Capacitative Ca2+ entry in enteric glia induced by thapsigargin and extracellular ATP

George A. Sarosi, Douglas C. Barnhart, Douglas J. Turner, and Michael W. Mulholland

Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109-0331

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mobilization of intracellular Ca2+ stores is coupled to Ca2+ influx across the plasma membrane, a process termed capacitative Ca2+ entry. Capacitative Ca2+ entry was examined in cultured guinea pig enteric glia exposed to 100 µM ATP, an inositol trisphosphate-mediated Ca2+-mobilizing agonist, and to 1 µM thapsigargin, an inhibitor of microsomal Ca2+ ATPase. Both agents caused mobilization of intracellular Ca2+ stores followed by influx of extracellular Ca2+. This capacitative Ca2+ influx was inhibited by Ni2+ (88 ± 1%) and by La3+ (87 ± 1%) but was not affected by L- or N-type Ca2+ channel blockers. Pretreatment of glia with 100 nM phorbol 12-myristate 13-acetate for 24 h decreased capacitative Ca2+ entry by 48 ± 2%. Chelerythrine (0.1-10 µM), a specific antagonist of protein kinase C (PKC), dose dependently inhibited capacitative Ca2+ entry. The nitric oxide synthase inhibitor NG-nitro-L-arginine (1 mM) decreased Ca2+ influx by 42 ± 1%. Capacitative Ca2+ entry was inhibited to a similar degree by the guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Capacitative Ca2+ entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by PKC and nitric oxide.

glial cell; endothelin; myenteric plexus

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ENTERIC NERVOUS SYSTEM (ENS) is located between the muscular layers of the wall of the digestive tract and extends from the esophagus to the internal anal sphincter. The ENS is the largest division of the peripheral nervous system and modulates a wide variety of gastrointestinal functions, including motility, mucosal secretion, and blood flow (9). Within the ENS, glial cells outnumber neurons two to one. Enteric glia were originally thought to play a structural or supportive role for enteric neurons, but evidence is emerging to suggest that they may have a functional role in signaling by the ENS. Enteric glia respond to a variety of neuroligands with Ca2+ signaling and also demonstrate dye coupling between glia, suggesting they participate in enteric information transfer (7, 10, 26).

Ca2+ is an important second messenger in a variety of cell types, transducing extracellular signals into intracellular events. In nonexcitable cells, most Ca2+ signaling occurs via receptor-mediated activation of the phospholipase C-inositol phosphate system. Typically, Ca2+ signals occur in two phases: 1) an initial release of intracellular Ca2+ stores mediated by the interaction of inositol 1,4,5-trisphosphate (IP3) with the intracellular IP3 receptor, followed by 2) influx of extracellular Ca2+ (19). The mechanisms underlying the initial process of Ca2+ release have been intensively studied. The mechanisms of subsequent Ca2+ entry are less well defined. In several cell types, depletion of internal Ca2+ stores causes the activation of a Ca2+ entry pathway, termed capacitative Ca2+ entry, leading to the influx of Ca2+ from the extracellular space. Ca2+ entry refills intracellular stores, allowing for further Ca2+ signaling. Capacitative Ca2+ entry appears to modulate a variety of biological processes ranging from enzyme activation to cell mitogenesis (1).

We postulated that enteric glia, like other nonexcitable cells that utilize Ca2+ signal transduction mechanisms, display capacitative Ca2+ entry in response to depletion of internal Ca2+ stores, and we sought to examine the mechanisms regulating Ca2+ entry. The current studies demonstrate that cultured enteric glia display capacitative Ca2+ entry in response to both agonist-evoked and pharmacological depletion of internal stores. Ca2+ entry occurs via a lanthanum-inhibitable channel and is regulated by both protein kinase C (PKC) and nitric oxide (NO).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NG-nitro-L-arginine (L-NNA) and sodium nitroprusside (SNP) were obtained from Calbiochem (San Diego, CA). Fura 2-AM and fura 2-free acid were from Molecular Probes (Eugene, OR). Adenosine triphosphate, collagenase type V, thapsigargin, trypsin-EDTA, soybean trypsin inhibitor (type I-S), penicillin-streptomycin solution, and HEPES were from Sigma Chemical (St. Louis, MO). omega -Conotoxin GVIA, phorbol 12-myristate 13-acetate (PMA), 4alpha -phorbol 12-myristate 13-acetate (4alpha -PMA), verapamil, and diltiazem were obtained from Research Biochemicals International (Natick, MA). Hanks' balanced salt solution (HBSS), medium 199, FCS, and L-glutamic acid were from GIBCO BRL (Grand Island, NY). 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) was obtained from Tocris Cookson (St. Louis, MO). One-day-old male Duncan-Hartley guinea pigs were obtained from Simonsen Labs (Gilroy, CA).

