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The Journal of Physiology logoLink to The Journal of Physiology
. 2002 Mar 1;539(Pt 2):503–510. doi: 10.1113/jphysiol.2001.013071

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

X W Fu *, C A Nurse *, V Wong *, E Cutz *
PMCID: PMC2290169  PMID: 11882682

Abstract

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.


Oxygen availability plays a pivotal role in many cellular processes, and therefore it is not surprising that most biological systems elaborate a variety of mechanisms for sensing oxygen and maintaining PO2 homeostasis (Semenza 1999; Lopez-Barneo et al. 2001). In neuronal cells, responses to a decrease in oxygen availability or hypoxia include both facilitation and inhibition of neurotransmitter release (Gibson & Peterson, 1981; Gibson et al. 1991). For example, hypoxia may increase catecholamine release (Hirsch & Gibson, 1984; Peterson & Gibson, 1984), or inhibit acetylcholine release (Gibson & Peterson, 1981; Freeman et al. 1987) from brain cells. In a peripheral chemosensory organ, the mammalian carotid body, hypoxia stimulates catecholamine release from specialized O2-chemoreceptor (glomus) cells, whether present in the intact organ (Fidone et al. 1982; Donnelly, 1993), in tissue slices (Pardal et al. 2000), or as isolated cells or cell clusters in vitro (Urena et al. 1994; Montoro et al. 1996; Jackson & Nurse, 1997). Hypoxia also stimulates catecholamine release from neonatal adrenal chromaffin cells (Mojet et al. 1997; Thompson et al. 1997) and from PC-12 cells (Taylor & Peers, 1998; Kumar et al. 1998), an O2-sensitive cell line derived from the adrenal medulla. In particular, hypoxia causes inhibition of K+ channels, leading to increased membrane depolarization or action potential frequency, entry of extracellular calcium and amine secretion (Lopez-Barneo et al. 2001).

The neuroepithelial bodies (NEB) are specialized pulmonary structures composed of clusters of innervated amine- and peptide-containing cells, widely distributed within airway mucosa of human and animal lung (Lauweryns et al. 1972; Sorokin & Hoyt, 1989; Cutz et al. 1997). NEB cells are preferentially located at or near airway bifurcations, a site ideally suited for sensing changes in airway gas concentration. It has been reported that NEB cells express a membrane-bound O2 sensor protein, NADPH oxidase, which appears to function in association with certain K+ channels (Wang et al. 1996; Fu et al. 1999). In these specialized receptor cells, hypoxia inhibits Ca2+-dependent and Ca2+-independent K+ channels, leading to an increase in cell firing or a facilitation of membrane depolarization (Youngson et al. 1993; Fu et al. 1999). These events are proposed to stimulate neurotransmitter release, which in turn activates second order vagal chemoafferent neurons (Fu et al. 2001). Though the predominant amine in mammalian and human NEB cells is 5-HT, as revealed by immunohistochemistry, its function in lung physiology is unknown. The secretory status of these cells is further complicated by the presence of other regulatory peptides including bombesin, calcitonin gene related peptide (CGRP), cholecystokinin (CCK) and substance P (Cutz et al. 1997). Previous studies have provided indirect evidence suggesting that acute hypoxia leads to 5-HT release from NEB cells both in vivo and in vitro. Lauweryns et al. (1978) reported that acute airway hypoxia, but not hypoxaemia, caused increased exocytosis of dense core granules from neonatal rabbit NEB cells, as revealed by electron microscopy and by the spectrofluorometric decrease in amine fluorescence. Studies in our laboratory, using cultures of NEB cells isolated from late fetal rabbit lungs, have suggested that these cells show graded responses to acute hypoxia, based on determination of their 5-HT content by HPLC (Cutz et al. 1993).

