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MS 8518 Received 21 July 1998; accepted after revision 1 October 1998.
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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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INTRODUCTION |
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O2 sensing is a property of numerous tissue types and responses to reduced O2 levels can be divided into two classes in terms of the rapidity of response. Rapid responses to acute hypoxia are exhibited by a variety of cell types, and were first described in the chemosensory carotid body type I cell, where acute hypoxia leads to rapid inhibition of certain K+ channel types (Lopez-Barneo et al. 1988; Peers, 1990; Stea & Nurse, 1991; Buckler, 1997). Similar responses were subsequently reported in tissues as diverse as pulmonary smooth muscle cells (see Weir & Archer, 1995), neuroepithelial cell bodies (Youngson et al. 1993) and central neurones (Jiang & Haddad, 1994). In each tissue type, K+ channel inhibition by hypoxia leads to important physiological responses, such as release of neurotransmitters from type I carotid body cells (Gonzalez et al. 1994; Urena et al. 1994) or constriction of pulmonary smooth muscle cells (Weir & Archer, 1995).
Slow responses to hypoxia include altered expression of specific proteins known to be important in oxygen homeostasis. In particular, induction of erythropoietin mRNA in kidney and liver cells is stimulated in hypoxia. This arises primarily from increased levels of a protein termed hypoxia-inducible factor 1 (HIF-1) which acts to promote transcription (Wang & Semenza, 1993; Ratcliffe et al. 1998; Caro & Salceda, 1998; see Bunn & Poyton, 1996, for review). Similarly, the steady-state levels of tyrosine hydroxylase mRNA in the carotid body are greatly enhanced following prolonged hypoxia (Czyzyk-Krzeska et al. 1992). Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine production, and so prolonged hypoxia can enhance release of this transmitter from the carotid body in part through increased production arising from increased transcription.
Rat phaeochromocytoma (PC12) cells represent an ideal model system for studying the mechanisms underlying both fast and slow reponses to hypoxia: they respond rapidly to acute hypoxia by depolarizing as a consequence of K+ channel inhibition, an effect which causes a rise of [Ca2+]i (Zhu et al. 1996) and subsequent release of catecholamines (Kumar et al. 1998; Taylor & Peers, 1998). In addition, slow responses of PC12 cells to hypoxia include increased tyrosine hydroxylase levels (Czyzyk-Krzeska et al. 1994) and expression of the Shaker Kv1.2 gene (Conforti & Millhorn, 1997). This latter observation is of particular interest since the Kv1.2 K+ channel appears to be selectively inhibited in a rapid manner by acute hypoxia (Conforti & Millhorn, 1997).
In the present study, we have investigated the functional effects of chronic hypoxia in PC12 cells by examining their secretory responses to acute hypoxia following a prolonged hypoxic episode. To do this, we have used carbon fibre microelectrodes for amperometric monitoring of quantal catecholamine release from PC12 cells in response to acute hypoxia.
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METHODS |
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PC12 cells were originally obtained from the American Tissue Type Cell Collection (Rockville, MA, USA) and stored in liquid nitrogen. To grow cells in culture, an aliquot was thawed rapidly at 37°C, diluted 1: 5 with RPMI 1640 culture medium (containing L-glutamine) supplemented with 20 % fetal calf serum and 1 % penicillin-streptomycin (from Gibco) and incubated at 37°C for 24 h in a humidified atmosphere of 5 % CO2-95 % air. Following this period, cells in suspension culture were removed from the flask, centrifuged at 70 g for 10 min, resuspended in fresh medium and re-seeded in flasks at low density. This preparation of cells was designated passage 1, and cells were used for experiments for up to 20 passages. Each passage was conducted after 7 days when the cells were resuspended in fresh medium and diluted 1 : 2. The prolonged period without medium change was believed to enhance evoked catecholamine release (Takashima & Koike, 1985). Cells used for experiments were transferred to smaller flasks in 10 ml of medium and 1 µM dexamethasone (Sigma, from a stock solution of 1 mM in ultrapure water) was applied for 72-96 h to enhance catecholamine secretion further (Tischler et al. 1983). Cells exposed to chronic hypoxia were treated identically, except that for 21-26 h prior to experiments they were transferred to a humidified incubator equilibrated with 10 % O2, 5 % CO2 and 85 % N2. Following this period in chronic hypoxia, cells were exposed to room air for no longer than 1 h before experimentation.
