Corresponding author M. Inoue: Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-01, Japan. Email: firstname.lastname@example.org
1The perforated patch method and amperometry were used to determine whether the adrenal medullary cell itself is capable of sensing hypoxia and, if so, how such sensation is transduced to secretion of catecholamines (CA).
2Exposure to hypoxia, cyanide (CN), or muscarine facilitated CA secretion from dissociated chromaffin cells. The CN-induced secretion was not affected by removal of glucose, indicating that the CN release is due to chemical hypoxia.
3The secretions induced by CN and muscarine were markedly diminished by removal of Ca2+ ions or by application of Cd2+ or methoxyverapamil (D-600).
4Cyanide and muscarine produced depolarizations with generation of action potentials and increased intracellular Ca2+ concentrations determined using the acetoxymethyl (AM) ester form of fluo-3 in the presence of external Ca2+ ions, but not in their absence.
5Hypoxia and CN produced inward currents at an equilibrium potential for Cl− ions, irrespective of whether or not Na+ ions were present in the cells, and substitution of N-methyl-D-glucamine for 134 mM Na+ ions in the perfusate inhibited the CN current by 71 %. The reversal potential for the CN current was −24 mV in the standard perfusate.
6The hypoxia-, CN- and muscarine-induced currents decreased in parallel with hyperpolarizations, and exposure to CN prevented muscarine, but not nicotine, from inducing a further inward current.
7We conclude that hypoxia and CN induce CA secretion through depolarization and the subsequent activation of voltage-dependent Ca2+ channels and that this depolarization is due to opening of cation channels, which are possibly identical to muscarinic cation channels.
Catecholamines (CA) secreted from the adrenal medulla play a central role in the defence against various stressors, such as haemorrhage, hypoglycaemia, and hypoxia (Axelrod & Reisine, 1984). The released CA increases both cardiac output and the availability of metabolic substrates to peripheral tissues, thereby allowing the body to deal with life-threatening events (Cryer, 1980). Such stressors are generally detected outside the adrenal medulla and the secreting information is transmitted neuronally (Lewis, 1975). There is good evidence that adrenal medullary cells themselves are capable of sensing hypoxia at least in the perinatal period (Lagercrantz & Bistoletti, 1977; Seidler & Slotkin, 1985; Mojet, Mills & Duchen, 1996; Thompson, Jackson & Nurse, 1997) and the secreted CA increases survival potential of newborn rats and infants. How a decrease in O2 tension is sensed and transduced to CA release remains to be determined.
Exposure to cyanide (CN), an electron transport inhibitor (Albaum, Tepperman & Bodansky, 1946) or to hypoxia induces an increase in concentration of intracellular Ca2+ ions ([Ca2+]i) in glomus or type I cells of rabbit (Montoro, Ureña, Fernández-Chacón, Alvarez de Toledo & López-Barneo, 1996) and rat (Buckler & Vaughan-Jones, 1994) carotid bodies, cells which act as sensors for O2 tension in the blood (Fishman, Greene & Platika, 1985; Ureña, Fernández-Chacón, Benot, Alvarez de Toledo & López-Barneo, 1994). This increase was initially considered to be mainly due to a release of Ca2+ ions from mitochondria (Biscoe & Duchen, 1990), but accumulating evidence indicates that the increase in [Ca2+] and consequent secretion of dopamine result from suppression of K+ channels and the subsequent activation of the voltage-dependent Ca2+ influx (Montoro et al. 1996; López-Barneo, 1996). The type of K+ channel involved in hypoxia sensing is voltage dependent in the rabbit carotid body cell and voltage independent in the rat (Buckler, 1997). In contrast, the hypoxia-sensing mechanism in adrenal medullary cells has not been investigated so thoroughly. In chromaffin cells obtained from newborn rats, hypoxia suppressed voltage-dependent K+ channels (Thompson et al. 1997), as was noted in rabbit carotid body cells. However, it remains to be determined whether the suppression of this channel is responsible for hypoxia-induced depolarization.
We did studies to determine whether hypoxia and metabolic suppression with CN would induce CA secretion from dissociated adrenal chromaffin cells of adult guinea-pigs and, if so, what mechanism is involved. Hypoxia and metabolic inhibition were found to induce an increase in CA release through activation of a cation channel.