In the present study, we investigated the effects of acute hypoxia on 5-HT release from intact NEB cells using neonatal rabbit lung slices and carbon fibre amperometry. The latter technique can resolve amine release from individual dense core vesicles at the single cell level (Alvarez de Toledo et al. 1993). We obtained direct evidence that hypoxia-induced 5-HT secretion is an intrinsic property of NEB cells in newborn rabbit lung. We further characterized the cellular mechanisms underlying this secretory response, including the response to graded hypoxia, the involvement of voltage-gated Na+ and Ca2+ channels, as well as the effect of 5-HT3 autoreceptors on hypoxia-induced 5-HT release from intact NEB clusters.

METHODS

Lung slice preparation

For electrophysiological recordings, lung tissues from newborn New Zealand white rabbits (1–6 days of age) of both sexes were used. All experiments were carried out with the approval of the local ethics committee and in accordance with the Institutional Guidelines for Animal Care. The rabbits were killed by an intraperitoneal injection of Euthanyl (100 mg kg−1; Bimeda-MTC, Cambridge, Ontario, Canada). The lung slices were prepared after embedding the tissue in 2 % agarose (FMC Bioproducts, Rockland, ME, USA) as previously described (Fu et al. 1999). Sectioning was performed with tissue immersed in ice cold Krebs solution that had the following composition (mm): 140 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl3, 10 Hepes and 5 glucose adjusted to pH 7.3 with NaOH. Transverse lung slices (200–300 μm) were cut with a Vibratome (Ted Pella, Inc., Redding, CA, USA).

5-HT immunohistochemistry

For immunohistochemical localization of 5-HT in NEB cells, lungs from neonatal rabbits (2 days old) were fixed in 4 % paraformaldehyde for 5 h, immersed in phosphate buffered solution (PBS) and stored at 4 °C until use. Lung slices (50–60 μm) were cut using a Vibratome. To block non-specific staining, the slices were incubated with 3 % H2O2 in absolute methanol followed by normal rabbit serum. The slices were then incubated overnight at 4 °C with a monoclonal antibody against 5-HT (1:100; Medicorp, Montreal, Canada). Biotinylated rabbit-anti rat IgG (Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody. The reaction was visualized using ABC-Kit (Vector) and DAB as a chromogen. The slices were mounted in water soluble universal mount (Research Genetics, Inc.)

Amperometric detection of 5-HT secretion from NEBs

For amperometric measurements of 5-HT secretion from NEB cells, fresh lung slices were transferred to a recording chamber mounted on the stage of a Nikon microscope (Optiphot-2UD, Nikon, Tokyo, Japan). The perfusing Krebs solution had the following composition (mm): 130 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 10 NaHCO3, 5 Hepes and 10 glucose at pH 7.35 −7.4. To identify NEB cells in fresh lung tissue, the slice was incubated with the vital dye neutral red (0.02 mg ml−1) for 15 min at 37 °C as previously described (Youngson et al. 1993; Fu et al. 1999). All recordings were made from submerged lung slices at room temperature. Carbon fibre microelectrodes (ProCFE, Dagan Corporation, 5 μm diameter) used in this study consisted of polypropylene-insulated carbon fibre designed for the detection of oxidizable analytes. Detectable neurotransmitters include catecholamines (adrenaline (epinephrine), noradrenaline (norepinephrine) and dopamine), and indolamines (5-HT). The ProCFE microelectrode oxidizes these compounds within a voltage range of 600–900 mV. The microelectrodes were positioned adjacent to NEB cells and were polarized to +700 mV, a potential more positive than the oxidation potential of 5-HT released by NEB cells. All potentials are reported with respect to an Ag-AgCl reference electrode. The carbon fibre microelectrode was filled with 3 m KCl as the internal solution and calibrated by adding known amounts of 5-HT to the medium. Quantal secretion was detected as transient upward current deflections, each arising from the oxidation of the released contents from a single vesicle containing 5-HT (Alvarez de Toledo et al. 1993). Currents were recorded using an Axopatch 200B amplifier (with extended voltage range), filtered at 1 kHz and digitized at 2 kHz before storage on a personal computer. All data acquisitions were performed using a Digidata 1200 interface and Fetchex software from pCLAMP 6.04 (Axon Instruments, Foster City, CA, USA). Exocytosis was expressed as the frequency of quantal events, determined with the aid of the Mini Analysis Program (Synaptosoft Inc., Leonia, NJ, USA) by counting the number of secretory events over a 60 s period, beginning 15 s after switching to the test solution. All data were expressed as means ± s.e.m. and statistical comparisons were made using the unpaired t test, with the level of significance set at P < 0.05.

