Cathy Vollmer

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.

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.

GABA Mediates Autoreceptor Feedback Inhibition in the Rat Carotid Body Via Presynaptic GABAB Receptors and TASK‐1

Background K+ channels exert control over neuronal excitability by regulating resting potential and input resistance. Here, we show that GABAB receptor‐mediated activation of a background K+ conductance modulates transmission at rat carotid body chemosensory synapses in vitro. Carotid body chemoreceptor (type I) cells expressed GABAB(1) and GABAB(2) subunits as well as endogenous GABA. The GABAB receptor agonist baclofen activated an anandamide‐ and Ba2+‐sensitive TASK‐1‐like background K+ conductance in chemoreceptor cell clusters, but was without effect on voltage‐gated Ca2+ channels. Hydroxysaclofen (50 μm), 5‐aminovaleric acid (100 μm) and CGP 55845 (100 nm), selective GABAB receptor blockers, potentiated the hypoxia‐induced receptor potential; this effect was abolished by pre‐treatment with pertussis toxin (PTX; 500 ng ml‐1), an inhibitor of Gi, or by H‐89 (50 μm), a selective inhibitor of protein kinase A. The protein kinase C inhibitor chelerythrine chloride (100 μm) was without effect on this potentiation. GABAB receptor blockers also caused depolarisation of type I cells in clusters, and enhanced spike discharge in spontaneously firing cells. In functional co‐cultures of type I clusters and petrosal sensory neurones, GABAB receptor blockers potentiated hypoxia‐induced postsynaptic chemosensory responses mediated by the fast‐acting transmitters ACh and ATP. Thus GABAB receptor‐mediated activation of TASK‐1 or a related channel provides a presynaptic autoregulatory feedback mechanism that modulates fast synaptic transmission in the rat carotid body.

Expression of Multiple P2X Receptors by Glossopharyngeal Neurons Projecting to Rat Carotid Body O2-Chemoreceptors: Role in Nitric Oxide-Mediated Efferent Inhibition

In mammals, ventilation is peripherally controlled by the carotid body (CB), which receives afferent innervation from the petrosal ganglion and efferent innervation from neurons located along the glossopharyngeal nerve (GPN). GPN neurons give rise to the “efferent inhibitory” pathway via a plexus of neuronal nitric oxide (NO) synthase-positive fibers, believed to be responsible for CB chemoreceptor inhibition via NO release. Although NO is elevated during natural CB stimulation by hypoxia, the underlying mechanisms are unclear. We hypothesized that ATP, released by rat CB chemoreceptors (type 1 cells) and/or red blood cells during hypoxia, may directly activate GPN neurons and contribute to NO-mediated inhibition. Using combined electrophysiological, molecular, and confocal immunofluorescence techniques, we detected the expression of multiple P2X receptors in GPN neurons. These receptors involve at least four different purinergic subunits: P2X2 [and the splice variant P2X2(b)], P2X3, P2X4, and P2X7. Using a novel coculture preparation of CB type I cell clusters and GPN neurons, we tested the role of P2X signaling on CB function. In cocultures, fast application of ATP, or its synthetic analog 2′,3′-O-(4 benzoylbenzoyl)-ATP, caused type I cell hyperpolarization that was prevented in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium. These data suggest that ATP released during hypoxic stress from CB chemoreceptors (and/or red blood cells) will cause GPN neuron depolarization mediated by multiple P2X receptors. Activation of this pathway will lead to calcium influx and efferent inhibition of CB chemoreceptors via NO synthesis and consequent release.

P2Y2 receptor activation opens pannexin-1 channels in rat carotid body type II cells: potential role in amplifying the neurotransmitter ATP.

Carotid body (CB) chemoreceptor complexes consist of receptor type I cells, intimately associated with glia‐like type II cells whose function is poorly understood. We show that type II cells in the rat CB express gap junction‐like proteins, pannexin‐1 (Panx‐1) channels, which form non‐selective pores permeable to ions and large molecules such as ATP, a key CB neurotransmitter. Activation of purinergic P2Y2 receptors on type II cells led to a rise in intracellular Ca2+, and a prolonged membrane depolarization due to opening of Panx‐1 channels. In a CB co‐culture model, where purinergic P2X2/3‐expressing petrosal neurones served as a reporter or biosensor of ATP release, we show that selective activation of P2Y2 receptors on type II cells can lead to ATP release via Panx‐1 channels. We propose that type II cells may function as amplifiers of the neurotransmitter ATP during chemotransduction, via the mechanism of ATP‐induced ATP release.

