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MS 9385 Received 15 March 1999; accepted after revision 2 July 1999.
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ABSTRACT |
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-conotoxin GVIA (1 µM) or -agatoxin GIVA (200 nM). The two toxins applied together almost completely abolished (> 97 %) acid-evoked secretion.
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INTRODUCTION |
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The carotid body is the major arterial chemoreceptor and responds to physiological stimuli (hypoxia, hypercapnia, acidosis) by increasing the discharge frequency of afferent chemosensory neurons, thereby initiating corrective changes in breathing pattern (Fidone & Gonzalez, 1986; Gonzalez et al. 1994). Type I (glomus) cells within the carotid body are central to this chemoreceptive process, releasing transmitters - particularly catecholamines - in response to physiological stimuli in a manner which correlates well with increased chemosensory nerve activity (reviewed by Gonzalez et al. 1994). Patch-clamp recordings have shown that type I cells possess O2-sensitive K+ channels whose activity is reduced under hypoxic conditions (Lopez-Barneo et al. 1988; Delpiano & Hescheler, 1989; Peers, 1990a; Stea & Nurse, 1991). Such an effect causes membrane depolarization, opening of voltage-gated Ca2+ channels (Buckler & Vaughan-Jones, 1994a; Wyatt & Peers, 1995) and thus transmitter release (Urena et al. 1994). In the rat model, evidence suggests that acidic/hypercapnic stimuli evoke transmitter release via a similar mechanism (Peers, 1990b; Peers & Green, 1991; Buckler & Vaughan-Jones, 1994b) although in the rabbit model, a completely different mechanism has been put forward to account for transduction of acidic stimuli (Rocher et al. 1991).
Hypoxic and acidic stimuli are well-known to be multiplicative in their ability to increase afferent chemosensory nerve discharge (Fitzgerald & Parks, 1971; Lahiri & Delaney, 1975), an effect which may account for the well-known postnatal maturation of this sensory organ (Pepper et al. 1995). However, no explanation has been forwarded to account for this interactive effect at the cellular level, and it remains unknown whether this interaction occurs within the type I cell or involves other cellular elements of the carotid body.
Recently, evidence has emerged that the rat phaeochromocytoma (PC12) cell line responds to hypoxia in a manner which is remarkably similar to that of the type I carotid body cell. Thus, hypoxia inhibits K+ channels in these cells, causing membrane depolarization and a subsequent rise in [Ca2+]i (Zhu et al. 1996; Conforti & Millhorn, 1997). In addition, we have shown that hypoxia evokes quantal secretion of catecholamines from PC12 cells, which is entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels and is inhibited by > 90 % following treatment of cells with the N-type Ca2+ channel blocker -conotoxin GVIA (Taylor & Peers, 1998). Furthermore, PC12 cells respond to prolonged hypoxia by increasing tyrosine hydroxylase mRNA levels (Czyzyk-Krzeska et al. 1994) in a manner that compares extremely well with responses observed in type I cells (Czyzyk-Krzeska et al. 1992). There are no available data to date, however, to suggest that PC12 cells act (like type I cells) as acid chemoreceptors. The present study was therefore conducted to investigate the ability of acidic stimuli to evoke transmitter release from individual PC12 cells, and to determine whether acidic and hypoxic stimuli were interactive in their ability to evoke secretion.
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METHODS |
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PC12 cells obtained from the American Tissue Type Cell Collection (Rockville, MA, USA) were cultured as described previously (Taylor & Peers, 1998) in RPMI 1640 culture medium (containing L-glutamine) which was supplemented with 20 % fetal calf serum and 1 % penicillin-streptomycin (all from Gibco). Cells were incubated at 37°C in a humidified atmosphere of 5 % CO2-95 % air and used for up to 20 passages. Each passage was conducted after 7 days, when the cells were resuspended in fresh medium and diluted 1:2. This prolonged period without medium change has previously been shown to enhance evoked catecholamine release from PC12 cells (Takashima & Koike, 1985). Cells used for experiments were transferred to smaller flasks in 10 ml of medium to which was added 1 µM dexamethasone (Sigma; from a stock solution of 1 mM in Ultrapure water) and were cultured for a further 72-96 h to enhance catecholamine secretion further (Tischler et al. 1983).
