INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

COORDINATED CONTRACTIONS OF visceral smooth muscle organs depend on a complicated array of regulatory processes. Key to normal motor behavior is the regulation provided by autonomic and/or enteric motor neurons. The efferent motor outflow in most visceral organs is organized into both excitatory and inhibitory neurons. In the gastrointestinal (GI) tract neural excitatory regulation comes from the release of acetylcholine (ACh) and neuropeptides such as substance P or neurokinin A (cf. Ref. 20). ACh impinges on muscarinic receptors (M₂ and M₃ isoforms; see Refs. 35 and 36) in postjunctional cells, and activation of these receptors initiates a complex series of events that lead to enhanced contractile force. Prominent among the many responses attributable to postjunctional muscarinic stimulation of GI muscles are: 1) activation of nonselective cation channels (3, 14-16, 27, 32) and 2) G protein-dependent stimulation of phopholipase C and generation of inositol 1,4,5-trisphosphate (IP₃; e.g., Ref. 4). The former is thought to depolarize GI muscles and increase the entry of Ca²⁺ through L-type Ca²⁺ channels. IP₃ production initiates Ca²⁺ release from IP₃ receptor-operated stores, a common feature of most smooth muscles. Ca²⁺ release from stores is thought to summate with Ca²⁺ entering cells to augment the strength of contraction. Recent studies, however, have provided an expanded view of the role of Ca²⁺ release from internal stores. When localized to specific membrane areas, Ca²⁺ release can activate Ca²⁺-dependent conductances in the plasma membrane (24, 26, 29, 38), and activation of these channels can transduce the release of Ca²⁺ (which would normally be an excitatory signal) to inhibitory events leading to hyperpolarization and relaxation (see Ref. 17). Both ryanodine and IP₃ receptors have been linked to localized Ca²⁺ release and regulation of Ca²⁺-dependent conductances in the plasma membrane (1, 2, 26).

In GI smooth muscles G protein-coupled receptors mediate excitatory (e.g., muscarinic or neurokinin receptors) and inhibitory responses (e.g., P_2Y receptors) that couple to responses through IP₃-dependent pathways and Ca²⁺ release. How coupling through similar second messenger pathways can elicit opposite responses is not understood. We have reported that stimulation of P_2Y receptors of colonic smooth muscles cells is coupled to inhibitory responses via IP₃ production and localized Ca²⁺ release (1, 19). Stimulation of P_2Y receptors increased the occurrence and amplitude of spontaneous Ca²⁺ transients and spontaneous transient outward currents (STOCs). The STOCs were due to activation of large-conductance Ca⁺-activated K⁺ channels (BK channels) and small-conductance Ca²⁺-activated K⁺ channels (SK channels) (1, 19). Thus localized Ca²⁺ release from stores in these cells led to membrane hyperpolarization. Stimulation of muscarinic receptors also increases production of IP₃. Since enhanced IP₃ production greatly increases the occurrence of spontaneous Ca²⁺ transients and STOCs, we hypothesize that another mechanism must be present and additively coupled to muscarinic stimulation to suppress the increase in Ca²⁺ transients and/or coupling between Ca²⁺ transients and activation of Ca²⁺-activated K⁺ channels. Previous studies have demonstrated that muscarinic stimulation can reduce the open probability of BK channels (11), but there is no known inhibitory regulation of SK channels mediated by muscarinic receptor stimulation. Thus it would seem that, to suppress the K⁺ conductances activated by IP₃ receptor-operated Ca²⁺ release, a mechanism to suppress localized Ca²⁺ release from stores may exist.

In the present study we have characterized localized Ca²⁺ transients in response to muscarinic stimulation and investigated the coupling of these responses to activation of outward currents. We have observed that an increase in basal Ca²⁺ that occurs via activation of receptor-operated conductances in the plasma membrane suppresses the natural tendency for muscarinic stimulation to increase localized Ca²⁺ transients in colonic myocytes. In addition to depolarization, suppression of spontaneous Ca²⁺ transients is a new mechanism attributed to the receptor-operated channels activated by muscarinic stimulation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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

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, allow the cells to tightly adhere to the bottom of the chambers, and to complete the deesterefication of fluo 3. All measurements were made within 45 min after restoring extracellular Ca²⁺.