Experiments were performed on chromaffin cells enzymatically isolated from adrenal medullae. The cell isolation procedure is described elsewhere (Inoue & Imanaga, 1995). Briefly, female guinea-pigs weighing 250–350 g were killed by a blow to the neck, and the adrenal glands removed and immediately put into ice-cooled Ca2+-free balanced salt solution in which 1.8 mM CaCl2 was simply removed from standard saline (see below). Adrenal medullae were cut into three to six pieces and incubated for 30 min with 0.25 % collagenase dissolved in the Ca2+-free solution. After the incubation, the tissues were washed three or four times in the Ca2+-free solution and gently dissociated with a fire-polished Pasteur pipette. Catecholamine release from dissociated cells was measured using amperometry (Chow, von Rüden & Neher, 1992; Zhou & Misler, 1995). A carbon fibre electrode (ProCFE; Axon Instruments) was carefully placed on a chromaffin cell and +600 mV was applied to the electrode under voltage clamp conditions. The current due to oxidation of CA at the tip of the electrode was recorded using an Axopatch 200A (Axon Instruments) and stored on videotape after digitization with an analog-to-digital converter. The signals were low-pass filtered at 500 Hz and digitized at a sampling interval of 1 ms using AxoData software (version 1.2; Axon Instruments). The total charge of evoked currents was measured using AxoGraph software (version 3.0; Axon Instruments). The bath was continuously perfused with standard saline containing (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.53 NaH2PO4, 5 D-glucose, 5 Hepes, and 2.6 NaOH. To study the effects of CN, the bath was perfused with CN solution which had essentially the same composition as standard saline except 5 mM NaCl was replaced with 5 mM NaCN and the glucose was removed. To examine the effects of external Ca2+, 3.6 mM MgCl2 was substituted for 1.8 mM CaCl2. The pH of all external solutions was adjusted to 7.4.
Measurement of Ca2+ concentration
Dissociated cells were loaded with 10 μM of the acetoxymethyl (AM) ester form of fluo-3 for 40 min and then kept for 20 min in standard saline without the dye. The dish in which the loaded cells settled was placed on a Zeiss Axiovert microscope (× 63 objective lens) attached to a Zeiss LSM 410 laser confocal scanning unit (Carl Zeiss, Germany). Illumination with 488 nm was provided by an argon laser and emission was monitored between 510 and 560 nm. Fluorescence images were acquired every 4 s and the fluorescence intensity was averaged over the entire cell, the transmitted (differential interference contrast) image of which was obtained prior to the fluorescence. To study the effects of CN or muscarine, half the 2 ml solution in the dish was replaced with a test solution containing 10 mM CN or 6 μM muscarine, and the administration was completed within 8 s. On the other hand, the Ca2+ ionophore A-23187 was dissolved in 2 ml DMSO and 20 μl of this stock solution (100 μM) was added directly to the dish solution. When Ca2+ ions were removed from the dish solution, the standard solution in the dish was replaced three to four times with the Ca2+-free, high Mg2+ solution.
The whole-cell current was recorded using the perforated patch method to diminish washout of cellular components (Horn & Marty, 1988). Dissociated chromaffin cells were left for a few minutes to facilitate attachment to the bottom of the bath before being constantly perfused with the standard saline at a rate of ∼1 ml min−1. The current was recorded using an Axopatch 200A amplifier and then fed into a brush recorder after low-pass filtering at 5 Hz and onto a videotape after digitizing using an analog-to-digital converter. The standard pipette solution contained (mM): 120 potassium isethionate, 20 KCl, 10 NaCl, 10 Hepes and 2.6 KOH. In low Cl− solution, 17 mM KCl was replaced equimolarly with potassium isethionate and in low Cl−, Na+-free solution, a further substitution of 10 mM KCl for 10 mM NaCl was made. When CN-induced current-voltage (I-V) relationships were investigated, the low Cl− solution was used and 1 mM tetraethylammonium was added to the bath solution to reduce Ca2+-dependent K+ currents (Neely & Lingle, 1992). Thus, voltage errors due to the series resistance (20–30 MΩ) did not exceed 3 mV in I-V curves shown. The pulse protocol consisting of a 50 ms negative pulse, 300 ms interval, and a 50 ms positive pulse was applied every 5 s 5–8 times in 10 mV steps from a holding potential of −62 mV. At the same time, currents were stored on a computer diskette at a sampling interval of 0.1 ms after filtering at 3 kHz. On the day of experiment, nystatin was added to the pipette solution at a final concentration of 100 μg ml−1. The pH of all pipette solutions was adjusted to 7.2. The membrane potential was corrected for a liquid junction potential of −12 mV between the nystatin solutions and the standard saline. To study the effects of hypoxia, the bath solution, with (severe hypoxia) or without (mild hypoxia) 0.5 mM Na2S2O4 added, was bubbled with 100 % N2. The O2 pressure in the bath, measured using an acid-base analyser ABL 30 (Radiometer, Copenhagen, Denmark), was 20 and 58 mmHg in the presence and absence of Na2S2O4, respectively. All experiments were carried out at 23–25°C. Data are expressed as means ±s.d. unless otherwise stated of n cells, and statistical significance was determined using Student's t test or, for CA release, the sign test.