Normoxic solutions were equilibrated with room air. Test solutions were equilibrated with the appropriate gas mixture by bubbling for at least 30 min before perfusion of the slices. The solution flow rate was ∼6 ml min−1 and the pH remained relatively constant. The carbon fibre microelectrode was calibrated by equating the recorded current in saline equilibrated with air to a PO2 of 159 mmHg, and by assuming that the electrode passes no current during true anoxia (Mojet et al. 1997). The PO2 levels tested in these experiments included 95 mmHg (mild hypoxia), 75 mmHg (moderate hypoxia) and 18 mmHg (severe hypoxia). Drugs were applied via the perfusate and their delivery to the cells was controlled by separate valves. TEA (tetraethylammonium), 4-AP (4-aminopyridine) and TTX (tetrodotoxin) were obtained from Sigma (Oakville, Ontario, Canada). Nifedipine, ω-conotoxin GVIA toxin (ω-CgTx), and ICS 205 930 (3-tropanyl-indole-3-carboxylate) were obtained from RBI (Research Biochemicals Inc., Natick, MA, USA). Stock solutions of all drugs were prepared in distilled water on the day of the experiment and diluted with Krebs solution to the desired final concentration before use.

RESULTS

Immunohistochemical localization of 5-HT in NEB cells

In lung slices immunostained for 5-HT, NEB appeared as well-defined organoid cell clusters localized within the airway epithelium (Fig. 1). The overall size, distribution and appearance of NEBs identified by 5-HT immunostaining in fixed lung tissue sections corresponded to those observed in fresh lung slices stained by the vital dye neutral red. In agreement with previous studies, NEB cells were preferentially located near or at airway branch points and their apical surface was exposed to the airway lumen (Fig. 1, inset).

Figure 1. Low magnification view of neonatal rabbit lung slice immunostained for 5-HT.

Figure 1

A small bronchiole (BR) with NEB cell cluster stongly positive for 5-HT located at airway bifurcation(arrow). PA, pulmonary artery. (Immunoperoxidase method for 5-HT; scale bar represents 100 μm). Inset, higher magnification of airway epithelium with two 5-HT immunoreactive NEBs facing each other with their apical surfaces exposed to airway lumen (arrowhead). (Immunoperoxidase method for 5-HT; scale bar represents 5 μm.)

Hypoxia-induced 5-HT secretion from NEB cells

The secretory activity of individual NEB cells within a cluster was studied by measuring the current resulting from the oxidation of released 5-HT molecules. The secretory response to hypoxia during recordings from an intact NEB cell in a rabbit lung slice is illustrated in Fig. 2A. When slices were perfused under normoxic conditions with solution containing normal extracellular K+ (5 mm), few or no secretory events were detected in NEB cells (n = 105). However, after switching to a hypoxic solution (PO2 ∼18 mmHg), NEB cells responded with a progressive increase in amperometric spike frequency. A similar secretory pattern was reported for carotid body type 1 cells (Pardal et al. 2000) and PC-12 cells (Taylor & Peers, 1998). The relationship between PO2 and 5-HT secretion for NEB cells is shown in Fig. 2B. There was a graded increase in 5-HT release as PO2 decreased, with the highest release occurring at the lowest PO2 (18 mmHg) tested. The mean spike frequency was 0.0054 ± 0.003 Hz (n = 30), 0.08 ± 0.008 Hz (n = 8), 0.15 ± 0.02 Hz (n = 8), and 0.38 ± 0.05 Hz (n = 18) when the PO2 was 155, 95, 75 and 18 mmHg, respectively.

Figure 2. Amperometric recording from individual NEB cells.