On each experimental day, PC12 cells were plated onto poly-L-lysine-coated coverslips and allowed to adhere for approximately 1 h. Fragments of coverslip were then transferred to a recording chamber (volume, 80 µl) which was continuously perfused under gravity (flow rate, 1-2 ml min-1) with a solution of composition (mM): NaCl, 135; KCl, 5; MgSO4, 1·2; CaCl2, 2·5; Hepes, 5; and glucose, 10 (pH 6·8-7·4, osmolarity adjusted to 300 mosmol l-1 with sucrose, 21-24°C). Ca2+-free solutions contained 1 mM EGTA and no added Ca2+. All drugs were applied in the perfusate except in the cases of -conotoxin GVIA (-CgTX) and -agatoxin GIVA (-Aga-IVA). The effects of these agents were investigated by pre-incubation of cells in extracellular solution containing these agents for at least 10 min. Experiments were conducted within 3 min of transfer of these cells to the perfused recording chamber. The effects of nifedipine (Sigma) were investigated at low light intensity, and nifedipine was added to the perfusate from a stock solution of 20 mM in ethanol, made fresh each day. Ethylisopropylamiloride (EIPA; Sigma) was added to the perfusate from a 30 mM stock in dimethylsulphoxide. Hypoxic solutions were obtained by continuously bubbling one or more of the reservoirs supplying the recording chamber with N2 as required. Hypoxic reservoirs were pre-equilibrated with N2 for at least 30 min before being applied to cells.
Carbon fibre microelectrodes (proCFE; Axon Instruments) with a diameter of 5 µm were positioned adjacent to individual PC12 cells using a Narishige MX-2 micromanipulator and were polarized to +800 mV to allow oxidation of released catecholamine. Resulting currents were recorded using an Axopatch 200A amplifier (with extended voltage range; Axon Instruments), filtered at 1 kHz and digitized at 2 kHz before storage on computer. All acquisition was performed using a Digidata 1200 interface and Fetchex software from the pCLAMP 6.0.3 suite (Axon Instruments). The same equipment was also used to monitor PO2 levels in the recording chamber, except that the polarity of the microelectrode was reversed to -800 mV (see Mojet et al. 1997). The time course of the fall in PO2 in the recording chamber was highly reproducible for any given degree of hypoxia.
Unless otherwise stated, each experiment consisted of current recordings of a control period during which cells were only perfused with normoxic external medium. This was then exchanged for a test solution and amperometric signals were recorded for a further period of 1-4 min. Catecholamine secretion was apparent as discrete spike-like events, each corresponding to the released contents of a single vesicle of catecholamine (Wightman et al. 1991; Chow & Von Ruden, 1995). We did not distinguish between dopamine and noradrenaline, both known to be released from PC12 cells (e.g. Kumar et al. 1998), but secretory events were never seen unless the electrode was polarized and adjacent to a cell. Quantification of release was achieved by determining spike number or frequency using Minian 16 software (Jaejin Software, Columbia, NY, USA). This allowed visual inspection of each event so that artefacts (due, for example, to solution switches) could be rejected from analysis. We did not integrate events in order to estimate quantal size (Taylor & Peers, 1999), since pH is known to affect catecholamine levels within vesicles, and the rate of release of catecholamines (Jankowski et al. 1994); this would preclude comparison between different levels of acidic stimulation and between acidic and hypoxic stimulation.
Results are presented as individual examples or means ± standard error of the mean and statistical comparisons were made using Student's unpaired t test.
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RESULTS |
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Under normoxic conditions at physiological pH (7·4), no secretion was detected from PC12 cells (e.g. Fig. 1A; representative of > 50 cells). However, reduction of pH to between 7·2 and 6·8 caused a graded increase in the appearance of exocytotic events (Fig. 1B-D), indicating that acidosis was an effective secretagogue in these cells. To quantify the amount of exocytosis evoked by exposure of cells to different levels of acidity, the mean cumulative number of events detected at each pH level studied was plotted (Fig. 2). It is clear that even a modest acidification (to pH 7·2) could evoke substantial secretion, and that the effects of acidosis approached saturation at pH 6·8. It is noteworthy that the release rate declined with time at higher stimulation levels, an observation which is likely to be attributable to the finite available pool of releasable vesicles.
