Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, United Kingdom
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ABSTRACT |
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The toxic actions of scrapie prion protein (PrPsc) are poorly understood. We investigated the ability of the toxic PrPsc fragment 106-126 to interfere with evoked catecholamine secretion from PC-12 cells. Prion protein fragment 106-126 (PrP106-126) caused a time- and concentration-dependent augmentation of exocytosis due to the emergence of a Ca2+ influx pathway resistant to Cd2+ but sensitive to other inorganic cations. In control cells, secretion was dependent on Ca2+ influx through L- and N-type Ca2+ channels, but after exposure to PrP106-126, secretion was unaffected by N-type channel blockade. Instead, selective L-type channel blockade was as effective as Cd2+ in suppressing secretion. Patch-clamp recordings revealed no change in total Ca2+ current density in PrP106-126-treated cells or in the contribution to total current of L-type channels, but a small Cd2+-resistant current was found only in PrP106-126-treated cells. Thus PrP106-126 augments secretion by inducing a Cd2+-resistant Ca2+ influx pathway and alters coupling of native Ca2+ channels to exocytosis. These effects are likely contributory factors in the toxic cellular actions of PrPsc.
regulated exocytosis; pheochromocytoma; amperometry; ion channels; toxic peptides
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INTRODUCTION |
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CELLULAR PRION PROTEINS (PrPc) are naturally occurring, copper-binding glycoproteins found primarily on neuronal and glial plasma membranes as well as those of many other cell types (2, 9, 11). Their physiological function(s) is not well established, although available evidence [based largely on studies employing PrPc-deficient mice (reviewed in Ref. 11)] suggests that they can modulate neuronal excitability, synaptic transmission, and intracellular Ca2+ homeostasis (6, 7, 12) as well as alter sleep patterns (33), learning, and memory processes (22).
Prion diseases are characterized by neurodegeneration, gliosis, and accumulation of plaquelike, extracellular deposits in the brain. These diseases include scrapie (in sheep), bovine spongiform encephalopathy (in cattle), and Creutzfeldt-Jakob disease (in humans). Collectively, these disorders are referred to as transmissible spongiform encephalopathies and arise from infection with the protease-resistant scrapie form of prion (PrPsc). After infection, the presence of PrPsc permits conversion of PrPc to PrPsc, leading to a progressive accumulation of PrPsc that is believed to underlie neurodegenerative processes manifested in the above-mentioned diseases (11).
The cellular mechanisms by which PrPsc causes neuronal
dysfunction are currently unclear but are the subject of much interest. These studies, commonly employing the PrPsc fragment
106-126 [which is functionally extremely similar to the
full-length peptide (9)], suggest that PrPsc
has marked effects on Ca2+ homeostasis, altering
transmembrane Ca2+ flux possibly by interacting with
voltage-gated Ca2+ channels (23, 30, 31). In
addition, prion protein fragment 106-126 (PrP106-126) has
been demonstrated to form nonselective ionic channels in planar lipid
bilayers (17, 18), which could permit Ca2+
entry into cells, although others have attempted and failed to reproduce such an effect (20). This notwithstanding, the
possible channel-forming property of PrP106-126 is remarkably
similar to one action of Alzheimer's amyloid -peptides (A
Ps)
(1, 14). We have previously shown that A
Ps can form
Ca2+-permeable channels in pheochromocytoma (PC-12) cells
(a widely used neurosecretory model cell system; reviewed in Ref.
13) that contribute to the excessive catecholamine
secretory response of these cells when they are depolarized with
high-extracellular [K+]-containing solutions
(25). Such similarities in the functional effects of
A
Ps and PrPsc have prompted us to examine the effects of
PrP106-126 on the secretory responses of PC-12 cells. Our results
indicate that this peptide enhances evoked catecholamine secretion by
inducing a novel Ca2+ influx pathway that is distinct from
known voltage-gated Ca2+ channels. We suggest that
this influx may contribute to the neurodegenerative effects of
PrPsc.
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MATERIALS AND METHODS |
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The PC-12 cells used in this study were obtained within the last year from American Type Culture Collection (Rockville, MD) and cultured as previously described (25, 26) in RPMI 1640 culture medium that contained L-glutamine and was supplemented with 20% fetal calf serum and 1% penicillin/streptomycin (GIBCO, Paisley, Strathclyde, United Kingdom). Cells were kept at 37°C in a humidified atmosphere of 5% CO2-95% air, passaged every 7 days, and used for up to 20 passages. The prolonged period without medium change was believed to enhance evoked dopamine release (24). Cells used for experiments were transferred to smaller flasks in 10 ml of medium, and 1 µM dexamethasone (Sigma, Poole, UK; from a stock solution of 1 mM in ultrapure water) was applied for 72-96 h to further enhance dopamine secretion (32).