Myenteric plexus isolation. Dispersed primary cultures of guinea pig myenteric plexus were prepared on collagen-coated coverslips and used for experiments within 8 days postplating. The taenia coli from 1-day-old male Duncan-Hartley guinea pigs were removed and placed in HBSS solution plus 0.1% collagenase for 16-20 h at 4°C. After a 35-min incubation at 37°C, the muscle layers of the taenia coli were separated from the myenteric plexus with the use of a dissecting microscope. The myenteric plexus was trypsinized for 30 min at 37°C using 0.1% trypsin-EDTA solution, triturated with siliconized flamed pasteur pipettes of decreasing tip diameter, and plated on collagen-coated coverslips. Cultures were exposed to complete medium 199 plus 10% FCS and 0.001% trypsin inhibitor. Penicillin-streptomycin solution was added for the first 48 h at a 2% concentration. Antimitotic agents were not added. Medium was changed every other day. The cultures were incubated at 37°C with 5% CO2.

Solutions. All experiments were performed in standardized solutions except when noted. Standard control buffer was a modified Krebs-Ringer solution at pH 7.40 containing (in mM) 118 NaCl, 4.7 KCl, 1.8 CaCl2, 10 HEPES, 15 NaHCO3, 11 glucose, 0.9 NaH2PO4, and 0.8 MgSO4. In Ca2+-free control buffer, the CaCl2 was omitted and 0.5 mM EGTA was added. In experiments in which Ba2+ was used, HCO-3, S2O2-4, and H2PO-4 were removed from all solutions, because they form insoluble salts with Ba2+.

Loading and cell preparation for imaging. Cultured myenteric plexus was incubated at 37°C in fresh warmed media containing 1-3 µM fura 2-AM for 30 min. Loaded coverslips were washed and then stored in control buffer and placed in a lucite superfusion chamber. The superfusion rate of the control buffer and experimental solutions was 1 ml/min at 37°C. For Ca2+-free conditions, CaCl2 was removed from the buffer and 0.5-1 mM EGTA was added.

Ca2+ measurements. A Zeiss Axiovert inverted microscope and an Attofluor imaging system (Rockville, MD) were used to determine single-cell intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i was calculated from the ratios of the fluorescence intensities of fura 2 at 334- and 380-nm wavelengths, with an emission wavelength of 540 nm, monitored by an intensified charge-couple device camera, and subsequently digitized. Calibration of the system was performed with the two-point standardization equation, using fura 2-free acid
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB>[(R − R<SUB>low</SUB>)/(R<SUB>high</SUB> − R)]<IT>b</IT>
where Kd is the dissociation constant of the Ca2+-fura 2 complex (225 nm), R is F334/F380, i.e., the fluorescence at 334 nm excitation divided by the fluorescence at 380 nm excitation, Rlow is the ratio at zero Ca2+ (1 mM EGTA), Rhigh is the ratio at high Ca2+ (1 mM CaCl2), and b is F380 (zero Ca2+)/F380 (saturating Ca2+). Frames were not averaged to obtain images. A ratio pair was taken every 1.5-3.0 s.

In some experiments, Ba2+ was substituted for Ca2+ to measure rates of ion entry into glial cells. Ba2+ causes fura 2 to produce fluorescence ratio changes in a fashion similar to Ca2+. However, unlike Ca2+, Ba2+ cannot be sequestered in internal stores and therefore provides an isolated measure of divalent cation entry in cells (8). In the Ba2+ experiments, the imaging system was not calibrated, and data are expressed as the ratio of F334 to F380.