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 40 or 70 mV (after corrections of junction potentials) using pCLAMP software (version 7.0, Axon Instruments). Currents were digitized at 1 kHz in dual recordings and were digitized at 500 Hz in experiments that were stepped repetitively between 40 and 70 mV. 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 KRB used in this study contained (in mM) 120.35 NaCl, 5.9 KCl, 2.5 CaCl, 1.2 MgCl₂, 15.5 NaHCO₃, 1.2 NaH₂PO₄, 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 110 mM potassium aspartate, 30 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, 0.05 mM EGTA (pH 7.2), and 250 µg/ml amphotericin B.

Analysis of data. Image analysis was performed using custom analysis programs using Interactive Data Language software (Research Systems, Boulder, CO), as previously described (1). Baseline fluorescence (F₀) was determined by averaging 10 images (of 600) with no activity. F₀ from control experiments was used to determine the ratio of the records of following drug treatments. Ratio images were then constructed and replayed for careful examination to detect active areas where sudden increases in F/F₀ occurred. To detect changes in basal Ca²⁺ concentration, areas free of Ca²⁺ transients were selected. F/F₀ vs. time traces were further analyzed in Microcal Origin (Microcal Software, Northampton, MA) and AcqKnowledge Software (Biopac Systems, Santa Barbara, CA) and represent the averaged F/F_o 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 (1, 29). 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) (1). As a result, the data could not be accurately analyzed simply as changes in transient amplitude or frequency. Therefore, we analyzed the data as the area of the Ca²⁺ transients above a baseline drawn between the end points of 20-s scans. Areas are expressed in terms of amplitude units (F/F₀) times horizontal units (time). Changes of basal Ca²⁺ concentration were calculated as the increase in average ratio in areas of interest. The amplitudes of STOCs were determined off-line using the Mini Analysis Program (Synaptosoft software, Leonia, NJ). The threshold of STOCs was set at three times the single Ca²⁺-activated-K⁺ channel amplitude at 40 mV or at 6 pA. The open probability of Ca²⁺-activated K⁺ channels in the absence of Ca²⁺ release events is very low at 40 mV (number of channels times open probability  10³; see Ref. 5), and the probability of three simultaneous openings is far less.

Statistical analysis. Results are expressed as means ± SE where applicable. All the statistical analysis was made with SigmaStat 2.03 software (Jandel Scientific Software, San Rafael, CA). ANOVA on Ranks test was used to compare the results from different treatments.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of ACh on spontaneous Ca²+ transients in murine colonic myocytes. Spontaneous, transient elevations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were observed in freshly dispersed murine colonic myocytes, as previously reported (1). These events occurred either as localized events (Ca²⁺ puffs) or as more widely dispersed, propagating events (Ca²⁺ waves). Previous studies have indicated that the spontaneous Ca²⁺ transients in colonic myocytes are mediated by Ca²⁺ release from IP₃-receptor operated channels (1).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effects of muscarinic stimulation on basal Ca2+ and localized Ca2+ transients. A: a cell loaded with fluo 3. A spontaneous localized Ca2+ event is pictured at the bottom, middle region of the cell. Areas of interest (AOI) are noted by arrows. B: same cell after exposure to ACh (10 µM for 5 min). A generalized increase in basal Ca2+ was observed. C: Ca2+ transients (fluorescence ratio F/F0) that occurred within AOI-SA (spontaneously active) before (left trace) and after (right trace) ACh (10 µM) during 20-s scans (600 images/20 s). D: F/F0 from AOI-NSA (not spontaneously active) before (left trace) and after (right trace) ACh during the same 20-s scans as in C (600 images/20 s). AOIs were the same before and after ACh. Note the increase in baseline Ca2+ in both AOIs. E: time-dependent changes in Ca2+ transients (solid bars) and basal Ca2+ (open bars) after muscarinic stimulation. The changes in ratio are relative to a point at time = 0 where ratio = 100% (control). Data like those shown in A-D were tabulated at 4 time points after addition of ACh (10 µM; n = 11 cells for each time point). There were significant reductions in Ca2+ transients within 1 min of addition of ACh, and these persisted through 10 min of constant exposure. Basal Ca2+ increased significantly within 1 min of exposure, and this effect also persisted throughout the exposure. All points are significant to at least P < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Responses to ACh were inhibited by atropine. A: spontaneous Ca2+ transients under control conditions in an AOI-SA (left trace) and an AOI-NSA (right trace). B: response to ACh of the cell after pretreatment for 10 min with atropine (10 µM). ACh did not reduce spontaneous transients or increase the level of basal Ca2+. Data from 7 cells (P > 0.1) pretreated with atropine and stimulated with ACh for 1 and 3 min are tabulated in C (solid bars, Ca2+ transients; open bars, basal Ca2+).