O2-sensitive K+ channels in immortalised rat chromaffin-cell-derived MAH cells.

The regulation of K+ channels by O2 levels is a key link between hypoxia and neurotransmitter release in neuroendocrine cells. Here, we examined the effects of hypoxia on K+ channels in the immortalised v‐myc, adrenal‐derived HNK1+ (MAH) cell line. MAH cells possess a K+ conductance that is sensitive to Cd2+, iberiotoxin and apamin, and which is inhibited by ≈24 % when exposed to a hypoxic perfusate (O2 tension 20 mmHg). This conductance was attributed to high‐conductance Ca2+‐activated K+ (BK) and small‐conductance Ca2+‐activated K+ (SK) channels, which are major contributors to the O2‐sensitive K+ conductance in adrenomedullary chromaffin cells. Under low [Ca2+]i conditions that prevented activation of Ca2+‐dependent K+ conductances, a rapidly activating and slowly inactivating K+ conductance, sensitive to both TEA and 4‐aminopyridine (4‐AP), but insensitive to 100 nm charybdotoxin (CTX), was identified. This current was also reduced (by ≈25 %) when exposed to hypoxia. The hypoxia‐sensitive component of this current was greatly attenuated by 10 mm 4‐AP, but was only slightly reduced by 10 mm TEA. This suggests the presence of delayed‐rectifier O2‐sensitive channels comprising homomultimeric Kv1.5 or heteromultimeric Kv1.5/Kv1.2 channel subunits. The presence of both Kv1.5 and Kv1.2 α‐subunits was confirmed using immunocytochemical techniques. We also demonstrated that these K+ channel subunits are present in neonatal rat adrenomedullary chromaffin cells in situ. These data indicate that MAH cells possess O2‐sensitive K+ channels with characteristics similar to those observed previously in isolated chromaffin cells, and therefore provide an excellent model for examining the cellular mechanisms of O2 sensing in adrenomedullary chromaffin cells.

Role of ET-1 in hypoxia-induced mitosis of cultured rat carotid body chemoreceptors.

The mammalian carotid body (CB) contains O2-chemoreceptors, i.e. glomus cells, which display increased mitoses and endothelin-1 (ET-1) expression during chronic hypoxia. To investigate whether endogenous ET-1 might mediate these mitogenic effects, we quantified bromodeoxyuridine (BrdU) uptake by tyrosine hydroxylase (TH)-positive glomus cells in rat CB cultures using double-label immunofluorescence. In normoxia (20% O2), 2-day exposure to ET-1 (10-1000 nM) caused a dose-dependent increase in BrdU uptake which peaked (approximately 55% of TH+ cells) at around 500 nM ET-1. In chronic hypoxia (5% O2) alone, BrdU uptake was stimulated (approximately 46% of TH+ cells) relative to normoxia (approximately 30%), but the effect was abolished in the presence of specific (BQ 123) or non-specific (PD 142893) ETA receptor antagonists (10(-5) M). Thus paracrine/autocrine release of ET-1 in the hypoxic carotid body may promote glomus cell mitosis via ET(A) receptors.

Postsynaptic action of GABA in modulating sensory transmission in co‐cultures of rat carotid body via GABAA receptors

