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Cardiovascular Research 2004 64(3):536-543; doi:10.1016/j.cardiores.2004.08.002
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Copyright © 2004, European Society of Cardiology

Amyloid β peptides mediate physiological remodelling of the acute O2 sensitivity of adrenomedullary chromaffin cells following chronic hypoxia

Stephen T. Brown, Rosalyn P. Johnson, Rhandi Senaratne and Ian M. Fearon*

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

* Corresponding author. Current address: School of Biological Sciences, The University of Manchester, G. 38 Stopford Building, Oxford Road, Manchester, M13 9PT, UK. Tel.: +44 (0) 161 275 5496; fax: +44 (0) 161 275 5600. Email address: ian.fearon{at}man.ac.uk

Received 27 May 2004; revised 4 August 2004; accepted 5 August 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The non-neurogenic response of the neonatal adrenal medulla is vital in cardiovascular and respiratory development and to the survival of newborns exposed to hypoxic stress. Here, we examined the acute hypoxic response of immortalised rat adrenomedullary chromaffin cells following exposure to chronic hypoxia (CH; 6% O2 for 24 h).

Methods: Ca2+ and K+ channel currents were recorded using by whole-cell patch-clamp.

Results: Following incubation in CH, the acute O2 sensitivity of K+ current in immortalised adrenomedullary chromaffin (MAH) cells was enhanced due to a selective increase in the density of an O2-sensitive Ca2+-dependent K+ current, secondary to ROS-mediated augmentation of voltage-gated Ca2+ currents. The effect of CH on Ca2+ currents was not additive to exogenous Aβ1–40 and was blocked by the {gamma}-secretase inhibitors {gamma}-X and {gamma}-VI, demonstrating a role for amyloid β peptide (AβP) production. Ca2+ current enhancement was abolished in the presence of the transcription inhibitor actinomycin D but unaffected by the vacuolar H+ ATPase inhibitor bafilomycin A1.

Conclusion: AβP production and transcriptional regulation during CH regulated the properties of a peripheral chemosensory cell, defining a role for these enigmatic peptides in the signalling pathway of a physiological response to CH in the developing cardiovascular system.

KEYWORDS Hypoxia/anoxia; Ca-channel; K-channel; Developmental biology


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Due to its pivotal role in the production of ATP, O2 is a molecule fundamental to the survival of aerobic organisms. Not surprisingly, low O2 triggers tissue and cellular responses designed to counteract the cause of hypoxia or to minimize its deleterious effects. Broadly speaking, responses to hypoxia can be classified as acute and chronic, based on their relative time scales. Acute hypoxic responses are generally mediated via the regulation of plasmalemmal ion channels and ion and glucose transporters [1]. The first demonstration of an O2-sensitive ion channel was made in chemosensory carotid body type I cells [2], where acute hypoxia inhibited K+ current. Since that time, there have been numerous reports of hypoxic inhibition of various K+ and Ca2+ channel subtypes [3–5] in a host of different tissues and which contributes to the physiological regulation of O2 homeostasis. In immature adrenomedullary chromaffin cells in the period prior to sympathetic innervation to the adrenal medulla [6], hypoxia induces an increase in [Ca2+]i levels and catecholamine secretion [7], initiated by inhibition of both Ca2+-activated and delayed-rectifier K+ channels [8,9]. This release of catecholamines is critical for the development of the cardiovascular and respiratory systems in neonates and promotes their ability to survive hypoxic stress [6,10].

Responses to chronic hypoxia (CH) may be mediated via enhanced gene expression, the products of which mediate adaptive regulatory processes such as glucose metabolism, erythropoiesis and angiogenesis [1]. Such gene expression occurs due to the hypoxic accumulation of the {alpha} subunits of the hypoxia-inducible factor (HIF) family of transcription factors [11]. Recent evidence also suggests that in astrocytes [12] and PC12 cells [13,14], responses to CH are mediated by the accumulation of amyloid β peptides (AβPs). These neurotoxic proteins accumulate in the brains of patients with Alzheimers' disease and are causative of numerous aspects of neuronal dysfunction in this dementia [15]. While this pathological role is well documented, there is little information concerning a physiological role for AβPs [16], although non-toxic forms of AβPs control ion channel function in cerebellar granule neurons [17]. Furthermore, secretase inhibitors and antibodies raised against Aβ have recently been shown to promote the survival of primary cortical neurons [18], suggesting a role for AβPs in normal neuronal function.

