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Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada
Submitted 13 November 2003; accepted in final form 23 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Thus in response to a fall in arterial PO2, or an increase in PCO2 or acidity, there is a compensatory increase in sensory discharge in the afferent carotid sinus nerve (CSN), which projects to the central pattern generator in the brain stem. Activation of this reflex leads to an increase in ventilation and restoration of blood PO2 and PCO2/pH. There is a consensus that CB glomus or type I cells are the sensory receptors for these three chemostimuli (Gonzalez et al. 1994; Lopez-Barneo 1996; Peers and Buckler 1995). In the case of low PO2 or hypoxia, the depolarizing receptor potential in type I cells arises primarily from the inhibition of several classes of voltage-independent (background) and voltage-dependent K+ conductances, which vary among different species (Buckler et al. 2000; Lopez-Barneo et al. 2001; Peers and Buckler 1995; Prabhakar 2000). This depolarization leads to calcium entry through voltage-dependent Ca2+ channels and secretion of neurotransmitters that excite CSN afferent nerve terminals.
Although the transduction mechanisms for hypercapnic and acidic stimuli in type I cells have received less attention, there is strong evidence that these stimuli also produce inhibition of voltage-dependent K+ channels (Lopez-Lopez et al. 1989; Peers 1990; Peers and Green 1991; Stea et al. 1991) and cause membrane depolarization and voltage-gated Ca2+ entry (Buckler and Vaughan-Jones 1993, 1994; Roy et al. 1997). The effects of hypercapnia have long been considered to be due secondarily to acidification of intracellular pH or pHi (Buckler and Vaughan-Jones 1994; Buckler et al. 1991; Iturriaga et al. 1991, 1993; Travis 1971); however, more recent studies on rabbit type I cells suggest that CO2 may also have direct effects on enhancing L-type Ca2+ currents and presumably neurotransmitter release independent of changes in pHi (Summers et al. 2002). The neurotransmitters that mediate the excitatory effects of hypercapnic and acidic stimuli in the CB are poorly understood, although it is known that both stimuli evoke catecholamine secretion from type I cells (Buerk et al. 1998; Gonzalez et al. 1994). In the rat CB, the effects of the main catecholamine, dopamine, generally appear to be inhibitory and certainly not essential for hypoxic chemoexcitation (Donnelly 1996). Recent evidence from our laboratory based on a co-culture model of rat CB type I cell clusters and dissociated petrosal neurons, favor co-release of ATP and ACh as the principal mechanism for mediating hypoxic chemoexcitation (Prasad et al. 2001; Zhang et al. 2000).
In the present study, we exploit the advantages of the co-culture model, particularly the ability to record and analyze subthreshold events in postsynaptic neurons situated close to a type I cluster, to elucidate the neurotransmitter mechanisms underlying hypercapnic and acidic chemotransmission. We present evidence that as for hypoxia, co-release of ATP and ACh appears to be an important mechanism. In addition, given that the rat CB petrosal terminals express P2X2-P2X3 purinergic receptors (Prasad et al. 2001), which are known to be pH sensitive (Stoop et al. 1997), we tested the hypothesis that acidic chemoexcitation within the physiological range of extracellular pH may consist of a postsynaptic component involving increased sensitivity of these purinergic receptors. Finally, because recent studies in rat CB suggest that cholinergic markers may be absent from type I cells in situ (Gauda et al. 2004), we used confocal immunofluorescence to investigate whether or not vesicular acetylcholine transporter (VAChT), a cholinergic protein marker, is expressed in rat type I cells in situ.
