Lamotrigine and phenytoin, but not amiodarone, impair peripheral chemoreceptor responses to hypoxia.

Amiodarone, lamotrigine, and phenytoin, common antiarrhythmic and antiepileptic drugs, inhibit a persistent sodium current in neurons (I(NaP)). Previous results from our laboratory suggested that I(NaP) is critical for functionality of peripheral chemoreceptors. In this study, we determined the effects of therapeutic levels of amiodarone, lamotrigine, and phenytoin on peripheral chemoreceptor and ventilatory responses to hypoxia. Action potentials (APs) of single chemoreceptor afferents were recorded using suction electrodes advanced into the petrosal ganglion of an in vitro rat peripheral chemoreceptor complex. AP frequency (at Po(2) approximately 150 Torr and Po(2) approximately 90 Torr), conduction time, duration, and amplitude were measured before and during perfusion with therapeutic dosages of the drug or vehicle. Hypoxia-induced catecholamine secretion within the carotid body was measured using amperometry. With the use of whole body plethysmography, respiration was measured in unanesthesized rats while breathing room air, 12% O(2), and 5% CO(2), before and after intraperitoneal administration of amiodarone, lamotrigine, phenytoin, or vehicle. Lamotrigine (10 microM) and phenytoin (5 microM), but not amiodarone (5 microM), decreased chemoreceptor AP frequency without affecting other AP parameters or magnitude of catecholamine secretion. Similarly, lamotrigine (5 mg/kg) and phenytoin (10 mg/kg) blunted the hypoxic but not the hypercapnic ventilatory response. In contrast, amiodarone (2.5 mg/kg) did not alter the ventilatory response to hypoxia or hypercapnia. We conclude that lamotrigine and phenytoin at therapeutic levels impair peripheral chemoreceptor function and ventilatory response to acute hypoxia. These are consistent with I(NaP) serving an important function in AP generation and may be clinically important in the care of patients using these drugs.     

Spontaneous action potential generation due to persistent sodium channel currents in simulated carotid body afferent fibers.

The mechanism by which action potentials (APs) are generated in afferent nerve fibers in the carotid body is unknown, but it is generally speculated to be release of an excitatory transmitter and synaptic depolarizing events. However, previous results suggested that Na(+) channels in the afferent nerve fibers play an important role in this process. To better understand the potential mechanism by which Na(+) channels may generate APs, a mathematical model of chemoreceptor nerve fibers that incorporated Hodgkin-Huxley-type Na(+) channels with kinetics of activation and inactivation, as determined previously from recordings of petrosal chemoreceptor neurons, was constructed. While the density of Na(+) channels was kept constant, spontaneous APs arose in nerve terminals as the axonal diameter was reduced to that in rat carotid body. AP excitability and pattern were similar to those observed in chemoreceptor recordings: 1) a random pattern at low- and high-frequency discharge rates, 2) a high sensitivity to reductions in extracellular Na(+) concentration, and 3) a variation in excitability that increased with AP generation rate. Taken together, the results suggest that an endogenous process in chemoreceptor nerve terminals may underlie AP generation, a process independent of synaptic depolarizing events.     

Time-dependent modulation of carotid body afferent activity during and after intermittent hypoxia