In the current study, we have examined the effect of exposure to CH on the acute O2 sensitivity of immortalised adrenomedullary chromaffin (MAH) cells. Ca2+ currents were up-regulated following CH, leading to an enhanced O2-sensitive KCa current and also to augmentation of the magnitude of acute hypoxic inhibition of K+ current. Chronically hypoxic enhancement of Ca2+ currents was mimicked by exogenous Aβ1–40 and inhibited by pharmacological blockade of {gamma}-secretase, a key enzyme in the cleavage of amyloid precursor protein into its biologically active forms [19]. These data demonstrate the ability of the enigmatic AβPs to regulate the physiological function of a chemosensory cell during chronic hypoxia, and further demonstrate a physiological role for AβPs in the development of the cardiovascular system.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Culture of MAH cells
Experiments were carried out on rat adrenomedullary chromaffin-derived MAH cells [20]. Cells were grown in modified L-15/CO2 medium supplemented with 0.6% glucose, 1% penicillin/streptomycin, 10% fetal bovine serum and 5 µM dexamethasone as previously described [9]. Normoxic cultures were grown in a CO2 incubator (Forma) in a humidified atmosphere of 95% air/5% CO2 at 37 °C. In chronic hypoxia studies, cells were transferred for either 6 or 24 h into a separate incubator (Forma) with an atmosphere of 6% O2/5% CO2/89% N2.

2.2 Electrophysiology
Dishes with attached cells were transferred to a continually perfused (approximately 2 ml min–1) recording chamber and either whole-cell or nystatin (500 µg/ml) perforated patch-clamp recordings [21] were made using patch pipettes of resistance 4–6 M{Omega}. Recordings were made at room temperature (22±2 °C). Current traces were filtered at 5 kHz, digitized at 10 kHz and stored on a PC for later analysis. Capacitative transients were minimized by analogue means (residual transients have been truncated for illustrative purposes) and corrections for leak current were made off-line by the appropriate scaling and subtraction of the average leak current evoked by small hyperpolarising and depolarizing steps (≤5 mV). All analyses and voltage protocols were performed using a Multiclamp 700 amplifier in combination with a Digidata 1322A interface and pCLAMP 9.0 software (Axon Instruments). Current densities were calculated by dividing the evoked current by the cell’s capacitance. Cells were voltage-clamped at –80 mV (Ca2+ currents) or –60 mV (K+ currents), and currents evoked by step depolarizing the membrane to various test potentials for 100 ms at a frequency of 0.1 Hz. In some studies (see, e.g., Fig. 1A), membrane potential was ramped between –100 and +50 mV over a period of 1 s.


Figure 1
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  		Fig. 1 CH up-regulated an O2-sensitive KCa current and enhanced the acute O2 sensitivity of MAH cells. (A) Current–voltage (I–V) relationships demonstrating the increase in total K+ current in MAH cells exposed to chronic hypoxia (CH; 6% O2 for 24 h; representative of 40 such recordings) compared to normoxic controls (nox; 21% O2; representative of 37 such recordings). The mean reversal potential (Erev) of currents evoked in normoxic cells was –37.8±1.6 mV (n=40), a value not significantly different from that obtained in chronically hypoxic cells (–39.7±2.2 mV, n=37; P>0.05, unpaired Student's t-test). Cell capacitances were 7.76 pF (nox) and 7.68 pF (CH). Inset: This increase in K+ current was quantified by comparing currents evoked at +30 mV (holding potential, –60 mV) in cells exposed to nox and CH. Data are means (±S.E.M.) taken from the number of cells indicated in parentheses. Current density (pA/pF) was calculated by dividing the evoked current by the capacitance of the cell. (B) Mean magnitudes of inhibition of K+ current by 200 µM Cd2+, 5 mM 4-AP or 300 µM Zn2+ in cells cultured in normoxia (nox, open columns) and chronic hypoxia (CH, shaded columns), indicating that CH selectively up-regulated a Ca2+-dependent K+ current. Inset: Up-regulation of this O2-dependent current9 enhanced the O2 sensitivity of total K+ current. *P<0.05; NS, P>0.05; unpaired Student's t-tests.