Effects of ACh on spontaneous transient outward currents. We investigated the effects of ACh on STOCs in 11 experiments. In the first series of experiments six cells were stepped repetitively between 40 and 70 mV for 15-s intervals. As reported previously (1, 19), colonic myocytes held at 40 mV displayed STOCs. STOCs were not observed at 70 mV. Application of ACh (10 µM) reduced the amplitude and occurrence of STOCs (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of ACh on spontaneous transient outward currents (STOCs). A: a continuous record before and during a response to ACh (10 µM). After ACh there was a decrease in the amplitude and frequency of STOCs. B and C: STOC amplitude histograms before (B) and after (C) 5-min exposure to ACh (n = 8; P < 0.001).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Spontaneous Ca2+ transients were coincident with STOCs. A: a cell with 2 active AOI during control scans. After ACh, a more generalized increase in basal Ca2+ was observed (pairs of images at 1 and 3 min after addition of ACh). B: Ca2+ transients from the 2 AOI depicted in A. Note the occurrence of multiple Ca2+ transients at each site during the 20-s scan. Traces to the right show Ca2+ transients after exposure to ACh for 1 and 3 min. Note the rise in basal Ca2+ at each AOI and decrease in the Ca2+ transients above baseline. C: simultaneous recordings of whole cell currents during the fluorescence measurements. STOCs are coincident with Ca2+ transients, but the relationship between Ca2+ transients at different AOI and STOC amplitude differs, suggesting nonhomogenous coupling between Ca2+ transients and activation of Ca2+-activated K+ channels.

Participation of IP₃-producing mechanism on effects of ACh. As reported previously (1) an inhibitor of phospholipase C (PLC), U-73122 (2.5 µM), which inhibits IP₃ production, significantly reduced spontaneous Ca²⁺ transients (P < 0.05). In six experiments, we investigated the effects of U-73122 on the effects of ACh. Figure 5A shows that inhibition of PLC did not prevent the increase of basal Ca²⁺, suggesting that the rise in basal Ca²⁺ may be largely due to Ca²⁺ influx (P < 0.05).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effects of various agents on spontaneous Ca2+ transients and responses to ACh. A: spontaneous Ca2+ transients were inhibited by U-73122 as previously reported [see Bayguinov et al. (1)]. After U-73122, ACh (3- and 5-min time points tabulated) caused further reduction in spontaneous Ca2+. The increase in basal Ca2+ was not significantly reduced by pretreatment with U-73122. Data are summarized from 6 cells. B: xestospongin C (Xe-C) inhibited spontaneous Ca2+ transients as previously reported (see Ref. 1) but had no significant effect on baseline Ca2+. In the presence of Xe-C, ACh had no effect on Ca2+ transients. The increase in basal Ca2+ was not significantly reduced by pretreatment with Xe-C. Data are summarized from 6 cells. C: nicardipine (Nicard) had no significant effect on Ca2+ transients and basal Ca2+. After nicardipine ACh continued to cause an increase in basal Ca2+ and a reduction in Ca2+ transients. Data are summarized from 6 cells. D: effects of Cd2+ (200 µM). Cd2+ did not reduce Ca2+ transients or basal Ca2+ and did not significantly affect responses to ACh. *Significance of at least P < 0.05. Solid bars, Ca2+ transients; open bars, basal Ca2+.

Influence of Ca²+ channel blockers on the action of ACh. We have previously reported that dihidropyridines (e.g., nicardipine, 1 µM) or inorganic Ca²⁺ channels blockers [e.g., Ni²⁺ (200 µM) or Cd²⁺ (200 µM)] did not affect spontaneous Ca²⁺ transients in murine colonic myocytes (1). Pretreatment of cells with nicardipine (1 µM) or Cd²⁺ (200 µM) in the present study did not affect responses to ACh (10 µM). Figure 5, C and D, shows summaries of results from experiments on 12 cells in which the effects of nicardipine (n = 6, P < 0.05) and Cd²⁺ (n = 6, P < 0.05) were tested on responses to ACh.

