J Biol Chem, Vol. 274, Issue 44, 31217-31222, October 29, 1999

Hypoxic Enhancement of Quantal Catecholamine Secretion
EVIDENCE FOR THE INVOLVEMENT OF AMYLOID -PEPTIDES^*

Shafeena C. Taylor, Trevor F. C. Batten, and Chris Peers

From the Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Prolonged exposure to hypoxia (10% O₂) enhanced quantal catecholamine release evoked from O₂-sensing pheochromocytoma (PC12) cells, as monitored using single-cell amperometric recordings. The enhancement of exocytosis was apparent after 12 h of hypoxia and was maximal at 24 h. Elevated levels of secretion were due to the emergence of a Ca²⁺ influx pathway that persisted during complete blockade of known voltage-gated Ca²⁺ channels. Secretion triggered by this Ca²⁺ influx was severely reduced by known inhibitors of Alzheimer's amyloid -peptides (APs), including an N terminus-directed monoclonal antibody. The enhancing effect on secretion of chronic hypoxia was mimicked closely by direct application of AP to cells under normoxic conditions, although the effects of AP were more rapid at onset, being maximal after only 6 h. The present results suggest that prolonged hypoxia can induce formation of Ca²⁺-permeable AP channels and that such induction can lead directly to excessive neurosecretion. This is a potential contributory factor to AP pathophysiology following cerebral ischemia.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dementias in general and Alzheimer's disease in particular are more prevalent following periods of cerebral ischemia caused by cardiovascular dysfunctions such as stroke (1-3). Ischemia is a complex condition, causing perturbations in several parameters that are essential to neuronal survival such as lack of substrates, accumulation of metabolic products, acidosis, and reduction of oxygen levels. However, the specific contributions of each of these factors to cellular dysfunction and death are presently unknown.

A fundamental feature of Alzheimer's disease is the accumulation of fibrillar deposits consisting of amyloid -peptides (APs)¹ (reviewed in Ref. 4). APs are 40-42-amino acid peptides and are cleavage products derived from amyloid precursor protein (APP) (5, 6). APP is one of only a few gene products whose expression is increased following a period of cerebral ischemia (7, 8). Since APP is generally perceived to be neuroprotective, this increased expression of APP can be considered a defense mechanism against ischemia. However, increased APP levels seen in ischemia would also permit increased formation of APs, which cause neuronal damage and death (4); and indeed, AP production is increased following both mild and severe ischemia (9, 10). Thus, a clear link exists between ischemic insult and elevation of damaging AP levels. However, it remains completely unknown how ischemia might increase AP levels, and the mechanism(s) by which APs cause cellular dysfunction and death remain speculative.

Reduction of available O₂ (hypoxia) is a key feature of ischemia. Hypoxia is known to exert a diverse range of responses in cells, each of which serves a specific physiological purpose (11). Acute hypoxia can evoke extremely rapid responses such as selective, membrane-delimited inhibition of ion channels (12, 13). Prolonged (chronic) hypoxia can alter gene expression and so, for example, permit increased production of erythropoietin (14) or, in electrically excitable cells, tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis (15, 16). In this study, we have utilized the catecholamine-secreting pheochromocytoma (PC12) cell to investigate the effects of both acute and chronic hypoxia on exocytosis, as determined in real time using amperometry (17). PC12 cells represent a well defined, model excitable cell system, which has been used extensively to study the effects of both acute and chronic hypoxia on various cellular processes, including ion channel activity and gene expression (16, 18-20). PC12 cells have also been extensively characterized as a model system for studying exocytosis (21).

