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Am J Physiol Cell Physiol 279: C126-C135, 2000;
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Vol. 279, Issue 1, C126-C135, July 2000

Intracellular calcium events activated by ATP in murine colonic myocytes

Orline Bayguinov1, Brian Hagen1, Adrian D. Bonev2, Mark T. Nelson2, and Kenton M. Sanders1

1 Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557; and 2 Department of Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca2+-activated K+ (SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca2+ release. Isolated myocytes of the murine colon produced spontaneous, localized Ca2+ release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca2+ transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca2+ events in colonic myocytes "Ca2+ puffs." The P2Y receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca2+ puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca2+ puffs. Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, a P2 receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca2+ transients and the effects of 2-MeS-ATP on Ca2+ puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP3 receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca2+. These data suggest that localized Ca2+ release modulates Ca2+-dependent ionic conductances in the plasma membrane. Localized Ca2+ release may contribute to the electrical responses resulting from purinergic stimulation.

calcium puffs; local calcium transients; P2Y receptors; enteric neurotransmission


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ENTERIC INHIBITORY NEURONS express and utilize multiple neurotransmitters to regulate relaxation of gastrointestinal muscles. Numerous studies have demonstrated that nitric oxide (NO) is a key enteric inhibitory neurotransmitter (see Ref. 35 for review); however, in many gastrointestinal muscles, there are multiple components to the inhibitory response (see Refs. 15, 23, 36, 39). A number of reports suggest that, in addition to NO, ATP serves as a primary inhibitory neurotransmitter released from enteric motor neurons, and it is thought that ATP works by activation of an apamin-sensitive ionic conductance (13, 20). Recent studies have identified small-conductance, Ca2+-activated K+ (SK) channels in gastrointestinal muscles that are activated by purinergic stimulation (24, 41), but the mechanism of coupling receptor activation to channel opening is not understood.

Colonic muscles express P2Y receptors that are thought to mediate relaxation responses to ATP (12, 42). Several isoforms of P2Y receptors, but not all, have been shown to couple to activation of phospholipase C (PLC) and to stimulate production of inositol 1,4,5-trisphosphate (IP3; see Refs. 3, 11, 31). Ahn and co-workers (1) proposed that some of the effects mediated by activation of P2Y receptors in gastrointestinal smooth muscles may be mediated by release of Ca2+ from intracellular stores. Release of Ca2+ from IP3 receptor-operated stores may be a common mechanism by which ATP initiates intracellular signaling via P2Y receptors. For example, others have demonstrated ATP-dependent Ca2+ release from stores mediated by P2Y receptors in striatal and neurohypophysial astrocytes (14, 40).

Upon first consideration, it is unclear how inhibitory responses in gastrointestinal muscles could be mediated by Ca2+ release, but it is now apparent that localized Ca2+ release can occur in smooth muscles without significant effects on global cytoplasmic Ca2+ concentration. Localized Ca2+ release events (sparks) were first observed in vascular smooth muscle cells (30), and these events were associated with spontaneous transient outward currents (STOCs) that result from activation of large-conductance, Ca2+-activated K+ channels (BK channels; see Refs. 4 and 43). Periodic Ca2+ sparks in multicellular tissues yield a hyperpolarizing influence on vascular muscles (22). Recent studies have shown that a variety of smooth muscles manifest Ca2+ sparks or periodic Ca2+ waves, and these events regulate the open probabilities of Ca2+-dependent conductances in the plasma membrane (17, 33, 37, 43). Ca2+ release mediated by IP3 receptor-operated channels has also been reported in a variety of cell types (5, 8, 25). These events, termed Ca2+ blips (i.e., elementary Ca2+ release events) or Ca2+ puffs (i.e., release from clusters of IP3 receptors), could also be involved in regulating membrane ionic conductances (26), but coupling of IP3-dependent Ca2+ release to regulation of membrane conductances has not been demonstrated in smooth muscles. If localized Ca2+ transients have a net effect of activating K+ conductances, then these events could couple inhibitory responses in gastrointestinal smooth muscles to G protein-coupled receptors.