(±)-Muscarine chloride, A-23187, methoxyverapamil (D-600), tetraethylammonium (TEA) chloride, and nystatin were obtained from Sigma; fluo-3 AM from Dojin (Japan); collagenase from Yakult (Japan); NaCN from Hayashi Pure Chemical Industries (Japan); Na2S2O4 from Nacalai Tesque, Inc. (Japan).
Hypoxia evokes CA release
Figure 1A shows CA secretions in response to 3 μM muscarine, mild hypoxia, and 5 mM CN from a dissociated chromaffin cell. To compare the potency in enhancing CA secretion, amounts of muscarine- and mild hypoxia-evoked secretion, obtained by subtracting the amount of secretion for 100–140 s before stimulation from that for the corresponding period of stimulation, were compared with that of secretion induced by CN in the same cells: the muscarine-sensitive secretion thus obtained was 330 ± 248 % (n= 8) of the CN-induced secretion. On the other hand, the mild hypoxia-induced secretion in three of six cells tested was 52 ± 33 % of the CN, and in the remaining three cells responding to CN, mild hypoxia did not induce any noticeable release for up to 6 min. Thus, the rank order of the facilitating potency for secretion was 3 μM muscarine > 5 mM CN >> mild hypoxia. Another difference is a slow onset of CA secretion in response to mild hypoxia and CN (Fig. 1A and B). In six cells where CA release by muscarine occurred with very little delay (6 ± 1 s, a time which may reflect that for exchange of a perfusate), the facilitation by CN was significantly delayed in most of the cells (40 ± 16 s). A similar difference was also noted at the termination of stimulation: the muscarine-induced release subsided rapidly after washout, whereas the CN-induced release generally continued for 27 ± 13 s after washout (see Fig. 2). Other characteristics of the CN-induced secretion are that the spontaneous secretion sometimes ceased for a few tens of seconds following CN enhancement (Fig. 1, n= 3), whereas such cessation did not occur even with application of high concentrations of muscarine (see Fig. 2B). To determine whether the CN enhancement reflects the effect of chemical hypoxia, the extent of CA secretion evoked by CN for 140–180 s was compared in the presence and absence of 5 mM glucose. On average, the amount of CN-sensitive secretion in the presence of glucose was 114.6 ± 72.6 % of that in its absence. Thus, the enhanced secretion reflects a response to chemical hypoxia. In the following experiments, chemical hypoxia was used to stimulate secretion since CA secretion in response to mild hypoxia was inconsistent (see Discussion).
The CA secretion in response to various stimuli is thought to be mediated by an increase in [Ca2+]i. Thus, we investigated the role of external Ca2+ ions in secretion by CN and muscarine. Catecholamine release triggered by both agents was diminished markedly by substitution of 3.6 mM Mg2+ for 1.8 mM Ca2+ ions in the perfusate (Fig. 2A): amounts of CN- and 3 μM muscarine-sensitive secretion in the absence of Ca2+ ions were −1.8 ± 22.5 % (n= 6) and 5.0 ± 6.1 % (n= 5), respectively, of those in their presence (P < 0.05), thereby indicating the critical role of Ca2+ influx in the secretion. Thus, the possible involvement of voltage-dependent Ca2+ channels was explored. The application of 0.1 mM Cd2+ ions, an inorganic Ca2+ channel blocker, reduced CA secretion by CN and by muscarine to 10.4 ± 6.3 % (n= 6) and 5.3 ± 6.3 % (n= 5) of controls (P < 0.05; Fig. 2B and C). Similarly, administration of 10 μM D-600, an organic Ca2+ channel blocker, decreased the secretion by CN and by muscarine to 7.8 ± 1.1 % (n= 5) and 6.5 ± 4.5 % (n= 5) of controls (P < 0.05; Fig. 2D). These results suggest that CA releases in response to CN and to muscarine occur as a consequence of opening of voltage-dependent Ca2+ channels. If this thesis is tenable, then CN should induce membrane depolarization with the consequent generation of action potentials. Figure 3 shows that this is indeed the case: depolarizations with a robust generation of action potentials were consistently evoked by application of CN as well as muscarine. In contrast to CA release, exposure to CN and 3 μM muscarine induced similar amplitudes of depolarization (CN, 11.3 ± 4.6 mV, n= 6; muscarine, 13.4 ± 2.5 mV, n= 4; P > 0.05), without a substantial delay. While the muscarine-induced depolarization subsided rapidly after washout, the CN-induced depolarization outlasted the exposure.
