Colin A. Nurse

Co‐release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors

1 Using functional co‐cultures of rat carotid body (CB) O2 chemoreceptors and ‘juxtaposed’ petrosal neurones (JPNs), we tested whether ATP and ACh acted as co‐transmitters. 2 Perforated‐patch recordings from JPNs often revealed spontaneous and hypoxia‐evoked (PO2≈5 mmHg) excitatory postsynaptic responses. The P2X purinoceptor blocker, suramin (50 μM) or a nicotinic ACh receptor (nAChR) blocker (hexamethonium, 100 μM; mecamylamine, 1 μM) only partially inhibited these responses, but together, blocked almost all activity. 3 Under voltage clamp (‐60 mV), fast perfusion of 100 μM ATP over hypoxia‐responsive JPNs induced suramin‐sensitive (IC50= 73 μM), slowly‐desensitizing, inward currents (IATP) with time constant of activation τon= 30.6 ± 4.8 ms (n= 7). IATP reversed at 0.33 ± 3.7 mV (n= 4), and the dose‐response curve was fitted by the Hill equation (EC50= 2.7 μM; Hill coefficient ≈0.9). These purinoceptors contained immunoreactive P2X2 subunits, but their activation by α,β‐methylene ATP (α,β‐meATP; EC50= 2.1 μM) suggests they are P2X2/P2X3 heteromultimers. 4 Suramin and nAChR blockers inhibited the extracellular chemosensory discharge in the intact rat carotid body‐sinus nerve preparation in vitro. Further, P2X2 immunoreactivity was widespread in rat petrosal ganglia in situ, and co‐localized in neurones expressing the CB chemo‐afferent marker, tyrosine hydroxylase (TH). P2X2 labelling in the CB co‐localized with nerve‐terminal markers, and was intimately associated with TH‐positive type 1 cells. 5 Thus ATP and ACh are co‐transmitters during chemotransduction in the rat carotid body.

Developmental loss of hypoxic chemosensitivity in rat adrenomedullary chromaffin cells.

1. We investigated whether adrenomedullary chromaffin cells (AMCs) derived from neonatal (postnatal day (P) 1‐P2) and juvenile (P13‐P20) rats, and maintained in short‐term culture (1‐3 days), express O2‐chemoreceptive properties. 2. In whole‐cell recordings, the majority (approximately 70%; n = 47) of neonatal AMCs were sensitive to hypoxia. Under voltage clamp, acute hypoxia (PO2 approximately 40 mmHg) suppressed voltage‐dependent K+ current by 25.1 +/‐ 3.4% (mean +/‐ S.E.M.; n = 22); under current clamp, acute hypoxia caused a membrane depolarization of 14.1 +/‐ 1.3 mV (n = 13) from a resting potential of ‐54.8 +/‐ 2.8 mV (n = 13), and this was often sufficient to trigger action potentials. 3. Exposure of neonatal AMC cultures to a moderate (PO2 approximately 75 mmHg) or severe (PO2 approximately 35 mmHg) hypoxia for 1 h caused a dose‐dependent stimulation (approximately 3 or 6 times normoxia, respectively) of catecholamine (CA) release, mainly adrenaline, determined by HPLC. This induced CA release was abolished by the L‐type calcium channel blocker, nifedipine (10 microM). 4. In contrast to the above results in neonates, hypoxia had no significant effects on voltage‐dependent K+ current, membrane potential, or CA release in juvenile AMCs. 5. We conclude that rat adrenal chromaffin cells possess a developmentally regulated O2‐sensing mechanism, similar to carotid body type I cells.

Neurotransmission and neuromodulation in the chemosensory carotid body.