Data presentation and calculation. Capacitative Ca2+ entry was experimentally defined as the rise in [Ca2+]i that occurred after return of Ca2+ to the extracellular buffer following depletion of internal stores in Ca2+-free buffer. This was assessed by measuring the change in [Ca2+]i before reintroduction of Ca2+ relative to the peak value observed with the return of Ca2+.

Results are means ± SE. Data were analyzed using ANOVA with Fishers post hoc or Student's t-test. Significance was accepted as P < 0.05.

Dissection techniques, tissue preparation, media, and reagent vendors remained constant throughout the study. In this study, n equals the number of glial cells examined. At least three coverslips were used for each experimental condition. All experimental conditions were examined on glial cells derived from cell preparations performed on at least two different days.

Results have been calculated only from those responding glia having basal [Ca2+]i levels <150 nM, a criteria met by >95% of glial cells. Glia with a high [Ca2+]i before any addition of agonist were considered damaged or leaky and were excluded from the study. Only one microscope field was examined per coverslip. Peak [Ca2+]i was measured as the highest [Ca2+]i achieved during agonist exposure.

At the time of the experiments, two criteria were used to determine whether cells of interest were glial cells, as opposed to neurons. 1) Morphology. Myenteric neurons 2-7 days postplating are compact and phase-bright, with few or no processes. Enteric glia have a larger, dense nucleus with wide surrounding cytoplasm. 2) KCl depolarization. At the end of each experiment, the coverslip was superfused with 55 mM KCl. Enteric glia do not exhibit increments in [Ca2+]i on exposure to 55 mM KCl.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mobilization of internal Ca2+ stores causes capacitative Ca2+ entry. The baseline [Ca2+]i of enteric glia was 55 ± 2 nM. This value agrees with previously published reports (7, 26). To study Ca2+ entry in enteric glia, a standard protocol for mobilization of internal Ca2+ stores followed by reintroduction of extracellular Ca2+ was used. In this protocol, the cells began the experiment in Ca2+-free control buffer. After a period of observation to ensure that baseline [Ca2+]i was stable, the cells were then exposed to either ATP or thapsigargin, resulting in the release of internal Ca2+ stores. [Ca2+]i was allowed to return to baseline, and the cells were then exposed to Ca2+-containing buffer, allowing capacitative Ca2+ entry to occur. Exposure of glial cells to Ca2+-free buffer alone for periods of up to 10 min did not result in the depletion of internal Ca2+ stores.

Mobilization of internal Ca2+ stores by exposure of cells to 100 µM ATP, an IP3-mediated Ca2+-mobilizing agonist, for 150 s reliably produced capacitative Ca2+ entry with a mean [Ca2+]i increment of 177 ± 4 nM (n = 297 cells) (Fig. 1A). The pharmacological agent thapsigargin, an inhibitor of the microsomal Ca2+-ATPase, was also used to mobilize internal Ca2+ stores (6). Use of thapsigargin allowed assessment of whether agonist-independent mobilization of internal stores would produce capacitative Ca2+ entry. Exposure of enteric glia to 1 µM thapsigargin for 250 s produced capacitative Ca2+ entry with a mean [Ca2+]i increment of 152 ± 3 nM (n = 380 cells) (Fig. 1B). Mobilization of internal Ca2+ stores by either mechanism produced Ca2+ increments of similar magnitude and provided a model to study Ca2+ entry in enteric glial cells.


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Fig. 1.   Capacitative Ca2+ entry is independent of the method of intracellular Ca2+ depletion. A: application of ATP (100 µM) in Ca2+-free buffer induced release of Ca2+ from intracellular stores and activation of Ca2+ influx, as indicated by elevation of intracellular Ca2+ concentration ([Ca2+]i) after addition of 1.8 mM Ca2+ to the perfusion buffer. Tracing is representative of 297 cells. B: mobilization of intracellular Ca2+ stores by thapsigargin (1 µM) also induced Ca2+ entry of a magnitude similar to that observed with ATP. Tracing is representative of 380 cells.