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At pH 7·4, no secretion was detected from PC12 cells (A), but as the pH was reduced (B-D), a graded increase in secretion was observed. Each trace (A-D) was obtained from a single PC12 cell using a 5 µm diameter carbon fibre electrode polarized to +800 mV. In B-D, the arrow indicates the time point at which the perfusate was changed from one of pH 7·4 to solution at the pH levels indicated. The cells remained exposed to the acidic solution for the remainder of the period illustrated. Scale bars apply to all traces. | ||
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Mean cumulative numbers (with vertical S.E.M. bars) are plotted every 10 s for each pH level indicated, and were determined from between 9 and 10 cells except recordings made at pH 7·4 (where no release was observed) which were made in over 50 cells. Exchange of solution from control (pH 7·4) to the values indicated was at t = 5 s. | ||
In the carotid body type I cell, as in many cell types, a reduction in pHo causes a fall in pHi, and in the absence of significant CO2 or HCO3- in the perfusate, restoration of pHi is reliant on the activity of the Na+-H+ exchanger (Buckler et al. 1991a, b). We hypothesized, therefore, that secretion evoked by reduced pHo arose due to a resultant fall in pHi. To test this idea we examined the effects of the Na+-H+ exchange blocker EIPA (30 µM). When applied alone, EIPA evoked a modest amount of secretion (Fig. 3A), probably due to intracellular accumulation of protons. When EIPA was applied in a solution of pH 7·2, as shown in the example trace and average cumulative secretion plot of Fig. 3A, secretion evoked by the pH 7·2 solution was dramatically enhanced. In further support of the idea that extracellular acidification evokes secretion by causing intracellular acidification, we also found that application of sodium propionate (10 mM), which causes selective intracellular acidosis (see e.g. Peers & Green, 1991), was also extremely effective in evoking secretion from PC12 cells (Fig. 3B).
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A, left: example recording from a PC12 cell exposed (from the time point indicated by the arrow) to a solution of pH 7·2 containing 30 µM EIPA. Right, mean (with vertical S.E.M. bars) cumulative number of exocytotic events determined in cells exposed (at t = 5 s) as indicated to a solution of pH 7·2 in the absence ( | ||
Figure 4A (representative of 7 cells) shows ongoing secretion from a PC12 cell exposed to a solution of pH 6·8. For the period indicated by the horizontal bar, the acidic pH was maintained but extracellular Ca2+ was replaced with 1 mM EGTA; this caused complete and reversible inhibition of secretion. Similarly, if instead of removing extracellular Ca2+ the non-selective blocker of voltage-gated Ca2+ channels Cd2+ (200 µM) was applied, secretion was again fully inhibited (Fig. 4B, representative of 6 cells). Taken together, these data suggest that acid-evoked secretion from PC12 cells is fully dependent on Ca2+ influx through voltage-gated Ca2+ channels.
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A and B, ongoing secretion evoked from 2 different PC12 cells by exposure to solution of pH 6·8. For the periods indicated by the horizontal bars, either Ca2+ was removed from the perfusate and replaced with 1 mM EGTA (A), or Cd2+ (200 µM) was applied in the continued presence of Ca2+ (B). Scale bars apply to both traces. C, bar graph showing mean number of secretory events (with vertical S.E.M. bars) recorded over a 145 s period, evoked by reducing external pH from 7·4 to 6·8 in the absence of Ca2+ channel blockers (control), in Ca2+-free solution (containing 1 mM EGTA), in the presence of 200 µM Cd2+ or 2 µM nifedipine, or following a 10 min pretreatment period with 1 µM | ||
PC12 cells are known to possess at least three classes of voltage-gated Ca2+ channels (see Discussion). In order to evaluate their relative contribution to acid-evoked secretion, we examined the effects of selective blockers for these channels. The results are summarized in Fig. 4C. The L-type Ca2+ channel blocker nifedipine (2 µM) was completely without effect on acid-evoked secretion, but pretreatment of cells with either the N-type Ca2+ channel blocker -CgTX (1 µM) or the P/Q-type Ca2+ channel blocker -Aga-IVA (200 nM) caused significant reductions (P < 0·01 and P < 0·001, respectively) in secretion evoked by a reduction in extracellular pH to 6·8. Pretreatment of cells with both toxins almost completely inhibited the evoked secretion (Fig. 4C). Thus, Ca2+ influx via both N- and P/Q-type Ca2+ channels appears to be of primary importance for acid-evoked secretion from PC12 cells.
