Nitric oxide and carotid body chemoreception

RODRIGO ITURRIAGA

Laboratorio de Neurobiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago Chile

Correspondence to: Dr. Rodrigo Iturriaga. Laboratorio de Neurobiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Casilla 114-D, Santiago 1, Chile. Fax: (56-2) 222-5515. E-mail: riturria@genes.bio.puc.cl

Received: March 5, 2001. Accepted: July 10, 2001

ABSTRACT

Nitric oxide (NO) has been proposed as an inhibitory modulator of carotid body chemosensory responses to hypoxia. It is believed that NO modulates carotid chemoreception by several mechanisms, which include the control of carotid body vascular tone and oxygen delivery and reduction of the excitability of chemoreceptor cells and petrosal sensory neurons. In addition to the well-known inhibitory effect, we found that NO has a dual (dose-dependent) effect on carotid chemoreception depending on the oxygen pressure level. During hypoxia, NO is primarily an inhibitory modulator of carotid chemoreception, while in normoxia NO increased the chemosensory activity. This excitatory effect produced by NO is likely mediated by an impairment of mitochondrial electron transport and oxidative phosphorylation, which increases the chemosensory activity. The recent findings that mitochondria contain an isoform of NO synthase, which produces significant amounts of NO for regulating their own respiration, suggest that NO may be important for the regulation of mitochondrial energy metabolism and oxygen sensing in the CB.

Key terms: Carotid body; chemoreceptor; cytochrome oxidase; hypoxia; nitric oxide; oxidative phosphorylation

INTRODUCTION

The carotid body (CB) senses the partial pressure of respiratory gases and pH in the arterial blood and contributes to the respiratory and cardiovascular reflex regulation. It is well known that hypoxia, hypercapnia and acidosis increase the frequency of carotid chemosensory discharges in the carotid nerve, while hyperoxia, hypocapnia and alkalosis markedly decrease it (González et al., 1994). The CB is mainly composed of two kinds of cells, glomus (type I) cells and sustentacular (type II) cells. The glomus cells are innervated by afferent chemosensory petrosal neurons. In response to natural stimuli, glomus cells are expected to release one (or more) excitatory Ca²⁺-dependent transmitter(s), which in turn increases the frequency of discharge in the nerve terminals of chemosensory petrosal neurons.

Two hypotheses have been suggested to explain how glomus cells sense the oxygen pressure. The first hypothesis states that the mitochondrial metabolism of the glomus cells participates in the oxygen sensing. It has been proposed that a heme or redox sensitive protein such as the cytochrome oxidase a₃ is the primary oxygen sensor. The second hypothesis states that hypoxia inhibits a K⁺ channel, which is responsible for the depolarization of the glomus cells. The depolarization induced by hypoxia raises the intracellular Ca²⁺ and releases the excitatory transmitter(s). In addition to the excitatory transmitters, other molecules produced within the CB can regulate the chemosensory process. Among several putative modulators, the gaseous molecule nitric oxide (NO) has been proposed to be an inhibitory modulator of carotid chemosensory responses induced by hypoxia (Prabhakar et al., 1993; Chugh et al., 1994; Wang et al., 1994, 1995Trzbeski et al., 1995; ; Prabhakar, 1999).

Inhibitory effects of NO on carotid chemoreception to hypoxia

Physiological and immunocytochemical evidence supports the proposal that NO produces inhibition of carotid chemosensory discharges induced by hypoxia (Prabhakar et al., 1993; Grimes et al., 1994; Wang et al., 1994; Prabhakar, 1999;). Indeed, the administration of the NO precursor L-arginine or NO donors (Chugh et al., 1994; Wang et al., 1994) and more recently of NO gas (Iturriaga et al., 2000a) to the cat CB perfused in vitro partially reduces the chemosensory response to hypoxia. On the contrary, the inhibition of nitric oxide synthase (NOS) activity increases basal chemosensory discharges in the cat and rat CB in situ and in vitro (Chugh et al., 1994; Wang et al., 1994; Trzebsky et al., 1995) and moderately enhances the responses to cytotoxic-hypoxia in the cat CB in situ (Iturriaga et al., 1998). In vitro, we have found that superfusion with Tyrode supplemented with the NO donor sodium nitroprusside (SNP) did not raise basal chemosensory activity, but reversibly reduced the responses elicited by NaCN and nicotine (Alcayaga et al., 1997). Therefore, our results indicate that in addition to the inhibitory effect of NO on chemosensory responses induced by PO₂, NO also modulates the chemosensory response to other excitatory stimuli acting as a broad inhibitory modulator.

