells. We measured cytoplasmic Ca 2+ concentration in individual glomus cells, isolated in clusters from rat carotid bodies, in response to hypoxia (P O 2 = 15 mmHg) and to acidosis at pH 6.8. More than two-thirds (68%) were sensitive to both hypoxia and acidosis, 19% were exclusively sensitive to hypoxia and 13% exclusively sensitive to acidosis. Those sensitive to both revealed significant preferential sensitivity to either hypoxia or to acidosis. This uncoupling and reciprocity was recapitulated in a mouse model by altering the expression of the acid-sensing ion channel 3 (ASIC3) which we had identified earlier in glomus cells. Increased expression of ASIC3 in transgenic mice increased pH sensitivity while reducing cyanide sensitivity. Conversely, deletion of ASIC3 in the knockout mouse reduced pH sensitivity while the relative sensitivity to cyanide or to hypoxia was increased. In this work, we quantify functional differences among glomus cells and show reciprocal sensitivity to acidosis and hypoxia in most glomus cells. We speculate that this selective chemotransduction of glomus cells by either stimulus may result in the activation of different afferents that are preferentially more sensitive to either hypoxia or acidosis, and thus may evoke different and more specific autonomic adjustments to either stimulus.

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