Characteristics of 5-HT-containing chemoreceptor cells of the chicken aortic body

Abstract

1. Voltage-dependent and oxygen-sensitive currents in 5-HT-containing epithelioid cells isolated from chicken thoracic aorta were examined using the whole-cell patch clamp technique. 5-HT immunoreactive cells were identified with Neutral Red. The release of 5-HT from chicken thoracic aorta in the presence of excess KCl and veratridine was also examined using HPLC.

2. At a holding potential of −70 mV with CsCl pipette solution, depolarizing steps between −30 and +60 mV produced inward currents that were blocked by tetrodotoxin (0.2 μm). In the presence of tetrodotoxin and BaCl₂ (5 mm), depolarizing steps evoked slow inward currents that were sensitive to CoCl₂ (2 mm). Nifedipine (1 μm) decreased the currents to 79.4 ± 1.7%, and ω-conotoxin GVIA (1 μm) to 20.2 ± 3.8%.

3. When KCl pipette solution was used, depolarizing potentials positive to −40 mV caused outward currents that were inhibited by tetraethylammonium chloride. The K⁺ currents evoked by depolarizing steps to +20 mV were reduced to 90.3 ± 0.8% by hypoxia in five out of seven cells. Two cells failed to respond to hypoxia. The K⁺ current response was partly decreased by Neutral Red (20 μm).

4. Excess KCl (60 mm) and veratridine (30 μm) both caused the release of 5-HT from aortic strips. 5-HT outputs induced by both stimuli were partly inhibited by nifedipine (1 μm) and by ω-conotoxin GVIA (1 μm), and were abolished by these drugs in combination and by extracellular Ca²⁺ removal.

5. These results suggest that epithelioid cells containing 5-HT act as chemoreceptor cells in the chicken aortic body, having voltage-dependent Na⁺, K⁺, and L- and N-type Ca²⁺ channels, and oxygen-sensitive K⁺ channels.

It is well known that there are two main chemoreceptor organs in the peripheral tissue, the carotid body located in the carotid bifurcation and the aortic body located in the wall of the aorta. Evidence suggests that chemoreceptor cells in the carotid body, type I cells, sense changes in plasma P_O2, P_CO2 and pH and then release transmitters which activate the sensory nerve endings of the carotid sinus nerve. Catecholamines such as dopamine and/or noradrenaline in the type I cells may act as transmitters to the sensory nerve endings (see review by Gonzalez et al. 1994).

The carotid bodies of the rat, cat and human have been shown to contain not only catecholamines but also biogenic indoleamine, 5-HT (Chiocchio et al. 1967; Hellström, 1977; Perrin et al. 1986; Wang et al. 1992). Neuroendocrine cells of the airway neuroepithelial bodies, considered to be airway chemoreceptors, contain 5-HT but not dopamine (Cutz et al. 1993). In chicken carotid bodies, 5-HT has been reported to be dominant (Pearce et al. 1973; Yamamoto et al. 1989; Kameda, 1990).

The release of dopamine from rabbit carotid bodies in response to hypoxia or excess KCl has been shown to be dependent on extracellular Ca²⁺ (González et al. 1992), and chemoreceptor cells of the rabbit carotid body are excitable cells with voltage-dependent Na⁺, K⁺ and Ca²⁺ channels (Duchen et al. 1988; López-Barneo et al. 1988). Similar voltage-dependent channels are also seen in the neuroendocrine cells of the airway neuroepithelial bodies (Youngson et al. 1993).

The mechanisms by which the type I cells of the carotid body respond to changes in P_O2 remain uncertain. In type I cells, a voltage-activated K⁺ current has been proposed to be inhibited by hypoxia (oxygen-sensitive K⁺ current) (López-Barneo et al. 1988; Delpiano & Hescheler, 1989; Peers, 1990a). A similar oxygen-sensitive K⁺ current has been found in the neuroendocrine cells of the airway neuroepithelial bodies (Youngson et al. 1993), neonatal adrenal chromaffin cells (Thompson et al. 1997) and PC12 cells (Conforti & Millhorn, 1997). Quite recently, however, a novel oxygen-sensitive K⁺ current was reported in rat carotid body type I cells, in which hypoxia inhibited voltage-insensitive resting K⁺ conductance (Buckler, 1997).

Epithelioid cells containing 5-HT are aggregated into clusters and form a band of about 1 mm in width in the wall of the chicken thoracic aorta (Miyoshi et al. 1995). We have suggested that these cells are arterial chemoreceptors corresponding to the mammalian aortic body because hypoxic stimulation causes the release of 5-HT from pieces of chicken thoracic aorta (Ito et al. 1997). In comparison with the knowledge of properties of carotid chemoreceptor cells, less is known about the characteristics of chemoreceptor cells in the aortic body. Leech neurons containing catecholamines or 5-HT have been shown to be stained with a vital dye, Neutral Red (Stuart et al. 1974). Using this dye, it is possible that epithelioid cells containing 5-HT could be identified after dissociation from the chicken thoracic aorta.