Hypoxic and acidic stimuli are greater than additive in their ability to excite the carotid body, as determined by recordings of afferent chemosensory discharge (Fitzgerald & Parks, 1971; Lahiri & Delaney, 1975). However, it remains unknown whether this multiplicative effect is also reflected in the release of transmitters from these cells. Figure 5 indicates that under acidic (pH 6·8) and hypoxic (PO2, 6 mmHg; Fig. 5B) conditions, secretion is far greater than under hypoxic, alkaline (pH 8·0; Fig. 5C) conditions. To quantify the potentially interactive effect of these two stimuli in greater detail, we tested the effects of three levels of hypoxia at three different levels of pH. The two stimuli were applied and removed at the same time point, and since the bath PO2 level takes over 60 s to stabilize, we measured the frequency of occurrence of exocytotic events over a 60 s period, 90 s after switching to hypoxic-acidic solutions. Results are presented in Fig. 6. Exocytotic frequency at the control pH level of 7·4 increased with increasingly severe hypoxia, in excellent agreement with our previous study (Taylor & Peers, 1999). It is clear that hypoxia-evoked secretion was enhanced under acidic conditions and suppressed under alkaline conditions, and that the effects of these two stimuli were greater than additive. To quantify these effects in greater detail, we used analysis of covariance (with DMstat2.1; Dr D. A. S. G. Mary, University of Leeds) to determine statistically significant differences in the slopes of these plots. Regression analysis indicated that at each pH level tested, the relationship between secretion rate and PO2 was linear over the range examined, and yielded slope values of -0·065 ± 0·013, -0·034 ± 0·007 and -0·012 ± 0·004 Hz mmHg-1 at pH levels of 6·8, 7·4 and 8·0, respectively. Slopes determined at pH levels of 6·8 and 7·4 were significantly steeper than that determined at pH 8·0 (single-tailed probability values, F = 0·001 and 0·002, respectively), and the slope at pH 6·8 was significantly steeper (F = 0·04) than that at pH 7·4. These findings suggest that the effects of hypoxia and acidity to evoke secretion are synergistic.
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A, recording of perfusate PO2 before, during and after application of solution pre-equilibrated and continuously bubbled with N2. B, secretory response of a representative PC12 cell to the hypoxic challenge at pH 6·8. C, secretory response of another PC12 cell to the hypoxic challenge at pH 8·0. Scale bars apply to both B and C. | ||
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Plot of exocytotic frequency (determined over a 1 min period, 90 s after switching to hypoxic solution) as a function of final bath PO2 at three different pHo levels, as indicated. Each point shows the mean (with vertical S.E.M. bars, n = 6-18 cells in each case) exocytotic frequency determined from experiments such as those shown in Fig. 5. | ||
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DISCUSSION |
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The carotid body chemoreceptor, in addition to acting as an O2 sensor, is also an effective sensor of arterial CO2 and pH. All of these stimuli share the common feature that they evoke transmitter release from type I cells in a manner that correlates well with increased sensory nerve discharge (reviewed by Gonzalez et al. 1994). Microfluorimetric measurements of pHi in isolated type I cells have revealed that these cells, despite possessing various plasma membrane processes for regulating pHi (Buckler et al. 1991b), have a very steep pHi-pHo relationship (Buckler et al. 1991a). Furthermore, distinct changes in pHi in response to simulated respiratory or metabolic acidosis, or to isohydric hypercapnia, are temporally matched by the pattern of discharge of chemosensory neurons in the carotid sinus nerve (Grey, 1968; Buckler et al. 1991a). This suggests strongly that changes in pHi are of central importance in the functioning of the carotid body as an acid/CO2 sensor (see also Hanson et al. 1981; Rigual et al. 1991).
The present study indicates that acidosis is an effective secretagogue for PC12 cells, which have already been proposed as an excellent model system for studying O2 chemoreception (Zhu et al. 1996; Taylor & Peers, 1998). A modest reduction in pHo from 7·4 to 7·2 was capable of evoking quantal secretion, and the response to acidosis approached saturation at pH 7·0-6·8. Two lines of evidence suggested that this effect of reducing pHo was mediated by an intracellular acidosis. Firstly, application of acidic solutions in the presence of EIPA to inhibit the Na+-H+ exchanger increased secretion at the submaximally effective pHo level of 7·2 (Fig. 3A). In the absence of CO2 and HCO3- in the perfusate, Na+-H+ exchange is likely to be the major mechanism for pHi regulation and inhibition of this exchanger with EIPA would therefore prevent proton extrusion and thus promote intracellular acidification. Indeed, application of EIPA alone (pHo 7·4) induced secretion, presumably due to intracellular accumulation of protons (Fig. 3A). Secondly, the effects of reducing pHo were mimicked by application of propionate (Fig. 3B), a weak acid which causes selective intracellular acidosis (see Peers & Green, 1991). Our data are therefore consistent with the idea that extracellular acidity stimulates exocytosis from PC12 cells by causing an intracellular acidosis, as has been proposed for carotid body type I cells.