On each experimental day, aliquots of PC12 cells were plated onto poly-L-lysine-coated coverslips and allowed to adhere for about 1 h. Fragments of coverslip were then transferred to a recording chamber (volume ca 80 µl) which was continually 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 7·4, osmolarity adjusted to ca 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 case of the toxins 
-conotoxin GVIA toxin (-CgTX) and -agatoxin IVA (-Aga-IVA). The effects of these agents were investigated by pre-incubation of cells in extracellular solutions containing these agents for at least 10 min. Experiments were conducted within 3 min of transfer of these cells to the perfused recording chamber. Experiments investigating the effects of nifedipine were conducted at low light intensity, and nifedipine was added to the perfusate from a stock solution of 20 mM in ethanol, made fresh each day. Hypoxic solutions were obtained by continually 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), 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 fall in PO2 in the recording chamber (e.g. Fig. 1A) was highly reproducible for any given degree of hypoxia.
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A, measurement of bath PO2 determined with a carbon fibre microelectrode (applied potential -800 mV). B, amperometric recording (applied potential +800 mV) from a representative PC12 cell before, during and after exposure to acute hypoxia as shown in A. C, as B except that the recording was made from a PC12 cell that had previously been exposed to chronic hypoxia (10 % O2, 24 h). Scale bars apply to both B and C. | ||
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 acheived by determining spike 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 the analysis. In some experiments, events were integrated to obtain the charge, Q, in order to estimate quantal size as previously described (Finnegan et al. 1996):
Q = nFCV,
where n is the number of electrons released on oxidation of a molecule of catecholamine (n = 2 for both dopamine and noradrenaline), F is Faraday's constant, C is the vesicular concentration of catecholamine, and V is the vesicular volume. Thus, if C is assumed constant, Q is proportional to V and so Q1/3 is proportional to the vesicular radius. Alternatively, if V is assumed constant, Q1/3 is proportional to vesicle catecholamine concentration.
Results are presented as individual examples or means ± standard error of the mean and statistical comparisons were made using Student's unpaired t test or ANOVA, as indicated.
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RESULTS |
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Figure 1 shows representative secretory responses obtained in response to acute hypoxia (upper trace; final PO2 level 8 mmHg) from a control PC12 cell (Fig. 1B) and from a PC12 cell which had previously been cultured for 24 h in 10 % O2 environment (chronically hypoxic (CH); Fig. 1C). Exocytosis following exchange to a hypoxic solution usually began earlier during recordings from CH PC12 cells, when the bath PO2 was still falling towards a final value (Fig. 1A). It is also noteworthy that the amplitude of exocytotic events appeared larger in recordings from CH PC12 cells compared with controls cultured under normoxic conditions.
In order to quantify the time course of secretory responses to acute hypoxia, Fig. 2 plots the mean cumulative number of exocytotic events (measured at 10 s intervals; solution was exchanged for a hypoxic one at t = 5 s) recorded from control and CH PC12 cells in response to acute hypoxia (final PO2 30 mmHg). Clearly, the initial release rate and total number of secretory events was greatly enhanced in CH PC12 cells compared with controls (P < 0·0001, one-way ANOVA test). This finding suggested strongly that a 24 h period of chronic hypoxia resulted in an increased sensitivity of PC12 cells to acute hypoxia. This possibility was investigated further by examining the secretory responses of the two groups of cells to a range of PO2 levels. Figure 3 plots the mean exocytotic frequency versus bath PO2 level for both control and CH PC12 cells. To obtain this plot, we measured exocytotic frequency over a 55 s period, 90 s after switching the perfusate to the hypoxic solution (to allow PO2 levels to reach a near-stable final level). Clearly, the PO2 sensitivity of exocytosis frequency in response to acute hypoxia was enhanced in CH PC12 cells compared with controls (P < 0·001 when comparing data over the entire range of hypoxic levels using one-way ANOVA), and this was statistically significant (P < 0·01-0·05, unpaired t test) at each level of hypoxia studied, except at PO2 levels of 57 and 40 mmHg.