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METHODS |
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Separate cultures of dispersed CB cells or of dissociated petrosal ganglia were prepared by combined enzymatic and mechanical dissociation of the tissue as previously described (Stea and Nurse 1991, 1992; Zhong et al. 1997). The tissues were obtained from 9- to 14-day-old Wistar rat pups (Charles River, Quebec; Harlan, Madison, WI) after they were killed by decapitation after a blow to the head. All procedures for animal handling and care were carried out according to regulations of the Canadian Council on Animal care (CCAC) and institutional guidelines. Co-cultures were usually produced by first preparing monolayers containing CB type 1 cell clusters and then adding an overlay of dissociated petrosal neurons 3–5 days later. These procedures were identical to those described in detail elsewhere (Zhang et al. 2000; Zhong et al. 1997). Cultures were grown in F-12 nutrient medium supplemented with various additives (Zhong et al. 1997), at 37°C in a humidified atmosphere of 95% air-5% CO2. Electrophysiological recordings from separate petrosal (alone) or CB type I cell cultures were carried out within 3–5 days after plating; co-cultures were usually examined 3–6 days after the neurons were plated.
Electrophysiology
Nystatin perforated-patch, whole cell recording was used to measure membrane potential (current clamp) or ionic currents (voltage clamp) with the aid of an Axopatch 1D patch clamp amplifier and a Digidata 1200 A-D converter (Axon Instruments, Foster City, CA). The procedures are described in detail elsewhere (Zhang et al. 2000; Zhong et al. 1997). Current- and voltage-clamp protocols, data acquisition and analysis were performed using pCLAMP software (versions 5.5 and 8.0.1, Axon Instruments) and Axotape (version 2.02., Axon Instruments). All recordings were carried out at 
34°C, and the control (normocapnic) extracellular solution consisted of a bicarbonate/CO2-buffered saline of the following composition (in mM) 115 NaCl, 24 NaHCO3, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 12 sucrose at pH 7.4, maintained by bubbling 95% air-5% CO2. The chemosensory stimuli, i.e., isohydric or acidic hypercapnia (and in a few cases, hypoxia), were applied by a "fast-perfusion" system using a double-barreled pipette assembly as previously described (Zhong et al. 1997). In the case of isohydric hypercapnia, the pH was kept constant at 7.4 as the CO2 tension was increased by elevating Na bicarbonate (substituted for same amount of NaCl) as follows: 10% CO2 (48 mM NaHCO3); 15% CO2 (72 mM NaHCO3); and 20% CO2 (96 mM NaHCO3). In the case of acidic hypercapnia, the bicarbonate concentration was held constant at 24 mM as the CO2 tension was increased or, in a few cases, the bicarbonate concentration was reduced as the CO2 tension was held constant at 10%. Hypoxia (PO2 = 5 mmHg) was obtained by bubbling N2 gas into the control extracellular perfusate as previously described (Zhang et al. 2000; Zhong et al. 1997). Results are expressed in the text as means ± SE. For paired and multiple comparisons of current density (pA/pF) and membrane potentials, Student's paired t-test or ANOVA was used as appropriate; the nonparametric Mann-Whitney test or ANOVA was used for comparison of ratios and percentages. The level of significance was set at P < 0.05.
Drugs
Mecamylamine, hexamethonium, suramin, and acetazolamide were obtained from Sigma-Aldrich (Oakville, ON, Canada).
Confocal immunofluorescence
Cryostat sections of the carotid bifurcation from 2- to 3-wk-old rat pups were processed for immunoreactivity against tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). Animals were first anesthetized by intraperitoneal administration of Somnotol (pentabarbital sodium, 65 mg/kg) before perfusion via the aorta with phosphate-buffered saline (PBS) followed by PBS containing 4% paraformaldehyde. In a few experiments, the perfusate consisted of Streck tissue fixative (STF; Streck Laboratories, LaVista, Nebraska). The excised carotid bifurcation was then washed in PBS (3 x 5 min each) or Tris-buffered saline (TBS) and incubated overnight in 30% sucrose at 4°C. Sections (thickness, 15–18 µm) of the bifurcation containing the CB were cut in a cryostat and collected on glass slides coated with 2% silane (Sigma). After air drying, sections were stored at –20°C until ready for immunostaining. After rehydration in PBS (formaldehyde-fixed tissue) or TBS (STF tissue), sections were blocked for 30 min in 2% horse serum at room temperature and then incubated overnight at 4°C with primary antisera diluted in 1% BSA/PBS; 