Ca2+ influx in response to CN
To examine the involvement of Ca2+ influx in the muscarine- and CN-induced secretion further, change in [Ca2+]i was measured using the Ca2+-sensitive probe fluo-3 AM. In single or clustered cells loaded with fluo-3 AM, the addition of 1 ml of 6 μM muscarine or 10 mM CN-containing solution (final concentration, 3 μM or 5 mM) to the dish solution reversibly produced an increase in the fluorescence signal in the presence of 5 mM glucose, and at the end of the experiment, the administration of 20 μl A-23187 solution (final concentration, 1 μM) further enhanced the fluorescence (Fig. 4). On the other hand, such an increase rarely occurred in unloaded cells (n= 3). These results indicate that an increase in the signal reflects an increase in [Ca2+]. On average, maximum increases of the signal by muscarine and CN in the presence of external Ca2+ were 80.7 ± 47.0 % (n= 17) and 52.3 ± 33.8 % (n= 17) of the intensity before stimulation, and the signals became maximal 6 ± 4 and 29 ± 29 s after administration, respectively. These Ca2+ signals were abolished by the substitution of 3.6 mM Mg2+ for 1.8 mM Ca2+ (relative fluorescence change at 1 min of stimulation, 0.4 ± 8.7 % (n= 13) for muscarine and 2.2 ± 6.3 % (n= 8) for CN). Results obtained using the Ca2+ probe further support the notion that exposure to CN induces an increase in [Ca2+]i by facilitating the influx of Ca2+ ions.
Activation of cation channels by CN
The foregoing results indicate that the depolarization-induced Ca2+ influx is primarily responsible for the CA secretion by chemical hypoxia. Thus, we examined the effects of severe hypoxia and CN on whole-cell currents to identify the channel involved in the depolarization. Figure 5A shows a whole-cell current recorded at −62 mV, an equilibrium potential for Cl− ions, with the low Cl−, Na+-free pipette solution. Compared with development of the current evoked by muscarine, exposure to severe hypoxia and CN resulted in slow developments of the current with little delay, and the maximum amplitude of currents evoked by severe hypoxia and CN was 68 ± 34 % (n= 5) and 114 ± 44 % (n= 8) of the muscarinic current in the same cells, respectively. After a switch to normoxia or washout of CN, the evoked current diminished slowly. To confirm that this effect of CN is due to chemical hypoxia, whole-cell currents were recorded at −62 mV using the low Cl− solution and 5 mM CN was applied for 2 min in the presence of 10 mM glucose or 10 mM sucrose. In nine cells, the maximum amplitudes of CN-induced currents in the presence of sucrose were 90.7 ± 9.5 % of those in glucose, indicating no effect of glucose on the CN current. Next, we explored whether chemical hypoxia produced the current in a graded manner (Fig. 5B). As the concentration of applied CN decreased, development of the current and a maximum amplitude became slow and small, respectively, but the onset of development did not seem to be altered. In Fig. 5C, maxima of the inward current were plotted against concentrations of CN; the relationship exhibits a good fit to a Michaelis-Menten type equation with an ED50 of 0.34 mM.
