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J Appl Physiol 96: 1206-1215, 2004. First published December 2, 2003; doi:10.1152/japplphysiol.00879.2003
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HIGHLIGHTED TOPICS
Oxygen Sensing in Health and Disease

Facilitation of dopamine and acetylcholine release by intermittent hypoxia in PC12 cells: involvement of calcium and reactive oxygen species

Dong-Kyu Kim,1 Niranjana Natarajan,1 Nanduri R. Prabhakar,2 and Ganesh K. Kumar1

1Department of Biochemistry, and 2Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

Submitted 19 August 2003 ; accepted in final form 24 November 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
We have investigated the effects of preconditioning pheochromocytoma (PC12) cells with intermittent hypoxia (IH) on transmitter release during acute hypoxia. Cell cultures were exposed to either alternating cycles of hypoxia (1% O2 + 5% CO2; 30 s/cycle) and normoxia (21% O2 + 5% CO2; 3 min/cycle) for 15 or 60 cycles or normoxia alone (control) for similar durations. Control and IH cells were challenged with either hyperoxia (basal release) or acute hypoxia (PO2 of ~35 Torr) for 5 min, and the amounts of dopamine (DA) and acetylcholine (ACh) released in the medium were determined by HPLC combined with electrochemical detection. Hypoxia augmented DA (~80%) but not ACh release in naive cells, whereas, in IH-conditioned cells, it further enhanced DA release (ranging from 120 to ~145%) and facilitated ACh release (~30%). Hypoxia-evoked augmentation of transmitter release was not seen in cells conditioned with sustained hypoxia. IH-induced increase in DA but not IH-induced ACh release during hypoxia was partially inhibited by cadmium chloride (100 µM), a voltage-gated Ca2+ channel blocker. By contrast, 2-aminoethoxydiphenylborate (75 µM), a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, and N-acetyl-L-cysteine (300 µM), a potent scavenger of reactive oxygen species, either attenuated or abolished IH-evoked augmentation of transmitter release during hypoxia. Together, the above results demonstrate that IH conditioning increases hypoxia-evoked neurotransmitter release from PC12 cells via mechanisms involving mobilization of Ca2+ from intracellular stores through activation of IP3 receptors. Our findings also suggest that oxidative stress plays a central role in IH-induced augmentation of transmitter release from PC12 cells during acute hypoxia.

intracellular calcium mobilization; inositol 1,4,5-trisphosphate receptors

CHRONIC INTERMITTENT HYPOXIA (IH) associated with recurrent apnea has been implicated in the development of pathophysiological conditions such as hypertension and stroke (15, 16, 38). From studies in humans as well as in animal models, it has been suggested that the circulating levels of catecholamines are increased during IH possibly by mechanism(s) involving increased synthesis and/or release (16, 52). Toward unraveling the cellular mechanism(s) associated with IH, as an initial step, our laboratory developed and reported a method (29) wherein cells can be exposed to alternating cycles of hypoxia (1% O2, 30 s) and normoxia (21% O2, 3 min), simulating the hypoxic duration seen during recurrent apneas. Using this method, we have initiated studies on pheochromocytoma (PC12) cells, derived from the adrenal chromaffin cells, to investigate whether preconditioning of cells with IH affects neurotransmitter metabolism, including synthesis, storage, and release. Recently, our group (29) showed that IH activates tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines in PC12 cells, with a concomitant increase in catecholamine levels. Furthermore, our group showed that IH-induced TH activation is, in part, due to an increase in phosphorylation of serine residues, especially that of serine 40. These findings suggest that IH facilitates the synthesis of catecholamines in PC12 cells. However, whether the increase in catecholamine synthesis by IH is coupled to augmented catecholamine release is not known.