Nitric oxide synthase immunoreactivity has been found in the CB and in the petrosal ganglion. In the cat CB, NOS immunoreactivity is present in endothelial cells (Grimes et al., 1994), in autonomic neurons innervating the CB blood vessels and in sensory C fibers encircling the glomus cells (Wang et al., 1994), but is presumably absent in glomus cells, type II cells, or smooth muscle cells (Wang et al., 1994). Consequently to the distribution of NOS in the CB tissue, Wang et al., 1994; 1995) suggested that two cGMP-dependent mechanisms are involved in the inhibitory actions of NO on hypoxic chemoreception. The first mechanism involves the release of NO from parasympathetic neurons located in the CB that control the vascular tone. An effect of NO on CB blood flow and O₂ delivery is supported by the observation of Wang et al (1995) that the NO synthase inhibitor, N-w-nitro-L-arginine methyl ester (L-NAME) evoked a larger increase of basal discharges in the perfused cat CB preparation than in the superfused one, where vascular effects are absent. Buerk and Lahiri (2000) found that SNP increases cat CB tissue PO₂ and reduces basal chemosensory discharges while L-NAME reduces tissue PO₂ and increases chemosensory activity. These observations suggest a significant contribution of the vascular effect of NO on CB chemoreception.

Wang et al. (1995) suggested another mechanism to explain the inhibitory effect of NO on hypoxic chemoreception. They proposed that NO released from petrosal sensory C fibers would increase cGMP levels in glomus cells inhibiting hypoxic chemoreception. However, it is not clear how an increase of cGMP or NO levels may reduce the oxygen-dependent excitability of the glomus cells, because it is known that cGMP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) did not change the dependency of the K⁺- currents to PO₂ in rat (Haton and Peers, 1996) and rabbit glomus cells (Summers et al., 1999). On the contrary, the latter group found that SNP and spermine inhibit L-type Ca²⁺ currents in rabbit glomus cells through a cGMP-independent mechanism, probably mediated by an alteration of the thiol groups of the calcium channel proteins.

Another site for the action of NO is the petrosal ganglion. The petrosal ganglion contains the somas of the sensory neurons that innervate glomus cells in the CB. Alcayaga et al (1999) found that SNP and L-NAME modulate the acetylcholine-induced activity in petrosal ganglion neurons, which project through the carotid nerve. SNP reduced the sensitivity and amplitude of the dose-dependent increase of the frequency of discharge induced by acetylcholine in the carotid nerve, while L-NAME slightly enhanced the response (Alcayaga et al (1999). These results indicate that NO may play a role as modulator of petrosal chemosensory neurons. These neurons, which likely innervate the CB, are selectively activated by the putative excitatory transmitter acetylcholine (Alcayaga et al., 1998).