Our recent work has demonstrated that acute hypoxia evokes catecholamine release from PC12 cells by causing membrane depolarization and subsequent Ca²⁺ influx, primarily through N-type voltage-gated Ca²⁺ channels (20). Furthermore, chronic mild hypoxia (10% O₂ for 24 h) enhances this secretory response of PC12 cells to acute hypoxia (22). This enhancement is in part due to increased expression of O₂-sensitive K⁺ channels that influence membrane potential, but is also due to the emergence of a Ca²⁺ influx pathway that is resistant to known blockers of voltage-gated Ca²⁺ channels (22). The present study was aimed at identifying this induced Ca²⁺ influx pathway, and our results indicate that chronic hypoxia increases evoked exocytosis by increasing the production of APs that form Ca²⁺-permeable membrane channels that are tightly coupled to exocytosis. Given the known increased incidence of Alzheimer's disease following ischemia as described above, these findings are likely to be of widespread importance in understanding causes and effects of increased dementias following ischemic insult.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PC12 cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as described previously (20, 22) in RPMI 1640 medium (containing L-glutamine) supplemented with 20% fetal calf serum and 1% penicillin/streptomycin (all from Gibco, Paisley, Strathclyde, United Kingdom). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air, passaged every 7 days, and used for up to 20 passages. Cells used for experiments were transferred to smaller flasks in 10 ml of medium, to which was added 1 µM dexamethasone (Sigma, Poole, UK; from a stock solution of 1 mM in ultrapure water), and were cultured for a further 72-96 h to enrich catecholamine stores (23). Cells exposed to chronic hypoxia were treated identically, except that for 6-24 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.

Each experimental day, PC12 cells were plated onto poly-L-lysine-coated coverslips and allowed to adhere for ~1 h under either normoxic or hypoxic (10% O₂) conditions, as required. Fragments of coverslip were then transferred to a recording chamber (volume of 80 µl), which was continually perfused under gravity (flow rate of 1-2 ml/min) with a solution of 135 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 5 mM Hepes, and 10 mM glucose (pH 7.4; osmolarity adjusted to 300 mosM with sucrose, 21-24 °C). Ca²⁺-free solutions contained 1 mM EGTA and no added CaCl₂. All drugs were applied in the perfusate except Congo red and the 3D6 antibody. Congo red was added to cells in culture for the final 3-4 h before plating. The 3D6 antibody was diluted to a final concentration of 5 µg/ml in standard extracellular perfusate, and cells were exposed to this solution for 1 h at 37 °C. Exposure of cells to antibody-free perfusate under the same conditions was without effect on the ability of cells to secrete catecholamines in response to depolarization (data not shown). 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 experiments.

Carbon fiber microelectrodes (proCFE, Axon Instruments, Inc., Foster City, CA) 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 computer storage. All acquisition was performed using a Digidata 1200 interface and Fetchex software from the pClamp 6.0.3 suite (Axon Instruments, Inc.). 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 (24). The time course of fall in PO₂ in the recording chamber was highly reproducible and was stable after 1 min (between 17 and 23 mm Hg), and analysis of the effects of acute hypoxia was conducted 90 s after exchange to hypoxic perfusate.

Unless otherwise stated, each experiment consisted of current recordings of a control period during which cells were perfused only 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 (17, 25). We did not distinguish between dopamine and noradrenaline, both known to be released from PC12 cells (26); but secretory events were never seen unless the electrode was polarized and adjacent to a cell. Quantification of release was achieved by determining spike number or frequency using the Mini Analysis Program (Synaptosoft Inc., Leonia, NJ). This allowed visual inspection of each event so that artifacts (due, for example, to solution switches) could be rejected from analysis. Results are presented as individual examples or means ± S.E., and statistical comparisons were made using unpaired Student's t test.

To monitor [Ca²⁺]_i, cells were incubated for 1 h at room temperature (21-24 °C) in control perfusate solution containing 4 µM Fura-2/AM. Cells were then placed in a perfusion chamber exactly as described for amperometric recordings, and changes in [Ca²⁺]_i were indicated by the fluorescence emitted at 510 nm due to alternate excitation at 340 and 380 nm, using a Joyce-Loebl PhoCal apparatus (Applied Imaging Inc., Rochester, NY). Data are presented as ratio signals.