ACh induces inward currents in colonic myocytes. Many previous reports have shown that cholinergic (muscarinic) stimulation of GI smooth muscle cells induces a nonselective cation conductance (cf. Refs. 3, 14-16, 27, 32). We found that ACh (10 µM) induced a persistent net inward current that averaged 26.3 ± 6.5 pA (holding potential 70 mV) in murine colonic myocytes (Fig. 6A).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Gd3+ and SKF-96365 inhibit inward current activated by ACh. A: voltage-clamp recordings from a cell stepped from 40 to 70 mV before (control) and after addition of ACh (10 µM). There was a persistent increase in inward current noted in the presence of ACh. B: effects of Gd3+. Gd3+ reduced the basal inward conductance of cells (middle trace; cell stepped from 40 to 70 mV) and blocked the increase in inward current after ACh (right trace). C: effects of SKF-96365. SKF-96365 also decreased basal inward current (middle trace; cell stepped from 40 to 70 mV) and inhibited inward current stimulated by ACh (right trace). Summary data are reported in the text. In some of the traces shown spontaneous outward currents (which were observed at 40 mV but not at 70 mV) can be seen just before the step to 70 mV. Dotted line denotes zero current in each panel.

Gd³+ and SKF-96365 reduce the rise in basal Ca²⁺ and block the inhibition of Ca²⁺ transients caused by ACh. Pretreatment of cells with Gd³⁺ (10 µM) caused no significant effect in Ca²⁺ transients (112 ± 13% of control; n = 6, P > 0.05), and application of ACh (10 µM) significantly increased the area of Ca²⁺ transients (to 129 ± 15% of control; P < 0.05). Basal Ca²⁺ remained unchanged in response to ACh in the presence of Gd³⁺ (i.e., 97 ± 2% of control; n = 6, P > 0.05; Fig. 7).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Gd3+ blocks the effects of ACh on spontaneous Ca2+ transients. A: Ca2+ transients during 20 s from a spontaneously active AOI (AOI-SA). Middle and bottom traces show Ca2+ transients from the same AOI-SA after addition of Gd3+ (10 µM) and ACh (10 µM). B: data during the same scans as in A from an AOI that was not spontaneously active (AOI-NSA). Note that ACh did not increase basal Ca2+ or inhibit Ca2+ transients in the presence of Gd3+. C: summary of 6 experiments in which ACh was added after Gd3+. There was no statistical change in basal Ca2+ (open bars) after ACh in the presence of Gd3+, and there was a significant increase in Ca2+ transients (solid bars). *P < 0.05 after ACh in the presence of Gd3+.

View larger version (44K): [in this window] [in a new window]   Fig. 8.   SKF-96365 blocks the inhibitory effects of ACh on spontaneous Ca2+ transients. A: a cell with 3 active AOI (numbered 1-3; arrows). One nonactive AOI is also noted. Addition of SKF-96365 did not significantly affect the occurrence of Ca2+ transients or basal Ca2+. Addition of ACh after pretreatment with SKF-96365 caused a significant increase in spontaneous Ca2+ transients in some cells, and in others localized Ca2+ transients developed into spreading waves (e.g., cell depicted in A; sequence of Ca2+ wave as numbered). B: fluorescence traces from the AOIs noted in A. Note the occurrence of localized Ca2+ transients in AOIs 1-3 and lack of activity in AOI 4. After ACh a large Ca2+ transient was recorded at each AOI as a wave spread through the cell. C: tabulations of Ca2+ transients (solid bars) and basal Ca2+ (open bars) after SKF- 96365 and after ACh (5- and 10-min exposures). *Significance of at least P < 0.05.