The findings that exposure to hypoxia or CN resulted in development of an inward current at an equilibrium potential for Cl− ions indicate that the current is not a Cl− current. Furthermore, the CN-induced current was not affected by removal of Na+ ions from the pipette solution. When recorded with the low Cl− solution and the low Cl−, Na+-free solution, application of CN evoked 3.4 ± 1.0 pA (n= 6) and 4.0 ± 2.1 pA (n= 5) of the inward current at −62 mV, respectively. This result would mean that inhibition of the Na+ pump is probably not responsible for generation of the current. To elucidate the ionic mechanism for the CN-induced inward current further, a reversal potential was measured (Fig. 6). The CN-induced current, which was obtained by subtracting control currents from currents in response to command pulses during application of CN, reversed polarity at −24 mV (B; −24.0 ± 3.9 mV, n= 7), suggesting that the CN-sensitive channel is permeable to Na+ and probably K+. This notion was supported by a decrease in the CN current in a Na+-deficient perfusate. When 134.3 mM Na+ ions were replaced with equimolar N-methyl-D-glucamine (NMG; [Na+], 5.3 mM), CN currents induced at −62 mV decreased by 70.7 ± 8.3 % (n= 4). The other characteristic of CN-sensitive channels was a voltage-dependent gating. The CN-induced current decreased with hyperpolarizations below −60 mV, and little current was elicited at −112 mV. This voltage dependence developed rapidly: the current either diminished or disappeared almost instantaneously upon hyperpolarizations (Fig. 6A, lower panel).
Similarity between CN-sensitive and muscarinic cation channels
The voltage-dependent properties of CN-sensitive cation channels are similar to those of channels activated by muscarinic agonists (Inoue & Kuriyama, 1991a; Inoue & Imanaga, 1996). This raises the possibility that exposure to CN activates muscarinic cation channels. To explore this possibility, the voltage dependence of CN-sensitive currents was compared with that of muscarine-induced currents in the same cells. Figure 7A shows both currents at various membrane potentials. As the membrane potential was hyperpolarized, inward currents in response to CN and to 3 μM muscarine decreased in a parallel manner. Figure 7B is a summary of relative amplitudes of CN- and muscarine-induced currents. Both currents had a similar voltage dependence, suggesting that the CN-induced current may be flowing through muscarinic cation channels. Furthermore, the inward current evoked by severe hypoxia had voltage dependence similar to that of the CN current: severe hypoxia and CN currents at −70 mV were 66.9 ± 10.7 % (n= 4) and 63.9 ± 23.9 % (n= 4; P > 0.05) of the currents at −62 mV, respectively.
The involvement of common channels was further suggested by the failure of muscarine to induce an inward current during exposure to CN (Fig. 8). The upper panel in Fig. 8A shows that muscarine produced inward currents in a dose-dependent manner. Such currents did not develop during generation of an inward current by CN. On the contrary, the nicotinic receptor-regulated current flowing through cation channels other than muscarinic ones (Inoue & Kuriyama, 1991b) was not altered by generation of the CN-sensitive current (Fig. 8B). Figure 8C is a summary of the muscarine and nicotine-induced currents in the absence and presence of CN. The mitochondrial inhibitor selectively and reversibly suppressed further production of an inward current by muscarine. This result might be accounted for by a block of signal transmission somewhere from a muscarinic receptor to the channel. For elucidation, we examined effects of CN on outward and inward currents in response to 6 μM muscarine. This outward current is due to Ca2+ ions mobilized from intracellular store sites, probably by inositol 1,4,5-trisphosphate (Inoue, Sakamoto & Imanaga, 1995). Figure 9A shows that during exposure to CN, application of muscarine elicited the outward, but not inward, current. The substantial decrease in outward current during exposure to CN may be due to a decrease in [Ca2+] of store sites, since caffeine-induced outward currents were also diminished under similar conditions. This selective action of CN was consistently observed in two other cells. Furthermore, the addition of 4 mM extra Mg2+ ions to the perfusate suppressed the CN-induced currents by 29.7 ± 1.4 % (n= 4; not shown), similar to the extent of diminution of muscarinic currents (25.8 ± 8.0 %, n= 4: Inoue & Imanaga, 1993a; P > 0.05). Finally, amplitudes of CN-evoked currents which were measured about 1 min after application were roughly equal to those of 3 μM muscarine-induced currents, irrespective of the variety of amplitudes in the cells tested (Fig. 9B).