 
2.3 Ca2+ currents
Cells were perfused with a solution of (in mM): NaCl, 95; CsCl, 5; MgC12, 0.6; BaCl2 20; HEPES, 5; D-glucose, 10; TEA–Cl, 20 (21–24 °C, pH adjusted to 7.4 with NaOH) and patch electrodes were filled with (in mM): CsCl, 120; TEA–Cl, 20; MgC12, 2; EGTA, 10; HEPES, 10; ATP, 2 (pH adjusted to 7.2 with CsOH). Current amplitudes were measured at their peaks during each step depolarization.

2.4 K+ currents
Cells were perfused with a solution of (mM): NaCl, 135; KCl, 5; MgCl2, 1.2; HEPES, 5; CaCl2, 2.5 and D-glucose, 10 (pH 7.4 with NaOH). Electrodes were filled with (mM): NaCl, 5; KCl, 135; HEPES, 10; CaCl2, 1 and nystatin 500 µg/ml (pH 7.2 with KOH).

To render perfusates hypoxic, extracellular solutions were bubbled with N2 gas for at least 30 min prior to use. Bubbling produced no change in pH. Bath Po2, measured with a depolarised carbon-fibre electrode [22], reached a stable level of ~10 mm Hg 30 s after switching solutions.

2.5 Chemicals and statistical analyses
Aβ1–40, 4-AP, ZnCl2, CdCl2, ascorbic acid, bafilomycin A1 and actinomycin D were from Sigma. The secretase inhibitors {gamma}-X and {gamma}-VI were from Calbiochem. Results are expressed as means±S.E.M., and statistical comparisons were made using paired and unpaired Student's t-tests as appropriate.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Chronic hypoxia enhances the acute O2 sensitivity of K+ current via selective up-regulation of an O2-sensitive Ca2+-dependent K+ conductance
Initially, we examined the effects of CH (6% 02 for 24 h) on whole-cell K+ currents in MAH cells. As depicted in Fig. 1A, exposure to CH enhanced evoked K+ currents at all activating test potentials. This increase in the basal outward current was not due to an increase in cell size, since cell capacitance was 6.5±0.3 pF (n=40) in normoxia and 7.2±0.3 pF following CH (n=37; P>0.05). To quantify the effect of CH on K+ currents, we compared current densities (calculated by dividing evoked current by cell capacitance) evoked at a test potential of +30 mV (holding potential –60 mV) between normoxic and chronically hypoxic groups. Following CH, mean current densities were increased, from 41.4±2.9 pA/pF (n=40) in normoxically cultured cells to 59.5±4.4 pA/pF (n=37) in cells exposed to CH (P<0.05; Fig. 1A, inset). CH caused a small but insignificant cell membrane hyperpolarisation (measured under current-clamp), from –34±2 mV (normoxia; n=21) to –37±2 mV (CH; n=14; P>0.05, unpaired Student's t-test). Up-regulation of K+ current was due to the selective enhancement of a Cd2+-sensitive component of the whole-cell current, since the magnitude of inhibition by 200 µM Cd2+ was significantly increased following incubation in CH (P<0.05; Fig. 1B). In contrast, responses to 5 mM 4-AP or 300 µM Zn2+, inhibitors of delayed-rectifier and background K+ channels expressed in MAH cells [9,23], were unaltered following exposure to CH (P>0.05 for each of 4-AP and Cd2+; Fig. 1B). Thus, CH selectively up-regulated a Ca2+-dependent K+ current while expression of delayed-rectifier and background K+ currents remained unchanged.

The acute O2 sensitivity of K+ current in MAH cells is due in part to hypoxic inhibition of a Ca2+-dependent K+ current [9]. Following CH, the augmentation of such a current described above enhanced the acute O2 sensitivity of MAH cells, since there was a significant increase in the magnitude of the O2-sensitive K+ current (IKO2; calculated by dividing the current evoked during exposure to acute hypoxia by that evoked during normoxia) following exposure to CH (P<0.05; Fig. 1B, inset). Thus, prolonged exposure to hypoxia modified the acute response to the same stimulus.