Ca2+ entry occurs by a non-L-, non-N-type Ca2+ channel. The channel through which capacitative Ca2+ entry occurs in enteric glia was characterized using a panel of Ca2+ channel inhibitors. The divalent metal ions La3+ and Ni2+ are inorganic, nonspecific Ca2+ channel antagonists that have been shown previously to block capacitative Ca2+ entry in other cellular systems (8). For cells in which intracellular Ca2+ stores had been mobilized by exposure to ATP, both La3+ and Ni2+ inhibited capacitative Ca2+ entry. La3+ produced dose-dependent inhibition when added to the extracellular buffer before reintroduction of extracellular Ca2+ (43% at 0.1 mM, 62% at 0.2 mM, 89% at 1 mM). Similarly, Ni2+ inhibited capacitative Ca2+ entry 63% at 0.2 mM and 88% at 2.5 mM (Fig. 2). Diltiazem and verapamil are specific inhibitors of L-type voltage-gated Ca2+ channels. The agent omega -conotoxin GVIA is a specific inhibitor of N-type voltage-gated Ca2+ channels. Neither diltiazem (50 µM, n = 56 cells), verapamil (50 µM, n = 29 cells), nor omega -conotoxin GVIA (1 µM, n = 49 cells) produced any inhibition of capacitative Ca2+ entry in enteric glia (Fig. 2).


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Fig. 2.   A: inhibition of ATP-stimulated capacitative Ca2+ entry by La3+. Superfusion with 1 mM La3+ inhibited the increase of [Ca2+]i when Ca2+ was added to the buffer after mobilization of intracellular Ca2+ stores by 100 µM ATP. Tracing is representative of 39 cells. B: summary of inhibitory effects of La3+, Ni2+, diltiazem, and omega -conotoxin.

Inhibition of PKC decreases capacitative Ca2+ entry. Earlier studies have suggested a role for PKC in the regulation of capacitative Ca2+ entry (3, 15, 22). Pretreatment of enteric glia with 100 nM PMA for 24 h resulted in a 48 ± 2% (n = 79 cells) decrease in the mean change in [Ca2+]i observed during capacitative Ca2+ entry (Fig. 3). Pretreatment of glia with the inactive isomer 4alpha -PMA for 24 h resulted in a 12 ± 2% (n = 75 cells) decrease in the mean [Ca2+]i increment observed during capacitative Ca2+ entry (Fig. 3).


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Fig. 3.   A: enteric glia were incubated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h before stimulation of capacitative Ca2+ entry. Tracings illustrate capacitative Ca2+ entry in control and treated cells exposed to 1 µM thapsigargin and are representative of 79 cells. B: summary of inhibitory effects of 24-h preincubation with 100 nM PMA or 100 nM 4alpha -phorbol 12-myristate 13-acetate (4alpha -PMA). * P < 0.05 vs. control or 4alpha -PMA.

Because long-term phorbol ester treatment has nonspecific inhibitory effects in addition to inhibition of PKC, we examined the effects of the specific PKC inhibitor chelerythrine on capacitative Ca2+ entry. Exposure of glial cells to chelerythrine (0.1-10 µM) beginning 60 s before intracellular Ca2+ store depletion resulted in dose-dependent inhibition of capacitative Ca2+ entry (Fig. 4). Maximal inhibition of 55% was noted at 10 µM chelerythrine. Because inhibition of PKC could affect the capacitative Ca2+ transient either by inhibiting Ca2+ entry or by accelerating Ca2+ sequestration into internal stores, we examined the effect of chelerythrine treatment on Ba2+ entry in enteric glia (Fig. 4). Ba2+ is not pumped into internal Ca2+ stores, and observed changes in fluorescence ratios in the presence of Ba2+ reflect only cation entry into cells. Treatment of glia with chelerythrine decreased the rate of the fluorescence ratio increase, confirming that PKC inhibition decreases Ca2+ entry. It is also possible that chelerythrine acts to inhibit K+ channels, depolarizing cells and reducing Ca2+ entry. To eliminate this possibility, experiments were performed in a medium containing 55 mM KCl (NaCl reduced to maintain osmolarity). Capacitative Ca2+ transients in the presence of high K+ were not significantly different relative to transients observed in normal K+ medium. Exposure to chelerythrine (1 µM) in the presence of high K+ inhibited capacitative Ca2+ entry by 76%.