GABA is expressed in carotid body (CB) chemoreceptor type I cells and has previously been reported to modulate sensory transmission via presynaptic GABAB receptors. Because low doses of clinically important GABAA receptor (GABAAR) agonists, e.g. benzodiazepines, have been reported to depress afferent CB responses to hypoxia, we investigated the potential contribution of GABAAR in co‐cultures of rat type I cells and sensory petrosal neurones (PNs). During gramicidin perforated‐patch recordings (to preserve intracellular Cl−), GABA and/or the GABAA agonist muscimol (50 μm) induced a bicuculline‐sensitive membrane depolarization in isolated PNs. GABA‐induced whole‐cell currents reversed at ∼−38 mV and had an EC50 of ∼10 μm (Hill coefficient =∼1) at −60 mV. During simultaneous PN and type I cell recordings at functional chemosensory units in co‐culture, bicuculline reversibly potentiated the PN, but not type I cell, depolarizing response to hypoxia. Application of the CB excitatory neurotransmitter ATP (1 μm) over the soma of functional PN induced a spike discharge that was markedly suppressed during co‐application with GABA (2 μm), even though GABA alone was excitatory. RT‐PCR analysis detected expression of GABAergic markers including mRNA for α1, α2, β2, γ2S, γ2L and γ3 GABAAR subunits in petrosal ganglia extracts. Also, CB extracts contained mRNAs for GABA biosynthetic markers, i.e. glutamate decarboxylase (GAD) isoforms GAD 67A,E, and GABA transporter isoforms GAT 2,3 and BGT‐1. In CB sections, sensory nerve endings apposed to type I cells were immunopositive for the GABAAR β subunit. These data suggest that GABA, released from the CB during hypoxia, inhibits sensory discharge postsynaptically via a shunting mechanism involving GABAA receptors.

Confocal immunofluorescence study of rat aortic body chemoreceptors and associated neurons in situ and in vitro

Aortic bodies (ABs) are putative peripheral arterial chemoreceptors, distributed near the aortic arch. Though presumed to be analogous to the well‐studied carotid bodies (CBs), their anatomical organization, innervation, and function are poorly understood. By using multilabel confocal immunofluorescence, we investigated the cellular organization, innervation, and neurochemistry of ABs in whole mounts of juvenile rat vagus and recurrent laryngeal (V‐RL) nerves and in dissociated cell culture. Clusters of tyrosine hydroxylase‐immunoreactive (TH‐IR) glomus cells were routinely identified within these nerves. Unlike the CB, many neuronal cell bodies and processes, identified by peripherin (PR) and neurofilament/growth‐associated protein (NF70/GAP‐43) immunoreactivity, were closely associated with AB glomus clusters, especially near the V‐RL bifurcation. Some neuronal cell bodies were immunopositive for P2X2 and P2X3 purinoceptor subunits, which were also found in nerve terminals surrounding glomus cells. Immunoreactivity against the vesicular acetylcholine transporter (VAChT) was detected in local neurons, glomus cells, and apposed nerve terminals. Few neurons were immunopositive for TH or neuronal nitric oxide synthase. A similar pattern of purinoceptor immunoreactivity was observed in tissue sections of adult rat V‐RL nerves, except that glomus cells were weakly P2X3‐IR. Dissociated monolayer cultures of juvenile rat V‐RL nerves yielded TH‐IR glomus clusters in intimate association with PR‐ or NF70/GAP‐43‐IR neurons and their processes, and glial fibrillary acidic protein‐IR type II (sustentacular) cells. Cocultures survived for several days, wherein neurons expressed voltage‐activated ionic currents and generated action potentials. Thus, this coculture model is attractive for investigating the role of glomus cells and local neurons in AB function. J. Comp. Neurol. 519:856–873, 2011.

Adenosine and dopamine oppositely modulate a hyperpolarization‐activated current Ih in chemosensory neurons of the rat carotid body in co‐culture

Adenosine and dopamine (DA) are neuromodulators in the carotid body (CB) chemoafferent pathway, but their mechanisms of action are incompletely understood. Using functional co‐cultures of rat CB chemoreceptor (type I) cells and sensory petrosal neurons (PNs), we show that adenosine enhanced a hyperpolarization‐activated cation current Ih in chemosensory PNs via A2a receptors, whereas DA had the opposite effect via D2 receptors. Adenosine caused a depolarizing shift in the Ih activation curve and increased firing frequency, whereas DA caused a hyperpolarizing shift in the curve and decreased firing frequency. Acute hypoxia and isohydric hypercapnia depolarized type I cells concomitant with increased excitation of adjacent PNs; the A2a receptor blocker SCH58261 inhibited both type I and PN responses during hypoxia, but only the PN response during isohydric hypercapnia. We propose that adenosine and DA control firing frequency in chemosensory PNs via their opposing actions on Ih.