Immunofluorescent labeling with the 3D6 antibody was performed with cells plated onto coverslips and subjected to normoxic or hypoxic conditions as described above. Cells were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min and then rinsed thoroughly in several changes of 0.1 M phosphate-buffered saline (PBS) over a period of 2 h. The coverslips were incubated with the 3D6 antibody (diluted to 0.5 or 2.0 µg/ml in PBS) in 24-well microtitration plates on a shaker for 18 h at 4 °C. After two 10-min rinses in PBS, the cells were incubated for 2 h in a 1:200 dilution of Cy2-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). After two additional 10-min rinses in PBS, the coverslips were mounted onto glass microscope slides with glycerol/PBS, and the edges of the coverslips were fixed with clear nail polish. The cells were examined using a Zeiss Axioskop epifluorescence microscope using a No. 10 (fluorescein) filter set. Photographs were taken using an Eastman Kodak MDS120 digital camera system.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Exposure of PC12 cells to elevated extracellular [K⁺] (50 mM) evoked quantal catecholamine secretion, which was apparent as transient oxidative currents detected with a carbon fiber microelectrode (Fig. 1A). Release evoked by 50 mM K⁺ has previously been shown to be due to membrane depolarization and Ca²⁺ influx through voltage-gated Ca²⁺ channels (20, 27, 28) and so could be completely inhibited by removal of extracellular Ca²⁺ or by application of the nonselective inhibitor of voltage-gated Ca²⁺ channels, Cd²⁺ (Fig. 1A). When cells were cultured in an environment of reduced O₂ (10% instead of the normal 21%) for 24 h, secretion evoked by the same stimulus (50 mM K⁺) was significantly enhanced (Fig. 1B), from an average exocytotic frequency of 0.83 ± 0.12 Hz (n = 26 cells) observed in control cells to 1.93 ± 0.16 Hz (n = 19; p < 0.001, unpaired t test). In both control cells (n = 70) and cells maintained in chronic hypoxia (n = 75), no secretion was detected until the cells were exposed to 50 mM K⁺ (data not shown). These findings for the effects of chronic hypoxia to enhance K⁺-evoked secretion are extremely similar to our previously reported effects of chronic hypoxia on the secretory responses to acute hypoxia (22).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Induction of Cd2+-resistant exocytosis by chronic hypoxia. A and B, amperometric detection of secretion evoked in control and chronically hypoxic PC12 cells, respectively, in response to 50 mM K+ (stimulus application began 10 s before beginning of traces). For the periods indicated by the horizontal bars, cells were exposed to either Ca2+-free perfusate or 200 µM Cd2+ in the presence of 2.5 mM Ca2+ (in the continued presence of 50 mM K+) as indicated. The horizontal scale bar (10 s) applies to all traces. Vertical scale bar = 5 pA for A and 10 pA for B. Note that in chronically hypoxic cells, 200 µM Cd2+ failed to inhibit secretion completely.

Enhancement of evoked secretion from cells cultured under chronically hypoxic conditions remained entirely dependent on the presence of extracellular Ca²⁺ since removal of Ca²⁺ from the perfusate (replaced with 1 mM EGTA) caused complete cessation of exocytosis (Fig. 1B, upper trace, representative of recordings from nine cells). However, Cd²⁺ (200 µM) was unable to prevent fully the secretory response to 50 mM K⁺ (Fig. 1B, lower trace, representative of 19 cells). In the presence of 200 µM Cd²⁺, secretion was significantly reduced (p < 0.002) to 0.72 ± 0.11 Hz. Thus, a period of chronic hypoxia induced an enhanced secretory response from PC12 cells that was entirely Ca²⁺-dependent, but ~37% of which was resistant to Cd²⁺ (i.e. was not mediated by Ca²⁺ influx through known voltage-gated Ca²⁺ channels).