Possible activation of muscarinic cation channels by hypoxia
Exposure to mild hypoxia enhanced CA secretion in half the cells that responded to CN, and the extent of mild hypoxia-induced secretion in the responsive cells was about 50 % of that elicited by CN. On the other hand, amplitudes of inward current evoked by severe hypoxia averaged about 70 % of those evoked by CN in all the cells tested and the hypoxia- and CN-induced currents revealed a similar voltage dependence. In addition, the enhancement of CA secretion and production of the inward current was not affected by the addition of glucose. These results indicate that the effects of CN are due to chemical hypoxia and subsequent dysfunction of the mitochondria. The difference in potency between mild hypoxia and severe hypoxia may be ascribed solely to the level of decreased O2 tension since in one cell responding to CN, mild hypoxia did not induce an inward current. We could not study effects of severe hypoxia on CA secretion since Na2S2O4 interfered with the carbon electrode.
Chemical hypoxia consistently produced inward currents at membrane potentials of −62 mV or more negative, irrespective of whether or not Na+ ions were present in the cells. This observation indicates that the current is not due to suppression of the Na+ pump, since the pump does not run forwards in the absence of intracellular Na+. The CN-sensitive current was evoked at the equilibrium potential for Cl− ions and was markedly suppressed by an equimolar replacement of 134.3 mM Na+ ions with NMG. The reversal potential for CN-sensitive currents under standard conditions was −24 mV, a membrane potential which is half-way between the equilibrium potentials for Na+ and K+. These results raise the possibility that CN would activate cation channels permeable to Na+ and K+ ions. This CN-sensitive cation channel may be identical to the muscarinic channel. First, conductance of the CN-sensitive channel decreased with hyperpolarizations and this voltage dependence developed almost instantaneously, as was noted with the muscarinic cation channel (Inoue & Imanaga, 1996). Secondly, generation of the inward current by CN apparently prevented muscarine, but not nicotine, from inducing a further inward current. This prevention is not likely to be due to a block of the signal transmission from the receptor to the channel since the outward current which high concentrations of muscarine induced putatively through generation of inositol trisphosphate was not abolished by CN. Thus, the apparent occlusion is more likely to be due to the involvement of common channels in CN- and muscarine-induced currents. Finally, the CN-induced inward currents were suppressed by external Mg2+ ions to an extent similar to that seen with inhibition of muscarinic currents, and amplitudes of muscarine- and CN-induced currents were closely correlated.
The finding that CN mimics the effects of hypoxia indicates that the cation channel itself does not sense O2 tension and that dysfunction of mitochondria may play a pivotal role in channel activation. In general, the suppression of oxidative phosphorylation in mitochondria results in a decrease in intracellular pH (Wang, Randall & Thayer, 1994), an increase in [Ca2+]i, and diminution in cellular ATP contents. Among these alternations, the Ca2+ increase is the result of activation of cation channels, as was evident with measurement of intracellular Ca2+ ions (Fig. 4). We favour the view that a decrease in pH might not play a primary role in channel activation. First, exposure to CN consistently produced an inward current under conditions of whole-cell voltage clamp, where intracellular pH was set at 7.2 with addition of 5 mM Hepes to the pipette solution. Secondly, a decrease in pH generally induces a suppression of channel activity, such as a voltage-dependent K+ channel (Hille, 1992), and diminishes exocytosis in endocrine cells (Thomas, Wong, Lee & Almers, 1993). Finally, if a decrease in pH were involved in channel activation, then the activation should be enhanced in the presence of glucose since more H+ ions are expected to be produced by glycolysis under hypoxic conditions. The more likely cause for activation of cation channels by CN is a decrease in ATP contents. The ATP content in rat hepatocytes dropped to < 95 % of normal within 5 min of onset of metabolic inhibition with 5 mM CN and 2.5 mM iodoacetate (Gores, Nieminen, Fleishman, Dawson, Herman & Lemasters, 1988). This decrease in ATP concentration by mitochondrial and/or glycolytic inhibitors was shown to disrupt reversibly the cortical actin network in renal tubule cells (Molitoris, Geerdes & McIntosh, 1991) and endothelial cells (Hinshaw, Burger, Miller, Adams, Beals & Omann, 1993). A short actin filament resulting from disruption of the F-actin filament might be responsible for openings of cation channels, as was reported for Na+ channels in the epithelial cell line A6 (Cantiello, 1995). Alternatively, a change in membrane stretch induced by disorganization of the cortical actin network might be involved in channel activation.