The ventilatory response to several minutes of hypoxia consists of various time-dependent phenomena, some of which occur during hypoxia (e.g., short-term depression), whereas others appear on return to normoxia (e.g., posthypoxic frequency decline). Additional phenomena can be elicited by acute, intermittent hypoxia (e.g., progressive augmentation, long-term facilitation). Current data suggest that these phenomena originate centrally. We tested the hypothesis that carotid body afferent activity undergoes time-dependent modulation, consistent with a direct role in these ventilatory phenomena. Using an in vitro rat carotid body preparation, we found that 1) afferent activity declined during the first 5 min of severe (40 Torr Po(2)), moderate (60 Torr Po(2)), or mild (80 Torr Po(2)) hypoxia; 2) after return to normoxia (100 Torr Po(2)) and after several minutes of moderate or severe hypoxia, afferent activity was transiently reduced compared with prehypoxic levels; and 3) with successive 5-min bouts of mild, moderate, or severe hypoxia, afferent activity during bouts increased progressively. We call these phenomena sensory hypoxic decline, sensory posthypoxic decline, and sensory progressive augmentation, respectively. These phenomena were stimulus specific: similar phenomena were not seen with 5-min bouts of normoxic hypercapnia (100 Torr Po(2) and 50-60 Torr Pco(2)) or hypoxic hypocapnia (60 Torr Po(2) and 30 Torr Pco(2)). However, bouts of either normoxic hypercapnia or hypocapnic hypoxia resulted in sensory long-term facilitation. We suggest time-dependent carotid body activity acts in parallel with central mechanisms to shape the dynamics of ventilatory responses to respiratory chemostimuli.     

Carotid chemoreceptor activity during acute and sustained hypoxia in goats

The role of carotid body chemoreceptors in ventilatory acclimatization to hypoxia, i.e., the progressive, time-dependent increase in ventilation during the first several hours or days of hypoxic exposure, is not well understood. The purpose of this investigation was to characterize the effects of acute and prolonged (up to 4 h) hypoxia on carotid body chemoreceptor discharge frequency in anesthetized goats. The goat was chosen for study because of its well-documented and rapid acclimatization to hypoxia. The response of the goat carotid body to acute progressive isocapnic hypoxia was similar to other species, i.e., a hyperbolic increase in discharge as arterial PO2 (PaO2) decreased. The response of 35 single chemoreceptor fibers to an isocapnic [arterial PCO2 (PaCO2) 38-40 Torr)] decrease in PaO2 of from 100 +/- 1.7 to 40.7 +/- 0.5 (SE) Torr was an increase in mean discharge frequency from 1.7 +/- 0.2 to 5.8 +/- 0.4 impulses. During sustained isocapnic steady-state hypoxia (PaO2 39.8 +/- 0.5 Torr, PaCO2, 38.4 +/- 0.4 Torr) chemoreceptor afferent discharge frequency remained constant for the first hour of hypoxic exposure. Thereafter, single-fiber chemoreceptor afferents exhibited a progressive, time-related increase in discharge (1.3 +/- 0.2 impulses.s-1.h-1, P less than 0.01) during sustained hypoxia of up to 4-h duration. These data suggest that increased carotid chemoreceptor activity contributes to ventilatory acclimatization to hypoxia.     

Hypoxia transduction by carotid body chemoreceptors in mice lacking dopamine D(2) receptors.

Hypoxia-induced dopamine (DA) release from carotid body (CB) glomus cells and activation of postsynaptic D(2) receptors have been proposed to play an important role in the neurotransmission process between the glomus cells and afferent nerve endings. To better resolve the role of D(2) receptors, we examined afferent nerve activity, catecholamine content and release, and ventilation of genetically engineered mice lacking D(2) receptors (D(2)(-/-) mice). Single-unit afferent nerve activities of D(2)(-/-) mice in vitro were significantly reduced by 45% and 25% compared with wild-type (WT) mice during superfusion with saline equilibrated with mild hypoxia (Po(2) approximately 50 Torr) or severe hypoxia (Po(2) approximately 20 Torr), respectively. Catecholamine release in D(2)(-/-) mice was enhanced by 125% in mild hypoxia and 75% in severe hypoxia compared with WT mice, and the rate of rise was increased in D(2)(-/-) mice. We conclude that CB transduction of hypoxia is still present in D(2)(-/-) mice, but the response magnitude is reduced. However, the ventilatory response to acute hypoxia is maintained, perhaps because of an enhanced processing of chemoreceptor input by brain stem respiratory nuclei.     

Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats

During ventilatory acclimatization to hypoxia (VAH), time-dependent increases in ventilation lower Pco(2) levels, and this persists on return to normoxia. We hypothesized that plasticity in the caudal nucleus tractus solitarii (NTS) contributes to VAH, as the NTS receives the first synapse from the carotid body chemoreceptor afferents and also contains CO(2)-sensitive neurons. We lesioned cells in the caudal NTS containing the neurokinin-1 receptor by microinjecting the neurotoxin saporin conjugated to substance P and measured ventilatory responses in awake, unrestrained rats 18 days later. Lesions did not affect hypoxic or hypercapnic ventilatory responses in normoxic control rats, in contrast to published reports for similar lesions in other central chemosensitive areas. Also, lesions did not affect the hypercapnic ventilatory response in chronically hypoxic rats (inspired Po(2) = 90 Torr for 7 days). These results suggest functional differences between central chemoreceptor sites. However, lesions significantly increased ventilation in normoxia or acute hypoxia in chronically hypoxic rats. Hence, chronic hypoxia increases an inhibitory effect of neurokinin-1 receptor neurons in the NTS on ventilatory drive, indicating that these neurons contribute to plasticity during chronic hypoxia, although such plasticity does not explain VAH.     

Ventilatory and carotid body chemoreceptor responses to purinergic P2X receptor antagonists in newborn rats

Adenosine triphosphate, acting through purinergic P2X receptors, has been shown to stimulate ventilation and increase carotid body chemoreceptor activity in adult rats. However, its role during postnatal development of the ventilatory response to hypoxia is yet unknown. Using whole body plethysmography, we measured ventilation in normoxia and in moderate hypoxia (12% fraction of inspired O?, 20 min) before and after intraperitoneal injection of suramin (P2X? and P2X? receptor antagonist, 40 mg/kg) in 4-, 7-, 12-, and 21-day-old rats. Suramin reduced baseline breathing (～20%) and the response to hypoxia (～30%) in all rats, with a relatively constant effect across ages. We then tested the effect of the specific P2X? antagonist, A-317491 (150 mg/kg), in rats aged 4, 7, and 21 days. As with suramin, A-317491 reduced baseline ventilation (～55%) and the hypoxic response (～40%) at all ages studied. Single-unit carotid body chemoreceptor activity was recorded in vitro in 4-, 7-, and 21-day-old rats. Suramin (100 μM) and A-317491 (10 μM) significantly depressed the sinus nerve chemosensory discharge rate (～80%) in normoxia (Po? ～150 Torr) and hypoxia (Po? ～60 Torr), and this decrease was constant across ages. We conclude that, in newborn rats, P2X purinergic receptors are involved in the regulation of breathing under basal and hypoxic condition, and P2X?-containing receptors play a major role in carotid body function. However, these effects are not age dependent within the age range studied.     

Exercise training improves peripheral chemoreflex function in heart failure rabbits.

An enhancement of peripheral chemoreflex sensitivity contributes to sympathetic hyperactivity in chronic heart failure (CHF) rabbits. The enhanced chemoreflex function in CHF involves augmented carotid body (CB) chemoreceptor activity via upregulation of the angiotensin II (ANG II) type 1 (AT(1))-receptor pathway and downregulation of the neuronal nitric oxide synthase (nNOS)-nitric oxide (NO) pathway in the CB. Here we investigated whether exercise training (EXT) normalizes the enhanced peripheral chemoreflex function in CHF rabbits and possible mechanisms mediating this effect. EXT partially, but not fully, normalized the exaggerated baseline renal sympathetic nerve activity (RSNA) and the response of RSNA to hypoxia in CHF rabbits. EXT also decreased the baseline CB nerve single-fiber discharge (4.9 +/- 0.4 vs. 7.7 +/- 0.4 imp/s at Po(2) = 103 +/- 2.3 Torr) and the response to hypoxia (20.6 +/- 1.1 vs. 36.3 +/- 1.3 imp/s at Po(2) = 41 +/- 2.2 Torr) from CB chemoreceptors in CHF rabbits, which could be reversed by treatment of the CB with ANG II or a nNOS inhibitor. Our results also showed that NO concentration and protein expression of nNOS were increased in the CBs from EXT + CHF rabbits, compared with that in CHF rabbits. On the other hand, elevated ANG II concentration and AT(1)-receptor overexpression of the CBs in CHF state were blunted by EXT. These results indicate that EXT normalizes the CB chemoreflex in CHF by preventing an increase in afferent CB chemoreceptor activity. EXT reverses the alterations in the nNOS-NO and ANG II-AT(1)-receptor pathways in the CB responsible for chemoreceptor sensitization in CHF.     