On each experimental day, aliquots of PC-12 cells were plated onto poly-L-lysine-coated coverslips and allowed to adhere for ~1 h. Fragments of coverslips with attached cells were then transferred to a recording chamber (vol ~80 µl) that was continually perfused under gravity (flow rate 1-2 ml/min) with a solution of the following composition (in mM): 135 NaCl, 5 KCl, 1.2 MgSO4, 2.5 CaCl2, 5 HEPES, and 10 glucose, pH 7.4 (osmolality adjusted to ~300 mosmol/kgH2O with sucrose, 21-24°C). For experiments using solutions of raised K+ concentration, the Na+ concentration was reduced accordingly to maintain isoosmolarity. Ca2+-free solutions contained 1 mM EGTA and no added Ca2+.
Drugs were applied in the perfusate except in the cases of
-conotoxin GVIA (
-CgTx) and
-agatoxin GIVA
(
-Aga-IVA). The effects of these agents were investigated by
preincubation of cells in extracellular solutions containing these
agents for at least 10 min as previously described (25,
27). Experiments were conducted within 3 min of transfer of
these cells to the perfused recording chamber. Experiments
investigating the effects of nifedipine were conducted at low-light
intensity, and nifedipine was added to the perfusate from a stock
solution of 20 mM in ethanol, made fresh each day. PrP106-126 and
its scrambled version (used for initial control experiments, see
RESULTS) were purchased from BaChem, dissolved in ultrapure
water at a concentration of 1 mM, and stored at
20°C in aliquots.
They were added directly to cells in culture at the concentrations
indicated in RESULTS for varying periods (1-48 h)
immediately before experiments.
Carbon fiber microelectrodes (Dagan Instruments) with a diameter of 5 µm were positioned adjacent to individual cells and polarized to +800 mV to allow oxidation of released catecholamine. Resulting currents were recorded using an Axopatch 200A amplifier (with extended voltage range), filtered at 1 kHz, and digitized at 2 kHz before storage on computer. All acquisition was performed using a Digidata 1200 interface and Fetchex software from the pCLAMP 6.0.3 suite (Axon Instruments). Unless otherwise stated, each experiment consisted of current recordings of a brief control period during which cells were perfused with standard external medium (5 mM K+ concentration). 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 spikelike events, each corresponding to the released contents of a single vesicle of catecholamine (5, 35). Quantification of release was achieved by counting spikes using Minian 16 software (Jaejin Software, Columbia, NY). This allowed visual inspection of each event so that artifacts (due, for example, to solution switches) could be rejected from analysis. Integration of individual amperometric events allowed measurement of quantal size (see Fig. 2), as previously described (25, 28).
To record Ca2+ channel currents, cells were perfused with a
solution of the following composition (in mM): 110 NaCl, 5 CsCl, 0.6 MgCl2, 20 BaCl2, 5 HEPES, 10 glucose, and 20 tetraethylammonium-Cl, pH 7.4. Osmolality of the perfusate was
adjusted to 300 mosmol/kgH2O by addition of sucrose. Patch
pipettes (5-7 M resistance) were filled with a solution of (in
mM) 130 CsCl, 1.1 EGTA, 2 MgCl2, 0.1 CaCl2, 10 NaCl, 10 HEPES, and 2 Na2ATP, pH 7.2. After the whole cell
configuration was established, cells were voltage clamped at
80 mV.
To evoke whole cell Ca2+ channel currents, 200-ms voltage
ramps were applied from
100 mV to +100 mV at a frequency of 0.2 Hz
(10). As for amperometric recordings, evoked currents were
filtered at 1 kHz, digitized at 2 kHz, and stored on computer for
offline analysis.
All results are presented as individual examples or means ± SE, and statistical comparisons were made using an unpaired Student's t-test unless stated otherwise.
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RESULTS |
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Secretory responses to high (50 mM) K+-containing
solutions of PC-12 cells are shown in Fig.
1, A-C. It is
apparent from these examples and from the mean exocytotic
frequency plot of Fig. 1D that 24-h exposure of cells to
PrP106-126 caused a concentration-dependent increase in the
secretory response of cells, reaching a maximal increase at a
PrP106-126 concentration of 1-3 µM, where the response to
50 mM K+ was increased more than twofold. That this effect
was specific to PrP106-126 was evidenced by the fact that a
scrambled version of the peptide was without significant effect on
K+-evoked secretion (mean exocytotic frequency 0.76 ± 0.15 Hz, n = 8; not significantly different from
control cells). In control solutions (containing 5 mM K+),
no secretory events were detected in control or PrP106-126-treated cells. Measurement of quantal size, obtained by integrating individual exocytotic events, revealed no statistical difference
(P > 0.2) between control and PrP106-126-treated
cells in terms of mean quantal size (Fig.
