Rodrigo Iturriaga

Chronic intermittent hypoxia enhances cat chemosensory and ventilatory responses to hypoxia

The carotid body (CB) chemoreceptors may play an important role in the enhanced hypoxic ventilatory response induced by chronic intermittent hypoxia (CIH). We studied the effects of cyclic hypoxic episodes of short duration on cat cardiorespiratory reflexes, heart rate variability, and CB chemosensory activity. Cats were exposed to cyclic hypoxic episodes (P  O 2 ∼ 75 Torr) repeated during 8 h for 2–4 days. Cats were anaesthetized with sodium pentobarbitone (40 mg kg−1i.p., followed by 8–12 mg i.v.), and ventilatory and cardiovascular responses to NaCN (0.1–100 μg kg−1i.v.) and several isocapnic levels of oxygen (P  O 2 ∼ 20–740 Torr) were studied. After studying the reflex responses, we recorded the CB chemosensory responses induced by the same stimuli. Results showed that CIH for 4 days selectively enhanced cat CB ventilatory (VT and VI) responses to hypoxia, while responses to NaCN remained largely unchanged. Similarly, basal CB discharges and responses to acute hypoxia (P  O 2 < 100 Torr) were larger in CIH than in control cats, without modification of the responses to NaCN. Exposure to CIH did not increase basal arterial pressure, heart rate, or their changes induced by acute hypoxia or hyperoxia. However, the spectral analysis of heart rate variability of CIH cats showed a marked increase of the low‐/high‐frequency ratio and an increase of the power spectral distribution of low frequencies of heart rate variability. Thus, the enhanced CB reactivity to hypoxia may contribute to the augmented ventilatory response to hypoxia, as well as to modified heart rate variability due to early changes in autonomic activity.

Carotid body and cardiorespiratory alterations in intermittent hypoxia: the oxidative link.

Intermittent hypoxia, a feature of obstructive sleep apnoea, potentiates ventilatory hypoxic responses, alters heart rate variability and produces hypertension, partially owing to an enhanced carotid body responsiveness to hypoxia. Since oxidative stress is a potential mediator of both chemosensory and cardiorespiratory alterations, we hypothesised that an antioxidant treatment may prevent these alterations. Accordingly, we studied the effects of ascorbic acid (1.25 g·L−1 drinking water) on plasma lipid peroxidation, nitrotyrosine and inducible nitric oxide synthase (iNOS) immunoreactivity in the carotid body, ventilatory and carotid chemosensory responses to acute hypoxia, heart rate variability and arterial blood pressure in male Sprague–Dawley rats exposed to 5% O2; 12 episodes·h−1; 8 h·day−1 or sham condition for 21 days. Intermittent hypoxia increased plasma lipid peroxidation, nitrotyrosine and iNOS expression in the carotid body, enhanced carotid chemosensory and ventilatory hypoxic responses, modified heart rate variability and produced hypertension. Ascorbic acid prevented the increased plasma lipid peroxidation and nitrotyrosine formation within the carotid body, and the enhanced carotid chemosensory and ventilatory responses to hypoxia, as well as heart rate variability alterations and hypertension. The present results support an essential role for oxidative stress in the generation of carotid body chemosensory potentiation and systemic cardiorespiratory alterations induced by intermittent hypoxia.

Contribution of endothelin-1 to the enhanced carotid body chemosensory responses induced by chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) enhances carotid body (CB) chemosensory responses to acute hypoxia. We tested the hypothesis that endothelin-1 (ET-1), an excitatory modulator of CB chemoreception may contribute to the enhanced CB chemosensory responses in cats exposed to cyclic hypoxic episodes repeated during 8 h for 4 days. Accordingly, we measured the ET-1 immunoreactivity (ET-ir) in the CB and plasma. Using a perfused CB preparation, we studied the effects of exogenous ET-1 and bosentan, a non-selective endothelin receptor type A and B antagonist, on the frequency of chemosensory discharges (f(x)) during normoxia, mild and severe hypoxia. We found that CIH increased ET-ir in the CB by approximately 10-fold leaving ET-1 plasma levels unchanged. Application of ET-1 to control and CIH-treated CBs produced long-lasting dose-dependent increases in f(x), although the dose-response curve showed a rightward-shift in the CIH-treated CBs. CIH increased baseline f(x) and hypoxic chemosensory responses, which were reduced by 50 microM bosentan in CBs from CIH-treated cats. Present results suggest that a local increase of ET-1 in the CB may contribute to the enhanced chemosensory responses induced by CIH predominantly through a vasomotor mechanism.