Effect of chronic hypoxia on cholinergic chemotransmission in rat carotid body.

Current views suggest that oxygen sensing in the carotid body occurs in chemosensory type I cells, which excite synaptically apposed chemoafferent nerve terminals in the carotid sinus nerve (CSN). Prolonged exposure in a low-oxygen environment [i.e., chronic hypoxia (CH)] elicits an elevated stimulus-evoked discharge in chemoreceptor CSN fibers (i.e., increased chemosensitivity). In the present study, we evaluated cholinergic chemotransmission in the rat carotid body in an effort to test the hypothesis that CH enhances ACh-mediated synaptic activity between type I cells and chemoafferent nerve terminals. Animals were exposed in a hypobaric chamber (barometric pressure = 380 Torr) for 9-22 days before evaluation of chemoreceptor activity using an in vitro carotid body/CSN preparation. Nerve activity evoked by ACh was significantly larger (P < 0.01) after CH, suggesting increased expression of cholinergic receptors. Approximately 80% of the CSN impulse activity elicited by ACh (100- or 1,000-microg bolus) in both normal and CH preparations was blocked by the specific nicotinic receptor antagonist mecamylamine (100 microM). CSN activity elicited by acute hypoxia or hypercapnia in normal preparations was likewise blocked (> or =80%) in the presence of 100 muM mecamylamine, but after CH the enhanced CSN activity elicited by acute hypoxia or hypercapnia was not reduced in the presence of 100 or 500 microM mecamylamine. A muscarinic receptor antagonist, atropine (10 microM), and a specific nicotinic receptor alpha7 subunit antagonist, methyllycaconatine (50 nM), blocked approximately 50% of the hypoxia-evoked activity in normal preparations but were ineffective after CH. Prolonged exposure to hypoxia appears to dramatically alter chemotransmission in the carotid body, and may induce alternative neurotransmitter mechanisms and/or electrical coupling between type I cells and chemoafferent nerve terminals.     

Adult carotid chemoafferent responses to hypoxia after 1, 2, and 4 wk of postnatal hyperoxia.

Exposing newborn rats to postnatal hyperoxia (60% O2) for 1-4 wk attenuates the ventilatory and phrenic nerve responses to acute hypoxia in adult rats. The goal of this research was to increase our understanding of the carotid chemoreceptor afferent neural input in this depressed response with different durations of postnatal hyperoxic exposure. Rats were exposed from a few days before birth to 1, 2, or 4 wk of 60% O2 and studied after 3-5 mo in normoxia. The rats were anesthetized with urethane. Whole carotid sinus nerve (CSN) responses to NaCN (40 microg/kg iv), 10 s of asphyxia and acute isocapnic hypoxia (arterial Po2 45 Torr) were determined. Mean CSN responses to stimuli after postnatal hyperoxia were reduced compared with controls. Responses in rats exposed to 1 wk of postnatal hyperoxia were less affected than those exposed to 2 and 4 wk of hyperoxia, which were equivalent to each other. These studies illustrate the importance of normoxia during the first 2 wk of life in development of carotid chemoreceptor afferent function.     