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Fig. 4.   A: dose-dependent inhibition of thapsigargin-stimulated capacitative Ca2+ entry. Cells were exposed to a single concentration of chelerythrine (0.1-10 µM) beginning 60 s before thapsigargin (1 µM). Each experimental group contained more than 50 cells. * P < 0.05 vs. control. B: chelerythrine (1 µM), applied 60 s before thapsigargin, inhibited Ba2+ influx. Tracing is representative of 78 cells.

Effects of NO inhibition on capacitative Ca2+ entry. Previous studies have suggested a role for the second messenger NO in the regulation of capacitative Ca2+ entry (2, 5). The NO synthase (NOS) inhibitor L-NNA was used to investigate the effects of NO on capacitative Ca2+ entry. Pretreatment of enteric glia with 1 mM L-NNA for 10 min before depletion of internal Ca2+ stores resulted in a 42 ± 1% (n = 187 cells) inhibition of the mean [Ca2+]i increment observed during capacitative Ca2+ entry (Fig. 5). Because the observed effect on [Ca2+]i could represent either a decrease in Ca2+ entry or an increase in the rate of sequestration of Ca2+ into internal stores, we examined the effects of L-NNA on Ba2+ entry evoked by internal store depletion. Pretreatment of enteric glia for 10 min with 1 mM L-NNA decreased the rate of Ba2+ entry relative to control cells. This observation confirms that the decrease in the [Ca2+]i increment is caused by a decrease in Ca2+ entry (Fig. 5). As seen in Fig. 6, the addition of the exogenous NO donor SNP after internal store depletion reversed the effects of NOS inhibition. Rescue of capacitative Ca2+ entry by SNP was dose dependent. The effects of NOS inhibition were also examined in a high-K+ medium, as outlined above. Pretreatment with 1 mM L-NNA inhibited capacitative Ca2+ entry in high-K+ and normal-K+ media to a similar degree.


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Fig. 5.   A: cultured enteric glial cells were pretreated with 1 mM NG-nitro-L-arginine (L-NNA) for 10 min before exposure to thapsigargin. Tracing is typical of 187 cells. B: inhibition of nitric oxide synthase by L-NNA inhibited Ba2+ entry into cells exposed to 1 µM thapsigargin.


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Fig. 6.   Rescue of L-NNA inhibition of capacitative Ca2+ entry by sodium nitroprusside (SNP). A: Ca2+ mobilization in enteric glia was stimulated by exposure to 1 µM thapsigargin. Cells were pretreated with either 1 mM L-NNA or L-NNA plus 20 µM SNP. B: effects of varying concentrations of SNP on L-NNA-inhibited capacitative Ca2+ entry.

In most systems NO is thought to mediate its effects through activation of soluble guanylate cyclase, resulting in the production of cGMP. cGMP acts as a second messenger, resulting in the activation of a variety of protein kinases and phosphodiesterases (11). Pretreatment of enteric glia with the guanylate cyclase inhibitor ODQ dose dependently inhibited capacitative Ca2+ entry induced by thapsigargin (23% at 10 µM, 41% at 50 µM). The combination of L-NNA (1 mM) and chelerythrine (1 mM) did not produce additive inhibitory effects (L-NNA 48 ± 2%, chelerythrine 58 ± 2%, L-NNA + chelerythrine 63 ± 3%).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study demonstrates that mobilization of cellular Ca2+ stores by both agonist-dependent and agonist-independent mechanisms results in Ca2+ entry in enteric glia. Our data suggest that entry occurs through a Ca2+ channel that is not an L- or N-type voltage-gated channel. Ca2+ entry is diminished by the inhibition of PKC. In addition, inhibition of NOS also negatively modulates Ca2+ entry triggered by store depletion in enteric glia.

When nonexcitable cells are exposed to a variety of hormones, neurotransmitters, or growth factors, a rise in [Ca2+]i occurs that is typically biphasic in nature. An initial rise in [Ca2+]i occurs as a direct effect of IP3 binding to receptors on organelles that store intracellular Ca2+. The release of intracellular Ca2+ is transient but is followed by a sustained elevation in [Ca2+]i due to influx of Ca2+ across the plasma membrane. In a variety of cells, entry of Ca2+ is coupled to processes that cause the depletion of intracellular Ca2+ stores. This process was first postulated by Putney (18) and termed capacitative Ca2+ entry by analogy to a capacitor in an electrical circuit. Capacitative Ca2+ entry appears to be independent of the membrane signaling event that initiates Ca2+ depletion. The observation that thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases, mimics the effects of a surface membrane, IP3-linked agonist such as extracellular ATP, provides compelling support for this concept.