A number of studies have examined the effects of hypoxia on neurotransmitter release (18-20, 26, 28, 30, 31, 39, 43, 44, 46, 47). Among them, studies of the mammalian carotid body, the primary peripheral chemoreceptor tissue (3-5, 11, 13, 22, 31, 46, 47), and of undifferentiated PC12 cells, as an oxygenresponsive, presynaptic neuronal model (30, 43, 44), suggest that acute hypoxia facilitates the release of dopamine (DA). In addition to DA, both the carotid body and PC12 cells are also known to synthesize and store other transmitters, including acetylcholine (ACh; Refs. 14, 23, 24, 27, 31). The effects of hypoxia on ACh release from the carotid body are complex in that hypoxia stimulates sustained ACh release in the cat carotid body (11, 14), whereas in the rabbit carotid body there is an initial facilitation followed by a sustained inhibition of ACh release (27). The inhibition of ACh release by hypoxia seems to be primarily due to hypoxia-induced activation of inhibitory muscarinic cholinergic receptors and dopaminergic D2 receptors (27). However, whether hypoxia also facilitates ACh release from PC12 cells has not yet been investigated. In addition, few studies have examined the mechanisms associated with stimulus-secretion coupling during hypoxia; findings from these few studies lend support to the view that hypoxiaevoked catecholamine release critically depends on the extracellular Ca2+ influx via activation of voltage-gated Ca2+ channels (VGCC; Refs. 30, 46, 47). Whether Ca2+-dependent mechanism(s) is also critical for ACh release during hypoxia is not known.

In the present study, our objectives were to determine whether hypoxia facilitates the simultaneous release of multiple transmitters from PC12 cells and also to assess whether IH conditioning alters transmitter release during subsequent exposure of cells to acute hypoxia. For these investigations, we opted to focus on the release of DA and ACh from naive as well as IH-conditioned cells. DA and ACh from the same release medium were measured with two dedicated HPLC systems combined with electrochemical detection. Because an increase in intracellular Ca2+ is intrinsically coupled to stimulus-secretion coupling, we also examined the contribution of extracellular Ca2+ entry as well as mobilization of intracellular Ca2+ in hypoxia-evoked neurotransmitter release from IHconditioned PC12 cells. There is also increasing evidence suggesting that reactive oxygen species (ROS) are formed, especially after IH conditioning (1, 36-38). Therefore, we also examined whether ROS is involved in hypoxia-induced neurotransmitter release in naive and IH-conditioned cells. Overall, our results suggest that hypoxia selectively augments the release of DA but not ACh in naive cells, whereas, in IH-conditioned cells, hypoxia further facilitates DA release and stimulates ACh release that involves mobilization of intracellular Ca2+ and ROS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Materials and Reagents

DA, cadmium chloride, N-acetyl-L-cysteine (NAC), and penicillin G were purchased from Sigma Chemical (St. Louis, MO). ACh, C-18 reverse-phase microbore column, immobilized enzyme reactor (IMER) column, glassy carbon electrode, peroxidase redox polymer, and Kathon CG (bactericide) were obtained from Bioanalytical Systems (West Lafayette, IN). Prodigy C-18 reverse-phase minibore column and 2-aminoethoxydiphenyl borate (2-APB) were from Phenomenex (Torrance, CA) and Calbiochem (San Diego, CA), respectively.

Cell Cultures

PC12 cells (original clone from Dr. L. Green) were cultured on polystyrene-coated Falcon tissue culture flasks (25 cm2) (BD-Falcon Biosciences, Lexington, TN) in a humidified chamber maintained with 5% CO2 and 21% O2 at 37°C as described previously (30). Briefly, cells were grown in DMEM supplemented with 10% horse serum, 5% fetal calf serum, and 100 U/ml of penicillin G. The medium was changed every 2 days. Experiments were performed with cells maintained between passages 3 and 10. Before the experiment, the cells were placed in serum-free DMEM for 16 h. All experiments were performed in serum-free medium.

Exposure of Cells to IH

Cell cultures were exposed to IH by using procedures described previously (29). Briefly, cells were placed in a humidified Lucite chamber at 37°C containing gas inlets and outlets and exposed alternately to hypoxia (1% O2 + 5% CO2, 30 s) and normoxia (21% O2 + 5% CO2, 3 min) for 15 or 60 episodes. The gas flow (2.5 l/min) into the chamber and the duration of gas exposure were regulated with the use of timed solenoid valves. The oxygen levels in the chamber and in the tissue culture medium were continuously monitored with an oxygen analyzer (Beckman LB2) and an oxygen electrode (Lazar), respectively, and recorded on a strip chart recorder. Typically, the ambient PO2 of the chamber and the medium was 110 and 70 Torr, respectively. At each cycle of hypoxic exposure, the PO2 of the chamber and the medium dropped to 20 and 50 Torr, respectively. During IH exposure, the pH of the culture medium remained at 7.4. Cells exposed to either normoxia or intermittent normoxia (i.e., cells placed in the same Lucite chamber but challenged with alternating cycles of 21% O2) served as controls.