Excitatory effects of NO on carotid chemoreception in normoxia

When we studied the effects of SNP on the carotid chemosensory responses to excitatory and inhibitory stimuli in paralyzed and artificially ventilated cats (Iturriaga et al., 1998), we found that SNP increased basal chemosensory activity. This was an unexpected result because we (Alcayaga et al., 1997) and others (Chugh et al., 1994; Prabhakar et al., 1993; Wang et al., 1994) had found that NO donors reduced or had no effect on basal chemosensory discharge in the cat CB superfused or perfused in vitro. The most relevant difference between in situ and in vitro preparations of the CB is the presence of large amounts of endothelium and vascular smooth muscle tissue in situ. Since endothelium and smooth muscle cells are required to activate SNP for releasing NO (Kowaluk et al., 1992), it is plausible that large amounts of NO released from SNP in situ may account for the increased chemosensory activity. We tested this hypothesis studying the effects of NO released by NO donors on cat CB chemosensory activity during normoxia and hypoxia. The CBs were excised from pentobarbitone-anaesthetised cats and perfused in vitro with Tyrode solution. We recorded the changes of neural discharges and NO concentration, measured by an amperometric method with NO-selective carbon fiber microelectrodes inserted in the CB. As we expected, the injection of SNAP and 6-(2-hydroxy-1-methyl-nitrosohydrazino)-N-methyl-1-hexanamine (NOC-9), transiently reduced the hypoxic-augmented chemosensory discharge in a dose-dependent manner. However, during normoxic perfusion the injection of NO donors increased the chemosensory activity in a dose-dependent manner, showing a dual effect of NO on carotid chemoreception depending on PO₂ levels (Iturriaga et al., 2000b). We proposed that a high concentration of NO or its metabolite peroxynitrite released from NO donors might account for the chemosensory excitation. The most probable molecular targets for the action of NO in the CB are the soluble enzymes guanylyl cyclase and the cytochrome oxidase a₃ because these enzymes are highly sensitive to NO (Brown 1999; Carreras et al., 2000). However, the activation of guanylyl cyclase in the CB vascular smooth muscle cells should cause vasodilatation and a concomitant reduction in chemosensory activity. In contrast, the impairment of cytochrome oxidase a₃ activity and oxidative phosphorylation produced by NO or peroxynitrite (Cassina and Radi, 1996; Brown, 1999; Carreras et al., 2000) is expected to increase the chemosensory activity. Indeed, it is well known that the injections of mitochondrial electron chain blockers (antimycin A, NaCN), uncouplers (FCCP) and oxidative phosphorylation inhibitors (oligomycin) produce chemosensory excitation (Mulligan et al, 1981; Mulligan and Lahiri, 1982; González et al., 1994). NO at low and medium concentration specifically and reversibly inhibits cytochrome a₃ in competition with O₂, decrease mitochondrial transmembrane potential, inhibits oxygen consumption and ATP synthesis (Brown 1999; Carreras et al., 2000). On the contrary, large doses of NO irreversibly block other respiratory complexes (Brown, 1999) producing protein and lipid oxidation and mitochondrial damage (Carreras et al., 2000). Incidentally, we found that the administration of several large doses of SNAP and NOC-9 increased basal chemosensory activity up to the maximal discharge in half of the CB studied (Iturriaga et al., 2000b). Similarly to our in situ findings (Iturriaga et al., 1998) the increased chemosensory activity returned to the baseline when the CBs were perfused with 100% O₂ suggesting that the CB oxygen sensing mechanism was impaired by large amounts of NO.

Physiological role for NO in the CB oxygen sensing

NO may modulate hypoxic chemoreception in the CB by several mechanisms. As mentioned already, NO may regulate the CB vascular tone and oxygen delivery to the chemoreceptor cells (Wang et al., 1995; Buerk and Lahiri, 2000), the excitability of glomus cells (Wang et al, 1995; Summers et al., 1999) and petrosal sensory neurons (Alcayaga et al., 1999). Nevertheless, our studies indicate that NO has a dual effect on carotid chemoreception depending on the oxygen and NO levels. During hypoxia, NO is predominantly an inhibitory modulator of carotid chemoreception, while in normoxia NO increases the chemosensory discharges. The excitatory effect produced by NO is likely mediated by an impairment of mitochondrial electron transport and oxidative phosphorylation, which is expected to increase chemosensory activity. The effect of NO supports the main role played by the mitochondrial metabolism and cytochrome oxidase function in CB chemoreception to oxygen. Furthermore, an unexplored possibility is that the mitochondrial NO/O₂ ratio may be crucial for regulating the mitochondria function, playing a physiological role in oxygen sensing. The recent finding showing that the mitochondria contain a NOS isoform that produces enough NO to regulate its own respiration (Ghafourifar and Richter, 1997; Giulivi 1998) suggests that NO may be important for the regulation of energy metabolism. It is thus plausible that the NO inhibition of cytochrome oxidase a₃ could be involved in the physiological regulation of the carotid oxygen sensing. Further studies are necessary, however, to understand the role played by NO in CB chemoreception.

ACKNOWLEDGEMENTS

This work was supported by FONDECYT grant 198-0965. I would like to thank Mrs. Carolina Larraín for her assistance in the preparation of the manuscript. I am indebted to Dr. Jaime Alvarez for his encouragement, critical review and assistance in writing my first scientific paper alone (ITURRIAGA R (1985) Microtubule density and size of axons in early diabetes:

Implication for nerve cell homeostasis. Exp Neurol 88: 165-175). Thanks, Jaime, for being an outstanding Maestro and a great human being.

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