Chronic intermittent hypoxia alters NMDA and AMPA-evoked currents in NTS neurons receiving carotid body chemoreceptor inputs

Chronic exposure to intermittent hypoxia (CIH) has been used in animals to mimic the arterial hypoxemia that accompanies sleep apnea. Humans with sleep apnea and animals exposed to CIH have elevated blood pressures and augmented sympathetic nervous system responses to acute exposures to hypoxia. To test the hypothesis that exposure to CIH alters neurons within the nucleus of the solitary tract (NTS) that integrate arterial chemoreceptor afferent inputs, we measured whole cell currents induced by activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in enzymatically dispersed NTS neurons from normoxic (NORM) and CIH-exposed rats (alternating cycles of 3 min at 10% O2 followed by 3 min at 21% O2 between 8 AM and 4 PM for 7 days). To identify NTS neurons receiving carotid body afferent inputs the anterograde tracer 4- (4-(dihexadecylamino)styryl-N-methylpyridinum iodide (DiA) was placed onto the carotid body 1 wk before exposure to CIH. AMPA dose-response curves had similar EC50 but maximal responses increased in neurons isolated from DiA-labeled CIH (20.1 +/- 0.8 microM, n = 9) compared with NORM (6.0 +/- 0.3 microM, n = 8) rats. NMDA dose-response curves also had similar EC50 but maximal responses decreased in CIH (8.4 +/- 0.4 microM, n = 8) compared with NORM (19.4 +/- 0.6 microM, n = 9) rats. These results suggest reciprocal changes in the number and/or conductance characteristics of AMPA and NMDA receptors. Enhanced responses to AMPA receptor activation could contribute to enhanced chemoreflex responses observed in animals exposed to CIH and humans with sleep apnea.     

Contribution of non-inactivating Na+ current induced by oxidizing agents to the firing behavior of neuronal action potentials: experimental and theoretical studies from NG108-15 neuronal cells

The effects of chemical injury with oxidizing agents on voltage-gated Na+ current (I(Na)) in differentiated NG108-15 neuronal cells were investigated in this study. In whole-cell patch-clamp recordings, the challenge of these cells with t-butyl hydroperoxide (t-BHP; 1 mM) decreased the peak amplitude of I(Na) with no modification in the current-voltage relationship. It caused a slowing of current inactivation, although there was no alteration in the activation time course of I(Na). Cell exposure to t-BHP also increased a non-inactivating I(Na) (I(Na(NI)) elicited by long-lasting ramp pulses. The t-BHP-induced increase of I(Na(NI)) was reversed by a further application of riluzole (10 microM) or oxcarbazepine (10 microM). When I(Na) was elicited by simulated waveforms of action potentials (APs), during exposure to t-BHP, the amplitude of this inward current was diminished, accompanied by a reduction in inactivation/deactivation rate and an increase in current fluctuations. Under current-clamp recordings, addition of t-BHP (0.3 mM) enhanced AP firing in combination with clustering-like activity and sub-threshold membrane oscillations. In the simulation study, when the fraction of non-inactivating Na(v) channels was elevated, the simulated window component of I(Na) in response to a long-lasting ramp pulse was reduced; however, the persistent I(Na) was markedly enhanced. Moreover, when simulated firing of APs was generated from a modeled neuron, changes of AP firing caused by the increased fraction of non-inactivating Na(v) channels used to mimic the t-BHP actions were similar to the experimental observations. Taken together, it is anticipated that the effects of oxidizing agents on I(Na(NI)) could be an important mechanism underlying their neurotoxic actions in neurons or neuroendocrine cells occurring in vivo.     

Chronic hypoxia upregulates connexin43 expression in rat carotid body and petrosal ganglion.

Recent studies have demonstrated that oxygen-sensitive type I cells in the carotid body express the gap junction-forming protein connexin43 (Cx43). In the present study, we examined the hypothesis that chronic exposure to hypoxia increases Cx43 expression in type I cells as well as in chemoafferent neurons in the petrosal ganglion. Immunocytochemical studies in tissues from normal rats revealed diffuse and granular Cx43-like immunoreactivity in the cytoplasm of type I cells and dense punctate spots of immunoreactive product at the margins of type I cells and near the borders of chemosensory cell lobules. Cx43-like immunoreactivity was not detectable in petrosal ganglion neurons from normal animals. After a 2-wk exposure to hypobaric (380 Torr) hypoxia, Cx43 immunostaining was substantially enhanced in and around type I cells. Moreover, chronic hypoxia elicited the expression of Cx43-like immunoreactivity in the cytoplasm of afferent neurons throughout the petrosal ganglion. Quantitative RT-PCR studies indicate that chronic hypoxia evokes a substantial increase in Cx43 mRNA levels in the carotid body, along with a marked elevation of Cx43 expression in the petrosal ganglion. Increased Cx43 expression and gap junction formation in type I cells and sensory neurons may contribute to carotid body adaptation during sustained stimulation in extreme physiological conditions.     