Extraction of DA and ACh from PC12 Cells

After exposure to either normoxia or IH, cells (~3 x 106) were separated from the medium by centrifugation (500 g, 5 min). The cell pellets were placed in 1,000 µl of 0.1 N HClO4 containing 0.25 mM EDTA-Na2 and extracted at 4°C by sonication (Sonics and Materials, Danbury, CT) for three times, 30 s each. The homogenates were centrifuged at 12,000 g for 15 min at 4°C. The clear supernatant as such was used for the analysis of DA, whereas, for ACh, two parts of the supernatant were first neutralized with one part of 0.1 N Na3PO4. Subsequently, DA and ACh were measured by two dedicated HPLC systems coupled with electrochemical detectors as described below.

Determination of DA and ACh

DA and ACh were measured by using procedures described previously (27-30). Briefly, DA was separated on a Prodigy C-18 reverse-phase minibore column by isocratic elution with a mobile phase consisting of 7% methanol, 0.1 M sodium acetate, 0.0125 M sodium citrate, 0.1 mM EDTA-Na2, and 0.215 mM sodium octyl sulfate, at pH 4.5. Under the experimental conditions, DA was eluted at ~9.0 min with an average recovery of ~82% as determined with an internal standard, 3,4-dihydroxybenzylamine. The detection limit for DA was 250 fmol.

ACh was measured with HPLC coupled to an IMER column containing acetylcholinesterase and choline oxidase combined with an electrochemical detector as described previously (27). Briefly, ACh was separated from its endogenous metabolite choline on a microbore C-18 column by reverse-phase HPLC and then converted to H2O2 enzymatically online in the IMER column. The amount of H2O2, a measure of ACh, was determined with peroxidase-coated glassy carbon electrode and an electrochemical detector with an oxidation potential of 100 mV vs. Ag/AgCl. For the isocratic separation of ACh from choline, a mobile phase containing 30 mM NaH2PO4, pH 8.5, and bactericide, Kathon (5 ml/l), was used at a flow rate of 130 µl/min. Under the experimental condition, ACh eluted at ~13 min with a detection limit of 80 fmol. The elution profiles of DA and ACh were recorded and analyzed with the use of a Hitachi D-2500 Chromato-Integrator. The concentrations of ACh and DA were determined with the use of standard curves correlating the concentration of either ACh or DA to the corresponding integrated peak area. Transmitter content was expressed in picomoles per carotid body.

Experimental Protocols for Transmitter Release

Before we started experiments, the serum-free medium was removed and cells were washed with Ca2+- and Mg2+-free Krebs Ringer bicarbonate (KRB) medium and placed in 1.5 ml of normal KRB medium. After exposure to either hyperoxia (95% O2 + 5% CO2) or hypoxia (1% O2 + 5% CO2 in N2; PO2 of ~35 Torr) for 5 min, the medium was separated from the cells by centrifugation, and aliquots (20 µl) of the medium were analyzed simultaneously for the release of DA and ACh as described above. DA and ACh release was expressed as picomoles per minute per milligram of protein. We determined protein concentration by the bicinchoninic acid method using bovine serum albumin as the standard (41). For the release studies, the following experimental protocols were used.

Series 1. To determine the effect of acute hypoxia on DA and ACh release in either naive or IH-conditioned cells (15 or 60 cycles), cell cultures were placed in the incubation medium that was previously equilibrated with hypoxic gas mixture (PO2 of ~35 Torr) and maintained at 37°C for 5 min. Cells exposed to hyperoxia for a similar duration served as controls. To assess the effects of hypoxia on cells preconditioned with sustained hypoxia (SH), cells were also exposed to hypoxia (PO2 of 40 Torr) continuously for 75 min, which is the cumulative duration of hypoxia during 60 cycles of IH episodes, and then subjected to 5 min of hypoxia.