We investigated the nature of Ca2+ release events in colonic muscles. We also investigated the hypothesis that P2Y receptors are coupled to localized Ca2+ transients via activation of PLC and release of Ca2+ from IP3 receptor-operated stores. Local Ca2+ transients might activate Ca2+-dependent conductances in the plasma membrane without significant changes in global cytoplasmic Ca2+ concentration. Local Ca2+-dependent regulation of ionic conductances might mediate the responses of gastrointestinal muscles to inhibitory purinergic neurotransmission.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. BALB/C mice (15-30 days old) of either sex were anesthetized with chloroform and were killed by decapitation. The large intestine was removed and opened along the mesenteric border, and the luminal contents were washed with Krebs-Ringer bicarbonate buffer (see Solutions and drugs). Tissues were pinned to the base of a Sylgard-coated dish, and the mucosa and submucosa were removed by peeling.

Colonic muscles were equilibrated in Ca2+-free solution for 60 min, and then the buffer was replaced with an enzyme solution containing collagenase F (Sigma, St. Louis, MO) to disperse single smooth muscle cells. The tissues were incubated with the enzyme at 37°C for 16 min without agitation. After three to four washes with Ca2+-free Hanks' solution to remove the enzyme, the tissues were triturated through a series of three blunt pipettes of decreasing tip diameter. Isolated smooth muscle cells were freed from the tissue matrix by trituration.

Confocal microscopy. Cell suspensions were placed in a specially designed 0.5-ml chamber with a glass bottom. The cells were incubated for 35 min at room temperature in Ca2+-free buffer containing fluo 3-AM (10 µg/ml; Molecular Probes, Eugene, OR) and pluronic acid (2.5 µg/ml; Teflabs, Austin, TX). Cell loading was followed by a 25-min incubation in a solution containing 2 mM Ca2+ to restore normal extracellular Ca2+ concentration and to complete the deesterification. All measurements were made within 15-45 min after restoring extracellular Ca2+.

An Odyssey XL confocal laser scanning head (Noran Instruments, Middleton, WI) connected to a Nikon Diaphot 300 microscope with a ×60 water immersion lens (numeric aperture = 1.2) was used to image the cells. The cells were scanned using INTERVISION software (Noran Instruments) running on an Indy workstation (Silicon Graphics, Mountain View, CA). Changes in the fluo 3 fluorescence (indicating fluctuations in cytosolic Ca2+) were recorded for 20-s test periods using T-series acquisition and a laser wavelength of 488 nm (excitation for FITC). Six hundred frames were acquired per test period (one frame every 33 ms), creating 20-s movie files.

Single cell measurements of ionic currents. Ionic currents were measured in isolated muscle cells using the whole cell, perforated-patch (amphotericin B) configuration of the patch-clamp technique. Average cell capacitance was 56.1 ± 4.2 pF. An Axopatch 200B amplifier with a CV 203BU headstage (Axon Instruments, Foster City, CA) was used to measure ionic currents and membrane potential. Membrane currents were recorded while holding cells at -30 or -40 mV using pCLAMP software (version 7.0; Axon Instruments). Currents were filtered at 1 kHz and were digitized at 2 kHz. In some experiments, patch-clamped cells were simultaneously scanned for fluorescence changes in cells preloaded with fluo 3 as described above. All experiments were performed at room temperature (22-25°C).

Solutions and drugs. The standard Krebs solution used in this study contained (in mM) 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 134 Cl-, 15.5 HCO3-, 1.2 H2PO4-, and 11.5 dextrose. This solution had a final pH of 7.3-7.4 after equilibration with 97% O2-3% CO2. The bathing solution used in confocal microscopy studies and patch-clamp studies contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4). The enzyme solution used to disperse cells contained 1.3 mg/ml collagenase F, 2 mg/ml papain, 1 mg/ml BSA, 0.154 mg/ml L-dithiothreitol, 134 mM NaCl, 6 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). The pipette solution used in patch-clamp experiments contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (pH 7.2). The pipette solution also contained 250 µg/ml amphotericin B.