In the present experiments, we firstly examined whether or not Neutral Red-positive cells isolated from the chicken thoracic aorta contained 5-HT, and then examined the types of voltage-activated channels and oxygen-sensitive channels in Neutral Red-positive epithelioid cells using the whole-cell patch clamp technique. We also investigated characteristics of 5-HT secretion evoked by veratridine and excess KCl from pieces of chicken thoracic aorta.

METHODS

Tissue preparation

All experiments were performed under the regulations of the Animal Research Committee of the Graduate School of Veterinary Medicine, Hokkaido University. Male chicks (white leghorn, 14-28 days after hatching) were anaesthetized by placing them in a small chamber in which diethylether bubbled with pure O₂ was flowing. Following deep anaesthesia, the chick was decapitated. The thoracic aorta was removed and put into chilled and oxygenated physiological salt solution (PSS; for composition, see below). The thoracic aorta was freed from surrounding tissues and cut longitudinally to open its lumen, and then the aortic strip containing epithelioid cells was prepared by cutting it to about 4 mm in length. The pieces of aortic tissue were kept in the chilled and oxygenated PSS until use.

Secretory experiments

The aortic tissue was placed in PSS (0.1 ml) of the following composition (mM): NaCl, 134; KCl, 6; CaCl₂, 2.5; MgCl₂, 1.2; glucose, 10; and Hepes, 10; pH 7.3 with NaOH. In solutions containing various concentrations of KCl, NaCl was reduced by corresponding amounts. The solutions containing veratridine (Sigma), nifedipine (Wako Pure Chemicals, Japan) and Bay K 8644 (Sigma) were prepared by the addition of stock solutions of the drugs dissolved in DMSO. The final concentration of DMSO was less than 0.1 %, which had no effect on 5-HT release. ω-Conotoxin GVIA, ω-agatoxin IVA (Peptide Institute, Japan) and tetrodotoxin (Wako Pure Chemicals) were prepared from concentrated stock solutions. For Ca²⁺-free PSS, CaCl₂ was omitted. In the secretory experiment, 0.5 mM EGTA was added to the Ca²⁺-free PSS. The aortic tissue was put into the solution containing drugs on ice, and then incubated at 37°C in order to stimulate the tissue. For examination of the effects of blockers or modulators on the release of 5-HT, the aortic tissue was pre-incubated with each drug at 37°C for 5 min and then chilled on ice. The aortic tissue was then transferred to another tube containing a secretagogue and the drug in combination, and again incubated at 37°C. The secretory response was terminated by placing the tube on ice. In order to measure the amount of 5-HT appearing in the incubation medium, the solution was acidified with perchloric acid giving a final concentration of 0.4 n after removal of the aortic tissue. The aortic tissue was put in PSS containing 0.4 n perchloric acid to measure the amount of 5-HT left in the tissue. After centrifugation, these acidified supernatants were treated with K₂HPO₄ at a final concentration of 290 mM. After removal of the potassium perchlorate by centrifugation, the clear supernatant was analysed by high-performance liquid chromatography (HPLC; JASCO Corp., Japan).

5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were separated by HPLC with an ODS-column (Catecholpak, JASCO Corp.) and detected by an electrochemical detector (Eicom, Japan). The composition of the mobile phase was: KH₂PO₄-H₃PO₄ buffer, 100 mM (pH 3.5); EDTA, 40 μM; sodium octasulfonic acid, 1.16 mM; and methanol, 15-17 %.

Cell preparation

5-HT-containing epithelioid cells were isolated from three thoracic aortae with collagenase. The tip of a syringe (1 ml) was inserted in the proximal end of an isolated thoracic aorta. Ca²⁺-free PSS containing 0.15 % collagenase (Type 1, Sigma), 0.15 % soybean trypsin inhibitor (Type 1, Sigma) and 0.5 % bovine serum albumin (fraction V, Boehringer-Mannheim) was put into the lumen of the aorta, and the other end of the aorta was tied. Subsequently, the aorta was incubated for 30 min at 37°C. After incubation, the end of the aorta was cut open and the Ca²⁺-free PSS was repeatedly passed in and out with the syringe to facilitate the release of the cells from the aortic wall into the lumen. The enzymatic digestion was repeated twice using fresh enzyme solution. The cells isolated with the second enzyme digestion were centrifuged to remove collagenase and resuspended in PSS containing 0.5 mM CaCl₂. The aliquot of the cell suspension was placed on about ten coverslips and stored on ice under a 100 % O₂ atmosphere until used.