Series 2. In another series of experiments, the effect of voltagegated Ca2+ channel blocker on DA and ACh release in either control or IH-conditioned cells during 5 min of acute hypoxia was determined. Cadmium chloride (100 µM) was added to the culture medium before exposure of the cell cultures to either hyperoxic or hypoxic gas mixtures as described above.

Series 3. To evaluate the role of Ca2+ mobilized from the intracellular stores during IH conditioning on DA and ACh release by hypoxia, either naive or IH-conditioned cells were preincubated with 75 µM of 2-APB for 5 min at 37°C. Subsequently, the cells were washed with Ca2+- and Mg2+-free KRB medium and exposed to either hyperoxia or hypoxia as described above. Either naive or IH-conditioned cells that were not pretreated with 2-APB served as controls.

Series 4. To examine the possible involvement of ROS on IH-induced DA and ACh release during hypoxia, cells were exposed to either intermittent normoxia (control) or IH (60 episodes) in the presence of 300 µM NAC, a potent scavenger of ROS, including , and then subsequently exposed to either hyperoxia or acute hypoxia for 5 min. Cells that were not treated with NAC served as controls.

Data Analysis

All data presented were obtained from at least three independent experiments that used different cell cultures of similar passage and are expressed as means ± SE. Statistical significance was evaluated by a paired t-test or one-way ANOVA for repeated measures. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Effects of IH on the Content of DA and ACh in PC12 Cells

PC12 cells express a variety of transmitters, including DA and ACh (23, 24). To determine whether IH affects the content of these transmitters, we measured DA and ACh in 0.1 N HClO4 acid extracts of naive and IH-conditioned cells. In naive cells, the contents of DA and ACh were 18.8 ± 1.5 and 0.7 ± 0.3 nmol/mg protein, respectively (Table 1); this is in accord with previously reported values (23, 24). Our analysis also showed that, in cells exposed to 60 cycles of IH, the DA level increased by ~30% (P < 0.01; n = 4) compared with the naive cells. By contrast, the ACh level was not significantly altered by IH conditioning (Table 1).


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  		Table 1. Effect of intermittent hypoxia on the content of neurotransmitters in PC12 cells

 

Effect of IH on DA and ACh Release from PC12 Cells During Acute Hypoxia

Previous studies have shown that hypoxia stimulates DA release from PC12 cells (30, 43). The release of DA and ACh was simultaneously measured with two dedicated HPLC systems. In unconditioned cells, the basal DA release during hyperoxia was 44.3 ± 3.5 pmol·min-1·mg protein-1 and hypoxia significantly increased DA release (~80%; P < 0.01, n = 4; Fig. 1A). IH only marginally affected the basal release (data not shown), whereas it augmented hypoxia-evoked DA release in an IH-cycle-dependent manner. Thus DA release during hypoxia was increased by ~125 and ~145% (P < 0.01, n = 4) after 15 and 60 episodes of IH, respectively (Fig. 1A). By contrast, hypoxia-evoked DA release was not altered in cells that were conditioned with either SH for 75 min, corresponding to the cumulative duration of hypoxia during 60 episodes of IH (Fig. 1A) or comparable duration of intermittent normoxia (data not shown).



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  		Fig. 1. Effects of preconditioning of PC12 cells with intermittent hypoxia (IH) on hypoxia-evoked dopamine (DA) and acetylcholine (ACh) release. Cell cultures (~3 x 106) were exposed to either 15 or 60 cycles of IH or sustained hypoxia (SH) for 75 min, representing the cumulative duration of hypoxia during 60 cycles of IH as described in MATERIALS AND METHODS. Cells exposed to normoxia (naive) for similar duration served as controls. Naive as well as IH- or SH-conditioned cells were exposed to either hyperoxia (basal) or hypoxia (PO2 of ~35 Torr) for 5 min. Aliquots of the release medium were analyzed simultaneously for DA and ACh with the use of HPLC-electrochemical detection. A: DA release. B: ACh release. Average data from 4 independent experiments are shown as means ± SE. ns, Not significant. *P < 0.05. **P < 0.01.