Drugs used. Nicardipine, cyclopiazonic acid (CPA), ryanodine apamin, charybdotoxin (ChTX), and 2-methylthio-ATP (2-MeS-ATP) were obtained from Sigma. Pyridoxal-phospate-6-azophenyl-2',4'-disculphonic acid tetrasodium (PPADS), U-73122, and U-73343 were obtained from RBI (Natick, MA). Xestospongin C (Xe-C) was obtained from Calbiochem. Concentrations of drugs used were determined from the literature or by empirical testing.

Analysis of data. Image analysis was performed using custom-written analysis programs using Interactive Data Language software (Research Systems, Boulder, CO), as previously described (33). Baseline fluorescence (F0) was determined by averaging 10 images (out of 600) with no activity. Ratio images were then constructed and replayed for careful examination to detect active areas where sudden increases in F/F0 occurred (33). F/F0 vs. time traces were further analyzed in Microcal Origin (Microcal Software, Northampton, MA) and AcqKnoledge Software (Biopac Systems, Santa Barbara, CA) and represent the averaged F/F0 from a box region of 2.2 × 2.2 µm centered in the active area of interest to achieve the fastest and sharpest changes. This box size (4.8 µm2) was determined empirically to be the best compromise between temporal and spatial precision of Ca2+ release events and the signal-to-noise ratio (33). Rise time of puffs was calculated as the time required to reach peak fluorescence from the baseline. The rate of spread of Ca2+ waves was calculated as the time required for the peak fluorescence to move 10 µm.

Fluorescence records from single colonic myocytes were composed of Ca2+ transients of multiple characteristics (i.e., single Ca2+ puffs, clusters of puffs, and Ca2+ waves), as described in RESULTS. Some drug treatments changed the characteristics of the Ca2+ transients from brief Ca2+ puffs to events of more extended duration. As a result, it was not accurate to analyze the data as simple changes in frequency. Therefore, we analyzed the data as the area of the Ca2+ transients above baseline during 20-s scans.

To determine the amplitude of the STOCs, analysis was performed off-line, using a Mini Analysis Program (Synaptosoft Software, Leonia, NJ). The threshold of STOCs was set at three times the single Ca2+-activated K+ (KCa) channel amplitude at -40 mV or at 6 pA. The activity of KCa channels in the absence of Ca2+ release events is very low at -40 mV (number of channels × open probability ~ 10-3; see Ref. 9), with the probability of three simultaneous openings being exceedingly low.

Statistical analysis. Results are expressed as means ± SE where applicable. All statistical analysis was made with SigmaStat 2.03 software (Jandel Scientific Software, San Rafael, CA). The Spearman rank order correlation test was used for correlation analysis.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of spontaneous Ca2+ transients and STOCs. Colonic myocytes loaded with fluo 3 produced spontaneous, transient elevations in intracellular calcium concentration ([Ca2+]i). These events occurred as either localized events or more widely dispersed, spreading events (Ca2+ waves; Fig. 1). Localized Ca2+ events were characterized by a rapid focal rise in [Ca2+]i (mean rise time was 160 ± 34 ms) and slower decay (mean time to half amplitude was 742 ± 87 ms; n = 60). Frequently, the Ca2+ transients were clustered into groups consisting of multiple events that did not fully relax to the resting level between events (see Fig. 1B, inset). Clusters of transients were highly variable in duration but often lasted for more than a second.


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  		Fig. 1.   Spontaneous Ca2+ transients in colonic myocytes. A: localized Ca2+ transients that occurred at 2 different sites during a single 20-s scan. Frames shown are representative images taken at the maximum of the Ca2+ transients. B: traces 1 and 2 show the temporal relationship between the 2 sites displaying spontaneous Ca2+ transients shown in A. Inset in B shows an expanded time scale of a Ca2+ transient at site 1. Ca2+ waves could be recorded from the same cells that generated Ca2+ localized transients. C: additional images of the same cell shown in A during the same 20-s scan. A Ca2+ wave was detected in the lower one-half of the cell. Colored circles represent sites at which fluorescence was monitored during the Ca2+ wave. The wave was first detected at the red site and spread through a significant part of the bottom one-half of the cell. D: superimposed traces from the points marked in C. Traces show the progression of the fluorescence maxima, indicating a spreading Ca2+ wave. F/F0, ratio of recorded fluorescene to baseline fluorescence.