Indirect immunohistochemistry for 5-HT

In order to determine whether or not Neutral Red-positive cells contain 5-HT, the cells isolated from the thoracic aortae were placed on a siliconized glass slide and treated with Neutral Red (Wako Pure Chemicals). The resulting red-coloured cells were collected with a siliconized glass pipette with a tip diameter of about 30 μm connected to a microinjector (Nanoject, Dramund, PA, USA). The collected cells were put on a non-siliconized glass slide. After attachment to the glass, the cells were subjected to an indirect immunoperoxidase staining for 5-HT (Kon et al. 1992). In brief, after fixation with 4 % paraformaldehyde, the cells were pretreated with 1 % non-immune goat serum for 30 min. Rabbit anti-5-HT antiserum (Sigma), diluted 1:5000 with phosphate-buffered salt solution (PBS) containing bovine serum albumin (5 mg ml⁻¹), was used as the primary antiserum. The cells were incubated with biotinylated goat anti-rabbit secondary antibody and then with avidin-biotinylated horseradish peroxidase complex (Vecstain ABC kit, Vector Labs, CA, USA). Subsequently, the complex binding to antibodies against 5-HT was detected with diaminobenzidine tetrahydrochloride, producing a reddish brown precipitate. As a control, normal rabbit serum or rabbit anti-5-HT antiserum pretreated with 5-HT (10 mM) was substituted for the serum with the specific antibody against 5-HT.

Membrane current measurement

The coverslip was placed in a chamber set on the stage of an inverted microscope (Diaphot 300, Nikon, Japan). The vital dye Neutral Red (20 or 30 μM) was applied for about 1 min before starting the experiment. Neutral Red was used only to identify the cells and then the dye was washed out. The cells were superfused with PSS at a flow rate of 2-3 ml min⁻¹. Although a large number of the cells were lost during perfusion, some cells attached spontaneously to the coverslip. The resulting red-coloured cells were subjected to whole-cell voltage clamp. All perfusion solutions were equilibrated with 100 % O₂. For hypoxic stimulation, glucose oxidase (10 μg ml⁻¹)-containing PSS equilibrated with 100 % N₂ was used. In Ba²⁺-containing PSS, BaCl₂ (5 mM) was added to the Ca²⁺-free PSS without EGTA. In some experiments, 5 mM tetraethylammonium chloride (TEA-Cl) was added to the Ba²⁺ solution. Ca²⁺ channel blockers, tetrodotoxin, TEA-Cl, Neutral Red and hypoxic solution were applied to the cells using a Y-shaped tube, of which one opening (250 μm in diameter) was placed about 1 mm away from the cell of interest. The tube was connected to a reservoir of the solution containing the drug or hypoxic solution through a magnetic valve. The solution thus flowed into the tube by gravity as long as the valve was open. When the valve was shut, the solution flowed out rapidly from the opening placed near the cell.

Membrane current recordings were made by the standard patch clamp technique (Hamill et al. 1981) using an Axopatch 200A amplifier (Axon Instruments). Whole-cell membrane currents were recorded with a heat-polished patch pipette of 3.5-5 MΩ resistance filled with an internal solution of the following composition (mM): KCl, 140; MgCl₂, 1.2; glucose, 10; Na₂ATP, 2; EGTA, 5; and Hepes, 10; pH 7.2 with KOH. In the Cs⁺-containing pipette solution, KCl was replaced with CsCl, and the EGTA concentration was increased to 10 mM. The access resistance and membrane capacitance were 8-16 MΩ and 2-6 pF, respectively, both of which were calculated by capacitative currents evoked by hyperpolarizing steps to -90 mV from a holding potential of -70 mV. The series resistance (75-85 %) and cell membrane capacitance were electronically compensated. The voltage steps were produced by a step command generated by a microcomputer in conjunction with a Digidata 1200 interface using pCLAMP software version 6.0 (Axon Instruments). Data sampled at 2-10 kHz were stored on the hard disk of the computer using this interface. Data were also stored on the cassette tape of a PCM data recorder (NF Electronic Instruments, Japan) and displayed on a pen-writing recorder (Recti-Horiz-8K, Sanei, Japan). Data are presented without correction for liquid junction potential error (about 3 mV). When I-V curves were constructed, the P/4 subtraction protocol of Clampex was used to subtract the leak currents (Axon Instruments). Experiments were performed at room temperature (about 25°C) within 4 h after cell isolation.

All data are expressed as means ±s.e.m., for n cells. Statistical analysis of the data was performed using Student's unpaired t test. Differences with P < 0.05 were considered to be significant.

RESULTS

Identification of epithelioid cells isolated from the thoracic aorta

Neutral Red has been reported to stain leech neurons containing catecholamines or 5-HT (Stuart et al. 1974), adrenal chromaffin cells containing catecholamines (Role & Perlman, 1980), and airway chemoreceptor cells containing 5-HT (Youngson et al. 1993). When the chicken thoracic aorta was incubated with Neutral Red (30 μM) for 5 min at room temperature, red-coloured cells were aggregated in clusters and formed a band about 1 mm in width in the lumen of the aorta. This arrangement of Neutral Red-positive cells was similar to that of 5-HT immunoreactive cells reported by Miyoshi et al. (1995). The epithelioid cells were isolated with collagenase together with endothelial cells, and they were treated with Neutral Red (20-30 μM) for 1 min. Only a small percentage of the cells attached to the coverslip were stained with Neutral Red. Therefore, these red-coloured cells were collected with a pipette and placed on a glass slide (Fig. 1A). The cells attached to the glass slide spontaneously. After being fixed with paraformaldehyde, the cells were subjected to indirect immunoperoxidase staining for 5-HT, seen as a brown precipitate (Fig. 1B). All Neutral Red-positive cells were 5-HT immunoreactive. As a control, normal rabbit serum or rabbit anti-5-HT antiserum pretreated with 5-HT was substituted for the serum with the specific antibody to 5-HT. There was no immunoreactive brown precipitate in the control cells (Fig. 1D), which were stained with Neutral Red (Fig. 1C). The results indicate that 5-HT-containing cells can be identified with a vital dye, Neutral Red. These red-coloured cells were subjected to voltage clamp experiments using the standard whole-cell patch clamp technique. Neutral Red was used only to identify the cells. After washout of the dye, the red colour was gradually removed.