 

Unlike the augmentation of DA release by hypoxia, hypoxia did not stimulate ACh release from the naive cells and was similar to the basal release during hyperoxia (12.1 ± 0.3 pmol·min-1·mg protein-1). However, in IH-conditioned cells, hypoxia increased ACh release in an IH-cycle-dependent manner (Fig. 1B). Thus the increase in ACh release during hypoxia after 15 and 60 episodes of IH exposure was ~115% (P < 0.05; n = 4) and ~135% (P < 0.01, n = 4), respectively. There was no further increase after 120 episodes of IH (data not shown). Notably, the basal release of ACh was unaffected in IH-conditioned cells. By contrast, neither SH for 75 min (Fig. 1B) nor intermittent normoxia (data not shown) affected ACh release during hypoxia. Together, the above results demonstrate that IH but not SH conditioning facilitates transmitter release during hypoxia.

Partial Attenuation of Acute Hypoxia-Evoked DA and ACh Release by Cadmium Chloride in IH-Conditioned Cells

Extracellular Ca2+ has been shown to play an important role in stimulus-secretion coupling during hypoxia (18, 19). To begin to define the cellular mechanisms associated with IH-induced augmentation of transmitter release during hypoxia, we examined the requirement of extracellular Ca2+ influx through voltage-gated Ca2+ channels on acute hypoxia-evoked DA and ACh release in IH-conditioned cells. Cadmium chloride (100 µM), a voltage-gated Ca2+ channel blocker, abolished hypoxia-evoked DA release in the naive control cells (P < 0.01, n = 3; Fig. 2, A and B), whereas it partially decreased acute hypoxia-evoked DA (by ~30%; P < 0.01, n = 3; Fig. 2, C and D) release in IH-conditioned cells. On the other hand, blockade of voltage-gated Ca2+ channels had no significant effect on ACh release during hypoxia in both naive and IH-conditioned cells (Fig. 3, A-D). Likewise, addition of cadmium chloride had no significant effect on the basal ACh in both naive and IH-conditioned cells. Collectively, the above findings indicate that entry of extracellular Ca2+ via activation of voltage-gated Ca2+ channels only plays a minor role in hypoxiaevoked DA release in IH-conditioned cells.



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  		Fig. 2. Effect of cadmium chloride on acute hypoxia-evoked DA release in naive and IH-conditioned cells. Naive and IH-conditioned (60 cycles) cells were exposed to either hyperoxic (basal) or hypoxic medium for 5 min containing cadmium chloride (100 µM), a voltage-gated Ca2+ channel blocker. Cells exposed to medium lacking cadmium chloride (CdCl2) served as controls. A and B: naive cells. C and D: IH-conditioned cells. In B and D, the net DA released during hypoxia (i.e., hypoxia release minus basal release) in the presence and absence of CdCl2 is shown. Average data from 3 independent experiments are shown as means ± SE. Other experimental details are as described in legend to Fig. 1. **P < 0.01.

 


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  		Fig. 3. Effect of CdCl2 on acute hypoxia-evoked ACh release in naive and IH-conditioned cells. Naive and IH-conditioned (60 cycles) cells were exposed to either hyperoxic (basal) or hypoxic medium for 5 min containing cadmium chloride (100 µM), a voltage-gated Ca2+ channel blocker. Cells exposed to medium lacking cadmium chloride served as controls. ACh released from the naive (A and B) and IH-conditioned (60 cycles; C and D) cells are presented. In B and D, the net ACh release during hypoxia (i.e., hypoxia release - basal release) in the presence and absence of cadmium chloride is shown. Average data from 3 independent experiments are shown as means ± SE.

 

Effect of 2-APB on Acute Hypoxia-Evoked DA and ACh Release in IH-Conditioned Cells

Studies from the central and peripheral nervous systems suggest that Ca2+ mobilization from the internal stores plays a significant role in neurotransmitter release (9, 25, 34). We assessed the possible involvement of Ca2+ mobilized from the internal stores, such as endoplasmic reticulum in IH-induced release of transmitters during acute hypoxia using 2-APB, a membrane-permeable inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor (2, 7, 42). 2-APB (75 µM) either markedly attenuated or abolished hypoxia-evoked DA release (by ~80%; P < 0.01, n = 3; Fig. 4, A and B) and ACh release (by ~95%; P < 0.01, n = 3; Fig. 5, A and B) in IH-conditioned cells. By contrast, 2-APB had no effect on the basal release of DA and ACh in the naive cells (data not shown). These observations suggest that Ca2+ mobilized from the intracellular stores plays a major role in regulating IH-induced transmitter release during hypoxia.