2), although quantal sizes had a somewhat
broader distribution in PrP106-126-treated cells than was seen in
controls. Mean ± SD values for controls were
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We have previously demonstrated that K+-evoked secretion of
catecholamines from PC-12 cells is entirely dependent on
Ca2+ influx through voltage-gated Ca2+
channels, since it can be abolished in Ca2+-free solutions
or by bath application of the nonselective Ca2+ channel
inhibitor Cd2+ (26, 27). In
PrP106-126-treated cells, K+-evoked secretion was also
fully and reversibly abolished by replacement of extracellular
Ca2+ with 1 mM EGTA (Fig.
3A; representative of 13 cells
examined). However, secretion was not abolished in the presence of 200 µM Cd2+ (Fig. 3B, representative of 13 cells
examined). Instead, ~40% of the secretory response remained (see
also Fig. 6). Release evoked in scrambled
PrP106-126-treated cells was completely abolished in the
presence of Cd2+ (data not shown). Thus complete blockade
of voltage-gated Ca2+ channels failed to prevent fully the
secretory response, strongly suggesting that exposure to
PrP106-126 induced a distinct Ca2+ influx pathway that
contributed to the secretory response. When PrP106-126 was applied
for 24 h in the continual presence of Congo Red (500 µM),
K+-evoked secretion was not significantly altered
(0.83 ± 0.19 Hz, n = 10), and
Cd2+-resistant secretion was reduced by ~80% (see Fig.
4) to 0.13 ± 0.05 Hz
(n = 10).
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The time course of emergence of the Cd2+-resistant secretory response, and indeed the enhancement of the total secretory response, was studied by exposing cells to 1 µM PrP106-126 for between 1 and 48 h. Results are plotted in Fig. 4, which shows that the time course of enhancement of total secretory response (closed circles) was followed closely by the time course of emergence of the Cd2+-resistant secretory pathway: in both cases, maximal enhancement was observed after a 6-h exposure period. This plot also reveals that the magnitude and time course of enhancement of total secretion can be accounted for by the emergence of the Cd2+-resistant component of secretion.
Our findings thus far suggested that PrP106-126-induced
potentiation of the secretory response of PC-12 cells was due to the formation of a Cd2+-resistant Ca2+ influx
pathway. To probe the properties of this pathway further, we
investigated the ability of other cations to block
Cd2+-resistant secretion. We tested two divalent cations
(Ni2+ and Zn2+) and two trivalent cations
(Gd3+ and La3+), all at a concentration of 1 mM
and in the additional presence of 200 µM Cd2+, to compare
results with those obtained when cells are treated with APs
(25). These agents are known to block various
Ca2+ influx pathways in other cell types (28).
Results are exemplified and summarized in Fig.
5. All four cations suppressed secretion more than Cd2+ did alone, although for Ni2+,
this effect was found with the other cations. The potency
order was La3+ > Gd3+ > Zn2+ > Ni2+ (Fig. 5E), the
same as that seen for A
P-treated cells (25).
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Control PC-12 cells possess L-, N-, and P/Q-type voltage-gated
Ca2+ channels (19, 27). Ca2+
influx through each of these channels can contribute to catecholamine release in a stimulus-specific manner
(25-29). We compared the ability of
selective inhibitors of these channels to modulate K+-evoked secretion from control and
PrP106-126-treated cells. Statistical analysis was performed using
ANOVA with both Dunnett's and Bonferroni's multiple comparison post
hoc tests (both of which produced the same significance values). As
shown in Fig. 6, release evoked from
control cells was mediated by Ca2+ influx through L- and
N-type channels, since nifedipine (an L-type channel blocker) and
-CgTx (an N-type blocker) each reduced the secretory response by
~50% (P < 0.05 and P < 0.01, respectively), whereas
-AgaTx (a P/Q-type blocker) was without
significant effect. Furthermore, exposure of cells to both nifedipine
and
-CgTx was almost as effective as Cd2+ in suppressing
the secretory response. Responses in PrP106-126-treated cells were
markedly different. In these cells, nifedipine was almost as effective
as Cd2+ in suppressing the secretory response
(P < 0.01), whereas
-CgTx was without significant
effect [as was
-AgaTx; Fig. 6, although significant reduction
(P < 0.05) was seen when both toxins were applied
together]. Thus, in addition to inducing a Cd2+-resistant
Ca2+ influx pathway coupled to secretion, PrP106-126
appeared to prevent coupling of N-type channels to secretion, whereas
coupling of L-type was promoted.