0.5% Triton-X100. The primary antisera were monoclonal mouse anti-TH antibody (1:50 dilution; Boehringer Mannheim, Montreal, Quebec, Canada) or polyclonal rabbit anti-TH antibody (1:1,000 dilution; Chemicon, Temecula, CA) and polyclonal goat anti-VAChT antibody (1:200; Catalog No. AB1578; Chemicon). After rinsing in PBS (3 x 10 min each), the sections were incubated in the dark for 1 h at room temperature with the secondary antibodies diluted in blocking solution (1% BSA/PBS; 0.5% Triton-X100). The following combinations of secondary antibodies were used: N-hydroxysuccinimidyl fluorophore (Cy3)-conjugated donkey anti-goat IgG (1:500 dilution) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:50; Cappel, Aurora, OH) or FITC-conjugated donkey anti-goat IgG (1:50 dilution) and Texas Red-conjugated goat anti-rabbit IgG (1:100; Jackson Research Laboratories, Westgrove, PA). Samples were washed in PBS (3 x 5 min each) and covered with Vectashield Mounting Medium (Vector Laboratories, Burlington, Ontario, Canada) before viewing under a Bio-Rad Microradiance 2000 confocal microscope, equipped with argon (2 lines, 488 and 514 nm) and helium/neon (543 nm). Lasersharp software was used for image acquisition. Two types of control experiments were carried out. First, sections were processed as described in the preceding text except that the primary antisera were omitted. No positive staining was observed in these cases. Second, the specificity of VAChT immunostaining was confirmed in experiments where the primary antibody was preincubated (24 h at 4°C) with 10x molar excess antigen or blocking peptide, corresponding to amino acids 511–530 of the rat VAChT sequence, before application to the sections. In these preabsorption experiments (n = 3), positive staining for VAChT immunofluorescence was abolished or markedly reduced.
Carbonic anhydrase (CAH) cytochemistry
The localization of carbonic anhydrase activity in carotid body cultures was carried out using a modification of Hansson's cobalt-precipitation technique. The procedures were identical to those described in detail elsewhere (Nurse 1990).
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RESULTS |
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Using perforated-patch whole cell recording, we first verified that after culture for several days, O2-sensitive type I cells in clusters displayed CO2 sensitivity as did freshly isolated single cells (Buckler and Vaughan-Jones 1994). For these studies, we examined type I cells that were members of a large cluster in which spontaneous spike activity and voltage fluctuations are commonly recorded (Zhang and Nurse 2000). As exemplified in Fig. 1A, both hypoxia and isohydric hypercapnia significantly increased spike frequency in such cells (n = 4), and when present together, the combined response was higher than that due to either stimulus acting alone. The stimulus-induced spike frequency ratio relative to control is shown for different cells exposed to one or more of these chemostimuli (Fig. 1B).
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We previously showed that functional chemosensory connections develop de novo between type 1 clusters and petrosal neurons (PN) in co-culture (Nurse and Zhang 1999; Prasad et al. 2001; Zhang et al. 2000; Zhong et al. 1997). To understand further the mechanisms underlying hypercapnic chemotransmission, the membrane potential of PN that were juxtaposed to type I clusters was monitored under current clamp during rapid perfusion of the hypercapnic stimulus. In 86% of such recordings (37/43), isohydric or acidic hypercapnia caused PN membrane depolarization that was sometimes accompanied by a burst of action potentials (Fig. 2, A and B). An increase in synaptic-like activity in the neuron was sometimes seen during stimulus application. This activity progressively increased as the CO2 tension was increased from 5 to 20% (pH = 7.4) and consisted of a complex waveform of spikes and variable-amplitude, subthreshold potentials that resembled postsynaptic potentials (Fig. 2D). These responses to hypercapnia were absent in petrosal neurons cultured alone (n = 13; Fig. 2C), suggesting they depended on chemical synaptic interactions with type I cells as previously observed for hypoxic chemotransmission (Zhong et al. 1997). Confirmation that the hypercapnia-induced responses in co-cultured PN depended on chemical transmission was obtained in experiments where the extracellular solution was switched to one containing low Ca2+ (0.1 mM) and high Mg2+ (6 mM). In all such cases (n = 7), the hypercapnic response in the neuron was markedly and reversibly inhibited (Fig. 2E) (see also Prasad et al. 2001).
