Julio Alcayaga

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.

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.

Dissociation of hypoxia-induced chemosensory responses and catecholamine efflux in cat carotid body superfused in vitro.

1. To examine the correlation between chemosensory response and dopamine release induced by hypoxic stimulation, we studied carotid bodies excised from anaesthetized cats. 2. The carotid bodies with their carotid (sinus) nerves were superfused in vitro with modified Tyrode solution (pH 7.40, at 37.5 degrees C) equilibrated with 20 or 100% O2. The PO2 of the superfusing channel was monitored polarographically. The frequency of chemosensory discharges (fx) was recorded from the whole carotid nerve. Catecholamine (CA) efflux‐mostly consisting of dopamine‐was measured by high‐speed chronoamperometry, through Nafion‐coated carbon electrodes placed on the carotid body tissue. Chemosensory stimulation was induced by intrastream injections of NaCN, by superfusion with 100% N2‐equilibrated saline (lowering PO2 to 25‐40 Torr) or by flow interruption. 3. Low doses of NaCN increased fx, but had no measurable effect on CA efflux, while larger doses produced fast increases in fx, preceding delayed and prolonged increases in CA efflux. Repeated injections of NaCN, still increasing fx, gave reduced CA effluxes. 4. Switching to hypoxic superfusion for 6‐8 min produced large and fast fx increases, but delayed and prolonged augmentations of CA efflux. 5. Administration of three to four boluses of dopamine (7‐15 micrograms; augmenting CA concentration by up to 35 microM) initially decreased fx, after which hypoxic stimulation resulted in enhanced and faster CA effluxes, without changing the speed and intensity of chemosensory responses. 6. Flow interruptions induced fast increases in fx and delayed increases in CA efflux. Repeated flow interruptions produced similar increases in fx but progressively attenuated CA effluxes. 7. Our results suggest that CA efflux is not essential for hypoxia‐induced chemosensory excitation in the cat carotid body. They also suggest the presence of two pools of releasable CAs in the carotid body, one of slow turnover and release, and another of recently incorporated dopamine and fast release, both pools being rapidly depleted by repeated stimulation of the carotid body.

Electrical and pharmacological properties of petrosal ganglion neurons that innervate the carotid body

The petrosal ganglion (PG) contains the somata of primary afferent neurons that innervate the chemoreceptor (glomus) cells in the carotid body (CB). The most accepted model of CB chemoreception states that natural stimuli trigger the release of one or more transmitters from glomus cells, which in turn acting on specific post-synaptic receptors increases the rate of discharge in the nerve endings of PG neurons. However, PG neurons that project to the CB represent only small fraction (roughly 20%) of the whole PG and their identification is not simple since their electrophysiological and pharmacological properties are not strikingly different as compared with other PG neurons, which project to the carotid sinus or the tongue. In addition, differences reported on the actions of putative transmitters on PG neurons may reflect true species differences. Nevertheless, some experimental strategies have contributed to identify and characterize the properties of PG neurons that innervate the CB. In this review, we examined the electrophysiological properties and pharmacological responses of PG neurons to putative CB excitatory transmitters, focusing on the methods of study and species differences. The evidences suggest that ACh and ATP play a major role in the fast excitatory transmission between glomus cells and chemosensory nerve endings in the cat, rat and rabbit. However, the role of other putative transmitters such as dopamine, 5-HT and GABA is less clear and depends on the specie studied.

Adenosine triphosphate-induced peripheral nerve discharges generated from the cat petrosal ganglion in vitro

Since nucleotides have been postulated as transmitters between glomus cells and chemosensory nerve endings in the carotid body, we studied the effects of their application to the petrosal ganglion, where the perikarya of carotid (sinus) nerve are located. Cat petrosal ganglia were superfused in vitro, while electrical activities of their peripheral processes (carotid nerve and glossopharyngeal branch) were recorded simultaneously. Adenosine triphosphate (ATP) evoked dose-dependent bursts of impulses in carotid nerve, while those in glossopharyngeal branch were less intense and consistent. Adenosine monophosphate was less effective than ATP. ATP-induced carotid nerve responses presented no temporal desensitization and persisted after applying P(2Y) receptor blocker Reactive Blue 2 to the ganglion. The results indicate that ATP has an excitatory effect on the perikarya of the population of petrosal ganglion neurons projecting peripherally through the carotid nerve.

