Muscarinic stimulation increases basal Ca2+ and inhibits spontaneous Ca2+ transients in murine colonic myocytes

Orline Bayguinov, Brian Hagen, and Kenton M. Sanders

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Localized Ca2+ transients in isolated murine colonic myocytes depend on Ca2+ release from inositol 1,4,5-trisphosphate (IP3) receptors. Localized Ca2+ transients couple to spontaneous transient outward currents (STOCs) and mediate hyperpolarization responses in these cells. We used confocal microscopy and whole cell patch-clamp recording to investigate how muscarinic stimulation, which causes formation of IP3, can suppress Ca2+ transients and STOCs that might override the excitatory nature of cholinergic responses. ACh (10 µM) reduced localized Ca2+ transients and STOCs, and these effects were associated with a rise in basal cytosolic Ca2+. These effects of ACh were mimicked by generalized rises in basal Ca2+ caused by ionomycin (250-500 nM) or elevated external Ca2+ (6 mM). Atropine (10 µM) abolished the effects of ACh. Pretreatment of cells with nicardipine (1 µM), or Cd2+ (200 µM) had no effect on responses to ACh. An inhibitor of phospholipase C, U-73122, blocked Ca2+ transients and STOCs but did not affect the increase in basal Ca2+ after ACh stimulation. Xestospongin C (Xe-C; 5 µM), a membrane-permeable antagonist of IP3 receptors, blocked spontaneous Ca2+ transients but did not prevent the increase of basal Ca2+ in response to ACh. Gd3+ (10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca2+ after ACh and increased the frequency and amplitude of Ca2+ transients and waves. Another inhibitor of receptor-mediated Ca2+ influx channels, SKF-96365, also prevented the rise in basal Ca2+ after ACh and increased Ca2+ transients and development of Ca2+ waves. FK-506, an inhibitor of FKBP12/IP3 receptor interactions, had no effect on the rise in basal Ca2+ but blocked the inhibitory effects of increased basal Ca2+ and ACh on Ca2+ transients. These results suggest that the rise in basal Ca2+ that accompanies muscarinic stimulation of colonic muscles inhibits localized Ca2+ transients that could couple to activation of Ca2+-activated K+ channels and reduce the excitatory effects of ACh.

calcium puffs; nonselective cation current; muscarinic receptors; enteric neurotransmission


    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., P2Y 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 P2Y receptors of colonic smooth muscles cells is coupled to inhibitory responses via IP3 production and localized Ca2+ release (1, 19). Stimulation of P2Y 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.

Strips of colonic muscle were equilibrated in Ca2+-free solution for 60 min, and then the tissues were digested to free single cells with an enzyme solution containing Collagenase F (Sigma, St. Louis, MO). During digestion the tissues were incubated 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 with blunt pipettes of decreasing tip diameter to mechanically free smooth muscle cells.

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+.

An Odyssey XL confocal laser scanning head (Noran Instruments, Middleton WI) connected to a Nikon Diaphot 300 microscope with a ×60 water immersion lens (numerical aperture = 1.2) was used to image the cells. The cells were scanned using INTERVISION software (Noran Instruments, Middleton WI) 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 -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.

ACh, atropine, cadmium chloride, gadolinium (III) chloride, and nicardipine were obtained from Sigma. (1-{6-[(17beta )-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)- 1H-pyrrole-2,5-dione (U-73122) was obtained from RBI. Xestospongin C (Xe-C) was obtained from Calbiochem. 1-{2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl}-1H- imidazole (SKF-96365 hydrocloride) was obtained from Tocris Coocson. FK-506 was a generous gift from Fiji Sawa. Concentrations of drugs used were determined from previous studies in the literature or by empirical testing of effective concentrations on murine colonic myocytes.

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/Fo 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 approx  10-3; 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).

We tested the effects of ACh (10 µM) on spontaneous Ca2+ transients in murine colonic myocytes. ACh reduced the area of Ca2+ transients to 46 ± 6% of control (P < 0.005; n = 11; Fig. 1, A-D). The reduction in Ca2+ transients was accompanied by a significant increase in the basal Ca2+ level (i.e., to 120 ± 5%) of control. Both of the observed effects (inhibition of the spontaneous activity and increase in basal Ca2+) began within 1 min after application of ACh, and the effects were stable for at least 10 min of exposure. Figure 1E summarizes the results of 11 experiments. It is unlikely that the inhibition of Ca2+ transients in response to ACh was due to simple unloading of stores because addition of caffeine (1 mM) caused a massive release of Ca2+ and contraction of the cells (data not shown).