3.2 Enhancement of KCa currents following CH occurred due to increased Ca2+ entry via regulation of the expression of voltage-gated Ca2+ channels
In PC12 cells, CH enhances Ca2+ entry via the up-regulation of L-type [13] and T-type [24] Ca2+ channels. To test the hypothesis that chronically hypoxic augmentation of KCa currents was dependent on increased Ca2+ entry, we examined the magnitude of K+ currents in normoxic and hypoxic cells at +100 mV, a potential at which no Ca2+ entry occurs (see Fig. 2B). These experiments were carried out in the presence of 5 mM 4-AP and 300 µM Zn2+ to block contaminating Kv and background K+ currents from recordings, and to isolate the KCa current [9,23]. At this potential, the mean magnitude of depolarization-evoked K+ current density was not significantly different between normoxic and chronically hypoxic groups (P>0.05; Fig. 2A). We further examined Ca2+ current (ICa) amplitudes in voltage-clamp studies in which all other ionic currents were inhibited, prior to and following exposure to CH. As illustrated in Fig. 2B, CH (6% 02 for 24 h) significantly enhanced voltage-dependent Ca2+ currents at all activating test potentials examined. For example, at a potential of 0 mV evoked Ca2+ currents were –9.1±1.1 pA/pF (n=7) in cells cultured under normoxic conditions and –12.3±1.5 pA/pF (n=11) following CH (P<0.05). The effects of CH on Ca2+ currents were abrogated by the antioxidant ascorbic acid (200 µM), such that Ca2+ currents under CH were significantly reduced in the presence of ascorbic acid than in its absence (P<0.05; Fig. 2C). This was not due to a non-specific action of ascorbic acid since the antioxidant was without significant effect in control (normoxic) cells (P>0.05; Fig. 2C). Collectively, these data demonstrate that enhanced KCa currents were due to enhanced Ca2+ entry, which occurred due to a ROS-mediated increase in Ca2+ currents during CH.


Figure 2
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  		Fig. 2 Enhanced KCa currents following CH were due to up-regulation of voltage-gated Ca2+ currents. (A) When step-depolarising to +100 mV (holding potential –60 mV), a test potential at which no Ca2+ entry occurs, evoked K+ current densities were not significantly different between normoxic (nox) and chronically hypoxic (CH) groups. Recordings were made in the presence of 5 mM 4-AP and 300 µM Zn2+ to block Kv and background currents and isolate KCa current [9,22]. Data were averaged from the number of cells indicated in parentheses above each column. Thus KCa current up-regulation due to CH occurred via enhanced Ca2+ entry. (B) Ca2+ currents in cells exposed to CH were increased compared to normoxic controls. Mean current density–voltage relationships from 7 cells (nox) and 11 cells (CH). The increase in Ca2+ current amplitudes was voltage-independent. Currents were evoked by 100 ms step depolarisations to the indicated potentials from a holding potential of –80 mV. The mean reversal potential (Erev) of currents measured under these conditions was not significantly altered following exposure to CH (nox, 45.0±5.3 mV; CH, 47.1±5.4 mV; P>0.05, unpaired Students t-test). (C) Mean Ca2+ current densities, calculated by dividing current evoked by a step depolarisation to 0 mV by the cell capacitance, in cells incubated in normoxia (nox), in the presence of 200 µM ascorbic acid (asc), in chronic hypoxia (CH; 6% O2 for 24 h) and in chronic hypoxia in the presence of ascorbic acid (CH+asc). *P<0.05; NS, P>0.05; unpaired Student's t-tests. Sample sizes are indicated in parentheses. (D) The magnitudes of the responses to 2 µM nifedipine (nif; indicative of the magnitude of L-type Ca2+ current) and 500 µM Ni2+ (indicative of T-type current) were increased following chronic hypoxia (CH) when compared to normoxic controls. *P<0.05, unpaired Student's t-tests. Sample size is indicated in parentheses. (E) The mean magnitude of the Cd2+-insensitive Ca2+ current in normoxia (nox) and following exposure to CH. NS, not significantly different (P>0.05, unpaired Student's t-test).

 
Since rat chromaffin cells express numerous functional Ca2+ channel subtypes [25,26], we used pharmacological agents to examine whether the effect of CH on the total ICa was non-selective or specific for channel subtypes. The magnitude of the inhibitory response to 2 µM nifedipine, a selective L-type Ca2+ channel blocker was increased, from –3.8±0.6 pA/pF (n=8) under control conditions to –7.9±1.6 pA/pF (n=8) following exposure to CH (Fig. 2D). We further examined the magnitude of inhibition due to the selective T-type Ca2+ channel blocker Ni2+ at a concentration (500 µM) sufficient to fully block all three splice variants of T-type channels [27]. Inhibition due to Ni2+ was also increased, from –4.3±0.8 pA/pF (n=8) in normoxically cultured cells to –6.0±2.0 pA/pF (n=8) following culture in CH (Fig. 2D). Together, these data suggest that CH up-regulated both L-type and T-type Ca2+ channels in MAH cells.