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As mentioned earlier, the amplitudes of individual exocytotic events appeared larger in CH PC12 cells than in control cells, suggesting an increased vesicle size. However, event amplitudes can be distorted by experimental variables such as the distance between recording electrode and site of release (the greater the distance, the smaller and slower the event appears; see Chow & Von Ruden, 1995). A more accurate method of estimating quantal size is to integrate each exocytotic event to obtain the charge, Q, the cube root of which is proportional to vesicle size (see Methods). Figure 4 plots distributions of Q1/3 evoked by acute hypoxia (PO2 12 mmHg) in control (Fig. 4A) and CH PC12 cells (Fig. 4B). In both groups of cells Q1/3 was normally distributed, but the mean value was significantly greater (P < 0·0001) in CH PC12 cells compared with controls. This finding strongly suggested that a period of chronic hypoxia increased either vesicle size or the concentration of catecholamine within each vesicle (see Discussion).
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Plots of the distribution of Q1/3 (determined by integration of exocytotic events evoked by a perfusate PO2 of 12 mmHg) in 8 control cells (A; total number of events, 298) and 8 chronically hypoxic cells (B; total number of events, 648). | ||
Previous studies have indicated that exocytosis from PC12 cells in response to acute hypoxia arises because hypoxia inhibits specific K+ channels, causing depolarization and Ca2+ influx via voltage-gated Ca2+ channels (Zhu et al. 1996; Taylor & Peers, 1998). The O2-sensitive K+ channel has also been shown to be inhibited by tetraethylammonium (TEA) and its expression is increased following chronic hypoxia. Such information might suggest that TEA would evoke secretion from PC12 cells and that CH PC12 cells might exhibit enhanced TEA-evoked secretion. Results presented in Fig. 5 indicate that this is the case. In both groups of cells TEA (0·1-10 mM) evoked secretion in a concentration-dependent manner, and at 1 mM and 10 mM TEA, exocytotic frequency was significantly greater (P < 0·05 and P < 0·0001, respectively) in CH PC12 cells compared with controls.
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A, representative example of the effects of 10 mM tetraethylammonium (TEA) applied to a control PC12 cell at the point indicated by the arrow. B, as A, except that the recording was made from a chronically hypoxic PC12 cell. Calibration bars apply to both traces. C, plot of mean (± | ||
Our previous study demonstrated that hypoxia-evoked secretion from PC12 cells could be fully abolished by removal of extracellular Ca2+, and could also be abolished by the non-selective blocker of voltage-gated Ca2+ channels, Cd2+ (Taylor & Peers, 1998). If the enhanced secretion observed following chronic hypoxia was due solely to increased levels of O2-sensitive K+ channels in PC12 cells, then the same inhibitory effects of Ca2+ removal or application of Cd2+ might be expected. Figure 6A (representative of 8 cells tested) shows that removal of extracellular Ca2+ (replaced by 1 mM EGTA) fully abolished release evoked by acute hypoxia (PO2 20 mmHg) in CH PC12 cells. By contrast, bath application of Cd2+ (200 µM) reduced, but did not fully abolish, secretion evoked by acute hypoxia (Fig. 6B). On average, Cd2+ produced a reduction in secretion of approximately 63 % (Fig. 6C). This suggested that most of the secretion from CH PC12 cells in response to acute hypoxia depended on Ca2+ influx via voltage-gated Ca2+ channels, and to investigate this in further detail we examined the effects of selective blockers of voltage-gated Ca2+ channels known to be present in PC12 cells. The results are presented in Fig. 6C. Nifedipine (2 µM), a blocker of L-type channels and -agatoxin IVA (200 nM), a blocker of P/Q-type channels were without significant effect, but the N-type Ca2+ channel blocker -conotoxin GVIA caused significant inhibition of hypoxia-evoked release, being only slightly less effective that Cd2+.