Ca²+ influx blocks spontaneous Ca²⁺ transients. The experiments with Gd³⁺ and SKF-96365 reveal a dual effect of ACh in murine colonic myocytes. It appears from our study that activation of inward current in colonic myocytes by ACh is responsible for the rise in basal Ca²⁺ after ACh. Additionally, G protein-dependent increases in IP₃ leads to release of Ca²⁺ from internal stores (4). Our experiments have shown that the latter couples to activation of K⁺ channels (i.e., STOCs). It is unclear how muscarinic stimulation overrides the activation of K⁺ channels to yield a dominantly excitatory response. We hypothesized that the rise in basal Ca²⁺ caused by ACh inhibits spontaneous Ca²⁺ transients and thus deactivates the coupling between spontaneous Ca²⁺ release and K⁺ channel activation. We tested this hypothesis by adding ionomycin, a compound that would tend to raise cytoplasmic Ca²⁺ levels without the concomitant stimulation of Ca²⁺ release by PLC-dependent increases in IP₃ production. In experiments on six cells we compared the effects of ACh with ionomycin (250 and 500 nM) and found that both compounds increased basal Ca²⁺ and inhibited spontaneous Ca²⁺ transients (Fig. 9, A and C).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Ionomycin and elevated external Ca2+ increased basal Ca2+ and inhibited spontaneous Ca2+ transients. A: spontaneous Ca2+ transients from an active AOI during control recording and after addition of ionomycin (500 nM). Note the increase in basal Ca2+ and inhibition of Ca2+ transients. Other spontaneously active AOI were similarly affected. B: similar effects when external Ca2+ was elevated to 6 mM. Data are shown after a 3-min exposure to 6 mM Ca2+. Elevated Ca2+ caused an increase in basal Ca2+ and inhibition of Ca2+ transients. C: summary of the average decreases in spontaneous transients (solid bars) and increases in basal Ca2+ (open bars) caused by ionomycin and elevated external Ca2+. All points were significant to at least P < 0.05.

Effects of ACh on Ca²+ transients were inhibited by FK-506. Our data suggest that the rise in basal Ca²⁺ may inhibit spontaneous Ca²⁺ transients in murine colonic myocytes. Part of the regulation of IP₃-dependent Ca²⁺ release may be due to FKBP12 tethering of calcineurin to IP₃ receptors (8, 9). We tested the effects of FK-506, which disassociates FKBP12 from its targets, on the effects of ACh in colonic myocytes. Pretreatment of six cells with FK-506 (1 µM) had no effect on spontaneous Ca²⁺ transients or basal Ca²⁺ (P > 0.5 for both parameters). Addition of ACh (10 µM) in the continued presence of FK-506 caused a normal increase in basal Ca²⁺ (e.g., 139 ± 11% after 5 min exposure, n = 6, P < 0.05); however, spontaneous Ca²⁺ transients were not inhibited (P > 0.05; Fig. 10). In the presence of FK-506, Ca²⁺ waves were generated after addition of ACh in two of six cells.

View larger version (28K): [in this window] [in a new window]   Fig. 10.   FK-506 prevents the inhibition of Ca2+ transients by ACh. A: Ca2+ transients from an active AOI. FK-506 had no significant effect on the occurrence of spontaneous Ca2+ transients (2nd trace). Addition of ACh did not inhibit spontaneous Ca2+ transients (3rd and 4th traces), and in some cells Ca2+ waves developed (5th trace). B: fluorescence traces from an inactive AOI. Note that FK-506 did not inhibit the increase in basal Ca2+ after addition of ACh. C: summary of data from 6 cells pretreated with FK-506 and then exposed to ACh (1-, 3-, and 5-min exposures). Solid bars, spontaneous transients; open bars, basal Ca2+. *Significance of at least P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Localized Ca²+ release couples to activation of K⁺ channels in colonic myocytes. Localized Ca²⁺ transients have been reported in vascular, tracheal, and small intestinal smooth muscles (12, 26, 29, 31, 38), and, in general, these events have been attributed to Ca²⁺ release from ryanodine receptors. In colonic smooth muscle cells ryanodine was found to have no effect on spontaneous Ca²⁺ transients, and it was recently reported that localized Ca²⁺ transients predominantly resulted from Ca²⁺ release from IP₃ receptor-operated stores (1, 19). Studies of localized Ca²⁺ transients in smooth muscle cells have supported the unorthodox concept that release of stored Ca²⁺ in smooth muscle cells may not always couple to contraction, but, in fact, Ca²⁺ release may be an inhibitory signal in many smooth muscles due to activation of Ca²⁺-dependent K⁺ currents in the plasma membrane (26, 29). For example, localized Ca²⁺ transients in colonic myocytes activate Ca²⁺-dependent ion channels, including BK and SK channels (1, 19), and activation of these channels results in membrane hyperpolarization and reduced excitability. Stimulation of colonic myocytes cells with ATP, via P_2Y receptors and activation of PLC, increased Ca²⁺ transients, increased the tendency for localized Ca²⁺ release to develop into Ca²⁺ waves, and increased coupling between Ca²⁺ transients and activation of BK and SK channels. Thus inhibitory stimulation by ATP (and the P_2Y agonist 2-methylthio-ATP) increased K⁺ channel open probability via a mechanism involving localized Ca²⁺ release.