Ca2+ transients, beginning at discrete sites within cells, often spread through part or all of the cell. From 40 analyzed records, 60% of the cells displayed spontaneous Ca2+ waves. Ca2+ spread with an average propagation velocity of 32 ± 3 µm/s (n = 9; Fig. 1, C and D). Localized Ca2+ transients and Ca2+ waves could be recorded from the same cell and were observed to originate from the same or from different sites (see Fig. 1, A and C).

In 11 experiments, fluo 3 fluorescence and whole cell membrane currents were recorded simultaneously. Cells were held at -30 mV while membrane currents and spontaneous Ca2+ transients were recorded. The current records showed STOCs in association with the Ca2+ transients (Fig. 2, A-E). A correlation between STOCs and localized Ca2+ transients was demonstrated by plots of STOC amplitude vs. the amplitude of the corresponding Ca2+ transients (Fig. 2F; correlation coefficient was 0.931; n = 56; P < 0.005). STOCs were reduced in amplitude and frequency by ChTX (200 nM; treatment for 15 min), as previously reported (30, 33); however, ChTX did not fully block STOC activity (Fig. 3). STOCs remaining after ChTX were further reduced in amplitude and frequency by apamin (n = 7; Fig. 3), suggesting that SK channels also contribute to STOCs in colonic muscles as previously reported (26).


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  		Fig. 2.   Ca2+ transients in colonic myocytes were associated with spontaneous transient outward currents (STOCs). A: cell with a Ca2+ transient that developed into a local wave. Frames represent images taken during the black bar in B. B-D: Ca2+ transients that were recorded from 3 areas of interest during a 20-s scan of the cell in A. E: STOCs recorded simultaneously from the same cell. There was a high degree of correlation between Ca2+ transients and STOCs. However, a few STOCs (denoted by asterisks) were not apparently associated with resolvable Ca2+ transients in the 3 areas of interest. These STOCs may have resulted from Ca2+ transients at nonimaged sites. F: plot of STOC amplitudes as a function of the amplitude of the Ca2+ transients. Data were fit to a straight line via linear regression analysis (r = 0.854; n = 56; P < 0.001).



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  		Fig. 3.   STOCs persist after charybdotoxin (ChTX). A: STOC activity under control conditions at a holding potential of -30 mV. B: STOCs after application of ChTX (200 nM). C: apamin (1 µM) reduced the amplitude and frequency of ChTX-resistant STOCs. D: histograms showing counts of STOCs under control conditions (filled bars) and after ChTX (open bars) and apamin (shaded bars).

The occurrence of spontaneous Ca2+ transients and the propagation of Ca2+ waves was not significantly affected by nicardipine (1 µM). The average area of Ca2+ transients was 138 ± 31% of control after nicardipine (n = 5; P > 0.5). Ca2+ transients were also not significantly affected by Ni2+ or Cd2+ (200 µM each); average areas of Ca2+ transients were 100 ± 16% for Ni2+ and 96 ±17% of control area for Cd2+, respectively (each n = 10 and P > 0.5). Incubation of cells in nominally Ca2+-free buffer for at least 30 min, however, resulted in complete blockade of spontaneous Ca2+ transients. Spontaneous Ca2+ transients recovered in cells that had been incubated in Ca2+-free buffer by the readdition of 2 mM Ca2+ to the bath solution.

Spontaneous Ca2+ transients were reduced by CPA (10 µM). After CPA, the area of Ca2+ transients was 24 ± 4% of the control area (n = 5; P < 0.01). STOCs were also blocked by CPA (n = 5). Ryanodine (10 µM; 15 min) had little effect on spontaneous Ca2+ transients; the average area of Ca2+ transients was 97 ± 8% of the control area (n = 10; P > 0.5), and ryanodine was shown to have no resolvable effect on STOCs. Figure 4 shows the effects of CPA and ryanodine on spontaneous Ca2+ transients and STOCs.