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; Gonzalez et al. 1994; Hanson et al. 1981; Iturriaga et al. 1991, 1993; Travis 1971). This enzyme activity is localized intracellularly in type I cells both in situ (Nurse 1990; Ridderstrale and Hanson 1984; Rigual et al. 1985) and in culture (Fig. 3D) (see also Nurse 1990) and catalyzes the hydration of CO2, producing very rapid changes in intracellular pH (pHi) in response to extracellular changes in PCO2 (Buckler et al. 1991). As exemplified in Fig. 3A, the receptor potential recorded in type I cells during brief (4 s) application of the hypercapnic stimulus was reversibly abolished in the presence of the permeable CAH inhibitor, 10 µM acetazolamide (ACZ; n = 9). Because the magnitude of this receptor potential is expected to determine the amount of neurotransmitter release from type I cells, we predicted that ACZ should also inhibit the postsynaptic afferent response in co-cultured PN. Indeed, as shown in Fig. 3, B and C, the increase in spike discharge induced by isohydric hypercapnia (10% CO2; pH =7.4) in co-cultured PN was reversibly abolished in the presence of 10 µM ACZ (n = 5).
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), it was of interest to contrast the effects of ACZ during a prolonged PCO2 stimulus. As illustrated in Fig. 4, A and B, the initial or transient response recorded in co-cultured neurons during hypercapnia was larger than that seen at the end of a prolonged stimulus (1 min). The maximum response, measured as an increase in spike frequency (Fig. 4A, inset) or a subthreshold depolarization (Fig. 4, B and C), usually occurred within 12 s of stimulus application and declined over the next 40–50 s. In the presence of 10 µM ACZ, the transient response was considerably reduced, whereas the response near the end of the prolonged stimulus was hardly affected (Fig. 4, A–C). These data are consistent with the notion that CAH activity contributes to the rapid onset and overshoot of the CO2 response in the carotid body (Buckler et al. 1991; Gonzalez et al. 1994; Iturriaga et al. 1991, 1993; Travis 1971).
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Hypoxic chemotransmission in similar co-cultures is mediated principally by co-release of ACh and ATP from type I cells (Zhang et al. 2000). To investigate whether the same is true for hypercapnia, we tested the effects of nicotinic and purinergic receptor blockers separately, and in combination, on the neuronal response in co-culture. Two different nicotinic blockers, 200 µM hexamethonium or 1 µM mecamylamine, partially inhibited the response to isohydric hypercapnia (10% CO2) as exemplified by the co-cultured neurons in Fig. 5, A and B, respectively. Additionally, the purinergic blocker suramin (50 µM) also partially inhibited the response (Fig. 5B, middle) in the same neuron that was inhibited by 1 µM mecamylamine (see also Prasad et al. 2001). Co-release of ATP and ACh appeared to be the major neurotransmitter mechanism because combined application of 50 µM suramin and 1 µM mecamylamine almost completely inhibited the hypercapnic response in this neuron (Fig. 5B, bottom). In 12 similar cases, the mean neuronal depolarization evoked by 10% CO2 was 12.3 ± 2.5 mV (control) compared with 4.2 ± 1.7 mV in the presence of 1 µM mecamylamine, 5.1 ± 1.9 mV in the presence of 50 µM suramin, and 0.8 ± 0.2 mV in the presence of both drugs. The same conclusion was reached in other co-cultured neurons that showed an increase in spike discharge during isohydric hypercapnia (n = 4). For example in Fig. 6 (top and middle), the CO2-induced spike discharge was reduced in a dose-dependent manner when the two blockers (suramin and mecamylamine) were applied separately. However, combined application of these blockers was more effective, producing complete inhibition of the discharge at concentrations that caused only partial inhibition when applied separately (Fig. 6; bottom). For this neuron, mean spike frequency was 11.2 ± 1.7 Hz (n = 3 trials) in control (10% CO2) versus 2.1 ± 0.4 Hz in 1 µM mecamylamine, 1.6 ± 0.5 Hz in 50 µM suramin, 0 Hz in 1 µM mecamylamine plus 50 µM suramin, and 7.2 ± 1.0 Hz after washout of both drugs.