A Cd²⁺-resistant Ca²⁺ entry pathway induced by 24 h of hypoxia was also indicated by microfluorometric recordings, as shown in Fig. 2. For control cells (Fig. 2A), bath application of solution containing 50 mM K⁺ caused a rapid and reversible rise of [Ca²⁺]_i that could be attributed to Ca²⁺ influx through voltage-gated Ca²⁺ channels since it was completely abolished by 200 µM Cd²⁺ (co-applied as indicated by the horizontal bars in Fig. 2). By contrast, rises of [Ca²⁺]_i measured in chronically hypoxic PC12 cells could only be reduced by ~60% in the presence of 200 µM Cd²⁺ (Fig. 2B). Mean changes in [Ca²⁺]_i evoked by 50 mM K⁺ in control (open bars) and chronically hypoxic (closed bars) PC12 cells are summarized in Fig. 2C.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Chronic hypoxia induces a Ca2+ influx pathway resistant to blockade by Cd2+. A and B, microfluorometric recordings of [Ca2+]i taken from representative control PC12 cells and PC12 cells previously kept chronically hypoxic (10% O2 for 24 h), respectively. In each trace, cells were exposed to a solution containing 50 mM K+ for the periods indicated by the horizontal bars. In the left-hand traces of A and B, no Cd2+ was present; but in the right-hand traces, Cd2+ (200 µM) was co-applied with the 50 mM K+ solution. C, mean (with S.E. bars) changes in [Ca2+]i determined from control PC12 cells (open bars) and PC12 cells maintained in 10% O2 for 24 h (closed bars) evoked by a perfusate containing 50 mM K+ either with or without Cd2+ (200 µM) as indicated. The numbers in parentheses indicate the number of recordings made under each condition.

To characterize further the Cd²⁺-resistant Ca²⁺ influx pathway coupled to secretion that was apparent after chronic hypoxia, we tested the effects of other known blockers of Ca²⁺ influx pathways in cells previously maintained in hypoxia for 24 h. We have previously found that Cd²⁺-resistant secretion evoked in these cells by acute hypoxia could be significantly reduced by inorganic cations (22). These cations (Zn²⁺ and La³⁺) also reduced Cd²⁺-resistant secretion evoked by 50 mM K⁺ in chronically hypoxic PC12 cells (Fig. 3). Furthermore, Cd²⁺-resistant release evoked by 50 mM K⁺ was also inhibited by Congo red (Fig. 3). Both Congo red and Zn²⁺ have been shown previously to inhibit AP-mediated Ca²⁺ fluxes (29, 30), suggesting the surprising possibility that a 24-h period of hypoxia induced formation of Ca²⁺-permeable AP channels that coupled closely to exocytosis. In further support of this, we found that exposure of cells to a monoclonal antibody (3D6) raised against the extracellularly located N' terminus of AP, anti-AP-(1-5) (30, 31), for 1 h at 37 °C also significantly suppressed the Cd²⁺-resistant component of the secretory response (Fig. 3). Similar inhibition of Cd²⁺-resistant secretion evoked in response to acute hypoxia (PO₂ ~ 20 mm Hg) was also found with Congo red and the 3D6 antibody (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Amyloid -peptide inhibitors suppress K+-induced enhancement of exocytosis. The bar graph shows mean (with vertical S.E. bars) exocytotic frequency recorded in cells previously exposed to 10% O2 for 21-26 h in response to 50 mM K+ in the presence of 200 µM Cd2+ alone (left-hand bar) or following blockade with Congo red (10 mM), La3+ (1 mM), Zn2+ (10 mM), or the 3D6 antibody (5 µg/ml) as indicated (all in the presence of 200 µM Cd2+). All blockers produced significant inhibition (p < 0.04 to 0.001) of Cd2+-resistant release.

Immunocytochemical labeling of cells with the 3D6 antibody demonstrated specific binding of this antibody to cells previously exposed to chronic hypoxia for 24 h (Fig. 4A). This was seen as diffuse fluorescence over most of the surface area of each cell. This was a consistent pattern of labeling in all cells examined (>100) at both concentrations of the antibody tested (0.5 and 2.0 µg/ml), and this is exemplified by comparison of the phase-contrast image of the same cells (Fig. 4B). Fluorescent labeling was detectable only at very low levels in the normoxic cells processed under identical conditions (Fig. 4C; phase-contrast view of the same image shown in Fig. 4D).