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  		Fig. 4.   Effects of ryanodine and cyclopiazonic acid (CPA) on Ca2+ puffs. A: spontaneous Ca2+ transients before (top) and after (middle) addition of ryanodine (10 µM; 15 min exposure to ryanodine before scan). There was no significant change in the area of the Ca2+ transients after ryanodine. Trace on bottom shows the same cell after exposure to 2-methylthio-ATP (2-MeS-ATP). Ryanodine inhibited the increase in Ca2+ transients typically observed in response to 2-MeS-ATP. B: spontaneous Ca2+ transients before (top) and after (middle) addition of CPA (10 µM; 5 min exposure to CPA before scan). CPA inhibited spontaneous Ca2+ and blocked the increase in the area of Ca2+ transients observed in response to 2-MeS-ATP (bottom). C: STOCs recorded continuously during exposure to ryanodine (10 µM) and CPA (10 µM). Ryanodine did not affect STOCs, but these events were greatly reduced by CPA.

Cells pretreated with ryanodine (10 µM; 15 min), which had no effect on spontaneous activity (i.e., 97 ± 8% of control Ca2+ transient area), were exposed to Xe-C (5 µM), a membrane-permeable antagonist of IP3 receptors (16). After addition of Xe-C, spontaneous Ca2+ transients were reduced to 33 ± 4% (n = 5; P < 0.01; Fig. 5A) of the control area. In additional experiments, we found that Xe-C (5 µM), added in the absence of ryanodine, also blocked spontaneous Ca2+ transients (n = 6; discussed below). In patch-clamped cells held at -30 mV, ryanodine did not affect STOCs, but Xe-C added after ryanodine reduced STOCs to an unresolvable level (Fig. 5B). Thus the localized Ca2+ transients in colonic muscle cells appeared to be due to release from IP3 receptor-operated stores and are referred to below as "Ca2+ puffs."


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  		Fig. 5.   Block of Ca2+ transients and STOCs by xestospongin C (Xe-C). A: lack of effect of ryanodine on spontaneous Ca2+ transients. After ryanodine, addition of Xe-C (5 µM) completely blocked spontaneous Ca2+ transients. B: another cell studied with the patch-clamp technique. Similar to the findings with fluorescence, ryanodine did not affect STOCs, but these events were greatly reduced in amplitude and frequency by Xe-C. The effects of Xe-C did not depend on ryanodine pretreatment, but this sequence was shown to demonstrate the effects of both drugs on the same cells.

With all of the spontaneous Ca2+ transients measured, we did not observe cell shortening, suggesting that, even in cells in which Ca2+ waves were observed, these events did not raise global Ca2+ to the threshold for contraction. Addition of caffeine (1 mM), however, caused significantly larger Ca2+ transients in cells and produced cell shortening (see Fig. 10).

Effects of 2-MeS-ATP on intracellular Ca2+ transients. In 98% of the cells that produced spontaneous Ca2+ transients, 2-MeS-ATP (200 µM) increased the frequency and amplitude of Ca2+ puffs (average increase to 200 ± 19% of control area; n = 15; P < 0.05; Fig. 6) and STOCs. In eight cells pretreated with ChTX (200 nM), we also found that 2-MeS-ATP enhanced the occurrence of ChTX-insensitive STOCs (Fig. 7, B-D). In 50% of the cells that produced only Ca2+ puffs under control conditions, the addition of 2-MeS-ATP generated propagating Ca2+ waves. In the other one-half of these cells, 2-MeS-ATP either increased the frequency of puffs from the same site or introduced new sites of puffs (Fig. 6, C and D). In cells that displayed propagating waves before 2-MeS-ATP, addition of this drug increased the area of propagation (i.e., spontaneous Ca2+ waves spread over an area of 112.6 ± 11.7 µm2 in control cells and 318.8 ± 84.6 µm2 after 2-MeS-ATP; n = 7, P < 0.005). Although stimulation of cells with 2-MeS-ATP increased Ca2+ transients and the spread of Ca2+, the level of Ca2+ reached was apparently below the threshold for contraction, and shortening of cells was not observed. As a positive control, subsequent addition of caffeine (1 mM) caused a relatively massive increase in global Ca2+ and cell shortening (n = 8; see Fig. 10). To control for nonspecific activation of P2X receptors, which may also be expressed by colonic myocytes, we tested alpha ,beta -methylene-ATP (200 µM), and this compound was without resolvable effects on Ca2+ transients (n = 5, P > 0.5).