Figure 1. Epithelioid cells isolated from the chicken thoracic aorta stained with Neutral Red, and immunoperoxidase staining for 5-HT.

Cells isolated from the chicken thoracic aorta with collagenase were treated with Neutral Red (20 μM). The resulting red-coloured cells were collected with a siliconized glass pipette and placed on a non-siliconized glass slide. After attachment to the glass, cells were stained with Neutral Red again (A and C). After fixing with paraformaldehyde, they were subjected to indirect immunohistochemical staining for 5-HT, seen as a brown precipitate (B). Normal rabbit serum and rabbit anti-5-HT antiserum pretreated with 5-HT were substituted for rabbit anti-5-HT antiserum as a control. No brown stain was apparent in the control treated with normal rabbit serum (D). The cells marked with arrows were lost during the course of the immunostaining. The epithelioid cells are about 10 μm in diameter.

Voltage-activated fast inward currents in epithelioid cells

At a holding potential of -70 mV with CsCl patch pipette solution containing 10 mM EGTA, the mean input resistance and membrane capacitance were 2.3 ± 0.2 GΩ and 4.3 ± 0.2 pF (n = 21), respectively. A sequence of depolarizing potentials for 15 ms between -50 and +60 mV in 10 mV increments with 15 or 20 s intervals was applied to cells after changing the holding potential to -90 mV for 3 ms. In some experiments, TEA-Cl (5 mM) was added to the bathing solution to inhibit the residual outward currents evoked by the depolarizing pulses. As shown in Fig. 2, the depolarizing steps evoked inward currents which started to appear at around -30 mV. A depolarizing step to +10 mV elicited a fast inward current in all Neutral Red-positive cells examined, which attained a peak within 0.5 ms after depolarization and subsided quickly. In response to depolarizing potentials between 0 and +30 mV, the fast inward current was followed by a sustained small inward current in some cells. Tetrodotoxin (0.2 μM) reversibly abolished the fast current but not the sustained one. The peak Na⁺ inward current-voltage relationship is shown in Fig. 2C. The mean amplitude of the peak Na⁺ current obtained with a depolarizing potential of +10 mV was 664 ± 83 pA (n = 10).

Figure 2. Voltage-dependent Na⁺ currents in the epithelioid cell.

An epithelioid cell was voltage clamped at a holding potential of -70 mV. A, representative recordings of current responses to depolarizing potentials for 15 ms between -50 and +60 mV in 10 mV increments after changing the holding potential to -90 mV for 3 ms before (Control) and during exposure to tetrodotoxin (TTX, 0.2 μM), and after its removal (Washout). B, the same recordings as shown in A, with a faster time scale. C, current-voltage relationships of peak Na⁺ currents were obtained from the cell before (▪) and during (•) exposure to tetrodotoxin, and after its removal (▴).

Voltage-activated slow inward currents in epithelioid cells

In order to characterize the sustained inward currents, at a holding potential of -70 mV, a sequence of depolarizing potentials for 25 or 30 ms between -60 and +60 mV in 10 mV increments with 20 s intervals was applied to the cells in the presence of tetrodotoxin (0.2 μM) and BaCl₂ (5 mM) instead of CaCl₂. In some experiments, TEA-Cl (5 mM) was added to the perfusion solution to decrease residual outward currents. The inward currents developed gradually and reached a maximum within 10 ms after the depolarizing steps (Fig. 3A). The inward currents were reversibly blocked by CoCl₂ (2 mM). The peak Ba²⁺ current-voltage relationship is shown in Fig. 3B. The mean amplitude of peak Ba²⁺ current evoked by a depolarizing step of +10 mV was 274 ± 22 pA (n = 7). The Ba²⁺currents failed to occur with depolarizing steps of +20 mV in about one-half of the Neutral Red-positive cells examined.

Figure 3. Voltage-dependent Ba²⁺ currents in the epithelioid cells.