View larger version (94K): [in this window] [in a new window]   Fig. 4.   Immunofluorescent labeling of chronically hypoxic cells using an AP-directed monoclonal antibody. A, fluorescence image of individual cells (arrows) that had previously been exposed to hypoxia (10% O2) for 24 h and exposed to the 3D6 antibody; B, phase-contrast image of the same field; C, fluorescence image of individual cells (arrows) that had previously been cultured under normoxic conditions; D, phase-contrast image of the same field. Scale bars = 50 µm. Processing of cells led to flattening, leading to a larger apparent diameter than when they were perfused with solutions used for electrochemical recordings.

If chronic hypoxia did indeed induce formation of AP channels that coupled to exocytosis, we anticipated that direct application of APs to normoxically cultured cells would mimic the effects of chronic hypoxia. Therefore, in a separate series of experiments, we exposed cells to 100 nM AP-(1-40) for 24 h under normoxic conditions (21% O₂). These AP-treated cells secreted catecholamines in response to both acute hypoxia and raised K⁺ levels (Fig. 5A). Like both control and chronically hypoxic cells, they showed no spontaneous exocytosis under normoxic, non-depolarizing conditions (n = 47 cells; data not shown). Secretion evoked by either 50 mM K⁺ or acute hypoxia was significantly greater (p < 0.001) than in control cells (Table I) and was wholly dependent on the presence of extracellular Ca²⁺, and a substantial fraction was resistant to Cd²⁺ (Fig. 5A; see also Table I); such effects of AP treatment were extremely similar, both qualitatively and quantitatively, to the effects of chronic hypoxia (see above). Furthermore, the Cd²⁺-resistant component of the secretory response displayed the same sensitivity to other blockers as was observed in cells kept chronically hypoxic for 24 h, including the 3D6 monoclonal antibody (Fig. 5B). These findings strongly support the idea that chronic hypoxia appeared to induce formation of APs tightly coupled to exocytosis.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Exposure of cells to AP-(1-40) mimics the effects of chronic hypoxia. A, amperometric detection of secretion evoked in cells exposed previously to 100 nM AP-(1-40) in response to either acute hypoxia (upper traces; PO2 ~ 20 mm Hg, application began 1 min before beginning of traces) or 50 mM K+ (lower traces; stimulus application began 10 s before beginning of traces). For the periods indicated by the horizontal bars, cells were exposed either to Ca2+-free perfusate (left-hand traces) or 200 µM Cd2+ in the presence of 2.5 mM Ca2+ (right-hand traces) in the continued presence of acute hypoxia or 50 mM K+ as indicated. The scale bars apply to all traces. B, bar graph showing mean (with vertical S.E. bars, determined from the number of cells indicated in parentheses) exocytotic frequency recorded in cells previously exposed to 100 nM AP-(1-40) in response to acute hypoxia (open bars) or 50 mM K+ (hatched bars) in the presence of 200 µM Cd2+ alone or together with other blocking agents as indicated (applied as described in the legend Fig. 2). All blockers produced significant inhibition (p < 0.02 to 0.001) of Cd2+-resistant release.

View this table: [in this window] [in a new window]   Table I Effects of -amyloid treatment on secretory responses and their modulation by Ca2+ removal or application of Cd2 Cells were exposed for 21-26 h to 100 nM AP-(1-40) under normoxic conditions. The mean ± S.E. exocytotic frequency, evoked by acute hypoxia (PO2 ~20 mm Hg) or 50 mM K+, was determined from the number of cells indicated. The incomplete inhibition of secretion caused by Ca2+ in AP-treated cells was statistically significant (see Footnotes a and b).