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Because the carotid body acts as a pH sensor, independent of the effects of CO2 (Biscoe et al. 1970; Buckler and Vaughan-Jones 1993; Lopez-Lopez et al. 1989; Peers 1990; Stea et al. 1991; Summers et al. 2002; Wilding et al. 1992), we tested whether or not this property is retained in the co-culture model. Indeed, at a constant CO2 tension, the chemoexcitatory neuronal response in co-culture progressively increased as the pH of the extracellular perfusate decreased after reduction of the bicarbonate concentration (Fig. 7A). In general, the frequency of postsynaptic activity (spikes plus subthreshold EPSPs) recorded in co-cultured neurons was enhanced during acid hypercapnia (10% CO2/pH = 7.2) relative to isohydric hypercapnia (10% CO2/pH = 7.4) as exemplified in Fig. 7, B and C. The ratio of the frequency of synaptic events during isohydric and acid hypercapnia relative to control (normocapnia; 5% CO2/pH = 7.4) for a group of 6 cells is shown in Fig. 7D. It is conceivable that the increase in frequency of detectable EPSPs under these acidic conditions may be due, at least in part, to the acid sensitivity of the postsynaptic P2X receptors on petrosal neurons (see following text).
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Acidity is known to affect differentially the sensitivity of P2X receptors to ATP and purinergic agonists depending on receptor subtype (Li et al. 1996; Stoop et al. 1997). We proposed that heteromeric P2X2-P2X3 receptors were likely to be the functional ones expressed by carotid body chemosensory neurons (Prasad et al. 2001; Zhang et al. 2000). Because the effect of ATP on these heteromeric receptors is potentiated by acidity in native nodose neurons (Li et al. 1996) and in heterologous expression systems (Stoop et al. 1997), we considered the possibility that the pH sensitivity of postsynaptic P2X receptors may contribute to the carotid body response during extracellular acidity. We first identified neurons that responded to acid hypercapnia in co-culture as in Fig. 10A and then tested the pH sensitivity of ATP-induced whole cell currents in the same neurons. The dose of ATP (10 µM) used in these experiments was subsaturating, based on previous studies on the ATP dose-response relation for the purinergic receptors on functional petrosal neurons (Zhang et al. 2000). As shown in Fig. 10, B and C, the ATP-induced inward current at a holding potential of –60 mV was potentiated by acidic pH (7, 7.2) and inhibited by alkaline pH (7.6) in identified chemosensory neurons. Thus the pH sensitivity of postsynaptic P2X receptors on petrosal afferents may contribute to acidic chemoreception in the carotid body.
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The evidence presented in the preceding text, together with that from previous studies on similar co-cultures and the isolated carotid body-sinus nerve preparation (Zhang et al. 2000), provide strong support for the involvement of ACh as a co-transmitter during both hypercapnic and hypoxic chemotransmission in the rat carotid body. This view was recently challenged on the basis of the absence of cholinergic markers in type I cells of sectioned rat CB using in situ hybridization and immunocytochemical techniques (Gauda et al. 2004). To validate that cholinergic protein markers are indeed expressed in rat type I cells in situ, we used confocal immunofluorescence to localize the vesicular acetylcholine transporter (VAChT), an established cholinergic marker (Eiden 1998). Using double-label immunofluorescence on tissue sections of 2-wk-old rat CB, we found that type I cell clusters identified by positive tyrosine hydroxylase (TH)-immunofluorescence (Alexa) werealso immunopositive positive for VAChT (Cy3) as illustrated in Fig. 11, A and B, respectively. This result was confirmed in tissue sections from five different animals, and in each case, visualization at higher power verified there was co-localization of TH and VAChT immunostaining. In control experiments (n = 3), a 24-h preincubation of the primary VAChT antiserum with 10x molar excess of competing or blocking peptide, corresponding to amino acids 511- 530 of the VAChT sequence, resulted in abolition of staining (Fig. 11C); TH immunostaining of type I cells is evident in the same section (Fig. 11D). In other control experiments, omission of either primary antibody alone resulted in abolition of all immunofluorescence even though the secondary antibodies were present. Taken together, these data demonstrate that cholinergic markers are expressed in (Wistar) rat type I cells in situ.