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  		Fig. 6.   Ca2+ puffs in colonic myocytes were associated with STOCs. A: cell with a single spontaneous Ca2+ puff site during control conditions. Frame shown represents the maximum intensity and spatial spread of the puff. B: simultaneous recordings of whole cell current and fluorescence. Note that the 3 puff events recorded during the 20-s scan were associated with STOCs. C and D: addition of 2-MeS-ATP increased the number of puff sites and increased the frequency and intensity of Ca2+ puffs from the original site. The frequency of STOCs was increased in association with the increase in Ca2+ puffs after 2-MeS-ATP. Note that two fluorescence traces are shown in D to describe the temporal changes in fluorescence at the 2 puff sites (areas of interest).



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  		Fig. 7.   2-MeS-ATP increased ChTX-insensitive STOCs. A: control STOCs. B: reduction in the number and amplitude of STOCs after ChTX (200 nM). C: in the presence of ChTX, 2-MeS-ATP (200 µM) increases ChTX-insensitive STOC activity. D: histogram describing the number of STOCs under control conditions (filled bars), after ChTX (open bars), and after 2-MeS-ATP (shaded bars).

The nonspecific Ca2+ entry blockers Ni2+ and Cd2+ (200 µM) had no significant effects on the increases in Ca2+ transients elicited by 2-MeS-ATP [i.e., 225 ± 38% (n = 5; P < 0.01) of control area for cells pretreated with Ni2+ and 213 ± 25% (n = 5; P < 0.01) for cells pretreated with Cd2+]. Pretreatment of cells with CPA blocked the effects of 2-MeS-ATP (i.e., no significant increase over the effect of CPA alone; 100.2 ± 12%; n = 5; P > 0.5). Ryanodine (10 µM) inhibited the amplification of spontaneous Ca2+ transients caused by 2-MeS-ATP (i.e., after ryanodine, 2-MeS-ATP caused no increase over the effects of ryanodine alone; 101 ± 8%; n = 5; P > 0.5). The effects of CPA and ryanodine on responses to 2-MeS-ATP are shown in Fig. 4.

Pretreatment of cells with PPADS (10 µM), a P2 receptor antagonist (12), for 15 min did not significantly affect Ca2+ transients (76 ± 21% of control area; n = 7; P > 0.1), but this compound blocked the increase caused by 2-MeS-ATP (95 ± 14%; n = 7; P > 0.5 of pre-2-MeS-ATP level in the presence of PPADS; Fig. 8A). An inhibitor of PLC (U-73122, 2.5 µM; see Ref. 38), which prevents IP3 production, reduced spontaneous calcium transients (36 ± 7% of control; n = 9; P < 0.01; Fig. 8B) and ChTX-insensitive STOCs (Fig. 9). After addition of U-73122, 2-MeS-ATP did not significantly affect spontaneous Ca2+ transients (i.e., 83 ± 9% of pre-2-MeS-ATP level in the presence of U-73122; n = 9; P > 0.5). The inactive analog U-73343 had no effect on control Ca2+ transients or the response to 2-MeS-ATP (not shown). After pretreatment of cells with Xe-C (5 µM; 10 min), 2-MeS-ATP had no effect on Ca2+ transients (108 ± 7%; n = 6; P > 0.5 of area for cells pretreated with Xe-C; Fig. 10). As a positive control, caffeine was added at the end of experiments. This caused a massive increase in Ca2+ and cell shortening (Fig. 10F).


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  		Fig. 8.   Effects of a P2Y receptor blocker and an inhibitor of phospholipase C on spontaneous and stimulated Ca2+ transients. A-C: spontaneous Ca2+ transients before (A) and after (B) addition of pyridoxal-phosphate-6-azophenyl-2',4'-disculfonic acid tetrasodium (PPADS; 10 µM). PPADS had no significant effect on spontaneous Ca2+ transients, but it blocked the increase in Ca2+ transients produced by 2-MeS-ATP (C). D-F: Ca2+ transients (D) were reduced by addition of U-73122 (2.5 µM; E). U-73122 blocked spontaneous Ca2+ puffs, suggesting that ongoing production of IP3 is required for these events. There was no response to 2-MeS-ATP in the presence of U-73122 (F).