Epithelioid cells were voltage clamped at -70 mV in the presence of tetrodotoxin (0.2 μM) and BaCl₂ (5 mM) instead of CaCl₂. A, representative recordings of current responses of an epithelioid cell to depolarizing potentials for 30 ms between -60 and +60 mV in 10 mV increments before (Control) and during exposure to CoCl₂ (2 mM), and after its removal (Washout). B, peak inward current-voltage relationships were obtained from the cell before (▪) and during (•) exposure to CoCl₂, and after its removal (▴). C, effects of nifedipine and ω-conotoxin GVIA on peak Ba²⁺ currents in an epithelioid cell. At -70 mV in the presence of tetrodotoxin (0.2 μM), TEA-Cl (5 mM) and BaCl₂ (5 mM), Ba²⁺ currents were evoked by depolarizing steps for 15 ms to +10 mV with 20 s intervals. The peak Ba²⁺ currents are expressed as percentages of the first Ba²⁺ current recorded in the absence of blockers and plotted against time. Nifedipine (1 μM) and ω-conotoxin GVIA (1 μM) were applied during the periods indicated by horizontal lines starting with an arrow. Inset, superimposed current traces with letters corresponding to those in the peak current trace. The dotted line indicates the zero current level.

Ca²⁺ channel antagonists were used to characterize the subtype of Ca²⁺ channels present in the epithelioid cell membrane. Depolarizing steps for 15 ms from -70 to +10 mV were repeatedly applied to epithelioid cells with 20 s intervals in the presence of tetrodotoxin (0.2 μM), TEA-Cl (5 mM) and BaCl₂ (5 mM). Representative results of the effects of Ca²⁺ channel blockers nifedipine and ω-conotoxin GVIA on Ba²⁺ current responses are illustrated in Fig. 3C. Nifedipine (1 μM), an L-type Ca²⁺ channel blocker, partly inhibited the Ba²⁺ currents, and the addition of ω-conotoxin GVIA (1 μM), an N-type Ca²⁺ channel blocker, blocked residual Ba²⁺ currents completely. When nifedipine and ω-conotoxin GVIA at a concentration of 1 μM were applied separately for 2 min, nifedipine decreased the amplitude of Ba²⁺ currents to 79.4 ± 1.7 % (n = 6) of the current before the application of the drug, and ω-conotoxin GVIA decreased it to 20.2 ± 3.8 % (n = 6).

Voltage-activated outward currents in epithelioid cells

The cells were voltage clamped at -70 mV using a patch pipette solution containing 140 mM KCl and 5 mM EGTA. Under these conditions, the mean input resistance and mean membrane capacitance were 1.9 ± 0.2 GΩ and 3.5 ± 0.1 pF (n = 10), respectively. A sequence of depolarizing potentials for 80 ms between -60 and +60 mV in 10 mV increments with 15 s intervals was applied to the cell (Fig. 4A). Depolarizing potentials between -10 and +20 mV produced transient inward currents. The inward current was blocked by tetrodotoxin (0.2 μM), and followed by an outward current which continued during the depolarizing step. The K⁺ current-voltage relationship is illustrated in Fig. 4B. The activation of K⁺ currents started to appear from a depolarizing step of around -30 mV. The amplitude of the currents was increased with increasing depolarizing potentials up to +60 mV. The mean amplitude of the K⁺ currents in response to a depolarizing potential of 0 mV was 387 ± 38 pA (n = 11). The K⁺ current was inhibited by TEA⁺ in a dose-dependent fashion (Fig. 4C).

Figure 4. Voltage-dependent K⁺ currents in the epithelioid cells.

Epithelioid cells were voltage clamped at a holding potential of -70 mV. A, representative recordings of current responses to depolarizing potentials for 80 ms between -60 and +60 mV in 10 mV increments before (Control) and during exposure to TEA-Cl (TEA⁺, 5 mM), and after its removal (Washout) in an epithelioid cell. B, outward current-voltage relationships were obtained for the cell before (▪) and during (•) exposure to TEA⁺, and after its removal (▴). The current amplitude just before the cessation of each depolarizing step is plotted against the depolarizing voltage. C, dose-dependent inhibition of peak K⁺ currents by TEA⁺ (n = 5). K⁺ currents were evoked by depolarizing steps to +20 mV for 100 ms in the presence of various concentrations of TEA⁺. The peak K⁺ currents are expressed as percentages of the current in the absence of TEA⁺ and are plotted against TEA⁺ concentration. Inset, current traces recorded from a cell in the absence and presence of TEA⁺ at concentrations of 2, 5 and 10 mM are superimposed.

Oxygen-sensitive currents in epithelioid cells

We have previously reported that hypoxia evokes the release of 5-HT from isolated thoracic aorta (Ito et al. 1997). In order to determine whether Neutral Red-positive cells containing 5-HT act as chemoreceptors, the effect of hypoxia on voltage-activated currents was investigated. Depolarizing steps for 400 ms to +20 mV were repeatedly applied to the cell with 15 s intervals at a holding potential of -70 mV using a patch pipette containing 140 mM KCl. Hypoxia (N₂-equilibrated solution containing 10 μg ml⁻¹ glucose oxidase) for 2 min produced a decrease in the amplitude of K⁺ currents in five out of seven cells. A representative result is shown in Fig. 5A and B. In two cells, hypoxia failed to cause any perceptible effects on voltage-activated K⁺ currents. The K⁺ currents decreased in amplitude to 90.3 ± 0.8 % (n = 5). In Cs⁺ pipette solution, hypoxia did not produce any inhibitory effects on fast (Na⁺) and slow (Ca²⁺) inward currents evoked by the depolarizing step to +10 mV for 15 ms (Fig. 5C). It also had no effect on voltage-activated Ba²⁺ currents. These results suggest that Neutral Red-positive cells of the chicken aorta possess oxygen-sensitive K⁺ channels like type I cells of the carotid body.