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DISCUSSION |
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; Zhang et al. 2000, 2003; Zhong et al. 1997). The main conclusion from the present studies is that CO2/pH signaling is dependent on co-release of ATP and ACh from type I cells, and, consistent with this view, these cells were shown to express the cholinergic marker, VAChT, in rat CB tissue sections in situ. Although expression of cholinergic markers was not detected in rat type I cells by in situ hybridization and immunocytochemical techniques (Gauda et al. 2004), for reasons that may be related to strain and/or methodological differences, there is recent evidence that both hypercapnia and acidity stimulate ACh release from whole rabbit CB as detected by HPLC and that ACh itself is detectable in type I cells by immunocytochemistry (Kim et al. 2004). Additionally, our data raise the possibility that the acid sensitivity of P2X2-P2X3 purinoceptors, present on petrosal neurons and their afferent terminals (Prasad et al. 2001), may contribute to the CB chemoexcitatory response evoked by extracellular acidity. Co-release of ATP and ACh was previously reported to be the main mechanism for signaling hypoxia in similar co-cultures as well as in the intact CB-sinus nerve preparation in vitro (Zhang et al. 2000). More recently, a similar mechanism was proposed for the mediation of chemosensory responses induced by stop flow and acidity in the cat CB (Varas et al. 2003). A pivotal role for ATP and the purinergic P2X2 subunit in the ventilatory response to hypoxia was recently confirmed using a transgenic mouse model (Rong et al. 2003). In the latter study, however, the response to hypercapnia was unaffected probably because central chemoreceptors were still functional. Mechanisms of CO2 signaling by afferent neurons
Prior to investigating the neurotransmitter basis for CO2/pH chemotransmission we first confirmed that rat type I cell clusters retained the ability to sense hypercapnia after several days in culture (see also Sato 1994). In larger clusters, where type I cells show voltage fluctuations and occasional spike activity (Zhang and Nurse 2000), isohydric hypercapnia (10% CO2/pH = 7.4) increased spike discharge. In co-culture, isohydric hypercapnia induced depolarizing responses (spikes and/or subthreshold potentials) in many petrosal neurons. Such responses were absent in petrosal neurons cultured alone, consistent with type I cells acting as the primary receptors for this stimulus. Frequently, increased CO2 caused a burst of action potentials in the co-cultured neuron within a few seconds of stimulus application. These responses were inhibited after reduction of the extracellular Ca2+:Mg2+ ratio as expected if chemical transmission was involved. Moreover, the transient or rapid neuronal response to an increase in CO2 was inhibited by the permeable carbonic anhydrase inhibitor, acetazolamide (ACZ; 10 µM), whereas the reduced response remaining after prolonged stimulus application was hardly affected. Carbonic anhydrase is expressed intracellularly in type I cells (Nurse 1990; Ridderstrale and Hanson 1984; Rigual et al. 1985) (see also Fig. 3D), and in the present study, ACZ also abolished the rapid type I cell depolarization due to isohydric hypercapnia, presumably via slowing the rate of fall of intracellular pH or pHi (Buckler et al. 1991). These data are consistent with the previously described roles of carbonic anhydrase in regulating the speed and magnitude of the initial responses of the carotid body to CO2 rather than the steady-state responses (Buckler et al. 1991; Gonzalez et al. 1994; Iturriaga et al. 1991, 1993).