Carotid body inflammation and cardiorespiratory alterations in intermittent hypoxia

Chronic intermittent hypoxia (CIH), a main feature of obstructive sleep apnoea (OSA), increases hypoxic ventilatory responses and elicits hypertension, partially attributed to an enhance carotid body (CB) responsiveness to hypoxia. As inflammation has been involved in CIH-induced hypertension and chemosensory potentiation, we tested whether ibuprofen may block CB chemosensory and cardiorespiratory alterations induced by CIH in a rat model of OSA. We studied the effects of ibuprofen (40 mg·kg−1·day−1) on immunohistochemical interleukin (IL)-1&bgr; and tumour necrosis factor (TNF)-&agr; levels in the CB, the number of c-fos-positive neurons in the nucleus tractus solitarii (NTS), CB chemosensory and ventilatory responses to hypoxia, and arterial blood pressure in male rats either exposed for 21 days to 5% O2 (12 episodes·h−1, 8 h·day−1) or kept under sham condition. CIH increased CB TNF-&agr; and IL-1&bgr; and c-fos-positive neurons in the NTS, enhanced carotid chemosensory and ventilatory hypoxic responses, and produced hypertension. Ibuprofen prevented CB cytokine overexpression and CIH-induced increases in c-fos-positive neurons in the NTS, the enhanced hypoxic ventilatory responses and hypertension, but failed to impede the CB chemosensory potentiation. Results suggest that pro-inflammatory cytokines may contribute to the CIH-induced cardiorespiratory alterations, acting at several levels of the hypoxic chemoreflex and cardiovascular control pathways.

Carotid body potentiation induced by intermittent hypoxia: implications for cardiorespiratory changes induced by sleep apnoea.

1 The most usual form of chronic hypoxia in humans is the intermittent hypoxia resulting from obstructive sleep apnoea (OSA). The OSA syndrome is a highly prevalent sleep breathing disorder that is considered an independent risk factor for hypertension and cardiovascular diseases. Endothelial dysfunction, oxidative stress, inflammation and sympathetic activation have been proposed as potential mechanisms involved in the onset of the hypertension. However, evidence for a unique pathogenic mechanism has been difficult to establish in OSA patients because of concomitant comorbidities. Thus, animal models have been developed to study the pathological consequences of exposure to chronic intermittent hypoxia (CIH). 2 Because OSA patients and animals exposed to CIH show augmented ventilatory, sympathetic and cardiovascular responses to acute hypoxia, it has been proposed that enhanced carotid body responsiveness to hypoxia is involved in the autonomic changes induced by OSA and in the development of the hypertension. Recently, this proposal has received further support from recordings of carotid body chemosensory neural discharges in situ and in vitro showing that exposure of animals to CIH increases basal carotid body chemosensory discharges and enhances the chemosensory response to hypoxia. 3 In the present brief review, we discuss the evidence supporting an important role for the carotid body in the progression of cardiorespiratory changes induced by OSA and the contribution of oxidative stress, endothelin‐1 and pro‐inflammatory molecules in the potentiation of the carotid body chemosensory function induced by CIH.

ACh and ATP mediate excitatory transmission in cat carotid identified chemoreceptor units in vitro.

Several molecules have been proposed as excitatory transmitters between glomus (type 1) cells and nerve terminals of petrosal ganglion (PG) neurons in the carotid body (CB). We tested whether ACh and ATP have a role to play as excitatory transmitters in the cat CB by recording intracellularly from identified PG neurons functionally connected to the CB in vitro. PG neurons projecting to the CB were classified according to their intracellular responses as: (a) neurons with humped action potentials (hAP neurons) responding phasically to long-lasting depolarizing pulses (53/67), and (b) neurons with smooth action potentials (non-hAP neurons) that fire tonically during long-lasting depolarizations (14/67). CB stimulation by stop flow and/or acidosis induced activity in 28 of 39 hAP-type neurons, being classified as chemosensory, but in none of the non-hAP neurons. Hexamethonium (10 microM) and suramin (100 microM) reversibly abolished the increased discharges evoked in chemosensory neurons (8/9) by stop flow or acidosis. Moreover, 24 of 27 chemosensory neurons responded to ganglionar application of ACh and ATP, while two neurons responded only to ACh and one to ATP. Mechanical deformation of the carotid sinus induced firing activity in 10 of 13 non-hAP neurons, but in none of the hAP neurons tested. Interestingly, 4/10 non-hAP neurons, which responded to carotid sinus mechanical stimulation also responded to ganglionar application of ATP, but were insensitive to ACh. Present results favor the hypothesis that ACh and ATP are excitatory transmitters in the cat CB, acting-at least-on the PG neuron terminals in the CB.