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.

Pretreatment with atropine (10 µM) eliminated both effects of ACh. In the presence of atropine 1) the areas of the Ca2+ transients after the 1st and 3rd min of exposure to ACh (10 µM) were 101 ± 10% and 102 ± 16% of the control Ca2+ transients (10 µM; n = 7; P > 0.1) and 2) basal Ca2+ levels were 102 ± 4% and 101 ± 3% of control at the same time points (Fig. 2).


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).

Five cells were held at -40 mV, and membrane currents and spontaneous Ca2+ transients were recorded simultaneously. The current records demonstrated that the STOCs were associated with the Ca2+ transients (Fig. 4). The reduction in Ca2+ transients and rise in basal Ca2+ caused by ACh were accompanied by significant reduction in STOCs.


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+.

As reported previously (1), Xe-C (5 µM), a membrane-permeable antagonist of IP3 receptors, reduces the magnitude and frequency of Ca2+ puffs and STOCs. In experiments on six cells we found that Xe-C inhibited spontaneous Ca2+ transients (P < 0.05) but did not prevent the increase in basal Ca2+ after ACh (P < 0.05; Fig. 5B). ACh had no significant effect on Ca2+ transients in the presence of Xe-C (P > 0.05; Fig. 5B).

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.

Gadolinium (Gd3+), a blocker of nonselective cation currents including the nonselective cation conductance activated by muscarinic stimulation (H. K. Lee, O. Bayguinov, and K. M. Sanders, unpublished observations), reduced basal inward current at -70 mV (i.e., from -22.6 ± 2.2 to -14.0 ± 1.5 pA; n = 4; P < 0.05) and inhibited the increase in basal current on exposure to ACh (10 µM; i.e., from -14.0 ± 1.5 pA with Gd3+ to -13.1 ± 1.4 pA with Gd3+ and ACh; n = 4; P > 0.5; e.g., Fig. 6B). Another inhibitor of receptor-mediated Ca2+ influx channels, SKF-96365 (22), also inhibited basal inward current at -70 mV (i.e., from -12.2 ± 5.4 to -6.0 ± 2.7 pA; n = 4; P < 0.01) and inhibited the inward current induced by ACh (i.e., from -6.0 ± 2.7 pA with SKF-96365 to -6.0 ± 1.3 pA with SKF-96365 and ACh; n = 4; P > 0.5; e.g., Fig. 6C).

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+.

Pretreatment with SKF-96365 (10 µM) had no significant effect on Ca2+ transients, and application of ACh (1 µM) caused a significant increase in Ca2+ transients over control activity (e.g., to 146 ± 7% of control in the 10th min after exposure; n = 6, P < 0.05; Fig. 8). Basal Ca2+ levels were unchanged by ACh in the presence of SKF-96365 (e.g., 100 ± 7% of control after 10 min; n = 6, P > 0.05).


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.

In addition to increasing Ca2+ influx, it is possible that ionomycin could also have unloaded internal Ca2+ stores and thereby affected Ca2+ transients. Therefore, we also tested the effects of increasing external Ca2+ on basal cytoplasmic Ca2+ levels and spontaneous Ca2+ transients. Exposure of cells to 6 mM (n = 6) caused a progressive substantial increase in basal Ca2+ (to 205 ± 33%; P < 0.001) and reduced Ca2+ transients (to 47 ± 8.6; P < 0.005). Figure 9, B and C, shows an example and a summary of the effects of increasing extracellular Ca2+ on basal Ca2+ and Ca2+ transients.

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.

We also tested the effects of FK-506 and cyclosporin A on the rise in basal Ca2+ and inhibition of Ca2+ transients observed when cells were exposed to elevated external Ca2+. Exposure of cells to 6 mM caused an increase in basal Ca2+ and reduced Ca2+ transients (see above). After pretreatment of cells with FK-506 (1 µM), increased external Ca2+ increased basal Ca2+ to 221 ± 24% of control (P < 0.005), but Ca2+ transients were not significantly affected (i.e., 97 ± 20% of control; n = 6; P = 0.64). Pretreatment of cells with cyclosporin A had similar effects. In the presence of cyclosporin A (1 µM) elevated external Ca2+ increased basal Ca2+ to 225 ± 20% of control (P < 0.005), but the elevation in basal Ca2+ was not accompanied by effects on Ca2+ transients (i.e., 139 ± 31% of control; P = 0.89).