Effect of p47phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells

Membrane potential in oxygen-sensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K(+) channels. Coupling of Po(2) to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing Po(2) and a subset of K(+) channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47(phox) gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 microM) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formyl-Met-Leu-Phe (1 microM), arachidonic acid (10 microM), and cytochalasin B (5 microg/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 microM). KO neutrophils did not respond; however, azide (5 microM) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K+ current and increased intracellular Ca2+ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca2+ and enhanced the depression of K+ current in low O(2). Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K+ channel activity in hypoxia.     

Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms

We tested the hypothesis that nitric oxide (NO) produced within the carotid body is a tonic inhibitor of chemoreception and determined the contribution of neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the inhibitory NO effect. Accordingly, we studied the effect of NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the selective nNOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to hypoxia (Po(2) approximately 30 Torr). CBs excised from pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at 38 degrees C and pH 7.40, and chemosensory discharges were recorded from the carotid sinus nerve. SNAP (100 microM) reduced the responses to nicotine and NaCN. l-NAME (1 mM) enhanced the responses to nicotine and NaCN by increasing their duration, but TRIM (100 microM) only enhanced the responses to high doses of NaCN. The amplitude of the response to hypoxia was enhanced by l-NAME but not by TRIM. Our results suggest that both isoforms contribute to the NO action, but eNOS being the main source for NO in the cat CB and exerting a tonic effect upon chemoreceptor activity.     

Blockade of persistent sodium currents contributes to the riluzole-induced inhibition of spontaneous activity and oscillations in injured DRG neurons

In addition to a fast activating and immediately inactivating inward sodium current, many types of excitable cells possess a noninactivating or slowly inactivating component: the persistent sodium current (I(NaP)). The I(NaP) is found in normal primary sensory neurons where it is mediated by tetrodotoxin-sensitive sodium channels. The dorsal root ganglion (DRG) is the gateway for ectopic impulses that originate in pathological pain signals from the periphery. However, the role of I(NaP) in DRG neurons remains unclear, particularly in neuropathic pain states. Using in vivo recordings from single medium- and large-diameter fibers isolated from the compressed DRG in Sprague-Dawley rats, we show that local application of riluzole, which blocks the I(NaP), also inhibits the spontaneous activity of A-type DRG neurons in a dose-dependent manner. Significantly, riluzole also abolished subthreshold membrane potential oscillations (SMPOs), although DRG neurons still responded to intracellular current injection with a single full-sized spike. In addition, the I(NaP) was enhanced in medium- and large-sized neurons of the compressed DRG, while bath-applied riluzole significantly inhibited the I(NaP) without affecting the transient sodium current (I(NaT)). Taken together, these results demonstrate for the first time that the I(NaP) blocker riluzole selectively inhibits I(NaP) and thereby blocks SMPOs and the ectopic spontaneous activity of injured A-type DRG neurons. This suggests that the I(NaP) of DRG neurons is a potential target for treating neuropathic pain at the peripheral level.     

Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors.

