Prion protein fragment 106-126 potentiates catecholamine secretion from PC-12 cells

S. C. Taylor, K. N. Green, I. F. Smith, and C. Peers

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -peptides (Abeta Ps) (1, 14). We have previously shown that Abeta 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 Abeta 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 omega -conotoxin GVIA (omega -CgTx) and omega -agatoxin GIVA (omega -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 MOmega 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 0.37 ± 0.10 pC<SUP><FR><NU>1</NU><DE>3</DE></FR></SUP> and for PrP106-126-treated cells were 0.38 ± 0.14 pC<SUP><FR><NU>1</NU><DE>3</DE></FR></SUP>.


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Fig. 1.   Evoked, quantal catecholamine release is potentiated in prion protein fragment 106-126 (PrP106-126)-treated PC-12 cells. A-C: representative amperometric recordings obtained in response to elevated extracellular [K+] from a control PC-12 cell (A), a cell treated for 24 h with 0.3 µM PrP106-126 (B), and one treated with 3 µM PrP106-126 for 24 h (C). Solution exchange to one containing 50 mM K+ occurred at the point indicated by the arrow in each case. Scale bars apply to all 3 traces. D: concentration-response relationship for the effects of 24-h pretreatment of PC-12 cells with PrP106-126 on exocytotic frequency, determined over a 60-s period beginning 10 s after onset of 50 mM K+ application. Each point represents the mean (with vertical SE bars) frequency determined from between 8 and 16 cells.



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Fig. 2.   PrP106-126 treatment does not alter quantal size. Plots of the distribution of Q<FR><NU>1</NU><DE>3</DE></FR> (determined by integration of exocytotic events evoked by 50 mM K+) in 9 control cells (A; total number of events 485) and 9 PrP106-126-treated cells (B; total number of events 952).

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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Fig. 3.   Ca2+ dependence of K+-evoked catecholamine release in PrP106-126-treated PC-12 cells. A: amperometric recording of ongoing secretion of catecholamines from a representative PC-12 cell in response to 50 mM K+-containing solution. For the period indicated by the bar, the perfusate was exchanged for one containing no added Ca2+ and 1 mM EGTA in the continued presence of 50 mM K+. B: as in A, except for the period indicated by the bar, when 200 µM Cd2+ was added to the perfusate in the continued presence of Ca2+ and 50 mM K+. Note that Cd2+ reduced, but did not abolish, secretion. Scale bars apply to both traces.



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Fig. 4.   Time course of enhancement of K+-evoked catecholamine secretion in PC-12 cells. Shown is a plot of mean exocytotic frequency determined in cells exposed to PrP106-126 for the periods indicated (up to 48 h). Exocytosis was evoked by exposure of cells to 50 mM K+-containing solution either in the absence () or presence (open circle ) of 200 µM Cd2+. Each point represents the mean (with vertical SE bars) frequency determined from between 7 and 16 cells.

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 Abeta Ps (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 Abeta P-treated cells (25).


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Fig. 5.   Effects of inorganic cations on Cd2+-resistant secretion from PrP106-126-treated cells. A-D: ongoing secretion evoked from cells by exposure to 50 mM K+. For the periods indicated by the bars in each trace, cells were exposed to a perfusate containing 200 µM Cd2+ plus 1 mM of Ni2+ (A), Zn2+ (B), Gd3+ (C), or La3+ (D). Scale bars apply to all 4 traces. E: mean (± SE) exocytotic frequency evoked by 50 mM K+ in PrP106-126-treated cells during exposure to 200 µM Cd2+ plus 1 of the 4 cations illustrated in A-D.

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 omega -CgTx (an N-type blocker) each reduced the secretory response by ~50% (P < 0.05 and P < 0.01, respectively), whereas omega -AgaTx (a P/Q-type blocker) was without significant effect. Furthermore, exposure of cells to both nifedipine and omega -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 omega -CgTx was without significant effect [as was omega -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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Fig. 6.   Pharmacological profile of K+-evoked exocytosis and its modulation by PrP106-126 treatment. Bar graph illustrating mean (±SE) exocytotic frequency evoked in control cells (open bars) and cells exposed to 1 µM PrP106-126 for 24 h (solid bars). Secretion was monitored in the absence or presence of the Ca2+ channel blockers indicated (200 µM Cd2+; 2 µM nifedipine; pretreatment with 1 µM omega -CgTx; or 200 nM omega -AgaTx). Numbers in parentheses above each bar indicate number of cells studied. nif, Nifedipine.

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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Fig. 7.   Whole cell Ca2+ channel currents in control and PrP106-126-treated cells. Currents were evoked by ramp depolarizations (see MATERIALS AND METHODS) in control cells (A; n = 8) and cells pretreated with 1 µM PrP106-126 for 24 h (B; n = 15) in the absence (solid symbols) or presence (open symbols) of 200 µM Cd2+. C: higher resolution plot of residual currents recorded in the presence of 200 µM Cd2+ in control (open symbols) and PrP106-126-treated cells (solid symbols). Data from open symbols of A and B, respectively. D: Cd2+-resistant currents recorded in PrP106-126-treated cells in the absence of other blockers (, taken from C) or in the additional presence of 1 mM Zn2+ (, n = 7) or 1 mM La3+ (triangle , n = 8). Each point plotted in A-D represents the mean ± SE.

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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Fig. 8.   Effects of nifedipine on Ca2+ channel currents in control and PrP106-126-treated cells. A: mean (±SE) current-voltage relationships from 8 control cells in the absence () and presence (open circle ) of 2 µM nifedipine. B: mean (±SE) current-voltage relationships from 8 cells pretreated with 1 µM PrP106-126 for 24 h in the absence () and presence (open circle ) of 2 µM nifedipine.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Abeta Ps that we have previously reported (25). Such similarities have been documented by others also. For example, in GT1-7 cells derived from hypothalamic tissue, Abeta 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 Abeta 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 Abeta Ps 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 Abeta 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 Abeta 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, omega -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.


    ACKNOWLEDGEMENTS

We are grateful to Drs. J. P. Boyle and D. A. S. G. Mary for advice with statistical analysis.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(6):C1850-C1857
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society




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