The molecular basis for capacitative Ca2+ entry is complex; the mammalian Ca2+ release-activating channel (CRAC) mediating this process has not been identified with certainty. The CRAC entry pathway is characterized by a very low conductance (28). In agreement with the current study, the CRAC channel has been noted to be blocked by bivalent and trivalent cations such as Ni2+ and La3+ and to be insensitive to L- or N-type Ca2+ channel blockers (25). Studies on visual signal transduction in Drosophila suggest that the transient receptor (trp) gene product may function as a capacitative Ca2+ entry channel (4). Mammalian homologues of trp have been described in humans and mice (12, 23). In COS cells, expression of full-length cDNAs encoding human trp homologues increases capacitative Ca2+ entry (27). Introduction of portions of these genes in an antisense orientation suppresses capacitative entry in L cells.

The mechanisms by which PKC regulates capacitative Ca2+ entry are unsettled. In the current study, inhibition of PKC activity decreased intracellular store depletion-induced Ca2+ entry. PKC activity was inhibited by long-term incubation with 100 nM PMA and by acute exposure to the specific PKC antagonist chelerythrine. Both approaches produced partial (~50%) inhibition of capacitative Ca2+ entry. Stimulatory and inhibitory effects of PKC activation on capacitative Ca2+ entry have been variously reported using human and rabbit neutrophils, human megakaryocytes, and rat thyroid, leukemia, and insulinoma cells (3, 12, 14, 21, 22, 24). Recent observations suggest that the actions of PKC may be dependent on the level of enzyme activation and on the degree of cellular Ca2+ depletion. In Xenopus oocytes, low-level activation of PKC potentiates capacitative Ca2+ entry by blocking inactivation of Ca2+ influx (16). In NIH/3T3 cells, PKC augments Ca2+ entry occurring with submaximal intracellular Ca2+ pool depletion (20). The current study is in agreement with prior reports in that it suggests that, in enteric glia, PKC activation promotes capacitative Ca2+ entry.

In enteric glial cells, Ca2+ entry stimulated by intracellular Ca2+ store depletion was regulated by NO. Four observations support this conclusion: 1) the NOS inhibitor, L-NNA, significantly suppressed [Ca2+]i during store repletion, 2) L-NNA inhibited entry of extracellular Ba2+ into Ca2+-depleted glial cells, 3) the NO donor SNP dose-dependently reversed the effects of L-NNA, and 4) the guanylate cyclase inhibitor ODQ suppressed capacitative Ca2+ entry to a degree nearly identical to L-NNA. In enteric glial cells, inhibition of NOS did not completely suppress capacitative Ca2+ entry, decreasing the [Ca2+]i observed during repletion by 42%. Similar findings with regard to the inhibitory effects of L-NNA on capacitative Ca2+ entry have been reported for pancreatic acinar cells and colonic epithelial cells (2, 13). Our observation that thapsigargin-induced capacitative Ca2+ entry was inhibited by the guanylate cyclase inhibitor ODQ contrasts with reports by Gilon and co-workers (5). These investigators, using rat pancreatic acinar cells, reported that the inhibitory effects of L-NNA on Ca2+ entry could not be reversed by preincubation with 8-bromo-cGMP (5). The mechanism responsible for these observations requires further elucidation.

In conclusion, depletion of internal Ca2+ stores in enteric glia by extracellular ATP or thapsigargin induces capacitative Ca2+ entry. Capacitative Ca2+ entry is mediated by a Ni2+- and La3+-sensitive channel that is not inhibited by L- or N-type channel blockers. The process of capacitative Ca2+ entry in glial cells is subject to regulation by PKC and NO.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41204.

    FOOTNOTES

Address for reprint requests: M. W. Mulholland, 2920 Taubman, 1500 E. Medical Center Dr., Ann Arbor, MI 48109-0331.

Received 3 November 1997; accepted in final form 24 April 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(3):G550-G555
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