The mammalian carotid body is a small chemosensory organ that helps maintain the chemical composition of arterial blood via reflex control of ventilation. Thus, in response to decreased PO2 (hypoxia), increased PCO2 (hypercapnia), or decreased pH (acidity), chemoreceptor glomus or type I cells become stimulated and release neuroactive agents that excite apposed sensory terminals of the carotid sinus nerve. The resulting increase in afferent discharge ultimately leads to corrective changes in ventilation so as to maintain blood gas and pH homeostasis. Recent evidence that the organ can also sense low glucose further emphasizes its role as a polymodal sensor of blood-borne stimuli. The chemoreceptors occur in organized cell clusters that receive sensory innervation from petrosal afferents and are intimately associated with the blood supply. Additionally, synaptic specializations between neighboring receptor cells allow for autocrine and paracrine regulation of the sensory output. Though not without controversy, significant progress has been made in elucidating the various chemotransductive pathways, as well as the neurotransmitter and neuromodulatory mechanisms that translate the receptor potential into an afferent sensory discharge. Progress in the latter has been hampered by the presence of a wide variety of endogenous ligands, and an even broader spectrum of receptor subtypes, that apparently help shape the chemoreceptor output and afferent discharge. This review will highlight recent advances in understanding the role of these neuroactive ligands in carotid body function.

Neuroepithelial oxygen chemoreceptors of the zebrafish gill

In aquatic vertebrates, hypoxia induces physiological changes that arise principally from O2 chemoreceptors of the gill. Neuroepithelial cells (NECs) of the zebrafish gill are morphologically similar to mammalian O2 chemoreceptors (e.g. carotid body), suggesting that they may play a role in initiating the hypoxia response in fish. We describe morphological changes of zebrafish gill NECs following in vivo exposure to chronic hypoxia, and characterize the cellular mechanisms of O2 sensing in isolated NECs using patch‐clamp electrophysiology. Confocal immunofluorescence studies indicated that chronic hypoxia (P  O 2 = 35 mmHg, 60 days) induced hypertrophy, proliferation and process extension in NECs immunoreactive for serotonin or synaptic vesicle protein (SV2). Under voltage clamp, NECs responded to hypoxia (P  O 2 = 25–140 mmHg) with a dose‐dependent decrease in K+ current. The current–voltage relationship of the O2‐sensitive current (I  KO 2 ) reversed near EK and displayed open rectification. Pharmacological characterization indicated that I  KO 2 was resistant to 20 mm tetraethylammonium (TEA) and 5 mm 4‐aminopyridine (4‐AP), but was sensitive to 1 mm quinidine. In current‐clamp recordings, hypoxia produced membrane depolarization associated with a conductance decrease; this depolarization was blocked by quinidine, but was insensitive to TEA and 4‐AP. These biophysical and pharmacological characteristics suggest that hypoxia sensing in zebrafish gill NECs is mediated by inhibition of a background K+ conductance, which generates a receptor potential necessary for neurosecretion and activation of sensory pathways in the gill. This appears to be a fundamental mechanism of O2 sensing that arose early in vertebrate evolution, and was adopted later in mammalian O2 chemoreceptors.

Neurotransmitter and neuromodulatory mechanisms at peripheral arterial chemoreceptors.

The control of breathing depends critically on sensory inputs to the central pattern generator of the brainstem, arising from peripheral arterial chemoreceptors located principally in the carotid bodies (CBs). The CB receptors, i.e. glomus or type I cells, are excited by chemical stimuli in arterial blood, particularly hypoxia, hypercapnia, acidosis and low glucose, which initiate corrective reflex cardiorespiratory and cardiovascular adjustments. Type I cells occur in clusters and are innervated by petrosal afferent fibres. Synaptic specializations (both chemical and electrical) occur between type I cells and petrosal terminals, and between neighbouring type I cells. This, together with the presence of a wide array of neurotransmitters and neuromodulators linked to both ionotropic and metabotropic receptors, allows for a complex modulation of CB sensory output. Studies in several laboratories over the last ∼20 years have provided much insight into the transduction mechanisms. More recent studies, aided by the development of a co‐culture model of the rat CB, have shed light on the role of neurotransmitters and neuromodulators in shaping the afferent response. This review highlights some of these developments, which have contributed to our current understanding of information processing at CB chemoreceptors.