Figure 2

A, example of exocytosis induced from NEB cells by perfusion with a hypoxia solution (PO2 = 18 mmHg). At the point indicated by the arrow, the perfusate was changed to hypoxia solution. A, bottom left panel, a large spike-like exocytotic event is shown on an expanded time base. Scale bars apply to traces A, C and D. A, bottom right panel, frequency distribution of the charge of secretory events evoked by hypoxia in six different cells. B, effects of 4 different levels of partial pressures of oxygen in solution on secretory response from NEB cells. Each point represents the mean values of between 8 and 18 cells. The vertical bars show the mean ± s.e.m. * P < 0.05. C, effect of TTX on hypoxia-induced secretory response. Note that exocytosis was abolished by hypoxia plus 1 μm TTX; all recordings are from one cell. D, secretory response of an NEB cell to high extracellular potassium (50 mm). E, bar graph showing mean frequency of exocytosis induced by hypoxia with 5 mm K+ and normoxia with 50 mm K+ extracelluar solution. Each bar represents mean ± s.e.m. determined from the number of cells indicated in parentheses.

The average (± s.e.m.) quantal charge of secretory events during hypoxia was 33.1 ± 2.4 femtocoulombs (fC; n = 157 events from 6 cells, range 2.3–183 fC, Fig. 2A, bottom right). This value was obtained from the time integral of selected spikes with the fast rising phase and slow decay (Fig. 2A, bottom left) typical of secretory events occurring at the membrane facing the amperometric electrode (Bruns & Jahn, 1995; Bruns et al. 2000; Pardal et al. 2000). Assuming that one 5-HT molecule contributes an average of 4 electrons (Bruns & Jahn, 1995), it is estimated that a single synaptic vesicle or quantum in NEB cells releases an average of 13 000 ± 971 (n = 157; 6 cells) molecules of 5-HT. These values are at the lower end of the range reported for large dense cored vesicles (15000–300000) but higher than small vesicles (∼ 4700) in leech 5-HT-secreting Retzius cells (Bruns & Jahn, 1995; Bruns et al. 2000).

It was previously shown that hypoxia increased action potential frequency in rabbit NEB clusters grown in tissue culture (Youngson et al. 1993). Therefore we investigated whether or not the hypoxia-induced increase in 5-HT release was sensitive to TTX, a blocker of Na+-dependent action potentials in these cells. As illustrated in Fig. 2C, under normoxic conditions, no secretory events were detected from NEB cells; application of the hypoxic stimulus (PO2 = 18 mm Hg) in the presence of 1 μm TTX also caused no detectable 5-HT release, though significant release occurred in the same cell when hypoxia was applied after wash out of TTX (0.29 ± 0.04 Hz, n = 7).

As expected for electrically excitable cells with voltage-dependent K+ and Ca2+ channels, all NEB cells exhibited secretory responses when exposed to high extracellular K+ (50 mm; Fig. 2D). The mean spike frequency in the presence of 50 mm K+ was 0.33 ± 0.07 Hz (n = 10; Fig. 2E), a value comparable to that seen during severe hypoxia (PO2 ∼18 mmHg; Fig. 2E). Taken together, these findings indicate that NEB cells increase 5-HT release during both hypoxia and perfusion with high extracellular K+. Further, secretion induced by hypoxia was particularly sensitive to PO2 over the range 95–18 mmHg and was mediated by Na+-dependent action potentials.

TEA and 4-AP induce 5-HT secretion from NEB cells

Previous studies reported that hypoxic sensitivity of NEB cells is mediated by inhibition of voltage-gated, Ca2+-dependent and Ca2+-independent K+ channels, which could also be blocked by TEA and 4-AP (Youngson et al. 1993; Fu et al. 1999). Therefore, if these channels are open in intact NEB cells under normoxic conditions, then exposure to either TEA or 4-AP should mimic hypoxia in evoking 5-HT release. As illustrated in Fig. 3A and B, both TEA (20 mm) and 4-AP (2 mm) separately induced a secretory response under normoxic conditions; the amperometric spike frequency was 0.23 ± 0.02 Hz (n = 8) for TEA and 0.2 ± 0.04 Hz (n = 5) for 4-AP. These data are reminiscent of those obtained in a recent study on intact O2 chemoreceptors from tissue slices of rat carotid body, where TEA (5 mm) potentiated the secretory response under normoxic conditions (Pardal et al. 2000).

Figure 3. Secretory response of NEB cells to TEA and 4-AP.

Figure 3

A, example of an amperometric recording showing excytosis in normoxic solution (5 mm K+) containing 20 mm TEA. B, example recording showing exocytosis in normoxic solution (5 mm K+) containing 2 mm 4-AP. C, example recording illustrating lack of potentiation of the effect of hypoxia by TEA. D, bar graph showing mean frequency of exocytosis evoked by TEA and 4-AP in the absence or presence of hypoxia (PO2 = 18 mmHg). Each bar represents mean ± s.e.m. determined from the number of cells indicated in parentheses.

We next examined whether hypoxia-evoked 5-HT secretion from NEB cells is inhibited by the presence of the K+ channel blockers TEA or 4-AP, as expected if the underlying K+ channels mediate hypoxic chemosensitivity. This was indeed the case as shown in Fig. 3C and D, where the presence of either drug occluded the stimulatory effect of hypoxia on secretion. For example in Fig. 3D, when hypoxia was combined with TEA (20 mm) the amperometric spike frequency was 0.24 ± 0.03 Hz (n = 7), a value not significantly different from that observed with TEA alone (0.23 ± 0.02 Hz, n = 8). Similarly in Fig. 3D, combination of hypoxia with 2 mm 4-AP induced a spike frequency of 0.21 ± 0.04 Hz (n = 7), which was not significantly different from 4-AP alone (0.2 ± 0.04 Hz, n = 5).

L-type Ca2+ channels mediate hypoxia-induced 5-HT release from NEB cells

In NEB cells hypoxia-induced closure of K+ channels is proposed to evoke membrane depolarization and increased cell firing, Ca2+ influx and 5-HT secretion (Youngson et al. 1993; Fu at al. 1999). In order to test that this secretion involves Ca2+ influx through voltage-gated Ca2+ channels, 5-HT release was studied in NEB cells exposed to hypoxia in the presence of extracellular solution containing the non-selective Ca2+ channel blocker Cd2+ (200 μm; Fig. 4A and D). In addition, NEB cells were exposed to Ca2+-free medium (containing 1 mm EGTA) during hypoxia (data not shown). In both cases, exocytosis was almost completely abolished, suggesting an absolute dependence of hypoxia-induced 5-HT release on Ca2+ influx. In order to investigate the type(s) of Ca2+ channels mediating the secretory response, hypoxia-induced 5-HT release was monitored while perfusing the slice preparation with 1 μm ω-CgTx (Fig. 4B), a specific N-type Ca2+ channel blocker, or 2 μm nifedipine, a specific L-type Ca2+ channel blocker. As shown in Fig. 4D, NEB cells exposed to hypoxia plus nifedipine showed a mean amperometric spike frequency of 0.016 ± 0.006 Hz (n = 7, P < 0.05), corresponding to ∼90 % inhibition of release relative to hypoxia alone. In contrast, exposure to hypoxia plus ω-CgTx (1 μm) caused only a slight suppression of secretion (0.31 ± 0.1 Hz, n = 7), which was not statistically significant (Fig. 4D). These studies indicate that hypoxia enhances Ca2+ entry into NEB cells and triggers exocytosis primarily through L-type Ca2+ channels.

Figure 4. Effects of Ca2+ channels blockers and 5-HT3-R blocker on hypoxia-induced secretory reponses.

Figure 4

A, example of an amperometric recording of exocytosis evoked from an NEB cell following exposure to hypoxia (PO2 = 18 mmHg). Excytosis was abolished by switching the solution to one of the same PO2, containing 200 μm Cd2+. B, example of amperometric recording of exocytosis evoked from NEB cell following exposure to L-type Ca2+ blocker, 1 μm ω-CgTx. C, example of an amperometric recording of exocytosis evoked from an NEB cell following exposure to the 5-HT3-R blocker, 50 μm ICS 205 930. D, bar graph showing the mean frequency of exocytosis evoked by hypoxia (no blockers) in the presence of different selective blockers of voltage-gated Ca2+ channels and 5-HT3-R blocker. Cells were exposed to Cd2+ (200 μm), nifedipine (2 μm), ω-CgTx (1 μm), or ICS 205 930 (50 μm), as indicated under each bar. Each bar represents mean ± s.e.m. determined from the number of cells indicated in parentheses. * P < 0.05.