Dopamine modulates carotid nerve responses induced by acetylcholine on the cat petrosal ganglion in vitro.

We have recently reported that application of acetylcholine (ACh) or nicotine to the petrosal ganglion-the sensory ganglion of the glossopharyngeal nerve-elicits a burst of discharges in the carotid nerve branch, innervating the carotid body and sinus, but not in the glossopharyngeal branch, innervating the tongue and pharynx. Thus, the perikarya of sensory neurons for the carotid bifurcation exhibit selective cholinosensitivity. Since dopamine (DA) modulates carotid nerve chemosensory activity, we searched for the presence of DA sensitivity at the perikarya of these neurons in the cat petrosal ganglion superfused in vitro. Applications of DA in doses of up to 5 mg to the ganglion did not modify the rate of spontaneous discharges in the carotid nerve. However, if DA was applied 30 s before ACh injections, ACh-evoked reactions were modified: low doses of DA enhanced the subsequent responses to ACh, while high doses of DA depressed the responses to ACh. This depressant effect of DA on ACh responses was partially antagonized by adding spiroperone to the superfusate. Our results show that the response to ACh of petrosal ganglion neurons projecting through the carotid nerve is modulated by DA acting on D(2) receptors located in the somata of these neurons. Thus, dopaminergic modulation of cholinosensitivity could be shared also by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.

Cat carotid body chemosensory responses to non-hypoxic stimuli are inhibited by sodium nitroprusside in situ and in vitro

We studied the effects of sodium nitroprusside, a nitric oxide donor, on the chemosensory responses to cyanide and nicotine in the cat carotid body. In situ, sodium nitroprusside infusion reduced the cyanide-evoked responses in a dose-dependent manner. In vitro, Tyrode containing nitroprusside reversibly reduced the cyanide- (by 59%) and nicotine-induced (by 45%) chemosensory responses. The present results suggest that chemosensory responses induced by cyanide and nicotine are reduced by increased nitric oxide content, similarly to the hypoxic chemosensory responses.

Modulatory effect of nitric oxide on acetylcholine-induced activation of cat petrosal ganglion neurons in vitro

The inhibitory effect of nitric oxide (NO) on carotid chemosensory responses to hypoxia has been attributed in part to an antidromic inhibition of chemoreceptor cells activity. However, NO may also modulate the activity of the primary sensory neurons because NO is produced in the soma of these neurons located in the petrosal ganglion. Since a population of petrosal neurons is selectively activated by acetylcholine (ACh), we studied the effects of NO-donor, sodium nitroprusside (SNP), and the NO-synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), on the responses evoked in the carotid sinus nerve (CSN) by ACh applied to the petrosal ganglion in vitro. ACh (1 microgram-1 mg) increased the frequency of action potentials recorded from the CSN in a dose-dependent manner. SNP (10-50 microM) reduced the sensibility and amplitude of the CSN response to ACh, although the maximal response appears less affected. The withdrawal of SNP from the superfusion medium increased the sensibility of the responses to ACh. l-NAME (1-2 mM) slightly increased the sensibility of the ACh-induced responses, effect that persisted after l-NAME withdrawal. These results suggest that NO may play a role as modulator in this autonomic primary sensory ganglion.

Responses to hypoxia of petrosal ganglia in vitro.

NaCN is a classical stimulus used to elicit discharges from carotid body chemoreceptors. The effect is assumed to be mediated by glomus (type I) cells, which release an excitatory transmitter for the excitation of carotid nerve endings. Since the sensory perikarya of the glossopharyngeal nerve (from which the carotid nerve branches) are located in the petrosal ganglion, we tested whether application of this drug to the petrosal ganglion superfused in vitro elicits antidromic discharges in the carotid nerve. NaCN did indeed cause an intense and prolonged burst of nerve impulses in the carotid nerve, while provoking a less intense and much briefer burst of discharges in the glossopharyngeal branch. Carotid nerve responses to NaCN were reduced and shortened by prior or following application of dopamine to the ganglion. Sodium azide applied to the petrosal ganglion evoked a less intense and much briefer burst of impulses in the carotid nerve. Ganglionar application of 2,4-dinitrophenol did not induce discharges in the carotid nerve. Switching the superfusion of the ganglion from a normoxic to a hypoxic solution did not evoke discharges in the carotid nerve. Therefore, the perikarya of carotid nerve neurons are sensitive to NaCN, but are not excited by reducing the pO(2) of the superfusing solution.