Fig. 6 illustrates the time course of emergence of Cd²⁺-resistant, K⁺-evoked secretion in cells either kept chronically hypoxic (open circles) or exposed to 100 nM AP-(1-40) (closed circles). It is clear from this figure that exposure to hypoxia for up to 6 h was without effect on the secretory responses to 50 mM K⁺ (closed circles). Thereafter, the Cd²⁺-resistant component of secretion increased steeply for cells exposed to chronic hypoxia for between 12 and 24 h. This component of the secretory response then declined gradually following 30-48 h of exposure to hypoxia. These findings indicate that enhancement of secretion caused by the ability of hypoxia to induce Cd²⁺-resistant, Ca²⁺-dependent secretion was transient in nature and was maximal after 24 h of chronic hypoxia. By contrast, in cells treated with AP-(1-40), Cd²⁺-resistant secretion was apparent even after 1 h of treatment, was maximal at 6 h, and remained approximately constant for up to 24 h, after which time point there was a gradual decline in this component of secretion. Thus, there was a clear lag in the emergence of Cd²⁺-resistant, K⁺-evoked secretion induced by hypoxia as compared with AP-(1-40)-treated cells.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Time course of development of Cd2+-resistant exocytosis induced by hypoxia and AP-(1-40). Shown is a plot of mean exocytotic frequency determined in cells pre-exposed to chronic hypoxia (open circles) or AP-(1-40) (100 nM; closed circles) for periods up to 48 h. Secretion was evoked always in the presence of 200 µM Cd2+ by exposure of cells to 50 mM K+. Each plotted point is the mean ± S.E. determined from 6-10 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Prolonged periods of hypoxia are well known to alter gene expression and hence functional expression of numerous proteins (11). Several groups have documented altered levels of ion channel expression (particularly K⁺ channel expression) following periods of chronic hypoxia in tissues such as pulmonary vascular smooth muscle (32) and the carotid body (33). In this study, we investigated the effects of prolonged hypoxia on the secretory responses of PC12 cells to both acute hypoxia and raised extracellular [K⁺] since these cells have been used extensively as a model system for studying the effects of chronic hypoxia on gene expression (16, 18). As anticipated from these previous studies, we found that PC12 cells released more vesicles of catecholamine when stimulated by either acute hypoxia or exposure to tetraethylammonium (via blockade of O₂-sensitive K⁺ channels) and that each vesicle contained greater amounts of catecholamine (22) due to increased levels of tyrosine hydroxylase (16). What was completely unexpected, however, was the emergence of a Ca²⁺ influx pathway coupled to secretion that was not attributable to any of the known voltage-gated Ca²⁺ channels (L-, N-, or P/Q-type (34)) present in PC12 cells (28). This study provides compelling evidence that this pathway is due at least in part to Ca²⁺-permeable channels formed by APs. Thus, in the presence of a supramaximal concentration of Cd²⁺ to block voltage-gated Ca²⁺ channels, residual secretion (constituting ~37% of total secretion in response to 50 mM K⁺), arising from Ca²⁺ influx as indicated by microfluorometric recordings (Fig. 2), was further reduced by known blockers of APs, including Zn²⁺, Congo red, and the 3D6 monoclonal antibody (Fig. 3).

APs are neurotoxic products associated with Alzheimer's disease (4). The mechanisms underlying neuronal toxicity caused by APs are complex and remain to be fully resolved, although most evidence indicates that toxicity involves AP disruption of intracellular Ca²⁺ homeostasis (4, 35). There is also strong evidence that toxicity is oxidative and involves free radical damage (36-38). An alternative (or perhaps additional) hypothesis indicates that APs disrupt intracellular Ca²⁺ homeostasis by forming Zn²⁺-sensitive, Ca²⁺-permeable channels (39, 40). Such an effect may account for increased central synaptic activity. Indeed, enhancement of long-term potentiation and elevated glutamate release have been demonstrated in hippocampal neurons exposed to APs in vitro (41, 42).