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  		Fig. 9.   STOCs were blocked by U-73122. A: control STOC activity in a cell held at -30 mV. B: U-73122, an inhibitor of phospholipase C, blocked STOCs. C: histogram describing the number of STOCs in control conditions (filled bars) and after U-73122 (open bars).



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  		Fig. 10.   A: image of a cell with spontaneous Ca2+ puff activity. C: fluorescence recorded from the puff site during a 20-s scan. D: significant reduction in the puff activity after exposure to Xe-C (5 µM). E: Xe-C also inhibited the response to 2-MeS-ATP. B and F: cells treated with Xe-C retained significant internal stores of Ca2+, however, and this was demonstrated by a large Ca2+ transient in response to caffeine (1 mM; B and F) and contraction of the cell (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study has demonstrated Ca2+ puffs and waves in isolated murine colonic myocytes. In contrast to the localized Ca2+ transients previously described in vascular, tracheal, and small intestinal smooth muscles (17, 30, 33, 37, 43), spontaneous Ca2+ transients in colonic muscle cells were not primarily mediated by ryanodine receptors. IP3 receptor-operated stores appear to be the main source of spontaneous Ca2+ transients in these cells. Enhanced Ca2+ release by this mechanism may be a major mechanism for coupling between G protein-regulated receptors and activation of Ca2+-dependent conductances in the plasma membrane. Showing that stimulation with the P2Y agonist, 2-MeS-ATP, increased the occurrence of Ca2+ puffs and waves supports this hypothesis. The data also suggest that stimulation with 2-MeS-ATP also "recruits" additional Ca2+ release from ryanodine receptors. A similar phenomenon was recently observed in rat portal vein myocytes in response to stimulation with norepinephrine (8). Stimulation with 2-MeS-ATP also increased the tendency of Ca2+ puffs to become regenerative and develop into Ca2+ waves. Despite the dynamic mechanisms to mobilize Ca2+ in response to P2Y receptor occupation, the Ca2+ transients did not raise global Ca2+ sufficiently to activate the contractile apparatus. Thus the Ca2+ puffs and waves in colonic myocytes appear to be compartmentalized in microdomains near the plasma membrane where Ca2+-dependent ionic conductances can be regulated.

Localized Ca2+ transients are an important mechanism for regulating ionic conductances in the plasma membrane in smooth muscles (30, 37, 43). We found that the Ca2+ puffs and waves in murine colonic myocytes were of sufficient magnitude to activate Ca2+-dependent conductances. STOCs (4), which have been related to the activation of BK channels, were correlated with spontaneous Ca2+ transients. We have previously suggested that additional Ca2+-dependent conductances may also be regulated by localized Ca2+ transients in colonic myocytes (26). This concept has also been demonstrated by studies of guinea pig tracheal myocytes in which individual Ca2+ sparks initiated both inward currents (via a Ca2+-activated Cl- conductance) and outward currents via activation of BK channels, depending on the holding potential (43). In the present study, Ca2+ puffs were associated with activation of ChTX-insensitive STOCs. These events were reduced by apamin, a blocker of SK channels. Because ATP-sensitive hyperpolarization responses are also reduced by apamin in gastrointestinal muscles (2, 13), it is possible that coupling between Ca2+ puffs and SK channel activation is the mechanism coupling ATP to postjunctional hyperpolarization in situ.