It is notable from the examples of Fig. 1 and more clearly from the averaged data of Fig. 2 that exocytosis evoked under strongly acidic conditions (pH 7·0 and pH 6·8) was initially rapid, then declined somewhat during prolonged exposure, yet at the less acidic level of pH 7·2 release was more sustained. This is consistent with the idea that PC12 cells have a finite, readily releasable pool of vesicles, which becomes significantly depleted more rapidly when the release rate is high. This was also the case when a solution of pH 7·2 in the presence of EIPA and when sodium propionate was applied, both forms of stimulation resulting in very large amounts of release (Fig. 3).
In the rat carotid body type I cell, acidic changes in pHi are closely mirrored by rises in [Ca2+]i (Buckler & Vaughan-Jones, 1993), and also inhibit an O2-sensitive K+ channel in these cells (Peers & Green, 1991). Such findings have led to the suggestion that acidosis, like hypoxia, evokes secretion from type I cells via membrane depolarization and subsequent Ca2+ influx through voltage-gated Ca2+ channels. However, such a mechanism has not yet been directly demonstrated, and there is evidence in the rabbit type I cell that acid-evoked transmitter release arises from accumulation of intracellular Na+ (due to stimulation of Na+-H+ exchange) which is sufficient to reverse Na+-Ca2+ exchange (Rocher et al. 1991), thereby allowing Ca2+ entry via transporters rather than ion channels. Thus, it was of importance to investigate the mechanism underlying acid-evoked secretion in PC12 cells. Clearly, acid-evoked secretion was fully prevented by removal of extracellular Ca2+ (Fig. 4A), but this observation alone does not distinguish between the two proposed mechanisms described above for the type I cell. However, acid-evoked secretion was fully abolished by the non-selective voltage-gated Ca2+ channel blocker Cd2+ (Fig. 4B), indicating an absolute requirement for Ca2+ influx via these channels. Furthermore, acid-evoked release was significantly reduced by pretreatment of cells with -CgTX or -Aga-IVA, suggesting that the primary routes for Ca2+ influx were through N- and P/Q-type Ca2+ channels. By contrast, blockade of L-type Ca2+ channels with nifedipine had no discernible effect on acid-evoked secretion. PC12 cells are well known to possess both L- and N-type Ca2+ channels (Janigro et al. 1989; Usowicz et al. 1990), but the presence also of P/Q-type channels has only recently been established (Liu et al. 1996), and their role in PC12 cell functioning has not been identified. Without nerve growth factor-induced differentiation, the contribution of N- and P/Q-type channels to whole-cell Ca2+ currents is less than that of L-type (Usowicz et al. 1990; Liu et al. 1996) and so it was perhaps surprising that only N- and P/Q-type channels contributed to Ca2+ influx coupled to secretion in response to acidosis (Fig. 4). However, both N- and P/Q-type channels are known to interact directly with secretory vesicle proteins (Leveque et al. 1994, 1998; Charvin et al. 1997) indicating that exocytotic sites at the plasma membrane are located extremely close to these channel types. Thus a local rise in [Ca2+]i due to influx through N- or P/Q-type channels is likely to evoke exocytosis even if these channels are only present at low density.