It is possible that hypoxic induction of APs enhanced secretion by altering the activity of native voltage-gated Ca²⁺ channels, acting in a manner comparable to an auxiliary subunit (43). However, we consider this unlikely; Cd²⁺ is a Ca²⁺ channel pore blocker (i.e. it binds to similar regions within the pore-forming  subunit of Ca²⁺ channels), and it is highly unlikely that interaction of APs with native Ca²⁺ channels could cause conformational changes that would render the pore Cd²⁺-insensitive, yet more permeable to Ca²⁺ (to allow greater influx to account for greater secretory responses). We have not addressed the possibility that APs could also act intracellularly to modulate secretory responses, and such studies are worthy of future investigation. The simplest interpretation of our present findings, particularly in light of the knowledge that APs have been demonstrated to form Ca²⁺ permeable channels in membranes (see above), is that chronic hypoxia causes formation, in the plasma membrane, of Ca²⁺-permeable AP channels, which permit Ca²⁺ entry sufficient to trigger exocytosis. This interpretation also raises the further question of gating: following chronic hypoxia or direct application of APs to PC12 cells, there was no detectable basal secretion, and cells required stimulation (by either acute hypoxia or 50 mM K⁺) before enhanced secretion was observed, indicating that this Ca²⁺ influx pathway was voltage-dependent. This finding contrasts with studies in which APs were inserted into planar lipid bilayers: the channels formed did not display marked voltage dependence (39). We cannot account for this discrepancy at present, but it should be noted that L-type Ca²⁺ channels (which always display strong voltage dependence in cells), when purified and inserted into lipid bilayers, also display very little voltage dependence over a wide range of potentials (44). Thus, marked functional differences in channel activity when observed in lipid bilayers compared with cell membranes are to be expected.

It is noteworthy that the known blockers of APs failed to inhibit completely the Cd²⁺-resistant secretion in cells previously exposed to chronic hypoxia (Fig. 3). This suggests that formation of Ca²⁺-permeable AP channels might not account fully for the Cd²⁺-resistant Ca²⁺ influx pathway. Interestingly, these blockers also failed to inhibit fully the Cd²⁺-resistant secretion observed in cells pretreated with AP-(1-40) (Fig. 5). We cannot at present account for this incomplete blockade. Nevertheless, our present findings indicate that APs provide a Ca²⁺ influx pathway that can specifically trigger exocytosis. It should be noted that only Ca²⁺ influx specifically through N-type voltage-gated Ca²⁺ channels, but not L- or P/Q-type channels, influences depolarization-evoked exocytosis in control PC12 cells (20, 22). This suggests the possibility of an intimate association of APs with vesicle docking/fusion sites in a manner that may compare with the interaction of known voltage-gated Ca²⁺ channels and synaptic proteins (43). Most important, we have demonstrated that reduced O₂ levels alone can induce formation of AP-mediated Ca²⁺ influx. As described in the Introduction, APs are the cleavage products derived from amyloid precursor protein (5, 6), and levels of this precursor have been shown to increase following ischemia (9, 10). The enhanced Ca²⁺ influx reported here may therefore be an important contributory factor to the increased incidence of dementias and amyloid deposition following cerebral ischemia, leading to neurotoxicity through excessive transmitter release.

	ACKNOWLEDGEMENTS

We thank P. J. Kemp, P. F. T. Vaughan, and H. A. Pearson (University of Leeds) for advice and discussion during these studies. We are also grateful to R. Rydel and S. L. Walker (Elan Pharmaceuticals, San Francisco, CA) for the kind gift of the 3D6 antibody.

	FOOTNOTES

^* The work was supported by the British Heart Foundation and the Leeds University School of Medicine.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.

To whom correspondence should be addressed. Tel.: 113-233-4173; Fax: 113-233-4803; E-mail: c.s.peers@leeds.ac.uk.

	ABBREVIATIONS

The abbreviations used are: APs, amyloid -peptides; APP, amyloid precursor protein; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES