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¹ Department of Pharmacology and Physiology
² and the Center for Oral Biology, University of Rochester Medical Center, Rochester, NY, USA

	Abstract

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ARC channels (arachidonate-regulated Ca²⁺-selective channels) are a novel type of highly Ca²⁺-selective channel that are specifically activated by low concentrations of agonist-induced arachidonic acid. This activation occurs in the absence of any depletion of internal Ca²⁺ stores (i.e. they are ‘non-capacitative’). Previous studies in HEK293 cells have shown that these channels provide the predominant pathway for the entry of Ca²⁺ seen at low agonist concentrations where oscillatory [Ca²⁺]_i signals are typically produced. In contrast, activation of the more widely studied store-operated Ca²⁺ channels (e.g. CRAC channels) is only seen at higher agonist concentrations where sustained ‘plateau-type’[Ca²⁺]_i responses are observed. We have now demonstrated the presence of ARC channels in both parotid and pancreatic acinar cells and shown that, again, they are specifically activated by the low concentrations of appropriate agonists (carbachol in the parotid, and both carbachol and cholecystokinin in the pancreas) that are associated with oscillatory [Ca²⁺]_i signals in these cells. Uncoupling the receptor-mediated activation of cytosolic phospholipase A₂ (cPLA₂) with isotetrandrine reduces the activation of the ARC channels by carbachol and, correspondingly, markedly inhibits the [Ca²⁺]_i signals induced by low carbachol concentrations, whilst those signals seen at high agonist concentrations are essentially unaffected. Interestingly, in the pancreatic acinar cells, activation by cholecystokinin induces a current through the ARC channels that is only approximately 60% of that seen with carbachol. This is consistent with previous reports indicating that carbachol-induced [Ca²⁺]_i signals in these cells are much more dependent on Ca²⁺ entry than are the cholecystokinin-induced responses.

(Received 23 February 2005; accepted after revision 7 March 2005; first published online 10 March 2005)
Corresponding author T. J. Shuttleworth: Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA. Email: trevor_shuttleworth{at}urmc.rochester.edu

	Introduction

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Agonist-activated Ca²⁺ signals in non-excitable cells can take many forms, ranging from the oscillatory signals typically seen at low agonist concentrations, to the sustained ‘plateau’ type of response at high agonist concentrations. In each case, the overall signal is made up of two processes – the InsP₃-induced release of Ca²⁺ from intracellular stores, and an enhanced entry of Ca²⁺ from the extracellular medium. Classically, the receptor-activated entry of Ca²⁺ was thought to occur exclusively via so-called capacitative or store-operated Ca²⁺ channels (SOC channels), whose activation is entirely dependent on the depletion of the intracellular agonist-sensitive Ca²⁺ stores (Putney, 1986, 1990). The prototypical version of these channels are the Ca²⁺-release activated Ca²⁺ channels (CRAC channels) first described in mast cells and Jurkat lymphocytes (Hoth & Penner, 1992; Hoth & Penner, 1993; Zweifach & Lewis, 1993). Several studies have demonstrated that Ca²⁺ entry through these channels and their relatives, determines the amplitude of the sustained Ca²⁺ signals induced by high agonist concentrations, and is responsible for refilling of agonist-sensitive stores following termination of the signal. However, it has been shown that the activation of CRAC channels, even following maximal depletion of the stores, is generally a slow process taking several tens of seconds or even minutes to complete (Hoth & Penner, 1993; Parekh et al. 1997; Huang & Putney, 1998). This, together with other evidence (Shuttleworth, 1999), raised the question as to whether such store-operated channels could effectively operate during the brief periods of repetitive Ca²⁺ release that are associated with the oscillatory Ca²⁺ signals typically generated by low agonist concentrations. Consistent with this, direct activation of the store-operated CRAC channels at such low agonist concentrations has proven to be very difficult to demonstrate. As a consequence, possible alternative, non-capacitative pathways for agonist-activated Ca²⁺ entry were sought with the result that several recent studies have revealed the presence of such pathways in a variety of different cells. In particular, several different cell types have been shown to possess a receptor-activated non-capacitative pathway whose activation is specifically dependent on the generation of arachidonic acid (Shuttleworth, 1996; Shuttleworth & Thompson, 1998; Munaron et al. 1997; Broad et al. 1999). Significantly, this pathway appears to play a particularly important role in the oscillatory [Ca²⁺]_i signals generated at low agonist concentrations (Shuttleworth, 1996). Given that it is widely accepted that these types of signal generally represent the physiologically relevant response for most cells, characterization of this pathway is clearly of some importance. In a search for the specific channel that may be responsible for this Ca²⁺ entry, a highly Ca²⁺-selective conductance whose activation is independent of store depletion, and is specifically activated by arachidonic acid, was described originally in HEK293 cells stably transfected with the m3 muscarinic receptor (m3-HEK cells) (Mignen & Shuttleworth, 2000, 2001). These channels are identified as ARC channels (arachidonate-regulated Ca²⁺-selective channels) and, although superficially similar to the CRAC-like SOC channels that co-exist in the same cells, they are entirely distinct entities with unique biophysical characteristics (Mignen & Shuttleworth, 2000). These ARC channels were subsequently shown to be present in several different cell types (Mignen et al. 2003), and the suggestion was that it is these channels that are primarily responsible for the agonist-induced entry of Ca²⁺ at low agonist concentrations. Consistent with this, the ARC channels in m3-HEK cells were shown to be uniquely activated by the same low concentrations of agonists that generate oscillatory Ca²⁺ signals (Mignen et al. 2001). It was concluded that it is the ARC channels, and not the SOC channels, that provide the predominant route of receptor-activated Ca²⁺ entry at low agonist concentrations in these cells.

However, this unique role of ARC channels has, to date, only been demonstrated in the m3-HEK cells – a cell line that stably overexpresses a muscarinic receptor. If this finding is to have any real physiological relevance, it is necessary to determine whether a similar specific activation of ARC channels by low agonist concentrations occurs following stimulation of endogenous receptors in primary cells, and what contribution the resulting Ca²⁺ entry plays in the [Ca²⁺]_i responses observed. The exocrine acinar cells of the pancreas and the parotid salivary gland are classic examples of non-excitable cells whose key physiological activities are known to be dependent on [Ca²⁺]_i signals that involve a component of Ca²⁺ entry (Tsunoda et al. 1990; Mertz et al. 1990). In this report, we show that ARC channels are present in both these cell types, and are specifically activated by low concentrations of agonists acting at the relevant endogenous receptors. Moreover, we demonstrate that the entry of Ca²⁺ via these channels plays a unique and critical role in modulating the specific [Ca²⁺]_i signals in these cells that are key to their physiological activities.

	Methods

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Materials

Basal Medium Eagle (BME), bovine serum albumen and penicillin/streptomycin were purchased from Gibco (Rockville, MD, USA). Liberase RI enzyme was from Roche Applied Science (Indianapolis, IN, USA). Fura-2 acetoxymethylester, potassium salt, was purchased from Molecular Probes (Eugene, OR, USA). Arachidonic acid and isotetrandrine were purchased from BioMol (Plymouth Meeting, PA, USA). All other materials were obtained from Sigma Chemical Co. (St Louis, MO, USA).

Preparation of mouse parotid and pancreatic acinar cells

All animals were fed ad libitum and handled in accordance with NIH policy and established protocols with the Division of Laboratory Medicine, University of Rochester Medical Center, USA. Mice (approximately 25 g) were killed by cardiac puncture immediately following CO₂ gas asphyxiation. Single acinar cells and small acinar cell clusters were isolated from the parotid glands by sequential digestion with trypsin and collagenase, essentially as previously described (Begenisich et al. 2004). Briefly, the parotid glands were dispersed in a digestion solution composed of BME containing 0.1% trypsin and 5 mM EDTA, and minced for 5 min with fine scissors. This was followed by resuspension in a solution composed of BME containing 0.2% trypsin inhibitor, 0.17 mg ml^–1 Liberase RI enzyme, 1% bovine serum albumin and 2 mML-glutamine at 35–37°C. Tissue was incubated in this solution for 20 min, with the cells dispersed by pipetting every 10 min. Cells were finally rinsed in BME containing 2 mML-glutamine and 200 U ml^–1 penicillin/streptomycin, and incubated at 37°C until ready for use. The pancreatic acinar cells were prepared using a technique modified from that previously described (Williams et al. 1978). Briefly, following removal of the pancreas, the tissue was enzymatically digested with collagenase type II in BME with 1% bovine serum albumin and 1 mg ml^–1 soybean trypsin inhibitor for 30 min. The cells were then dispersed by trituration before being filtered through a 100 µm nylon mesh. Following resuspension in 2% bovine serum albumin in BME equilibrated with 95% O₂ and 5% CO₂ and centrifugation at 100 g for 2 min, the cells were suspended in Hepes-buffered saline until use.

Electrophysiology

Patch-clamp recordings of macroscopic whole-cell currents were performed using an Axopatch-200B patch clamp amplifier (Axon Instruments, Foster City, CA, USA) essentially as previously described (Mignen & Shuttleworth, 2000). Acinar cells from either the mouse parotid or pancreas were plated onto 5 mm glass coverslips and placed in a perfusion chamber (Warner) immediately before experimentation. Only single individual cells were selected for these measurements. The standard pipette (internal) solution contained (mM): caesium acetate 140, NaCl 10, MgCl₂ 1.22, CaCl₂, 1.89, EGTA 5, Hepes 10 (pH 7.2). The free Ca²⁺ concentration of this solution was calculated to be 100 nM as computed with Maxchelator (Bers et al. 1994). In certain experiments (see Results), the Ca²⁺ buffer capacity of the internal solution was increased by using 10 mM BAPTA whilst maintaining the free Ca²⁺ concentration at 100 nM. The standard extracellular solution contained (mM): NaCl 140, MgCl₂ 1.2, CaCl₂ 10, CsCl 5, D-glucose 10, Hepes 10 (pH 7.4). Occasionally, NMDG chloride was substituted for the NaCl (see Results). Whole-cell currents were recorded using 250 ms voltage steps to –80 mV from a holding potential of 0 mV delivered every 2 s. Current–voltage relationships were recorded either by using 150 ms voltage ramps from –100 to +30 mV, or by pulsing to a series of potentials between –100 and +30 mV at 10 or 20 mV intervals. No significant difference was seen in the data obtained between these two methods. Currents were sampled at 20 kHz during the voltage steps and at 5.5 kHz during the voltage ramps, and digitally filtered off-line at 1 kHz. Initial current–voltage relationships obtained before activation of ARC currents, were averaged and used for leak subtraction of subsequent current recordings. All experiments were carried out at room temperature (20–22°C). Averaged data are expressed as means ±S.E.M. Fast inactivation was examined by recording the currents seen during 250 ms hyperpolarizing pulses to –80 mV from a holding potential of 0 mV. In order to eliminate contributions from the initial capacity transients on stepping to –80 mV, the transient recorded on returning to 0 mV at the end of the pulse was inverted and subtracted from the currents recorded on stepping to –80 mV.

Intracellular calcium measurements

Isolated parotid and pancreatic acinar cells, suspended in a Hepes-buffered physiological saline solution (Hepes-PSS) containing (mM) 5.5 glucose, 137 NaCl, 0.56 MgCl₂, 4.7 KCl, 1 Na₂HPO₄, 10 Hepes (pH 7.4), 1.2 CaCl₂, were incubated in the presence of 2 µM fura-2 AM for 30 min at room temperature. The loaded cells were then washed and resuspended in the above Hepes-PSS and kept on ice. Cells were allowed to adhere to a glass coverslip that formed the base of a microscope perfusion chamber and were continually perfused with Hepes-PSS. [Ca²⁺] imaging was performed using an inverted epifluorescence Nikon microscope with a 40 x oil immersion objective lens (numerical aperture, 1.3). Cells were alternately excited with light at 340 and 380 nm using a high-speed monochromator (TILL Polychrome IV). Fluorescence images were captured at an emitted wavelength of 510 ± 45 nm and digitized at 12-bit resolution using an interline progressive scan CCD camera (Sensicam QE). The monochromator and image acquisition by the camera were driven by Imaging Workbench software version 5 (Indec). Images were typically acquired every second with an exposure of 10 ms and stored immediately to hard disk. Background subtraction and calculation of the resulting 340/380 ratio images were performed off-line. All experiments were performed at room temperature.

	Results

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Identification of ARC channels in parotid and pancreatic acinar cells

To determine if parotid and pancreatic acinar cells possess ARC channels, we examined the effect of exogenous arachidonic acid on macroscopic currents in isolated single cells held in the whole-cell patch clamp configuration. To eliminate any contribution from store-operated channels the internal Ca²⁺ concentration was maintained at 100 nM (Mignen & Shuttleworth, 2000). Under these conditions, addition of a low concentration of exogenous arachidonic acid (8 µM) resulted in the activation of an inward current measured at –80 mV in both cell types (Fig. 1A and B). The magnitude of this current averaged 0.55 ± 0.03 pA pF^–1 in the parotid cells, and 0.42 ± 0.03 pA pF^–1 in the pancreatic cells (mean ±S.E.M., n= 3–5). The arachidonic acid-activated current in parotid acinar cells was essentially completely inhibited by La³⁺ (50 µM) (Fig. 1A). Examination of the current–voltage relationship of these currents revealed marked inward rectification and a positive reversal potential of > +30 mV (Fig. 1C and D). To test for any possible contamination from Ca²⁺-activated conductances in the observed currents, the Ca²⁺ buffering capacity of the intracellular solution in certain experiments was increased by inclusion of 10 mM BAPTA. This failed to significantly affect the overall magnitude of the arachidonic acid-activated currents (mean value in parotid acinar cells was 0.50 ± 0.03 pA pF^–1, n= 6). Moreover, in pancreatic acinar cells, complete substitution of external Na⁺ with impermeant NMDG⁺ also failed to significantly affect either the magnitude of the currents observed or the current–voltage relationship obtained (Fig. 1E), suggesting that the current did not reflect the activity of a non-selective cation channel, either Ca²⁺-activated or otherwise. These data, together with the features of the currents observed, suggest that the activated conductance is highly Ca²⁺ selective.

View larger version (27K): [in this window] [in a new window]   Figure 1.  Arachidonic acid-activated currents in parotid and pancreatic acinar cells A and B, representative traces showing the effect of exogenous arachidonic acid (8 µM, added at black arrow) on currents measured at –80 mV in single isolated parotid acinar cells (A), and isolated pancreatic acinar cells (B). In A, La3+ (50 µM) was added at the red arrow. C and D, representative current–voltage relationships of the currents activated by addition of arachidonic acid (8 µM) in parotid (C) and pancreatic acinar cells (D). E, comparison of the arachidonic acid-activated currents in pancreatic acinar cells measured in normal external solution (black symbols), and that measured in a solution in which external Na+ was replaced with the impermeant cation NMDG+ (red symbols). F, representative currents recorded during 250 ms pulses to –80 mV from a holding potential of 0 mV in isolated parotid acinar cells (top trace) and pancreatic acinar cells (lower trace) following activation with arachidonic acid (8 µM). Capacity transients were corrected as described in Methods.

To examine the function of these channels in intact cells we measured the effects of exogenous arachidonic acid on changes in [Ca²⁺]_i, as measured by fluorescence ratio determinations of intracellularly loaded fura-2. Addition of 4–8 µM arachidonic acid to intact isolated parotid and pancreatic acinar cells resulted in a slow progressive increase in [Ca²⁺]_i as measured by fluorescence ratio imaging of intracellularly loaded fura-2 (Fig. 2A and B). In parotid acinar cells, this increase in [Ca²⁺]_i was completely blocked by 50 µM La³⁺ (Fig. 2C) showing that it specifically reflects an increase in Ca²⁺ entry. Moreover, the absence of any detectible increase in [Ca²⁺]_i on addition of arachidonic acid in the presence of La³⁺ confirms that the arachidonic acid was not inducing any detectible release of internal Ca²⁺. These data indicate that low concentrations of exogenous arachidonic acid induce an activation of Ca²⁺ entry in these cells that is independent of store depletion, consistent with the observed activation of the currents described above.

View larger version (16K): [in this window] [in a new window]   Figure 2.  Effect of arachidonic acid on [Ca2+]i in parotid and pancreatic acinar cells A and B, representative traces showing the effect of adding arachidonic acid at the indicated concentrations (black arrow) on [Ca2+]i measured as the fluorescence ratio (F340/380) of intracellularly loaded fura-2 in isolated parotid acinar cells (A), and isolated pancreatic acinar cells (B). C, inhibition of the arachidonic acid-induced increase in [Ca2+]i in parotid cells by La3+. Arachidonic acid (8 µM) was added (black arrow) either in the absence (black trace) or presence (red trace) of 50 µM La3+. The La3+ was subsequently removed (red arrow).

Previous studies on the isolated mouse parotid acinar cells have shown that stimulation with low concentrations of the muscarinic agonist carbachol (100–300 nM) routinely induces [Ca²⁺]_i signals that consist of a transient initial spike, followed by a series of oscillations generally superimposed on an elevated baseline (Bruce et al. 2002). These oscillations are typically sinusoidal in shape, but their amplitude and frequency varies markedly in different individual cells. To examine whether the same low concentrations of carbachol are capable of activating the ARC channels in parotid cells, we exposed cells maintained under whole-cell patch clamp to 250 nM carbachol. This resulted in the activation of a clear inward current at –80 mV (Fig. 3A). The magnitude of this carbachol-activated inward current averaged 0.72 ± 0.18 pA pF^–1 (n= 5). Examination of the current–voltage relationship revealed marked inward rectification and a reversal potential > +30 mV (Fig. 3B). To determine whether this current reflected the activation of the ARC channels we examined the effect of the drug isotetrandrine. In the m3-HEK cells, the carbachol-activated generation of arachidonic acid is entirely dependent on a type IV cytosolic phospholipase A₂ (cPLA₂) (Osterhout & Shuttleworth, 2000). Isotetrandrine reversibly inhibits the receptor-mediated activation of this enzyme (Hashizume et al. 1991; Akiba et al. 1992) without affecting the simultaneous activation of phospholipase C (Shuttleworth & Thompson, 1998). Consequently, pre-incubation with isotetrandrine blocks the activation of ARC channels by low concentrations of carbachol in the m3-HEK cells, whilst currents through the co-existing SOC channels are unaffected (Mignen et al. 2001). To investigate whether the same might apply to the carbachol-dependent activation of ARC channels in the parotid cells, we examined the effect of isotetrandrine on macroscopic ARC channel currents activated by 250 nM carbachol. In the presence of isotetrandrine (10 µM), the carbachol-activated ARC channel currents were inhibited by approximately 40% at –80 mV (mean current density equals 0.44 ± 0.09 pA pF^–1, n= 4) (Fig. 3C). To confirm that this effect of isotetrandrine was specific to the agonist activation of ARC channels, we examined its effect on currents through store-operated channels activated in the same parotid acinar cells by addition of the SERCA pump inhibitor cyclopiazonic acid (30 µM) (Bruce et al. 2002). Currents through these channels measured in the presence of 10 µM isotetrandrine were 108.9 ± 6.2% (n= 4) of the corresponding currents in the absence of isotetrandrine, indicating that isotetrandrine is without effect on the parotid store-operated channels, and that the observed response is specific to the ARC channels. These data suggest that the receptor activation of a cPLA₂ is involved in the generation of arachidonic acid and subsequent activation of the ARC channels. However, it is clear that isotetrandrine does not completely inhibit the currents activated by 250 nM carbachol. This could reflect a contribution from store-operated Ca²⁺ channels, as these are insensitive to isotetrandrine (see above). A characteristic feature of such store-operated currents in this, and other, cell types is the presence of a marked fast inactivation during pulses to negative voltages. Examination of the kinetics of the residual, isotetrandrine-insensitive, current during 250 ms pulses to –80 mV shows no significant fast inactivation (Fig. 3D), indicating that contamination with store-operated currents is minimal. We therefore consider that the failure of isotetrandrine to completely inhibit the activation of the ARC channels most likely reflects a contribution of other, cPLA₂-independent, pathways of arachidonic acid generation in these cells.

View larger version (19K): [in this window] [in a new window]   Figure 3.  Activation of ARC channels by low concentrations of carbachol in parotid acinar cells A, representative trace showing the effect of carbachol (250 nM, added at arrow) on currents measured at –80 mV in single isolated parotid acinar cells. B, representative current–voltage relationship of the current activated by 250 nM carbachol in parotid acinar cells. C, representative current–voltage relationships of the currents activated by 250 nM carbachol in parotid acinar cells in the presence (red trace) and absence (black trace) of isotetrandrine (10 µM). D, representative current recorded during a 250 ms pulse to –80 mV from a holding potential of 0 mV in an isolated parotid acinar cell following activation with 250 nM carbachol measured in the presence of 10 µM isotetrandrine. Capacity transients were corrected as described in Methods.

View larger version (12K): [in this window] [in a new window]   Figure 4.  Effect of isotetrandrine on the [Ca2+]i signals activated by low and high carbachol concentrations in isolated parotid acinar cells [Ca2+]i was measured as the F340/380 ratio of intracellularly loaded fura-2 as described. At the first arrow, carbachol (300 nM) was added; at the second arrow the carbachol concentration was increased to 10 µM. The black bars indicate when isotetrandrine (10 µM) was present.

Pancreatic acinar cells possess a variety of different receptors, the most physiologically relevant of which are the muscarinic receptors and receptors for the peptide hormone cholecystokinin (CCK). Activation of either of these two distinct receptor types by appropriate concentrations of relevant agonists (e.g. carbachol and CCK-8, respectively) results in the generation of characteristic oscillatory [Ca²⁺]_i signals. However, several previous studies have shown that the nature of the [Ca²⁺]_i oscillations induced differs with the two agonists (Yule & Gallacher, 1988; Osipchuk et al. 1990; Yule et al. 1991; Petersen et al. 1991a; Lawrie et al. 1993). Activation of muscarinic receptors typically produces rapid transient ‘spikes’ on an elevated base line. Activation of the CCK receptors on the other hand produces rather broad, low frequency oscillations, with [Ca²⁺]_i returning to values close to resting levels between each transient. We therefore sought to examine the potential role of the ARC channels in either or both of these responses. Whole-cell patch clamp studies showed that exposing isolated pancreatic acinar cells to either carbachol or CCK-8 at the same low concentrations that produce oscillatory Ca²⁺ signals (100–200 nM and 10–20 pM, respectively), resulted in the activation of a current that was indistinguishable from that activated by exogenous arachidonic acid in the same cells (Fig. 5A and B). The magnitude of these agonist-induced currents were 0.52 ± 0.05 pA pF^–1 and 0.31 ± 0.01 pA pF^–1 (n= 3) for carbachol and CCK, respectively. The current–voltage relationship of these conductances displayed marked inward rectification and a very positive reversal potential (Fig. 5C). Importantly, examination of both the carbachol-activated and CCK-activated currents during pulses of 250 ms to –80 mV revealed a complete absence of fast inactivation (Fig. 5D), demonstrating that both of these agonists were capable of activating the ARC channels.

View larger version (25K): [in this window] [in a new window]   Figure 5.  Activation of ARC channels by carbachol and cholecystokinin in pancreatic acinar cells A and B, representative traces showing the effect of carbachol (200 nM) and CCK-8 (20 pM), respectively, on currents measured at –80 mV in single isolated pancreatic acinar cells. The agonists were added at the arrow in each case. C, mean (±S.E.M.) current–voltage relationships of the currents activated by carbachol (200 nM, black circles), and CCK-8 (20 pM, red circles) in isolated pancreatic acinar cells. D, representative currents recorded during 250 ms pulses to –80 mV from a holding potential of 0 mV in isolated pancreatic acinar cells in the presence of carbachol (200 nM, top trace) and CCK-8 (20 pM, lower trace). Capacity transients were corrected as described in Methods.

View larger version (20K): [in this window] [in a new window]   Figure 6.  Effect of isotetrandrine on the [Ca2+]i signals activated by carbachol and cholecystokinin in pancreatic acinar cells Changes in [Ca2+]i in isolated pancreatic acinar cells, measured as F340/380 of intracellularly loaded fura-2, following addition of 200 nM carbachol (A), or 20 pM CCK-8 (B). In each case, the agonists were added at the arrow. Subsequently, isotetrandrine (10 µM) was added as indicated by the black bar. Notice the different time scales of the responses.

	Discussion

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In addition, experiments in the parotid cells using the drug isotetrandrine to inhibit receptor activation of cPLA₂, have indicated that the predominant route for the entry of Ca²⁺ at low agonist concentrations is via the ARC channels, consistent with our previous findings on the m3-HEK cells (Mignen et al. 2001). In contrast, at high agonist concentrations when the internal Ca²⁺ stores become depleted and SOC channels are activated, Ca²⁺ entry via the ARC channels is markedly reduced. This observation is consistent with the phenomenon of reciprocal regulation of Ca²⁺ entry previously detailed in m3-HEK cells (Mignen et al. 2001). It should be noted that Watson et al. (2004) have described a different type of Ca²⁺ entry in parotid acinar cells that is activated by higher concentrations of arachidonic acid. This entry pathway involves the generation of nitric oxide and is apparently dependent on the discharge of ryanodine-sensitive intracellular Ca²⁺ stores. However, we could see no evidence for the activation of this pathway at either the low arachidonic acid concentrations, or agonist concentrations, used in our study.

With respect to the pancreatic acinar cells, we have demonstrated the specific activation of the ARC channels by low concentrations of appropriate agonists acting at both the endogenous muscarinic receptors and CCK receptors. As discussed, previous studies have demonstrated that, although both responses are absolutely dependent on activation of phospholipase C and the generation of InsP₃ (Yule & Williams, 1992), the entry of Ca²⁺ plays a much greater role in the signals generated by activation of the muscarinic receptors than those resulting from the activation of CCK receptors (Yule & Gallacher, 1988; Yule et al. 1991; Petersen et al. 1991b).

For example, the [Ca²⁺]_i oscillations induced by acetylcholine (90–100 nM) are rapidly abolished on removal of external Ca²⁺ whilst those induced by CCK-8 (60–70 pM) persist for many minutes in the absence of external Ca²⁺, although their amplitude and frequency generally decline progressively. Similarly, inhibition of Ca²⁺ entry by external La³⁺ (250 µM) induced only a slow progressive decline in the amplitude of the [Ca²⁺]_i oscillations induced by 15 pM CCK over a period of several minutes with little obvious effect on frequency (Yule & Williams, 1992). In a modelling analysis of the underlying causes of these distinct behaviours (LeBeau et al. 1999), it was argued that a key contributing factor was that stimulation via the muscarinic receptors increases Ca²⁺ entry to a significantly greater extent than stimulation via CCK receptors. Incorporation of this into the models of agonist-activated [Ca²⁺]_i signals faithfully predicted the observed differential dependence on Ca²⁺ entry. At the time, the mode of Ca²⁺ entry was considered to be via a capacitative pathway. However, as we have argued, activation of such a pathway is, at best, likely to be severely limited under conditions of oscillatory [Ca²⁺]_i signals. Thus, direct measurements of the [Ca²⁺] in the stores of pancreatic acinar cells have indicated only minimal and transient depletion during oscillatory [Ca²⁺]_i signals following stimulation with muscarinic agonists (Park et al. 2000). Based on the data presented here, we suggest that the predominant pathway of Ca²⁺ entry activated at the agonist concentrations that give rise to such oscillatory [Ca²⁺]_i signals occurs not via any store-operated conductance, but rather via the ARC channels. Importantly, in the studies presented here, we observed that muscarinic receptor activation induced a markedly greater current through these channels than that seen following activation of the CCK receptors. As such, this could explain the much more pronounced dependence of Ca²⁺ entry in the overall [Ca²⁺]_i signals observed following activation of muscarinic receptors, relative to those seen following activation by CCK, as discussed above.

In conclusion, our data demonstrate that the activity of the ARC channels in these exocrine acinar cells specifically provides the predominant pathway for receptor-mediated Ca²⁺ entry at low levels of stimulation where oscillatory [Ca²⁺]_i signals are typically observed. The physiological relevance of this is particularly clear in the pancreatic acinar cells, where activation of store-operated pathways for Ca²⁺ entry has been shown to lead to the inappropriate intracellular activation of trypsin, resulting in the pathological development of acute pancreatitis (Raraty et al. 2000). In addition, evidence is presented indicating that the reported differences in the relative importance of extracellular Ca²⁺ to the agonist-specific [Ca²⁺]_i signals generated in pancreatic acinar cells by muscarinic receptors versus CCK receptors can be explained by the respective abilities of agonists acting at these receptors to activate the ARC channels.

Together, these data demonstrate the physiological relevance of these novel ARC channels to overall agonist-activated [Ca²⁺]_i signals in native cells, and their specific contribution to the oscillatory signals generated at low agonist concentrations. It should be noted that the specificity of this role of the ARC channels in HEK293 cells has recently been disputed (Bird & Putney, 2005). These authors argue that it is capacitative Ca²⁺ entry, and not entry via ARC channels, that is responsible for the support of [Ca²⁺]_i oscillations in these cells. However, their study involved no direct measurements of the relevant conductances and, instead, relied on the use of fluorescence measurements and a pharmacological approach to selectively block store-operated channels. Of the pharmacological means used, we have already shown 1 µM Gd³⁺ inhibits ARC channels by at least 50% (Mignen et al. 2003), and 2-APB is known to inhibit a variety of processes involved in agonist-activated [Ca²⁺]_i signals in addition to store-operated channels, including phospholipase C activity, InsP₃-induced Ca²⁺ release, mitochondrial Ca²⁺ uptake, and plasma membrane Ca²⁺ pumps (Missiaen et al. 2001; Peppiatt et al. 2003; Wu et al. 2004). Clearly, if any particular conductance is claimed to be responsible for the entry of Ca²⁺ that underlies the [Ca²⁺]_i signals generated by specific concentrations of agonists then, we would argue, it is critical that the activation of this conductance be demonstrated at those same agonist concentrations. To our knowledge, the ARC channels remain the only Ca²⁺-selective conductance to date that has been shown to be specifically activated by agonists at the low concentrations at which oscillatory [Ca²⁺]_i signals are induced in these cells (Mignen et al. 2001). Importantly, the data presented here confirm that this is not a finding that is unique to the m3-HEK cell line or to activation via expressed receptors. As we have now shown, this specific activation of ARC channels is also observed with low concentrations of agonists acting at different endogenous receptor types in primary cells from both the exocrine pancreas and the parotid gland. As such, this conductance plays a critical role in modulating the physiological responses of these cells.

	References

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	Acknowledgements

 
We thank Pauline Leakey for excellent technical help, and Pamela McPherson and Mark Wagner of the Center for Oral Biology, University of Rochester Medical Center, for the isolated parotid cell preparations. This work was supported by National Institutes of Health Grants GM40457 (T.J.S.), DK56468 (D.I.Y.), and DE13539 (T.J.S. and D.I.Y.), and the Alfred and Eleanor Wedd Endowment.

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