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A, exocytosis recorded from a representative chronically hypoxic PC12 cell in response to acute hypoxia (PO2 20 mmHg). For the period indicated by the horizontal bar, the perfusate was switched to one of the same PO2 but without any added Ca2+ and also containing 1 mM EGTA. B, as A, except that, for the period indicated by the horizontal bar, the perfusate was switched to one containing 200 µM Cd2+. Note that secretion was reduced but not abolished. Calibration bars apply to both traces. C, bar graph showing mean frequency of exocytosis evoked from chronically hypoxic PC12 cells by acute hypoxia (PO2 20 mmHg) in the absence or presence of different selective blockers of voltage-gated Ca2+ channels. Cells were either exposed to nifedipine (Nif; 2 µM), or pretreated with | ||
Results obtained with these selective blockers of voltage-gated Ca2+ channels are in general agreement with the pattern of inhibition seen in control PC12 cells (i.e. a dominant role for Ca2+ influx via N-type channels; see Taylor & Peers, 1998) with the important exception that a significant amount of exocytosis was still present even in the presence of Cd2+. This finding implied that a Cd2+-resistant Ca2+ influx pathway was also contributing to secretion in response to acute hypoxia in CH (but not control) PC12 cells. To probe this potential Ca2+ influx pathway further, we tested the effects of a range of inorganic ions known to block Ca2+ entry pathways in other systems (see Discussion). The results are presented in Fig. 7, and in each case the inorganic ion was applied together with 200 µM Cd2+ to prevent Ca2+ influx through L-, N- and P/Q-type voltage-gated Ca2+ channels. La3+ was by far the most potent ion tested, reducing Cd2+-resistant secretion by over 50 % at a concentration of 10 µM. Both trivalent ions (La3+and Gd3+) were more potent than Zn2+, and Ni2+ was completely without effect even at a concentration of 10 mM. These findings indicate that in CH PC12 cells acute hypoxia triggers exocytosis mediated by Ca2+ influx via N-type voltage-gated Ca2+ channels and a distinct, Cd2+-resistant influx pathway that is particularly sensitive to blockade by trivalent inorganic ions.
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Previous studies have employed PC12 cells as a model system for investigating the effects of chronic hypoxia on the regulation of gene expression (Wang & Semenza, 1993; Czyzyk-Krzeska et al. 1994; Conforti & Millhorn, 1997). More recently, they have been used as a model system for examining the effects of acute hypoxia: as is the case in numerous other cell types (for example carotid body type I cells (Peers, 1990; Stea & Nurse, 1991; Buckler, 1997; reviewed in Peers, 1997), pulmonary smooth muscle cells (Weir & Archer, 1995) and neuroepithelial cell bodies (Youngson et al. 1993)), PC12 cells possess O2-sensitive K+ channels and in hypoxia channel activity is decreased, leading to cell depolarization, a rise of [Ca2+]i (Zhu et al. 1996) and subsequent release of catecholamines (Kumar et al. 1998; Taylor & Peers, 1998).
Conforti & Millhorn (1997) recently provided convincing evidence that the K+ channel in PC12 cells that is sensitive to inhibition by hypoxia is Kv1.2, a TEA-sensitive, voltage-gated K+ channel. Furthermore, chronic hypoxia (10 % O2 for 18 h) selectively increased the levels of Kv1.2 mRNA, and increased the degree of inhibition of whole-cell K+ currents caused by acute hypoxia (Conforti & Millhorn, 1997). In agreement with their observations, we have previously demonstrated that application of TEA (but not 4-aminopyridine) evokes exocytosis from PC12 cells (Taylor & Peers, 1998). Importantly, in the present study, we show that chronic hypoxia markedly enhances the secretory response of PC12 cells to TEA (Fig. 5), a finding which is consistent with increased expression of functional K+ channels that influence cell resting membrane potential. We suggest, therefore, that chronic hypoxia enhances the secretory response of PC12 cells to acute hypoxia in part by increasing the functional expression of the oxygen-sensitive K+ channel Kv1.2. However, increased expression of Kv1.2 cannot account fully for the enhanced secretory response observed following chronic hypoxia.