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  		Fig. 4. Influence of 2-aminoethoxydiphenyl borate (2-APB) on acute hypoxiaevoked DA release in IH-conditioned cells. Naive and IH-conditioned (60 cycles) cells were incubated with 75 µM 2-APB, a blocker of inositol 1,4,5-trisphosphate receptor for 5 min at 37°C before hypoxic (PO2 of ~35 Torr) exposure for an additional 5 min. A: hypoxia-evoked DA release in the absence and presence of 2-APB in the control and IH-conditioned cells. B: differences in hypoxia-evoked DA release between IH and naive cells in the presence and absence of 2-APB. Average data from 3 independent experiments are shown as means ± SE. *P < 0.01.

 


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  		Fig. 5. Effect of 2-APB on acute hypoxia-evoked ACh release in IH-conditioned cells. Naive and IH-conditioned (60 cycles) cells were incubated with 75 µM 2-APB, a blocker of inositol 1,4,5-trisphosphate receptor for 5 min at 37°C before hypoxic (PO2 of ~35 Torr) exposure for additional 5 min, and ACh in the release medium was measured by HPLC-electrochemical detection. A: hypoxia-evoked ACh release in the absence and presence of 2-APB in the control and IH-conditioned cells. B: differences in hypoxia-evoked ACh release between IH and naive cells in the presence and absence of 2-APB. Average data from 3 independent experiments are shown as means ± SE. **P < 0.01.

 

Influence of NAC on Acute Hypoxia-Evoked DA and ACh Release in IH-Conditioned Cells

It has been proposed that IH represents a form of oxidative stress wherein the generation of ROS is increased (36-38). We tested whether ROS is involved in IH-induced augmentation of neurotransmitter release during hypoxia in PC12 cells. To test this possibility, cells were treated with NAC, a potent scavenger of during IH conditioning. NAC (300 µM) markedly attenuated IH-induced DA (~75%, P < 0.01; n = 3; Fig. 6, A and B) and ACh (~ 70%, P < 0.01; n = 2; Fig. 7, A and B) release during hypoxia. However, lower doses of NAC had no significant effect (data not shown). In cells conditioned with intermittent normoxia, NAC only partially inhibited DA release (~20%, P < 0.05; n = 3) during hypoxia, whereas it had no discernible effect on ACh release.



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  		Fig. 6. Effect of N-acetyl-L-cysteine (NAC) on acute hypoxia-evoked DA release in IH-conditioned cells. Cells were preconditioned with 60 cycles of either IH or intermittent normoxia (IN) in the presence of 300 µM NAC, a reactive oxygen species (ROS) scavenger. Cells not exposed to NAC served as controls. Subsequently, cells were exposed to hypoxia (PO2 of ~35 Torr) for 5 min, and DA in the medium was analyzed by using HPLC combined with electrochemical detection. A: hypoxia-evoked DA release in the absence and presence of NAC in IH- and IN-conditioned cells. B: differences in hypoxia-evoked DA release between IH and IN (control) cells in the presence and absence of NAC. Average data from 3 independent experiments are shown as means ± SE. *P < 0.05. **P < 0.01.