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The altered pharmacology of the secretory response caused by
PrP106-126 led us to investigate Ca2+ influx pathways
more directly, using whole cell patch-clamp recordings. Figure
7, A and B,
compares mean current densities in control (Fig. 7A) and
PrP106-126-treated (Fig. 7B) cells. In both cases, current density vs. voltage relationships were bell-shaped, typical of
Ca2+ channel currents, and were maximal in amplitude at +20
mV. Importantly, and perhaps unexpectedly, mean current density was not
significantly different at any activating test potential in the two
groups of cells. Furthermore, Cd2+ (200 µM) caused near
complete inhibition of these currents (open symbols, Fig. 7,
A and B). However, as shown in Fig.
7C, closer comparison of the Cd2+-resistant
Ca2+ channel currents revealed that these residual inward
currents were much greater in amplitude in PrP106-126-treated
cells than in controls, indicating the presence of a small but
significant Cd2+-resistant Ca2+ channel in this
group of cells. Indeed, in control cells at higher test potentials,
currents were outward, due to either incomplete block of outward
K+ currents or outward Cs+ flow through
Ca2+ channels. The inward, Cd2+-resistant
currents seen in PrP106-126-treated cells could be blocked by both
Zn2+ and La3+ (both applied at a concentration
of 1 mM in the presence of 200 µM Cd2+), further
suggesting that this current contributed to Cd2+-resistant
secretion following exposure to PrP106-126.
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Given the different pharmacological profile of secretion following
PrP106-126 treatment (Fig. 6), we next investigated the contribution of L-type channels to the total whole cell
Ca2+ channel current in control and PrP106-126-treated
cells by examining their responses to nifedipine. Results are plotted
in Fig. 8, which reveals that in control
cells, nifedipine (2 µM) reduced current amplitudes by 58.9 ± 7.3% (as determined at +20-mV test potential, P < 0.002, n = 9 cells). In PrP106-126-treated cells, the same concentration of nifedipine reduced currents measured at
+20-mV test potential by 38.6 ± 5.4% (P < 0.02, n = 9). This value was not significantly different from
the degree of blockade observed in control cells, indicating that the
greater influence on secretion of L-type channels in
PrP106-126-treated cells (Fig. 6) was not due to a selective
enhancement of their expression or activity as determined at the level
of whole cell patch-clamp recordings.
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DISCUSSION |
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In this study, we examined the cellular effects of PrP106-126, the toxic fragment of PrPsc, in the well-defined neuroendocrine cell line PC-12. Such effects of this peptide have not been extensively studied to date, despite the attention that transmissible spongiform encephalopathies have received in recent years due to contamination of the food chain and resultant implications for public health. The major observation of the present study is that PrP106-126 markedly augments evoked catecholamine release from PC-12 cells, as determined in real time using amperometry (Fig. 1). Amperometry limits our study to specific release of catecholamine from these cells (5), but the similar distribution of vesicular sizes in control and PrP106-126-treated cells (Fig. 2) strongly suggests that the same vesicular pool is mobilized during K+ depolarization in both groups. Importantly, the enhancement of exocytosis can be accounted for by the emergence of a Cd2+-resistant Ca2+ influx pathway coupled to secretion that is not present in control cells (Figs. 3 and 4) or in cells treated with a scrambled version of the peptide.
Induction of a Cd2+-resistant Ca2+ influx
pathway by PrP106-126 is remarkably similar to the effects of
treating PC-12 cells with APs that we have previously reported
(25). Such similarities have been documented by others
also. For example, in GT1-7 cells derived from hypothalamic
tissue, A
Ps and PrP106-126 raise intracellular Ca2+
concentration ([Ca2+]i) by
stimulating Ca2+ influx (15). However, such
influx was observed in the absence of depolarizing stimuli. Our results
suggest that if PrP106-126 does raise basal
[Ca2+]i, this is not sufficient to evoke
catecholamine secretion per se. A depolarizing stimulus is still also
required. The reason for this discrepancy is unclear at present, but it
is noteworthy that Kawahara et al. (15) exposed cells to
high peptide concentrations for brief periods of time, whereas in the
present study, and in our previous investigation of the effects of
A
Ps (25), we employed much lower peptide
concentrations, applied for much longer periods of time. This
difference may lead to different patterns of peptide aggregation and
insertion into the plasma membrane. Interestingly, these effects of
PrP106-126 were almost completely inhibited by coincubation with
Congo Red, which prevents aggregation of PrP and related peptides,
indicating that aggregation is a requisite step in channel formation
(see Ref. 17 and references therein).