Figure 5. Effects of hypoxia on voltage-activated currents.

A, at a holding potential of -70 mV, K⁺ currents were evoked by depolarizing steps to +20 mV for 400 ms with 15 s intervals. Glucose oxidase (10 μg ml⁻¹)-containing solution equilibrated with 100 % N₂ was applied for a period of 2 min, as indicated by the open bar. B, superimposed K⁺ current traces with a faster time scale corresponding to the K⁺ currents marked with the same letters in A. C, at a holding potential of -70 mV using Cs⁺ pipette solution, the fast and slow inward currents, Na⁺ and Ca²⁺ currents, respectively, were evoked by depolarizing steps to 10 mV for 15 ms with 15 s intervals. Superimposed current traces were obtained before and during exposure to hypoxic stimulation for 2 min, and 2 min after removal of stimulation.

Effects of Neutral Red on voltage-activated currents in epithelioid cells

It has been proposed that membrane depolarization by the inhibition of oxygen-sensitive K⁺ channels plays an important role in the chemotransduction of hypoxic stimulation (Gonzalez et al. 1994). In the epithelioid cells of the chicken aorta, the hypoxia-induced inhibition of K⁺ currents was not as great as that in type I cells of the carotid body of the rabbit and rat (López-Barneo et al. 1988; Peers, 1990a; López-López et al. 1997), or in neuroendocrine cells of the neuroepithelial body (Youngson et al. 1993), in which hypoxia decreased voltage-activated K⁺ currents by over 20 %. We therefore investigated the effect of Neutral Red on voltage-activated currents in epithelioid cells of the chicken thoracic aorta. At a holding potential of -70 mV, voltage-dependent currents were repeatedly activated by depolarizing steps and Neutral Red (20 μM) was again applied for 2 min. Neutral Red decreased the amplitude of the K⁺ currents evoked by the depolarizing potential of +20 mV for 400 ms. The inhibitory effect on the amplitude of K⁺ current just before the end of the depolarizing pulses (20.6 ± 3.1 % of control, n = 5) was greater than that on its peak amplitude (53.4 ± 4.9 % of control). Complete recovery did not occur during the course of the experiment even after washout of the dye.

The effect of Neutral Red on voltage-activated Na⁺ and Ba²⁺ currents was investigated using CsCl patch pipette solution containing 10 mM EGTA. At a holding potential of -70 mV, Neutral Red at 20 μM decreased Ba²⁺ currents evoked by the depolarizing potential of +10 mV for 25 ms to 79.2 ± 3.4 % of control (n = 6) but did not decrease Na⁺ currents (n = 3). Ba²⁺ currents inhibited by Neutral Red also failed to recover completely after washout of the dye.

5-HT secretion from chicken thoracic aorta in response to excess KCl and veratridine

Neutral Red-positive cells containing 5-HT in the chicken thoracic aorta seem to have voltage-dependent Na⁺ and Ca²⁺ channels in their membranes like the type I cells of the carotid body. If this is the case, these cells would be expected to release 5-HT in response to excess KCl and veratridine. A piece of thoracic aorta was incubated in the presence of excess KCl (60 mM) or veratridine (0.1 mM) and in the absence of secretagogues for various times at 37°C to observe the time course of 5-HT appearance in the medium. In the absence of secretagogues (Fig. 6A), 5-HT and 5-HIAA increased linearly with time. It is likely that the increase in 5-HIAA reflects the degradation of 5-HT released in the medium, because the increase of 5-HIAA is always preceded by that of 5-HT and there is only a small amount of 5-HIAA in the tissue (Ito et al. 1997). A spontaneous release of total 5-HT (5-HT plus 5-HIAA) was less than 10 % of the total content of 5-HT in the tissues during a 10 min incubation period.

Figure 6. Characteristics of the output of 5-HT evoked by excess KCl and veratridine.

The output of 5-HIAA and 5-HT is expressed as a percentage of the total content of 5-HT plus 5-HIAA in the chicken thoracic aorta, and values are plotted against incubation time in the presence of excess KCl (B, 60 mM, n = 5-6) and veratridine (C, 0.1 mM, n = 5-6), and in their absence (A, n = 3-6). Symbols and vertical bars indicate the mean output of 5-HT plus 5-HIAA (SUM, ▪), 5-HT (•) and 5-HIAA (▴), and the s.e.m., respectively. D and E, effects of channel modulators and Ca²⁺ removal on the output of 5-HT induced by excess KCl (D, 60 mM) and veratridine (E, 30 μM) for 5 min. The output of 5-HT (5-HT plus 5-HIAA) is expressed as a percentage of the total content of 5-HT plus 5-HIAA in the chicken thoracic aorta. The channel modulators used were tetrodotoxin (TTX), Bay K 8644, ω-agatoxin IVA (ATX), ω-conotoxin GVIA (CTX), nifedipine (Nif), and nifedipine plus ω-conotoxin GVIA (Nif + CTX), all at 1 μM. Results are also shown for output in the absence of these drugs (Control) and in the absence of extracellular Ca²⁺ (Ca²⁺ free). Columns and vertical bars indicate the mean output of 5-HT and the s.e.m. (n = 5-6), respectively.