Effect of 5-HT3-receptor blockade on hypoxia-induced 5-HT release

In previous studies, we have shown co-expression of both 5-HT and 5-HT3 receptors (5-HT3-R) in the same NEB cells using immunocytochemistry (Fu et al. 2001). We have also shown that NEB cells express functional 5-HT3-R ligand-gated ion channels using the whole-cell patch clamp technique (Fu et al. 2001). These studies raise the possibility that 5-HT3-R in NEB cells may function as autoreceptors which modulate hypoxic signalling via autocrine or paracrine mechanisms. In order to test this idea, the secretory response of NEB cells exposed to hypoxia was studied in the presence of ICS 205 930 (50 μm), a specific 5-HT3-R blocker. As illustrated in Fig. 4C, the mean amperometric spike frequency was reduced to 0.12 ± 0.04 Hz (n = 7, P < 0.05) in hypoxia plus ICS 205 930 (Fig. 4D), corresponding to ∼68 % inhibition of the response to hypoxia alone. This finding suggests that during hypoxia activation of 5-HT3 autoreceptors facilitates 5-HT release from NEB cells by a positive feedback mechanism.

DISCUSSION

The present study is the first to demonstrate quantal release of 5-HT from intact NEB cells in response to acute hypoxia. We used the amperometric technique which utilizes polarized carbon fibre microelectrodes to oxidize electroactive species at their surface (Taylor et al. 1998). This high resolution technique can detect only certain transmitter species (particularly amines) but has advantages over membrane capacitance measurements, since it is non-invasive, and secretion does not overlap with endocytotic events (Wightman et al. 1997; Chow et al. 1992; Taylor et al. 1998; Pardal et al. 2000). Our findings demonstrate that hypoxia enhances 5-HT release from NEB cells in newborn rabbit lung. The partial pressure of oxygen in tracheal air is ∼149 mmHg, whereas in alveoli it is ∼104 mmHg (Rhoades & Pflanzer, 1989). Interestingly, the highest PO2 tested that caused significant release of 5-HT from NEB cells was ∼95 mmHg, a value near the physiological level. These data suggest that basal 5-HT release from intact NEB cells may well occur in vivo. Most NEB cells are found in the intrapulmonary airways and are located preferentially at or near airway bifurcation, a site ideally suited for sensing changes in airway gas concentration. As hypoxia-sensitive airway chemoreceptors, NEB cells appear highly sensitive to changes in PO2, and therefore may have important secretory functions in both physiological and pathological conditions. Since many NEBs are innervated (Van Lommel et al. 1998), it is conceivable that 5-HT, released from NEB cells during airway hypoxia, may act as an excitatory neurotransmitter in stimulating post-synaptic receptors on vagal afferent nerve terminals. Indeed, many vagal afferent neurons are known to express 5-HT3-R in several species including rabbit (Higashi & Nishi, 1982).

Recently, we demonstrated expression of 5-HT3-R mRNA in NEB cells in the lungs of different mammals including rabbit, hamster, mouse and human (Fu et al. 2001). Using dual immunocytochemistry (for 5-HT and 5-HT3-R) and confocal microscopy we have localized 5-HT3-R on the plasma membrane of NEB cells. With the aid of fresh lung slices and whole-cell recording, we have also shown that functional 5-HT3-R ligand-gated ion channels are expressed on the surface of NEB cells (Fu et al. 2001). These studies raise the possibility that 5-HT3-R in NEB cells may function as autoreceptors. In support of this idea, we observed in the present study that the 5-HT3-R blocker, ICS 205 930, inhibited hypoxia-induced 5-HT release from NEB cells by ∼68 %. Therefore, presynaptic 5-HT3 autoreceptors may play an important physiological role in stimulating 5-HT release from NEB cells during hypoxia by a positive feedback mechanism.

In a previous study we reported that the O2-sensitive K+ current in NEB cells, was partially inhibited by TEA, 4-AP and Cd2+, suggesting it consisted of both Ca2+-dependent and Ca2+-independent components (Fu et al. 1999). Molecular biological studies have suggested that O2-sensitive K+ channels in NEB cells belong to the KV3.3a family (Wang et al. 1996). Our demonstration that both TEA and 4-AP can evoke quantal 5-HT release indicates that at least some K+ channels, with the pharmacology expected of O2-sensitive K+ channels in intact NEB cells, are open under normoxic conditions. Therefore, their closure during hypoxia could plausibly account for the hypoxia-induced stimulation of amine secretion observed in the present study. This idea is further strengthened by the observation that the effects of hypoxia and TEA or 4-AP were non-additive. The effect of TEA on amine release in NEB cells was similar to that reported for PC-12 cells (Taylor & Peers, 1998) and rat carotid body glomus cells (Pardal et al. 2000). However in the latter study, hypoxia and 5 mm TEA showed additive responses when applied together. This may be due to the lower dose of TEA used, and/or the additional presence in glomus cells of hypoxia-sensitive, background (TASK-1-like) K+ channels which are TEA-insensitive (Buckler, 1997). Other more specific blockers of the O2-sensitive Ca2+-dependent K+ channels (iberiotoxin) have been reported to mimic hypoxia in causing dopamine secretion from clustered glomus cells in both tissue slices (Pardal et al. 2000) or monolayer cultures (Jackson & Nurse, 1997) of rat carotid body. Since the hypoxia-evoked 5-HT secretion was virtually abolished in the presence of TTX, a blocker of Na+-dependent action potentials, it appears that the hypoxic chemotransduction steps in NEB cells ultimately lead to an increase in cell firing (Youngson et al. 1993).

Finally, we also examined the pathway of calcium entry into NEB cells during hypoxic exposure. Voltage-gated Ca2+ channels control the entry of Ca2+ ions across the surface membrane of neurons, neuroendocrine and other cells, thereby influencing electrical activity and secretory functions, as well as diverse cellular responses. We previously described an inward voltage-dependent Ca2+ current in NEB cells with L-type Ca2+ channel characteristics (Fu et al. 1999). To determine the relative importance of these channels in the secretory functions of NEB cells, we tested the effects of selective Ca2+ channel blockers. L-type Ca2+ channels clearly played the predominant role in facilitating Ca2+ entry into these cells during hypoxia since ∼90 % of 5-HT release was blocked by the selective L-type blocker, nifedipine. These findings are in agreement with previous studies on carotid body glomus cells (Urena et al. 1994; Montoro et al. 1996; Jackson & Nurse, 1997) and PC-12 cells (Kumar et al. 1998; Taylor et al. 2000), implicating L-type Ca2+ channels in stimulus-secretion coupling during hypoxia.

In summary, the present study indicates that pulmonary NEB cells respond to hypoxia by releasing 5-HT in a dose-dependent manner. The signalling pathway appears to be mediated via the O2 sensor protein NADPH oxidase, coupled to inhibition of Ca2+-dependent and Ca2+-independent K+ channels (Fu et al. 1999; 2001). Closure of these channels in turn causes increased cell firing, Ca2+ entry through L-type calcium channels and secretion of 5-HT. The released 5-HT, acting via presynaptic 5-HT3 autoreceptors in a positive feedback manner (and possibly postsynaptic 5-HT3 receptors on vagal afferents), may play an important physiological role locally, or by activating reflex pathways during airway hypoxia in the perinatal period or during pulmonary disease (Cutz et al. 1997).

Acknowledgments

This work was supported by grants from the Nicole Fealdman SIDS research fund and Canadian Institutes of Health Research (MOP-12742 and MGP15270). We thank Dr J. Lopez-Barneo for advice on amperometric calculations and Dr I. O'Kelly for advice on amperometric technique.

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