Selective activation of carotid nerve fibers by acetylcholine applied to the cat petrosal ganglion in vitro

The petrosal ganglion innervates carotid body chemoreceptors through the carotid (sinus) nerve. These primary sensory neurons are activated by transmitters released from receptor (glomus) cells, acetylcholine (ACh) having been proposed as one of the transmitters involved in this process. Since the perikarya of primary sensory neurons share several properties with peripheral sensory endings, we studied the electrical responses of the carotid nerve and glossopharyngeal branch to ACh locally applied to the cat petrosal ganglion superfused in vitro. Ganglionar applications of AChCl (1 microg-1 mg) generated bursts of action potentials conducted along the carotid nerve, while only a few spikes were exceptionally recorded from the glossopharyngeal branch in response to the largest doses. Carotid nerve responses to ACh were dose-dependent, the higher doses inducing transient desensitization. Application of nicotine to the petrosal ganglion also evoked dose-dependent excitatory responses in the carotid nerve. Responses to ACh were reversibly antagonized by adding hexamethonium to the superfusate, more intense and prolonged block of ACh responses being produced by mecamylamine. Ganglionar applications of gamma-amino butyric acid and serotonin, in doses of up to 5 mg, did not induce firing of action potentials in any of the branches of the glossopharyngeal nerve. Our results indicate that petrosal ganglion neurons projecting through the carotid nerve are selectively activated by ACh acting on nicotinic ACh receptors located in the somata of these neurons. Thus, cholinosensitivity would be shared by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.

Effects of dopaminergic blockade upon carotid chemosensory activity and its hypoxia-induced excitation

The effects of domperidone, antagonist of D2 receptors, on arterial chemoreceptor activity were studied in spontaneously breathing and pentobarbitone anesthetized cats, in which recordings of chemosensory impulse activity were obtained simultaneously from both cut carotid (sinus) nerves. Intravenous injections of domperidone 50 micrograms/kg produced a maintained increase in the basal frequency of chemosensory discharges, after which hyperoxic tests (breathing 100% O2 for 30 s) evoked larger falls in the rate of chemosensory impulses. Chemosensory responses evoked by hypoxic hypoxia (100% N2 tests) and by cytotoxic hypoxia (i.v. injections of NaCN) reached higher impulse rates after domperidone treatment. The effects of domperidone reveal that a resting release of dopamine from glomus cells maintains a low level of basal chemosensory activity under normoxic conditions. Domperidone turns off such restraining dopaminergic control and enhances the transient chemosensory responses to hypoxic stimuli. Present data support a modulatory role for dopamine within the chemoreceptor process, but not its participation as excitatory transmitter between glomus cells and sensory nerve endings.

CO reveals dual mechanisms of O2 chemoreception in the cat carotid body.

The hypothesis that CO-binding pigments in the carotid body participate in O2 chemoreception was tested. The chemosensory nerve discharges of cat carotid body perfused and superfused in vitro at 36-37 degrees C with cell-free solution containing CO2-HCO3- (pH approximately equal to 7.39) were recorded to monitor O2 chemoreception. Several levels of PCO (60-550 Torr) at two levels of PO2 (50 Torr-140 Torr) were used. With high PCO of 500-550 Torr at any PO2 the discharge rate peaked promptly but the effect was significantly less than that to hypoxia. At any stage of the CO effect, exposure to light promptly attenuated or eliminated the response, as if the stimulatory effect of hypoxia was absent. Lower PCO of 60-70 Torr attenuated the response to hypoxia which was not suppressed by light. PCO of 140 Torr also attenuated the response to hypoxia and made the activity partially photolabile. During high PCO exposure the excitatory response to cyanide but not to nicotine was attenuated, consistent with the idea that the effects of nicotine are downstream from those of CO. Both inhibitory and excitatory effects of CO were promptly reversible. The results indicate that two types of CO-binding chromophores participate in O2 chemoreception in the carotid body.

Differential expression of pro-inflammatory cytokines, endothelin-1 and nitric oxide synthases in the rat carotid body exposed to intermittent hypoxia

The enhanced carotid body (CB) chemosensory response to hypoxia induced by chronic intermittent hypoxia (CIH) has been attributed to oxidative stress, which is expected to increase the expression of chemosensory modulators including chemoexcitatory pro-inflammatory cytokines in the CB. Accordingly, we studied the time-course of the changes in the immunohistological expression of TNF-α, IL-1β, IL-6, ET-1, iNOS, eNOS and 3-nitrotyrosine in the CB, along with the progression of enhanced CB chemosensory responses to acute hypoxia in male Sprague-Dawley rats exposed to CIH (5%O₂, 12 times/h per 8h) for 7, 14 and 21 days. Exposure to CIH for 7 days resulted in a sustained potentiation of CB chemosensory responses to acute hypoxia, which persisted until 21 days of CIH. The chemosensory potentiation was paralleled by an increased 3-nitrotyrosine expression in the CB. On the contrary, CIH produced a transient 2-fold increase of ET-1 immunoreactivity at 7 days, a decrease in eNOS immunoreactivity, and a delayed but progressive increase of TNF-α, IL-1β and iNOS immunoreactivity, which was not associated with changes in systemic plasma levels or immune cell invasion within the CB. Thus, present results suggest that the local expression of chemosensory modulators and pro-inflammatory cytokines in the CB may have different temporal contribution to the CB chemosensory potentiation induced by CIH.