 


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  		Fig. 7. Effect of NAC on acute hypoxia-evoked ACh release in IH-conditioned cells. Cells were preconditioned with 60 cycles of either IH or IN in the presence of 300 µM NAC, a ROS scavenger. Cells not exposed to NAC served as controls. Subsequently, cells were exposed to hypoxia (PO2 of ~35 Torr) for 5 min, and ACh in the medium was analyzed with the use of procedures described in MATERIALS AND METHODS. A: hypoxia-evoked ACh release in the absence and presence of NAC in IH- and IN-conditioned cells. B: differences in hypoxia-evoked ACh release between IH and IN (control) cells in the presence and absence of NAC. Average data from 3 independent experiments are shown as means ± SE. **P < 0.01.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
The objectives of the present study were to assess whether hypoxia stimulates the simultaneous release of DA and ACh from PC12 cells and to determine whether prior exposure of cells to IH affects transmitter release during a subsequent exposure to acute hypoxia. Our results demonstrated that hypoxia selectively facilitates the release of DA but not ACh from the naive, unconditioned cells, whereas it simultaneously augments the release of both DA and ACh from IH-conditioned cells. Furthermore, our results showed that Ca2+ mobilized from the internal stores as well as ROS contribute to the increase in hypoxia-evoked transmitter release from IH-conditioned cells.

One of our objectives in this study was to determine whether hypoxia facilitates the concurrent release of multiple transmitters or differentially increases the release of a specific transmitter. For this purpose, we investigated the release of DA and ACh during hypoxia from PC12 cells, a frequently used cell model for investigations on stimulus-secretion coupling (30, 40, 43, 44). Although the release of DA by hypoxia has been extensively investigated (30, 43), there is paucity of information on ACh release from PC12 cells during hypoxia. We have, therefore, simultaneously monitored the release of DA as well as ACh from the same release medium containing PC12 cells, exposed to medium preequilibrated with various gas mixtures. To achieve a high sensitivity of detection necessary for the simultaneous analysis of DA and ACh, we developed the following strategies. First, we used a minibore column and a microbore column coupled with an IMER column for the HPLC analyses of DA and ACh, respectively. The use of these columns permits optimal separation of DA from other catecholamines and also the resolution of the low abundant ACh from the abundant choline. Second, for the analyses of DA and ACh, we used highly sensitive electrochemical detection techniques based on glassy carbon electrodes and peroxidasecoated glassy carbon electrodes, respectively. Third, we were able to immediately analyze the release medium for DA and ACh using two dedicated HPLC-ECD systems, thus preventing any losses due to auto-oxidation of DA or degradation of ACh. From the simultaneous analysis of DA and ACh, we found that hypoxia selectively facilitates the release of DA but not ACh in the naive cells. Consistent with the above results are the findings that hypoxia preferentially facilitates the release of DA but not ACh in the striatal slice preparation (39).

How does hypoxia induce the release of DA but not ACh from PC12 cells? If DA and ACh are stored in the same vesicles in PC12 cells, then hypoxia is expected to facilitate the release of both the transmitters concurrently. However, immunolabeling of PC12 cells with antibodies specific to vesicular monoamine and ACh transporters, which serve as markers for dopaminergic and cholinergic vesicles, respectively, showed that DA is preferentially localized to large dense core vesicles that are in close proximity to the active zone, whereas ACh is exclusively associated with the clusters of small synaptic vesicles at sites distant from the plasma membrane (32, 49). These observations suggest that DA and ACh are perhaps stored in distinct vesicles in PC12 cells and further raise the possibility that hypoxia may selectively mobilize dopaminergic but not cholinergic vesicles, thus leading to the preferential release of DA but not ACh from PC12 cells. Alternately, hypoxia may differentially activate the inhibitory but not the excitatory cholinergic autoreceptors, thereby preventing the release of ACh during acute hypoxia. Supporting such a possibility is the finding that, in the rabbit carotid body, hypoxia inhibits ACh release via activation of the inhibitory muscarinic cholinergic receptors (27).

Interestingly, IH conditioning resulted in a marginal increase in the basal DA but not ACh release in PC12 cells. The enhanced basal release of DA could arise, in part, from the increase in the cellular level of DA after IH conditioning. In a previous study (29), our laboratory showed that IH activates TH, the rate-limiting enzyme in catecholamine biosynthesis, by increasing the phosphorylation of serine residues, especially that of serine 40. It is likely that IH-induced activation of TH contributes to the elevated cellular level of DA in IH-conditioned cells.