Co-release of ATP and ACh from type I cells appeared to be the main mechanism mediating CO2 chemotransmission in co-culture as previously reported for hypoxia (Zhang et al. 2000). In particular, separate application of nicotinic (mecamylamine or hexamethonium) and purinergic (suramin) blockers only partially inhibited the neuronal CO2 response in co-culture (see also Prasad et al. 2001), whereas combined application of both blockers abolished most or all of the response. The same conclusion was reached whether the neuronal response was sub- or suprathreshold, suggesting that conduction block in the nerve terminals was not a complicating factor. We previously reported that P2X2 and P2X3 purinoceptor subunits are expressed on petrosal chemoafferent nerve terminals in the rat CB in situ, where they appear to mediate the ATP component of the postsynaptic response during hypoxia via heteromultimeric P2X2-P2X3 receptors (Prasad et al. 2001; Zhang et al. 2000; see, however, Rong et al. 2003). Although the functional subunits of the nicotinic ACh receptors (nAChR) on petrosal afferents remain to be determined, there is evidence that these neurons express a variety of nAChR subtypes (Fitzgerald 2000) and are sensitive to ACh (Nurse and Zhang 1999; Varas et al. 2003; Zhang et al. 2000; Zhong and Nurse 1997). Therefore the simplest explanation of our results is that ATP and ACh, released from type I cells during hypercapnia, act on postsynaptic P2X2-P2X3 and nicotinic receptors to produce their excitatory effects. However, the possibility that during increased CO2 these same neurotransmitters are also involved in autocrine-paracrine modulation of type I cell responses cannot be excluded (Mokashi et al. 2003; Nurse and Zhang 1999; Xu et al. 2003).
Mechanisms of acidic chemotransmission
In this study, we also examined the effects of varying extracellular acidity under constant CO2 tension in co-culture. Acidity within the physiological range (pH = 7.0–7.2) increased the chemosensory response recorded in co-cultured neurons when the CO2 tension was maintained at 10%. In these cases, the enhanced neuronal response appeared as an increased frequency of action potentials and/or subthreshold potentials that resembled EPSPs seen at conventional chemical synapses. Moreover, the presence of acetazolamide did not noticeably affect the rapid responses induced by acid hypercapnia (10% CO2; pH = 7.1), suggesting the effects of acidity were independent of CO2.
The neurotransmitter mechanisms mediating the effects of acid hypercapnia appeared similar to those discussed in the preceding text for isohydric hypercapnia. In particular, the response was partially inhibited when either mecamylamine or suramin was present alone but was abolished when both drugs were present together. These data suggest that co-release of ATP and ACh from type I cells is the main mechanism mediating the effects of both acidic and isohydric hypercapnia in the rat CB. A similar mechanism was recently proposed as the basis for acidic chemoexcitation in the isolated cat sinus nerve-CB preparation (Varas et al. 2003), although in this case the acidic stimulus was severe (pH = 6.8 –7) and subthreshold postsynaptic events were not recorded. Additionally, our studies uncovered a potential postsynaptic mechanism for enhancing the CB response to extracellular acidity within the physiological range (pH = 7–7.3). In identified chemosensory neurons that responded to acidity in co-culture, we found that the ATP-evoked inward current at –60 mV was enhanced by acidic (pH = 7.0–7.2) and suppressed by alkaline (pH = 7.8) conditions. This reflects the expected pH sensitivity of homomeric P2X2 and heteromeric P2X2-P2X3 purinoreceptors (Stoop et al. 1997) of which the latter are the presumed functional ones in chemosensory petrosal afferent terminals (Prasad et al. 2001; Zhang et al. 2000). Therefore the pH sensitivity of these receptors represents a likely postsynaptic mechanism by which ATP released from type I cells contributes to acidic chemoreception in the CB. Recent studies suggest that ATP may also have presynaptic effects via autocrine-paracrine actions on neighboring type I or type II cells (Mokashi et al. 2003; Xu et al. 2003). It remains to be determined whether other CB neurotransmitters (e.g., 5-HT, GABA, dopamine) mediate autocrine-paracrine modulation of the chemosensory response during acid hypercapnia as recently reported for hypoxia (Fearon et al. 2003; Zhang et al. 2003).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. A. Nurse, Dept. of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario L8S 4K1, Canada (E-mail: nursec{at}mcmaster.ca).
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REFERENCES |
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) vs. acid (
) hypercapnia in cells exposed to the 3 treatments. Data are means ± SE and indicate significant (P < 0.01) effects of hypercapnia and acidity on neuronal activity. The resting potential was –73 mV in A and –67 mV in B.