In rat phaechromocytoma cells, CH induced functional expression of a Cd2+-resistant Ca2+ influx pathway alongside its potentiating effect on voltage-gated Ca2+ channels [13]. To examine whether such an influx pathway contributed to the augmented Ca2+ entry due to CH in MAH cells we examined the residual current following the application of 200 µM Cd2+, a potent blocker of all subtypes of classical voltage-gated Ca2+ channels. In all cells examined in normoxia, there remained a small amount of Ca2+ entry during exposure to Cd2+ which appeared voltage-dependent. The mean (±S.E.M.) magnitude of this current at it's peak potential (–20 mV) was –1.1±0.3 pA/pF (n=5). Following exposure of MAH cells to CH, the Cd2+-resistant current remained, and its magnitude at the potential which evoked peak current was not significantly different to that seen in normoxia (–1.6±0.5 pA/pF, n=7; P>0.05; Fig. 2E). Thus, CH did not induce a Cd2+-resistant Ca2+ influx pathway in MAH cells.

3.3 Effects of CH on Ca2+ currents were mimicked by Aβ1–40 and inhibited by {gamma}-secretase inhibitors
In neuronal PC12 cells, CH-induced augmentation of voltage-gated Ca2+ entry involves the production of AβPs [13,14]. In immortalised chromaffin cells, the effects of CH on Ca2+ currents were mimicked by exogenous Aβ1–40 (50 nM for 24 h in normoxia; Fig. 3A). The effects of both CH and Aβ1–40 were non-additive (Fig. 3B), suggesting that they were both acting via the same mechanism, i.e., CH caused AβP production which regulated Ca2+ channel function and/or expression. To further examine this hypothesised role for AβPs, MAH cells were incubated in CH in the presence of either 10 µM {gamma}-X or 100 nM {gamma}-VI, inhibitors of the {gamma}-secretase responsible for cleaving amyloid precursor proteins into their physiologically active forms [19]. When cells were exposed to CH in the presence of either secretase inhibitor, the response to CH was significantly reduced. For example, at a test potential of 0 mV, Ca2+ current amplitudes were –12.3±1.5 pA/pF (n=11) in cells exposed to CH, and –9.5±1.6 pA/pF (n=7) and –7.5±0.6 pA/pF (n=6) in cells exposed to CH with {gamma}-X or {gamma}-VI, respectively (Fig. 3C and D; in each case, P>0.05). The effects of secretase inhibitors were not due to inhibition of Ca2+ channel expression per se, since both {gamma}-X and {gamma}-VI were without effect on Ca2+ currents in normoxia (Fig. 3E). Similar to CH, the magnitude of the Cd2+-insensitive Ca2+ current following treatment of cells with AβP1–40 was not significantly different from controls (Fig. 3F). Collectively, these data demonstrate a role for AβP production in the enhancement of classical voltage-gated Ca2+ currents by CH.


Figure 3
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  		Fig. 3 Enhancement of Ca2+ currents by CH was mimicked by Aβ1–40 and ablated by selective inhibitors of {gamma}-secretase. (A–D) Mean current density–voltage relationships obtained from between 7 and 11 cells in each case. Ca2+ currents were evoked as described in Fig. 2. (A) Aβ1–40 (50 nM) enhanced Ca2+ currents in a voltage-independent manner, mimicking the effects of CH. (B) The effects of CH and Aβ1–40 were not additive, suggesting they enhance Ca2+ currents via a common mechanism of action. The increase in Ca2+ current due to CH was abrogated in the presence of (C) 100 nM {gamma}-X and (D) 10 µM {gamma}-VI, selective inhibitors of an enzyme involved in the production of active forms of amyloid peptides from their precursors [18]. (E) These inhibitors were without significant effect on Ca2+ current amplitudes when applied during normoxia. Columns indicate mean (±S.E.M.) current densities evoked by a step depolarisation to 0 mV (holding potential –80 mV) in the number of cells indicated in parentheses. P>0.05 for each inhibitor when compared to normoxic controls. (F) AβP1–40 was without significant effect on the magnitude of the Cd2+-insensitive Ca2+ current in MAH cells. Data are mean (±S.E.M.) current densities evoked by a step depolarisation to –20 mV, which the peak amplitude of these currents. Data were obtained from the number of cells indicated in parentheses. NS, P>0.05, Student's unpaired t-test.