A major finding of the present study is that, in IH-conditioned cells, hypoxia facilitates the release of both DA and ACh in a coordinated manner. This is in sharp contrast to the selective facilitation of DA but not ACh release by hypoxia in naive cells. Because extracellular Ca2+ influx via the activation of VGCC is critical for transmitter release, we have examined the role of VGCC in IH-evoked transmitter release during hypoxia. Although blockade of VGCC in naive cells nearly abolished hypoxia-evoked DA release, it only marginally attenuated IH-induced DA and ACh release during hypoxia. It has been increasingly recognized that mobilization of Ca2+ from the intracellular stores also participates during transmitter release. For instance, Ca2+ mobilized from the intracellular stores triggered the release of glutamate from the small synaptic vesicles of the presynaptic neuron (9) and that of ACh from the cholinergic synapses in Aplysia (34). In most neuronal and secretory cells, the major intracellular Ca2+ stores are associated with the endoplasmic reticulum/sarcoplasmic reticulum (8, 10, 45, 48), and Ca2+ mobilization from these stores occurs by a mechanism involving activation of IP3 receptors (6, 51). In IH-conditioned cells, preincubation with 2-APB, an antagonist of IP3 receptors, markedly attenuated DA release and completely abolished ACh release during acute hypoxia. These findings support the notion that Ca2+ released from the intracellular stores coupled to IP3 receptors plays a major role in IH-induced transmitter release during acute hypoxia and further raise the possibility that IP3 receptors are activated during IH. In addition to IP3, a novel Ca2+ binding protein (50) that belongs to the calmodulin superfamily of proteins has been reported to function as an alternative, high-affinity endogenous ligand for IP3 receptors. Future studies are necessary to further determine whether IH-induced activation of IP3 receptors involves activation or upregulation of phospholipase C, the enzyme that catalyzes the hydrolysis of phospholipids to generate IP3 (12, 33) or upregulation of the novel Ca2+ binding protein that serves as an endogenous ligand for IP3 receptors.

It is evident from the above observations that extracellular Ca2+ influx is critical for DA release during hypoxia in naive cells, whereas Ca2+ signaling arising from intracellular Ca2+ mobilization plays an important role in IH-induced augmentation of transmitter release during hypoxia. Furthermore, unlike IH, conditioning of cells with SH did not alter the transmitter release during hypoxia. The differences in hypoxia-induced transmitter release between IH- and SH-conditioned cells and in Ca2+-signaling mechanism between the naive and IH cells may, in part, be attributed to the periodic reoxygenation that occurs after each cycle of hypoxia, resembling ischemia-reperfusion wherein increased generation of ROS has been proposed. The activity of aconitase is sensitive to and inhibited by ROS, especially the superoxide anion (17). The degree of inhibition of aconitase was used as an in vivo measure of ROS production. Our finding that aconitase activity is strongly inhibited in IH-conditioned cells (1, 36) supports such a notion. Interestingly, NAC, a potent scavenger of ROS, markedly inhibited IH-induced augmentation of transmitter release by hypoxia, suggesting that ROS is involved in the modulation of hypoxia-evoked transmitter release during IH. It is also worth noting that NAC also partially attenuated (~20%) DA release during hypoxia in naive cells, suggesting a potential role for ROS in hypoxia-evoked DA release. It is likely that ROS may mediate the oxidative modification of several proteins associated with Ca2+ homeostasis affecting either extracellular Ca2+ influx (35, 45) or intracellular Ca2+ release (21) and may lead to augmentation of transmitter release during hypoxia. Cellular sources of ROS include mitochondria and various cytosolic and membrane-associated oxidases. However, it remains to be established whether one or more of these sources contribute to the increased generation of ROS during IH in PC12 cells.

In summary, our results show that acute hypoxia selectively augmented DA but not ACh release from PC12 cells. In addition, IH conditioning facilitated DA release and stimulated ACh release during acute hypoxia. The increase in neurotransmitter release by acute hypoxia after IH preconditioning appears to occur primarily via mechanisms associated with Ca2+ mobilization from the intracellular stores and also, in part, from voltage-gated Ca2+ influx. More importantly, ROS seem to play a vital role in the modulation of hypoxia-induced transmitter release in IH-conditioned cells.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-25830.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. K. Kumar, Dept. of Biochemistry, School of Medicine, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4935 (E-mail: kgk{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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