Regulating the frequency and amplitude of localized Ca2+ transients is an important means of coupling receptor activation to electrical responses. Several second messenger mechanisms have been shown to regulate Ca2+ release events in smooth muscles. Ca2+ sparks recorded from rat coronary and cerebral arteriole myocytes were increased in frequency by cAMP-dependent mechanisms (34) and were reduced by protein kinase C-dependent mechanisms (9). Spark frequency in these studies may have been modulated by affecting Ca2+ uptake in the sarcoplasmic reticulum (SR; i.e., modifying luminal Ca2+ content) or by changing the properties of ryanodine receptors, such as altering the sensitivity to Ca2+. In porcine tracheal muscles, discrete Ca2+ sparks developed into Ca2+ oscillations when cells were stimulated with ACh (37). In these studies, the Ca2+ oscillations were attributed to release from ryanodine receptors; however, it is also possible that amplification via IP3 receptors may have participated in the cholinergic responses, as described in studies of duodenal myocytes (8). The present study suggests that localized Ca2+ release is mediated by IP3-dependent mechanisms in murine colonic myocytes, and, as part of the response, regenerative responses involving Ca2+ release from IP3 receptors and ryanodine receptors may be important. Both Ca2+ release mechanisms are facilitated by cytoplasmic Ca2+ and thus are capable of regenerative responses (see Ref. 10). Factors such as luminal concentration of Ca2+ in the SR (29, 32), basal IP3 levels (27), basal Ca2+ levels in the microdomain near ryanodine and IP3 receptors (6, 18, 21, 28), receptor isoform and density, and the spatial relationship between receptors could all be important in determining the mechanism of local Ca2+ transients and the responses to agonist stimulation in specific types of smooth muscle. A recent study in which caged Ca2+ was released in portal vein smooth muscle cells also supports the concept of cooperativity between IP3 receptors and ryanodine receptors (7). These authors provided evidence that IP3-dependent Ca2+ release is amplified by ryanodine receptors, and this facilitates the development of Ca2+ waves.

The results of this study offer new insights into the mechanisms of enteric inhibitory regulation of gastrointestinal muscles and suggest that localized Ca2+ transients are a means of coupling receptors with inhibitory effectors such as plasma membrane K+ channels (Fig. 9). There is significant evidence that at least a portion of the inhibitory neural response in many species is due to release of ATP from enteric inhibitory neurons (13, 15, 20). Previous studies showed that ATP activates SK channels in murine colonic (24) and small intestinal (41) myocytes, and activation of these channels is likely to explain the apamin-sensitive hyperpolarization response to enteric inhibitory neurotransmission in intact gastrointestinal muscles. The actions of ATP appeared to be mediated via P2Y receptors because they were blocked by PPADS and mimicked by 2-MeS-ATP (24). The mechanism for coupling between P2Y receptors and SK channels, however, has not been previously described. Our studies suggest the following model: 2-MeS-ATP increases localized Ca2+ release via a mechanism involving P2Y receptors, PLC, and IP3 receptors. Because SK channels are highly sensitive to Ca2+, the increase in Ca2+ near the plasma membrane provides a plausible mechanism for increasing the open probability of SK channels. In the present study, Ca2+ puffs and waves were increased by 2-MeS-ATP. In association with the increase in Ca2+ transients, STOCs were increased. Ca2+ transients were correlated with ChTX-sensitive large-amplitude STOCs and ChTX-insensitive STOCs that were reduced by apamin. ChTX-insensitive STOCs increased in response to 2-MeS-ATP, and this occurred in parallel with increases in Ca2+ transients. Thus activation of apamin-sensitive K+ channels by localized Ca2+ release provides a mechanism for hyperpolarization responses (inhibitory junction potentials) caused by enteric inhibitory nerve stimulation in gastrointestinal muscles.


    ACKNOWLEDGEMENTS

We thank Dr. C. W. R. Shuttleworth and Julia Bayguinov for technical assistance with the confocal microscope and preparation of smooth muscle cells.


    FOOTNOTES

This study was supported by National Institutes of Health Grants DK-41315 (to K. M. Sanders, O. Bayguinov, and B. Hagen) and HL-44455 (to M. T. Nelson and A. D. Bonev). The Noran Confocal microscope was purchased by shared equipment Grant HL-44455

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Anderson Medical Bldg., Reno, NV 89557 (E-mail: kent{at}physio.unr.edu).

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. §1734 solely to indicate this fact.

Received 19 November 1999; accepted in final form 19 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 279(1):C126-C135
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