Whole-cell and perforated-patch recordings from O2-sensitive rat carotid body cells grown in short- and long-term culture

We are investigating transduction mechanisms in a major peripheral chemosensory organ, the rat carotid body, using short- and long-term dissociated cell cultures and patch-clamp, whole-cell recording. In this study membrane properties of cultured glomus or type I cells were characterized with conventional whole-cell recording and the new perforated-patch technique during control (160 Torr) and low-PO2 (20 Torr) conditions. These cells contained voltage-gated channels typical of electrically excitable cells and had large input resistances (approx. 2 GΩ). Under whole-cell voltage clamp the cells produced brief inactivating inward currents, which were largely abolished by 0.2–2.0 μM tetrodotoxin, followed by prolonged outward currents, which were reduced by 5 mM tetraethylammonium or abolished by the substitution of Cs+ ions for K+ ions in the pipette. On exposure to hypoxia the outward K+ current was reduced typically by 15%–20% with both conventional whole-cell and perforated-patch recording, which minimizes washout of the cells cytoplasm. This effect persisted in long-term culture and was specific, since the inward current was unaffected and, moreover, it did not occur in cultured small intensely fluorescent cells, which are closely related to glomus cells. These properties of cultured rat glomus cells are contrasted with those recently reported for freshly isolated rabbit glomus cells.

Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones : role in chemosensory signalling

1 Hypoxic chemotransmission in the rat carotid body (CB) is mediated in part by ATP acting on suramin‐sensitive P2X purinoceptors. Here, we use RT‐PCR, cloning and sequencing techniques to show P2X2 and P2X3 receptor expression in petrosal neurones, some of which develop functional chemosensory units with CB receptor clusters in co‐culture. 2 Single‐cell RT‐PCR revealed that hypoxia‐responsive neurones, identified electrophysiologically in co‐culture, expressed both P2X2 and P2X3 mRNA. 3 Isohydric hypercapnia (10% CO2; pH 7.4) caused excitation of chemosensory units in co‐culture. This excitation depended on chemical transmission, with ATP acting as a co‐transmitter, since it was inhibited by reduction of the extracellular Ca2+:Mg2+ ratio and by the purinoceptor blocker suramin (50–100 μm). 4 Hypoxia and isohydric hypercapnia could separately excite the same chemosensory unit, and together the two stimuli interacted synergistically. 5 Using confocal immunofluorescence, co‐localization of P2X2 and P2X3 protein was demonstrated in many petrosal somas and CB afferent terminals in situ. Taken together, these data indicate that ATP and P2X2–P2X3 purinoceptors play important roles in the peripheral control of respiration by carotid body chemoreceptors.

Neuroepithelial cells and associated innervation of the zebrafish gill: a confocal immunofluorescence study.

Peripheral chemoreceptors responsive to hypoxia have been well characterized in air‐breathing vertebrates, but poorly in water‐breathers. The present study examined the distribution of five populations of neuroepithelial cells (NECs), putative O2 chemoreceptors, and innervation patterns in the zebrafish gill using whole‐mounts and confocal immunofluorescence. Nerve bundles and fibers of the gill were labeled with zn‐12 (a zebrafish‐specific neuronal marker) and SV2 antisera and NECs were characterized by serotonin (5‐HT) immunoreactivity (IR), SV2‐IR and the purinoceptor P2X3‐IR. A zn‐12‐IR nerve bundle extended the length of the gill filament and gave rise to a nerve plexus surrounding the efferent filament artery (eFA) and a rich network of fibers that innervated both serotonergic and nonserotonergic NECs of the filament and lamellar epithelium. Three populations of serotonergic, SV2‐IR neurons intrinsic to the gill filaments are described, one of which provided innervation to NECs of the filament epithelium. Degeneration of nerve fibers in gill arches maintained in explant culture for 2 days revealed the extrinsic origin of nerve fibers of the plexus and lamellae and the innervation of filament NECs by both intrinsic and extrinsic fibers. Intrinsic innervation surrounding the eFA survived in explant cultures, suggesting a mechanism of local vascular control within the gill. In addition, NECs survived in explants after degeneration of extrinsic nerve fibers. Thus, NECs of the zebrafish gill are organized in a manner reminiscent of O2 chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation. J. Comp. Neurol. 461:1–17, 2003.