An intriguing observation arising from the present study, when compared with our previous report (Taylor & Peers, 1998), is that acid-evoked release is mediated by both N- and P/Q-type Ca2+ channels (Fig. 4), whereas hypoxia-evoked release is mediated almost exclusively via N-type channels. At present, we cannot categorically account for this difference but it should be noted that secretion evoked by pHo 6·8 was considerably higher than that evoked by hypoxia alone (Taylor & Peers, 1998). This suggests that pHo 6·8 promoted greater voltage-gated Ca2+ entry, presumably by causing greater depolarization than hypoxia. Acidosis is known to exert an inhibitory effect on high-voltage-activated Ca2+ channels (e.g. Tombaugh & Somjen, 1996) and, importantly, N-type channel activation is shifted to more positive potentials under acidic conditions (Zhou & Jones, 1996). Thus it is possible to account for the differential influence of N- and P/Q-type channels in mediating secretion evoked by hypoxia and acidity if N-type channels, at the control pH level of 7·4, have a more negative activation threshold than P/Q-type channels in these cells. If this were the case, then hypoxia-evoked release (due to more moderate depolarization than that caused by pHo 6·8) might activate only N-type channels, whereas pHo 6·8 could evoke Ca2+ entry through both N- and P/Q-type channels by causing greater depolarization; this would activate both classes of channel and overcome (at least in part) the known positive shift in activation of N-type channels caused by acidity. Such a scheme must remain speculative at present, since there is no detailed information concerning the differential effects of pH on different classes of Ca2+ channel within a given preparation. Furthermore, although hypoxia is known to regulate L-type Ca2+ channels (Fearon et al. 1997), its effects on other classes of Ca2+ channel remain completely unexplored.
Hypoxia and acidity have long been known to be greater than additive in their ability to increase afferent chemosensory nerve activity, since the slope of the linear relationship between CO2/acidosis and nerve activity increases with increasing degrees of hypoxia (Fitzgerald & Parks, 1971; Lahiri & Delaney, 1975). However, no mechanism has been demonstrated to account for this characteristic feature of arterial chemoreception. Indeed, it is not known whether interaction of these stimuli occurs at the level of the type I cell, or whether it involves other cellular elements within the carotid body (e.g. afferent nerve endings, type II cells). Here, we clearly demonstrate that hypoxia and acidity are interactive at the level of catecholamine release from PC12 cells (Figs 5 and 6), a finding which suggests that, within the carotid body, cellular elements other than type I cells may not be involved in the multiplicative effects of hypoxia and acidosis. The results of experiments with Ca2+ channel blockers (Fig. 4) indicate likely mechanisms for interaction of these two stimuli. In the present experiments, when both N- and P/Q-type Ca2+ channels were activated by acidosis, release of catecholamine was almost double the sum of that produced by N-type channels alone (in the presence of -Aga-IVA) and P/Q-type channels alone (in the presence of -CgTX). A non-linear response to an increased [Ca2+]i would be expected from the well-known co-operativity in the [Ca2+]i-induced release of neurotransmitters (e.g. Dodge & Rahamimoff, 1967). In PC12 cells, hypoxia-evoked exocytosis involves N-type Ca2+ channels almost exclusively, since -CgTX causes > 90 % inhibition of such release (Taylor & Peers, 1998). When acidosis accompanies hypoxia, the additional entry of Ca2+ through the acidosis-activated P/Q- and N-type Ca2+ channels (Fig. 4C) would greatly potentiate the secretion produced by hypoxia alone, when only the N-type channels are opened. The reduced sensitivity to hypoxia which accompanies alkalosis (Fig. 6) might be accounted for by the earlier observation that K+ currents in type I carotid body cells are enhanced at pH 8·0 (Peers & Green, 1991). Such an increase would suppress the depolarization produced by any degree of hypoxia, resulting in reduced Ca2+ influx through N-type channels and hence less catecholamine secretion.
In summary, we have demonstrated that acidity is an effective secretagogue in PC12 cells. Acid-evoked secretion involves intracellular acidification and activation of voltage-gated (N- and P/Q-type) Ca2+ channels, presumably via membrane depolarization. Importantly, acidity and hypoxia were found to be multiplicative in their ability to evoke secretion, a finding which may account for the interactive effects of these physiological stimuli on the carotid body chemoreceptors.
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REFERENCES |
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This work was supported by The British Heart Foundation. We are grateful also to Dr D. A. S. G. Mary (University of Leeds) for statistical advice and software.
Corresponding author
C. Peers: Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, UK.
Email: c.s.peers{at}leeds.ac.uk
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; n = 10; replotted from Fig. 2 for comparison) and presence (
; n = 7) of 30 µM EIPA. Also plotted is the cumulative number of events detected in 10 cells in response to application of EIPA alone (pHo 7·4; 
). B, left: example recording from a PC12 cell exposed (from the time point indicated by the arrow) to a solution containing 10 mM sodium propionate (pHo 7·4). Right, mean (with vertical S.E.M. bars) cumulative number of exocytotic events determined in 10 cells exposed to 10 mM sodium propionate from t = 5 s. Scale bars apply to both example traces.