 
Since ion channels may be regulated by CH in a post-transcriptional manner involving subunit trafficking [28], we examined the effects of the vacuolar-type H+-ATPase inhibitor bafilomycin A1 on the response to CH. At a concentration of 100 nM, this agent was without effect on the chronic hypoxic response of Ca2+ current (Fig. 4B). This indicated that the effects of CH in MAH cells were not due to post-transcriptional regulation of Ca2+ channel subunit expression, a finding which is consistent with the above data demonstrating that the effect of CH was abolished under conditions which preclude Ca2+ entry during cell depolarisation. In contrast to this lack of post-transcriptional regulation of KCa current, the effect of CH on voltage-gated Ca2+ current was reversed in the presence of 1 µg/ml actinomycin D (Fig. 4C), an inhibitor of transcription, suggesting a role for altered gene transcription in mediating altered Ca2+ channel expression.


Figure 4
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  		Fig. 4 Augmentation of Ca2+ currents was mediated by transcriptional but not post-transcriptional regulation. (A–C) Mean current density–voltage relationships obtained from between 6 and 11 cells in each case. Ca2+ currents were evoked as described in Fig. 2. (A) Ca2+ currents were enhanced following a 6-h exposure to hypoxia (6% O2). (B) Inhibition of the vacuolar-type H+-ATPase was without effect on Ca2+ current augmentation by CH, suggesting a lack of involvement of channel/subunit trafficking in the response to CH. (C) Enhancement of Ca2+ currents was abolished by incubation with the transcription inhibitor actinomycin D (1 µg/ml). Data are mean (±S.E.M.) current densities evoked by a step depolarisation to 0 mV (holding potential –80 mV) in the number of cells indicated in parentheses.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the neonatal adrenal medulla, prior to its sympathetic innervation this organ responds directly to hypoxia and releases catecholamines via non-neurogenic mechanisms, a response vital for the development of the cardiovascular and respiratory systems [6,10]. The well-characterised initiation of this acute hypoxic response is mediated via inhibition of Ca2+-dependent [8] and delayed-rectifier [9] K+ channels causing membrane depolarization, Ca2+ influx and subsequently Ca2+-dependent catecholamine release [10]. There is, however, little information concerning the responses of these chromaffin cells to more prolonged periods of hypoxia, although CH increased basal catecholamine secretion and enhanced responsiveness of these cells in the rainbow trout [29]. In numerous cell types, CH up-regulates various gene products involved in regulating homeostasis and abrogating the deleterious effects of hypoxia [1]. In many cases, this effect is mediated by the HIF family of transcriptional activators, the accumulation of which is enhanced during CH due to inhibition of O2-dependent degradation [11]. CH may also act via amyloid-mediated regulation of Ca2+ entry pathways [12–14], an effect which may link hypoxic episodes with Alzheimer's dementia [30]. In the present study, we demonstrated that CH up-regulated whole-cell voltage-gated Ca2+ conductance in immortalised adrenomedullary chromaffin cells, an effect not attributable to either a simple increase in cell size or a change in the reversal potential (Erev) since both the mean cell capacitance and the mean Erev were unaltered by CH. This effect of CH was abrogated by selective {gamma}-secretase inhibition and mimicked in normoxia by exogenous application of an amyloid β peptide, providing unequivocal evidence that amyloid production mediated Ca2+ current enhancement due to CH. The net effect of Ca2+ conductance up-regulation was an enhanced Ca2+-dependent K+ current, and due to the O2 sensitivity of this current [9] an accentuated response to acute hypoxia. Similar to Ca2+ currents, the effect of CH on K+ currents was not attributable to an altered Erev. Thus, exposure to CH modulated the responsiveness of a chemosensory cell to a physiological stimulus via direct and indirect up-regulation of numerous transmembrane ionic conductances, and the involvement of AβPs, which chromaffin cells are capable of synthesizing [31], demonstrates a physiological role for these enigmatic peptides in cardiovascular development [16].