Cyanide-induced release of CA
Catecholamine secretion by chemical hypoxia and by muscarine was either abolished or markedly diminished by removal of external Ca2+ ions or by application of Ca2+ channel blockers, and a sustained increase in [Ca2+]i in response to CN and muscarine was abolished by substitution of Mg2+ ions for external Ca2+. Furthermore, exposure to CN and 3 μM muscarine resulted in similar amplitudes of depolarization with a robust generation of action potentials, and both agents were similarly potent in activating cation channels. Thus, CA release by both agents is probably due to openings of cation channels and to the subsequent activation of voltage-dependent Ca2+ channels. Consistent with this notion, average time courses of an increase in [Ca2+] in response to CN and muscarine roughly parallel those of CA secretion. Since the input impedance at the resting membrane potential (−60 to −70 mV) in the chromaffin cell is high (Inoue & Imanaga, 1993b), the production of inward currents in the order of picoamps by muscarine or CN would be sufficient for the depolarization and subsequent firings.
Compared with CN, 3 μM muscarine facilitated three times the amount of CA secretion and produced a 1.5-fold increase in [Ca2+]. This discrepancy between the effects on secretion and [Ca2+] and on the cation channel would be in part due to a slow activation of the channel by CN (e.g. Figs 5 and 6). Another contribution would be an inhibitory action of CN on CA secretion. The major part of this suppression may not be due to inhibition of voltage-dependent Ca2+ channels, since CA was spontaneously secreted even in the absence of a voltage-dependent Ca2+ influx (absence of external Ca2+ ions or application of Ca2+ channel blockers). This spontaneous secretion ceased for a few tens of seconds following enhancement of secretion by CN (Fig. 2). Finally, a membrane current or potential might have been recorded from less damaged cells, in comparison with measurements for secretion or [Ca2+]. It was difficult to maintain whole-cell recordings from damaged cells, but this kind of difficulty principally does not exist in measuring secretion or Ca2+ ions.
The mechanism for facilitation of CA secretion by hypoxia in the chromaffin cell evidently differs from that for dopamine secretion by hypoxia in glomus cells of rat and rabbit carotid bodies. In the latter, an increase in [Ca2+]i was due to a voltage-dependent Ca2+ influx induced by suppression of K+ channels and the subsequent depolarization (López-Barneo, 1996; Buckler, 1997). In the chromaffin cell, exposure to CN also suppressed inwardly rectifying K+ channels (Inoue, Sakamoto, Yano & Imanaga, 1997), which contribute mainly to a resting membrane potential (Inoue & Imanaga, 1993b). This suppression, however, began to develop slowly about 3 min after CN application and was at most 50 % at 12 min, and thus the time course conspicuously differed from that of activation of cation channels. The inhibition of inward rectifier was due to depletion of cellular ATP and the subsequent dephosphorylation of the channel or regulator. Thus, such a long exposure to chemical hypoxia may alter activity of other channels, such as Ca2+-dependent K+ channels, since phosphorylation of the channels or regulators is known to decrease or increase it (Levitan, 1994). Because of these complicated actions on ion channels, mechanisms related to hypoxia-induced secretion might differ with short and long exposures. In the light of little latency and graded activation, however, there is no doubt that hypoxia sensing through cation channels plays a pivotal role for defence against the life-threatening events. This mechanism may not be confined to the guinea-pig adrenal medullary cell, since similar cation channels appear to be present in the rat (Herrington, Solaro, Neely & Lingle, 1995) and hypoxia was recently shown to enhance CA secretion from cells of new-born rats (Thompson et al. 1997). This capacity of the rat adrenal cell to sense hypoxia was lost by the second postnatal week, whereas that of the guinea-pig persists.
The present results demonstrate that in addition to hypoxia sensing by carotid body cells (Fishman et al. 1985) and brainstem neurons (Sun & Reis, 1994), the adrenal chromaffin cell itself is capable of detecting hypoxia through cation channels. This mechanism in the chromaffin cell may fully function in cases of extreme hypoxia or anoxia, since such conditions would likely impair neuronal transmission in the nervous system (Lipton & Whittingham, 1979). Finally, it should be noted that cation channels responding to hypoxia and to muscarinic stimulation may be one and the same. Thus, the effector involved in neuronally induced secretion may also be operative in hypoxia-induced secretion.
This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Ciba-Geigy Foundation (Japan).