Anoxia differentially modulates multiple K+ currents and depolarizes neonatal rat adrenal chromaffin cells

1 Using perforated‐patch, whole cell recording, we investigated the membrane mechanisms underlying O2 chemosensitivity in neonatal rat adrenomedullary chromaffin cells (AMC) bathed in extracellular solution containing tetrodotoxin (TTX; 0.5–1 μm), with or without blockers of calcium entry. 2 Under voltage clamp, low PO2 (0–15 mmHg) caused a graded and reversible suppression in macroscopic outward K+ current. The suppression during anoxia (PO2= 0 mmHg) was ∼35 % (voltage step from −60 to +30 mV) and was due to a combination of several factors: (i) suppression of a cadmium‐sensitive, Ca2+‐dependent K+ current, IK(CaO2); (ii) suppression of a Ca2+‐insensitive, delayed rectifier type K+ current, IK(VO2); (iii) activation of a glibenclamide‐ (and Ca2+)‐sensitive current, IK(ATP). 3 During normoxia (PO2= 150 mmHg), application of pinacidil (100 μm), an ATP‐sensitive potassium channel (KATP) activator, increased outward current density by 45.0 ± 7.0 pA pF−1 (step from −60 to + 30 mV), whereas the KATP blocker glibenclamide (50 μm) caused only a small suppression by 6.3 ± 4.0 pA pF−1. In contrast, during anoxia the presence of glibenclamide resulted in a substantial reduction in outward current density by 24.9 ± 7.9 pA pF−1, which far exceeded that seen in its absence. Thus, activation of IK(ATP) by anoxia appears to reduce the overall K+ current suppression attributable to the combined effects of IK(CaO2) and IK(VO2). 4 Pharmacological tests revealed that IK(CaO2) was carried predominantly by maxi‐K+ or BK potassium channels, sensitive to 50–100 nm iberiotoxin; this current also accounted for the major portion (∼60 %) of the anoxic suppression of outward current. Tetraethylammonium (TEA; 10–20 mm) blocked all of the anoxia‐sensitive K+ currents recorded under voltage clamp, i.e. IK(CaO2), IK(VO2) and IK(ATP). 5 Under current clamp, anoxia depolarized neonatal AMC by 10–15 mV from a resting potential of ∼‐55 mV. At least part of this depolarization persisted in the presence of either TEA, Cd2+, 4‐aminopyridine or charybdotoxin, suggesting the presence of anoxia‐sensitive mechanisms additionalto those revealed under voltage clamp. In Na+/Ca2+‐free solutions, the membrane hyperpolarized, though at least a portion of the anoxia‐induced depolarization persisted. 6 In the presence of glibenclamide, the anoxia‐induced depolarization increased significantly to ∼25 mV, suggesting that activation of KATP channels may function to attenuate the anoxia‐induced depolarization or receptor potential.

Hypoxia‐induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit

We examined the effects of hypoxia on the release of serotonin (5‐HT) from intact neuroepithelial body cells (NEB), presumed airway chemoreceptors, in rabbit lung slices, using amperometry with carbon fibre microelectrodes. Under normoxia (PO2∼155 mmHg; 1 mmHg ≈133 Pa), most NEB cells did not exhibit detectable secretory activity; however, hypoxia elicited a dose‐dependent (PO2 range 95–18 mmHg), tetrodotoxin (TTX)‐sensitive stimulation of spike‐like exocytotic events, indicative of vesicular amine release. High extracellular K+ (50 mm) induced a secretory response similar to that elicited by severe hypoxia. Exocytosis was stimulated in normoxic NEB cells after exposure to tetraethylammonium (20 mm) or 4‐aminopyridine (2 mm). Hypoxia‐induced secretion was abolished by the non‐specific Ca2+ channel blocker Cd2+ (100 μm). Secretion was also largely inhibited by the L‐type Ca2+ channel blocker nifedipine (2 μm), but not by the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (1 μm). The 5‐HT3 receptor blocker ICS 205 930 also inhibited secretion from NEB cells under hypoxia. These results suggest that hypoxia stimulates 5‐HT secretion from intact NEBs via inhibition of K+ channels, augmentation of Na+‐dependent action potentials and calcium entry through L‐type Ca2+ channels, as well as by positive feedback activation of 5‐HT3 autoreceptors.