Numerous similarities in the properties of ion channels formed from
cytotoxic peptides have been highlighted (17). In
accordance with this, our observation that PrP106-126 potentiates
evoked secretion in a manner comparable with that of APs indicates
further pathophysiological similarities between these peptides. We also report similar pharmacological properties. Figure 5 indicates that
Cd2+-resistant secretion is blocked by cations with a
potency order La3+ > Gd3+ > Zn2+ > Ni2+, identical to that for
A
P-mediated, Cd2+-resistant secretion induced by chronic
hypoxia (25, 28). Perhaps surprisingly, given the marked
influence on secretion of PrP106-126, when we investigated this
Ca2+ influx pathway more directly with patch-clamp
recordings, current amplitude was extremely small (<5% of total
Ca2+ channel current amplitude, Fig. 7). Once again,
however, this is comparable with the effects of A
Ps
(10). For such a small current to exert such a marked
influence on secretion suggests that there may be specific localization
of these channels to sites of exocytosis. For native Ca2+
channels, direct binding of channel proteins to proteins associated with the exocytotic apparatus has been demonstrated (3).
It is conceivable, therefore, that PrP106-126 might interact in a comparable way such that the small amount of Ca2+ that
enters the cell through these channels is targeted at release sites to
optimize exocytotic efficiency. In this regard, it is of interest to
note that PrPsc has been localized in
detergent-insoluble lipid "raft" domains within cells
(16, 21). Such rafts in PC-12 cells are also enriched with
soluble N-ethylmaleimide-sensitive factor attachment protein
target receptor proteins essential for regulated exocytosis (4). Thus there is likely a direct association of prion
proteins in the process of Ca2+-dependent exocytosis.
Modulation of existing, native voltage-gated Ca2+ channels
(particularly L-type channels) is a well-documented cellular effect of
PrP106-126. However, the specific modulation of such channels appears variable. Thus, in astrocytes and microglia, PrP106-126 potentiates L-type channel activity (23, 30) and appears
to enhance Ca2+ influx through voltage-gated
Ca2+ channels in synaptosomes (34). In
contrast, L-type channel activity appears to be inhibited by
PrP106-126 in neuroectodermal GH3 cells and in
isolated cerebellar granule neurons (8, 30, 31). The
present study indicates that in PC-12 cells, PrP106-126 does not
alter the activity of L-type Ca2+ channels, since their
contribution to total whole cell Ca2+ current density was
unaffected (Fig. 8). However, their influence on a specific cellular
function was dramatically altered. Thus, after treatment with
PrP106-126, nifedipine was virtually as effective as
Cd2+ in suppressing the secretory response to 50 mM
K+, and, furthermore, -CgTx was without significant
effect (Fig. 6). This observation suggests that PrP106-126 may act
intracellularly to promote coupling of exocytosis to Ca2+
influx through L-type channels (without affecting their activity, as
determined by patch-clamp recordings; Fig. 8) while inhibiting the
contribution to secretion of Ca2+ influx through N-type
channels. The mechanism(s) underlying such an effect remains to be
determined but has important potential implications for transmitter
release in other cells types, since Ca2+ channel expression
within a given cell is targeted so that specific channel subtypes are
present at transmitter release sites, such as nerve terminals or
boutons (3).
In summary, the present study indicates that PrP106-126 markedly augments evoked catecholamine secretion from PC-12 cells. This occurs largely by inducing a Cd2+-resistant Ca2+ influx pathway sensitive to blockade by divalent and trivalent cations. In addition, the coupling of native, voltage-gated Ca2+ channels is altered such that the influence of N-type channels on secretion is diminished and the influence of L-type is enhanced, without an increase in the contribution of these channels to total whole cell current. Our results provide novel data to indicate that PrP106-126 can exert multiple effects on secretory cells and have implications for understanding the pathophysiological actions of PrPsc at the cellular level.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. J. P. Boyle and D. A. S. G. Mary for advice with statistical analysis.
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FOOTNOTES |
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This work was supported by the Medical Research Council (through a PhD studentship to K. N. Green) and the Leeds University School of Medicine. I. F. Smith holds a Cooperative Award in Science and Engineering PhD studentship with Pfizer Central Research.
Address for reprint requests and other correspondence: C. Peers, Institute for Cardiovascular Research, Univ. of Leeds, Leeds LS2 9JT, United Kingdom (E-mail: c.s.peers{at}leeds.ac.uk).
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.
Received 29 May 2001; accepted in final form 16 August 2001.
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