Excess KCl (60 mM) caused a rapid increase in 5-HT in the incubation medium, which attained a maximum at 1 min. Although no further increase in 5-HT occurred during the 20 min incubation, 5-HIAA increased linearly until the end of the incubation. Consequently, the total amount of 5-HT (5-HT plus 5-HIAA) rapidly increased during the first 1 min of the incubation period and gradually increased thereafter (Fig. 6B). The increase in 5-HT output 5 min after the start of the incubation could be partly due to spontaneous increases in 5-HT output, because about 10 % of 5-HT in the tissue was released spontaneously for 20 min even in the absence of drugs under the present conditions (Fig. 6A). In contrast, the release of 5-HT induced by veratridine was slower in onset than that by excess KCl and attained a maximum at 5 min. The increase of 5-HIAA lagged behind that of 5-HT and developed slowly for 20 min (Fig. 6C). The total release of 5-HT (5-HT plus 5-HIAA) induced by veratridine attained a maximum at 5 min and release did not occur thereafter.

The effects of nifedipine, ω-conotoxin GVIA, ω-agatoxin IVA, Bay K 8644 and the removal of extracellular Ca²⁺ on 5-HT release induced by 60 mM KCl were examined (Fig. 6D). In order to avoid the extensive decomposition of released 5-HT in the medium, the secretory responses to excess KCl and veratridine were observed with a 5 min incubation. The content of 5-HT plus 5-HIAA in the incubation medium was expressed as the amount of 5-HT in the tissue. After pretreatment with these drugs at 1 μM or exposure to Ca²⁺-free solution for 5 min, the thoracic aorta was stimulated with excess KCl for another 5 min in the presence of the drug or in the absence of Ca²⁺. The release of 5-HT in response to excess KCl was blocked by the removal of extracellular Ca²⁺, indicating that the output of 5-HT in response to excess KCl depended upon extracellular Ca²⁺. The release of 5-HT induced by excess KCl was potentiated by Bay K 8644 (1 μM), an activator of L-type Ca²⁺ channels. Both nifedipine and ω-conotoxin GVIA, L- and N-type Ca²⁺ channel blockers, respectively, inhibited the 5-HT release induced by excess KCl, but ω-agatoxin IVA, a P/Q-type Ca²⁺ channel blocker, did not. When nifedipine was applied together with ω-conotoxin GVIA at the same concentration of 1 μM, the secretory response to excess KCl was decreased to 4.7 ± 0.8 % (n = 6), which was not significantly different from the spontaneous release of 5-HT for 5 min (4.1 ± 0.8 %, n = 6).

Veratridine (30 μM) elicited the release of 5-HT, which was blocked by the removal of extracellular Ca²⁺ and by tetrodotoxin (1 μM) (Fig. 6E). The secretory response to veratridine was partly inhibited by 1 μM of either nifedipine or ω-conotoxin GVIA and blocked by the two drugs in combination.

DISCUSSION

The present results indicate that epithelioid cells from the chicken thoracic aorta stained with Neutral Red contain 5-HT and possess voltage-dependent Na⁺, Ca²⁺ and K⁺ channels. It is suggested that veratridine and excess KCl indirectly cause the activation of voltage-dependent Ca²⁺ channels resulting in the release of 5-HT from the epithelioid cells of the chicken aorta. In the present experiments, hypoxia caused a decrease in voltage-dependent K⁺ currents in epithelioid cells. Quite recently, we reported that hypoxia caused the release of 5-HT from the chicken thoracic aorta (Ito et al. 1997). Taken together, these results suggest that 5-HT-containing epithelioid cells in the chicken thoracic aorta are chemoreceptors in the aortic body. The chemoreceptor cells in the carotid bodies have been shown to originate from the neural crest (Pearce et al. 1973) and to be excitable cells with voltage-dependent channels (Duchen et al. 1988; López-Barneo et al. 1988). Therefore, the epithelioid cells of the aortic body seem to have properties similar to those of the chemoreceptor type I cells of the carotid body.

It has been shown that voltage-dependent Ca²⁺ channels are present in type I cells of the carotid body (Fieber & McCleskey, 1993; Buckler & Vaughan-Jones, 1994; e Silva & Lewis, 1995). In the present experiments, the Ba²⁺ current responses to depolarizing steps and the release of 5-HT induced by excess KCl and veratridine were abolished by nifedipine and ω-conotoxin GVIA, L- and N-type Ca²⁺ channel blockers, respectively, in combination. These results indicate that at least L- and N-type voltage-dependent Ca²⁺ channels are present in the epithelioid cells of the chicken aorta, as reported in the chemoreceptor cells of the rat carotid body (e Silva & Lewis, 1995), and that the release of 5-HT by excess KCl and veratridine results from Ca²⁺ influx through both voltage-dependent Ca²⁺ channels. Nifedipine and ω-conotoxin GVIA inhibited the release of 5-HT to the same extent. In the voltage clamp experiments, however, the Ba²⁺ current response was more sensitive to ω-conotoxin GVIA than to nifedipine. In porcine adrenal chromaffin cells, it has been reported that a depolarizing step for 1 s caused greater inactivation of N-type Ca²⁺ channels than L-type Ca²⁺ channels (Kitamura et al. 1997). The difference in the inhibitory efficacy of nifedipine on Ba²⁺ current responses and secretory responses might be explained by the differences in time-dependent inactivation of N-type and L-type Ca²⁺ channels.

The release of 5-HT in response to excess KCl occurred during the first 1 min of incubation and then gradually increased for 20 min. The later slow phase of 5-HT output seemed to result from the increase in the spontaneous output of 5-HT, because about 10 % of 5-HT in the tissue was released spontaneously for 20 min even in the absence of drugs. If so, the real releasing response to excess KCl may terminate within 1 min. Similar rapid changes in the [Ca²⁺]_i response to excess KCl are shown in rat carotid type I cells (Buckler & Vaughan-Jones, 1994). In contrast, the releasing response to veratridine increased gradually and reached a maximum at 5 min. In adrenal chromaffin cells, it has been reported that excess KCl causes a rapid increase followed by a decrease in both catecholamine release (Ito et al. 1980) and [Ca²⁺]_i (López et al. 1995), and that veratridine causes a slow increase in catecholamine secretion (Ito et al. 1980; Ito, 1983) and oscillations of [Ca²⁺]_i (López et al. 1995). In the present study, the veratridine-induced 5-HT release was inhibited by tetrodotoxin, nifedipine or ω-conotoxin GVIA, indicating that the activation of voltage-dependent Na⁺ channels gives rise to depolarization which results in the activation of voltage-dependent L-type and N-type Ca²⁺ channels.

In the chemoreceptor type I cell of the rabbit carotid body, there are voltage- and Ca²⁺-activated K⁺ channels (Ureña et al. 1989), and K⁺ currents show some sensitivity to apamin or TEA⁺ (Duchen et al. 1988) and to charybdotoxin (Peers, 1990b). In the epithelioid cells of the chicken aorta, the depolarizing steps elicited outward currents sensitive to TEA⁺, suggesting the presence of TEA⁺-sensitive K⁺ channels. Furthermore, Neutral Red-positive cells responded to hypoxia with an inhibition of voltage-activated K⁺ current without any effects on Na⁺ and Ca²⁺ currents, suggesting that the epithelioid cells containing 5-HT are chemoreceptor cells with oxygen sensors. Similar voltage-activated and oxygen-sensitive K⁺ currents have been observed in the type I cells of mammalian carotid bodies (López-Barneo et al. 1988; Delpiano & Hescheler, 1989; Peers, 1990a; López-López et al. 1997), neuroepithelial body cells (Youngson et al. 1993), neonatal rat adrenal chromaffin cells (Thompson et al. 1997) and PC12 cells (Conforti & Millhorn, 1997).

There are some types of K⁺ channel that are sensitive to TEA⁺. However, we could not examine the properties of oxygen-sensitive K⁺ currents in the chicken aortic epithelioid cells because hypoxia inhibited the voltage-activated K⁺ current by only 10 %. The extent of current suppression seems to be smaller than that observed in the chemoreceptor type I cells of the carotid bodies (López-Barneo et al. 1988; Peers, 1990a; López-López et al. 1997). It is possible that the P_O2 in the bathing solution did not fall to sufficiently low levels. Another possibility is that the impairment of oxygen sensors may contribute to a diminished K⁺ current inhibition because we used freshly dissociated cells rather than cells in culture. Although cells stained with Neutral Red have been reported to retain normal electrical properties (Stuart et al. 1974), Neutral Red caused a partial inhibition of voltage-activated K⁺ currents in the present experiments. Neutral Red might affect K⁺ channel function. In rat carotid body type I cells, it has been reported that TEA⁺ fails to evoke a significant rise in [Ca²⁺]_i (Buckler, 1997) or change sinus nerve chemoreceptor activity (Donnelly, 1995) even if it reduces voltage-activated K⁺ currents. TEA⁺ effectively inhibited voltage-activated K⁺ currents in 5-HT-containing epithelioid cells but did not release 5-HT from pieces of aorta (S. Ito, T. Ohta & Y. Nakazato, unpublished observations). Further studies are required to characterize the voltage-dependent and oxygen-sensitive K⁺ channels in the chicken aortic epithelioid cells.