Muscarinic stimulation of colonic myocytes inhibits spontaneous Ca²+ release from IP₃ receptor-operated stores. In the present study we investigated how muscarinic stimulation, which is also coupled by G proteins to activation of PLC and IP₃ formation, can overcome the tendency for activation of IP₃ receptor-operated Ca²⁺ release to drive activation of K⁺ channels, which is predominantly an inhibitory response. The main implications of the present study are that muscarinic stimulation of colonic muscles includes at least two important phases: 1) IP₃ receptor-operated Ca²⁺ release generates Ca²⁺ transients, and 2) global Ca²⁺ rises, due at least in part to Ca²⁺ entry, and this suppresses Ca²⁺ transients and reduces coupling between localized Ca²⁺ release and Ca²⁺-activated K⁺ currents. We suggest that reducing the activation of Ca²⁺-activated K⁺ channels by this mechanism facilitates the development of excitatory responses to muscarinic stimulation. Deactivation of localized Ca²⁺ transients is a previously unreported aspect of muscarinic stimulation in GI smooth muscles.

Increased basal Ca²+ due to Ca²⁺ entry inhibits Ca²⁺ release from IP₃ receptor-operated stores. Muscarinic stimulation of colonic myocytes resulted in a conversion in the pattern of Ca²⁺ transients from spontaneous localized Ca²⁺ transients to a sustained elevation in global Ca²⁺. Conversion of the Ca²⁺ response pattern from localized Ca²⁺ transients to a global increase in Ca²⁺ in response to agonist stimulation has been observed in nonexcitable cells (e.g., Ref. 7). In these experiments conversion from local responses to global increases in Ca²⁺ were characterized by increased frequency, amplitude, and/or distribution of localized transients, and spatial and/or temporal summation of transients led to massive Ca²⁺ waves. The responses of colonic myocytes differed in that global Ca²⁺ increased tonically in response to ACh, not as repetitive waves. Second, there were two processes that contributed to the Ca²⁺ response. Ca²⁺ entry through receptor-operated cation channels was necessary for the global increase, and the global increase appeared to inhibit the IP₃-dependent Ca²⁺ transients. When Ca²⁺ entry was prohibited, ACh stimulation increased Ca²⁺ transients (much the same way that ATP increased Ca²⁺ transients via an IP₃-dependent mechanism; see Ref. 1). In some cells in which Ca²⁺ entry was blocked, Ca²⁺ waves occurred in response to ACh, and this response may have been equivalent to the changes observed in nonexcitable cells during agonist stimulation (7).

How does increased basal Ca²+ inhibit Ca²⁺ release from IP₃ receptor-operated stores? Our results suggest that the rise in basal Ca²⁺ that accompanies muscarinic stimulation inhibits IP₃ receptor-operated Ca²⁺ release events. The complex effects of cytoplasmic Ca²⁺ on the sensitivity of IP₃ receptors might explain the inhibition of Ca²⁺ release caused by a rise in basal Ca²⁺. Studies on skinned smooth muscle fibers have shown that release of Ca²⁺ from IP₃ receptor-operated stores is regulated by [Ca²⁺]_i in a biphasic manner with positive feedback operating below 300 nM [Ca²⁺]_i and negative feedback acting above 300 nM [Ca²⁺]_i (13). According to studies of single IP₃-receptor channels, the bell-shaped relationship between open probability (P_o) and [Ca²⁺]_i has a sharp peak when [Ca²⁺]_i is <1 µM and levels of IP₃ are low (i.e., <20 nM) (21). These studies suggested that the effects of [Ca²⁺]_i on P_o of IP₃ receptors would be to increase IP₃ sensitivity as [Ca²⁺]_i increases from low basal levels, but, with further increases in [Ca²⁺]_i, IP₃ sensitivity would decrease. Thus, at low levels of [Ca²⁺]_i, IP₃ levels may be sufficient to produce spontaneous Ca²⁺ transients, but, as basal Ca²⁺ rises, even though IP₃ levels may also rise in response to muscarinic stimulation, the spontaneous openings of IP₃ receptor-operated channels could be reduced by the negative feedback exerted by rising [Ca²⁺]_i.