The purpose of the present study was to investigate whether hypoxia influences acetylcholine (ACh) release from the rabbit carotid body and, if so, to determine the mechanism(s) associated with this response. ACh is expressed in the rabbit carotid body (5.6 +/- 1.3 pmol/carotid body) as evidenced by electrochemical analysis. Immunocytochemical analysis of the primary cultures of the carotid body with antibody specific to ACh further showed that ACh-like immunoreactivity is localized to many glomus cells. The effect of hypoxia on ACh release was examined in ex vivo carotid bodies harvested from anesthetized rabbits. The basal release of ACh during normoxia ( approximately 150 Torr) averaged 5.9 +/- 0.5 fmol.min-1.carotid body-1. Lowering the Po2 to 90 and 20 Torr progressively decreased ACh release by approximately 15 and approximately 68%, respectively. ACh release returned to the basal value on reoxygenation. Simultaneous monitoring of dopamine showed a sixfold increase in dopamine release during hypoxia. Hypercapnia (21% O2 + 10% CO2) as well as high K+ (100 mM) facilitated ACh release from the carotid body, suggesting that hypoxia-induced inhibition of ACh release is not due to deterioration of the carotid body. Hypoxia had no significant effect on acetylcholinesterase activity in the medium, implying that increased hydrolysis of ACh does not account for hypoxia-induced inhibition of ACh release. In the presence of either atropine (10 microM) or domperidone (10 microM), hypoxia stimulated ACh release. These results demonstrate that glomus cells of the rabbit carotid body express ACh and that hypoxia overall inhibits ACh release via activation of muscarinic and dopaminergic autoinhibitory receptors in the carotid body.     

Dependency of hypoxic chemotransduction in cat carotid body on voltage-gated calcium channels.

The hypothesis that the entry of extracellular calcium ions into some compartment, quite possibly the type I cells, through voltage-gated calcium channels (VGCC) is essential for hypoxic chemotransduction in the cat carotid body was tested using an in situ perfusion technique. The neural output of the carotid body of anesthetized, paralyzed, and artificially ventilated cats in response to perfusions with Krebs-Ringer bicarbonate solution (KRB), calcium-free KRB, KRB containing calcium channel blockers, or KRB containing BAY K 8644 was recorded. Selective perfusion of the carotid body with hypoxic calcium-free KRB significantly decreased carotid chemoreceptor activity, suggesting that extracellular calcium is essential for hypoxic chemotransduction. Selective perfusion of the carotid body with hypoxic KRB containing verapamil (10-100 microM), diltiazem (10-100 microM), or nifedipine (10-100 microM) dose dependently attenuated the increase in chemoreceptor activity produced by hypoxia, suggesting that VGCC need to be activated for hypoxic chemotransduction. The carotid body response to hyperoxic KRB containing the calcium channel agonist BAY K 8644 (10 microM) was 267 +/- 87% of hyperoxic control KRB, suggesting that an enhanced influx of calcium ions through VGCC stimulates carotid chemoreceptor activity. Selective perfusion of the carotid body with severely hypoxic KRB containing BAY K 8644 did not increase chemoreceptor activity above that produced by severe hypoxia alone. This suggests that severe hypoxia increases intracellular calcium in some compartment of the carotid body to achieve stimulatory maximum response and that further increase in intracellular calcium does not produce further elevation of neural activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-dependent effect of hypoxia on carotid body chemosensory function

The time-dependent effects of hypoxia on the discharge rate carotid chemoreceptors were measured in anesthetized cats. Hypoxic exposure of two different durations were used: a short-term exposure (2-3 h) was used to measure the response of the same carotid chemoreceptors; and a long-term exposure (28 days at inspired PO2 of 70 Torr) to study carotid chemoreceptor properties in one group of cats relative to those of a control group. In the chronically hypoxic and control groups, determinations were made of the 1) steady-state responses to four levels of arterial PO2 (PaO2) at constant levels of arterial PCO2; 2) steady-state responses to acute hypercapnia during hyperoxia; and 3) maximal discharge rates during anoxia. We found that the acute responses of carotid chemoreceptor afferents to a given level of hypoxia (PaO2 = 30-40 Torr) did not significantly change within 2-3 h. After long-term exposure the carotid chemoreceptor responses to hypoxia significantly increased, with no significant changes in the hypercapnic response and in the maximal discharge rate during anoxia. We conclude that isocapnic hypoxia may not elicit a sufficient cellular response within 2-3 h in the cat carotid body to sensitize the O2 responsive mechanism, but hypoxia of longer duration will sensitize such a mechanism, thereby augmenting the chemosensory activity.