The finding that TEA evokes greater secretion in CH PC12 cells than in control cells provides functional evidence to support the extensive investigations into altered mRNA production induced by periods of chronic hypoxia (Czyzyk-Krzeska et al. 1992, 1994; Bunn & Poyton, 1996; Caro & Salceda, 1998; Ratcliffe et al. 1998). The mRNA for tyrosine hydroxylase has also been shown to be increased by chronic hypoxia in PC12 cells, and our study of vesicle size - indicated by measurements of Q1/3 (Fig. 4) - also agrees with these studies. An increase in Q1/3 is indicative of either an increase of vesicular dimensions or an increase in the catecholamine content of vesicles. Further studies, such as electron micrograph measurements of vesicular diameter are required to clarify this issue, but given that tyrosine hydroxylase mRNA levels are increased by chronic hypoxia (Czyzyk-Krzeska et al. 1992, 1994), we would suggest that the observed increases in Q1/3 values reported here reflect an increase in catecholamine concentration within vesicles.
Our previous study showed that acute hypoxia evoked secretion from PC12 cells in a manner which was wholly dependent on the presence of extracellular Ca2+ and could be fully abolished by 200 µM Cd2+, a non-selective blocker of voltage-gated Ca2+ channels (Taylor & Peers, 1998). Furthermore, release could be almost fully blocked (< 90 %) by pretreatment of cells with the selective N-type Ca2+ channel blocker -CgTX (Taylor & Peers, 1998). This result contrasted with another recent report (Kumar et al. 1998) which suggested that most hypoxia-evoked catecholamine release from PC12 cells could be blocked with the L-type Ca2+ channel blocker nitrendipine. However, these workers applied this dihydropyridine at a concentration of 10 µM, and organic Ca2+ channel blockers (including dihydropyridines) are known to have marked inhibitory effects on other classes of neuronal, high voltage-activated Ca2+ channels in this concentration range (Diochot et al. 1995). Our finding, that hypoxia-evoked exocytosis is governed predominantly by Ca2+ influx through N-type Ca2+ channels, is consistent with the report that this class of Ca2+ channels is found in secretory granules of PC12 (and other) cells, and can be translocated in these vesicles into the plasma membrane (Passafaro et al. 1996), suggesting strongly that they are located in close proximity to release sites in PC12 cells.
Following a period of chronic hypoxia, secretion in response to acute hypoxia remained fully dependent on extracellular Ca2+ (Fig. 6A). However, unlike in control cells, bath application of 200 µM Cd2+ was unable to inhibit completely the secretory response to acute hypoxia, and approximately 37 % of the secretory response remained. If the response observed in the presence of Cd2+ is subtracted from those observed in the presence of selective Ca2+ channel blockers (Fig. 6C), then the effects of these agents are comparable to effects observed in control cells (Taylor & Peers, 1998) i.e. a dominant influence of N-type Ca2+ channels (based on the observation that -CgTX caused marked inhibition of release), with little or no influence from L-type or P/Q-type Ca2+ channels. However, perhaps the most important observation was that acute hypoxia evoked exocytosis from chronically hypoxic PC12 cells even in the presence of Cd2+ at a concentration which would be expected to fully inhibit Ca2+ influx through voltage-gated Ca2+ channels. Thus, acute hypoxia activates a Cd2+-resistant Ca2+ entry pathway which can trigger exocytosis in CH PC12 cells.