A recent study examined the response of K+ currents to chronic hypoxia in HEK293 cells expressing recombinant BKCa {alpha} and β K+ channel subunits [28]. Following exposure to CH, K+ current density was increased due to post-transcriptional up-regulation of auxiliary (β) subunit surface expression. Data in the present study do not support this finding, since enhancement of K+ currents was absent when examined under conditions precluding Ca2+ entry. This suggests that the number of functional channels and/or auxiliary subunits contributing to the KCa current was not altered by chronic hypoxia and that the increase in K+ current density observed was driven by an increase in Ca2+ currents. This increase was itself not due to channel trafficking since the effect of CH on Ca2+ currents was not responsive to bafilomycin A1. It was, however, ablated by actinomycin D, suggesting that the transcription of Ca2+ channel gene transcripts was involved in the hypoxic response. Furthermore, the present study was carried out in a cell line expressing endogenous voltage-gated and Cd2+-insensitive Ca2+ entry pathways, both of which are absent in HEK293 cells. Thus, our current finding of transcriptional regulation of Ca2+ channel expression mediating altered KCa currents would not be reproducible in such a recombinant system. Interestingly however, in the previous study [28], effects of CH were not observed in the time frame at which we observed transcriptionally mediated hypoxic up-regulation of ion channels. This gives rise to the possibility that further post-transcriptional up-regulation of BK channel subunits may supercede the transcriptional regulation of Ca2+ channels reported here, when the effects of hypoxia are examined over a more extended time period.

Alongside the involvement of AβPs, our data demonstrated a role for altered transcription in mediating the effects of CH since they were abolished in the presence of actinomycin D. In PC12 cells, the CH-mediated transcription of T-type Ca2+ channels was dependent on HIF stabilisation [24]. Although there is no evidence to date to suggest that the expression of L-type channels is regulated in a similar manner, collectively, these findings propose a link between HIF accumulation and the production of AβPs during CH. In both primary cortical neurons and neuronal cell lines, HIF levels were up-regulated by exogenous AβPs [32]. Thus, given the demonstrations here and elsewhere [13,14] that AβP production mediates chronically hypoxic enhancement of Ca2+ channels, a putative pathway would be the amyloid-dependent stabilisation of HIF{alpha} subunits and subsequently altered Ca2+ channel gene expression via activation of these transcription factors. The mechanism underlying CH-induced amyloid production in this pathway may be one of altered {gamma}- and β-secretase activity, which occurs during hypoxia [12,33]. Conversely, activation of HIF during CH caused activation of the presenilin gene [34], and thus HIF activation may precede the up-regulation of this component of the β-amyloid-producing {gamma}-secretase [35]. These possibilities necessitate further examination of the role of AβPs and HIF-mediated transcriptional regulation in mediating the effects of CH.

Our data demonstrating that the effects of CH on Ca2+ currents were abrogated by the antioxidant ascorbic acid provide direct evidence of a role for ROS production in causing Ca2+ current up-regulation, an occurrence strikingly similar to that reported in neuronal PC12 cells [14]. Whether such ROS production occurs prior to or precedes [36,37] AβP production remains to be fully elucidated. Further studies are required to clarify the roles and temporal sequence of ROS and amyloid production in enhancing functional Ca2+ channel expression.

In summary, during CH, AβP production mediated the up-regulation of a physiological response in MAH cells, providing new insight into the mechanisms underlying the chronic hypoxic regulation of the chemosensitivity of cells which are pivotal to the development of the cardiovascular system, while providing novel evidence of a role for the enigmatic AβPs in physiology as well as pathology.


   Acknowledgements
 
This research was supported by a Grant-in-Aid (Grant NA 5230) to IMF from the Heart and Stroke Foundation of Ontario. Equipment was provided by New Opportunities grants to IMF from the Canada Foundation for Innovation (7400) and the Ontario Innovation Trust. We are indebted to I. O'Kelly for constant technical support and assistance, and to S. Fearon for providing inspiration.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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J. Buttigieg, S. T. Brown, M. Lowe, M. Zhang, and C. A. Nurse
Functional mitochondria are required for O2 but not CO2 sensing in immortalized adrenomedullary chromaffin cells
Am J Physiol Cell Physiol, April 1, 2008; 294(4): C945 - C956.
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