Certain candidate pathways are worthy of consideration as mediators of the Cd2+-resistant Ca2+ entry pathway in CH PC12 cells. One possibility is a store-operated Ca2+ entry pathway (capacitative Ca2+ entry; see Parekh & Penner, 1997). In PC12 cells, as in a wide variety of other cell types, depletion of intracellular Ca2+ stores can activate Ca2+ influx across the plasma membrane and Koizumi & Inoue (1998) have recently demonstrated that this Ca2+ influx is sufficient to trigger catecholamine release from PC12 cells. However, we have no evidence that acute hypoxia causes store depletion in CH PC12 cells, and it is not clear why such a pathway should be activated by acute hypoxia in these cells. Furthermore, the order of potency of blockade by inorganic ions of release from CH PC12 cells evoked by acute hypoxia in the presence of Cd2+ (Fig. 7) does not agree with the observed effect of these ions with respect to inhibiting capacitative Ca2+ entry (Hoth & Penner, 1993).
Another possible Ca2+ entry pathway may be via non-selective cation channels intrinsic to P2X receptors: such receptors are present in PC12 cells, and are potently blocked by trivalent cations (Nakazawa et al. 1997). It is conceivable that ATP is co-released with catecholamines in response to acute hypoxia, and enhanced release from CH PC12 cells (due to the presence of more O2-sensitive K+ channels) may be sufficient to activate P2X receptors thereby activating non-selective cation channels and hence allowing further Ca2+ influx. However, we consider this possibility unlikely since experiments were conducted whilst cells were perfused at a high flow rate which might be expected to rapidly remove ATP from close to the cell surface.
Dopico & Treistman (1997) have recently identified a novel, large conductance non-selective cation channel in PC12 cells, and this could also underlie the Cd2+-resistant Ca2+ influx pathway triggering secretion in response to acute hypoxia from CH PC12 cells reported here. However, this channel was identified in cells which had not been previously exposed to chronic hypoxia, so there is no reason at present to expect it to contribute to exocytosis significantly in CH but not control PC12 cells. Similarly, Ca2+-permeable N-methyl-D-aspartate channels are present in PC12 cells (Casado et al. 1996), and these channels are severalfold more sensitive to inhibition by Zn2+ than by Cd2+ (Legendre & Westbrook, 1990). However, there is no evidence to support their involvement in stimulus-secretion coupling in CH but not control PC12 cells at present.
Acute hypoxia has recently been shown to evoke catecholamine release from isolated guinea-pig chromaffin cells (Inoue et al. 1998). This evoked release was attributable in part to activation of a cationic channel which could also be activated by cyanide and appeared to be the same type of channel as that coupled to muscarinic receptors. Interestingly, activation of this Ca2+ entry pathway was sufficient to evoke exocytosis even in the presence of 100 µM Cd2+, although no other pharmacological data concerning this pathway were provided. However, PC12 cells are derived from chromaffin cells, and it is at least conceivable that the cation channel described by Inoue et al. (1998) is similar to the Ca2+ entry pathway described here for CH PC12 cells. Clearly, further work would be required to establish such similarities.
In summary, we have shown that a period of chronic hypoxia increases the secretory response of PC12 cells to acute hypoxia. This increased response can be attributed in part to increased functional expression of O2-sensitive K+ channels, but is also partly due to the activation of a Cd2+-resistant Ca2+ entry pathway which is not observed (or at least is not sufficient to trigger exocytosis) in control PC12 cells. Our results provide functional effects which might be anticipated from previous studies on the effects of hypoxia on mRNA levels for specific proteins in PC12 cells, and also reveal a novel effect of chronic hypoxia: activation of a Cd2+-resistant Ca2+ influx pathway that is highly sensitive to trivalent cations. We suggest that amperometric recordings from PC12 cells represent a valuable system for studying the mechanisms underlying both slow cellular adaptations to chronic hypoxia and rapid responses to acute hypoxia.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This work was supported by The British Heart Foundation. We also thank Dr P. J. Kemp and Dr D. A. S. G. Mary for helpful discussion.
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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) and 8 chronically hypoxic cells (
). Solution exchange from normoxic to hypoxic was at t = 5 s.