    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 P2Y 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 P2Y 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).

There are many reports in the literature about the nonselective cation conductance activated in visceral smooth muscles by muscarinic stimulation (e.g., Refs. 6, 15, 16, 27, 33). The cation conductance activated by muscarinic stimulation requires the simultaneous activation of pertussis toxin-sensitive (M2) receptors and PLC (M3)-linked receptors (6, 15, 16, 27, 33). Although this conductance is permeable to Ca2+, it is unclear whether much Ca2+ entry occurs via this pathway in physiological Ca2+ gradients (14). Pacaud and Bolton (29) reported that the global Ca2+ transients in guinea pig jejunal muscle cells in response to muscarinic stimulation included an initial transient phase that was dependent on release of Ca2+ from IP3 receptor-operated stores and a second, more sustained phase that depended on Ca2+ entry by a non-L-type Ca2+ conductance. We confirmed that the rise in basal Ca2+ in response to ACh was not blocked by nicardipine. Ca2+ entry that was responsible for the increase in basal Ca2+ occurred via a conductance blocked by Gd3+ and SKF-96365; however, further studies will be necessary to fully determine the nature of this conductance and whether it is related to the nonselective cation conductance activated in other GI muscles.

In previous studies ACh transiently increased STOCS in tracheal [Wade and Sims (37), Saunders and Farley (34)], esophageal [Hurley et al. (14)], jejunal [Benham and Bolton (2)], and vascular smooth muscle cells. Reductions in or cessation of STOCs often followed the initial stimulation. These authors concluded that the initial burst of STOCs following ACh application was due to release of Ca2+ from intracellular stores, and the inhibitory phase was due to unloading of stores because a similar pattern of STOC activity was observed when high concentrations of caffeine were applied. Application of ACh to murine myocytes did not result in a significant initial burst of STOC activity, but, within a short period, we observed decreased localized Ca2+ transients and reduction in STOCs. The reasons for the lack of an initial increase in STOCs in murine myocytes are not entirely clear, but this may be due to a relatively lower store content in phasic colonic muscles vs. tonic muscles such as tracheal and esophageal muscles.

Our experiments suggest that the inhibitory effects of ACh on STOCs were directed at inhibition of the localized Ca2+ transients that underlie STOCs. We concluded that this effect was not due to store unloading because: 1) the inhibitory effects of ACh on Ca2+ transients were absent (and in fact we observed a sustained increase in Ca2+ transients) when the rise in global Ca2+ in response to ACh was blocked by Gd3+ and SKF-96365; and 2) addition of caffeine after inhibition of Ca2+ transients by ACh caused a massive increase in Ca2+ and contraction of cells. Blockade of the rise in basal Ca2+ converted muscarinic responses into effects that were similar to those observed previously via stimulation of P2Y receptors (i.e., enhancement in localized Ca2+ transients; and a tendency for localized Ca2+ transients to organize into Ca2+ waves; see Ref. 1). Furthermore, the rise in basal Ca2+ and inhibition of spontaneous Ca2+ transients and STOCs in response to ACh were mimicked by treatment of cells with ionomycin and elevated external Ca2+, more generic stimulants of Ca2+ entry that were unlikely to involve signaling via metabotrophic receptors. These observations led to the conclusion that the rise in basal Ca2+ promotes the inhibition of Ca2+ transients (and STOCs). These findings highlight some interesting similarities between two stimuli (i.e., ACh and ATP) that have not previously been explained: both agonists couple to responses through IP3 formation, but ACh and ATP elicit opposite responses in colonic smooth muscles. In the case of muscarinic stimulation, the superposition of a mechanism to enhance basal Ca2+ suppresses the underlying IP3-driven increase in localized Ca2+ transients that naturally elicit activation of outward currents.

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 (Po) 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 Po 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.

The expression and distribution of the different isoforms of IP3 receptors in visceral smooth muscles may influence the pattern of responses to changes in [Ca2+]i and IP3. Studies have shown that the three isoforms of IP3 receptors respond differently to cytoplasmic agonists such as IP3, Ca2+, and ATP (25), and expression of multiple isoforms can result in complex spatiotemporal patterns. Future studies will investigate relative isoform expression in GI smooth muscle that manifest spontaneous IP3-dependent Ca2+ transients to determine the unique combination of receptors responsible for the patterns of Ca2+ transients in these cells. IP3 receptor expression becomes an important issue because studies have now shown that Ca2+-release events via IP3 receptor-operated channels contribute to resting membrane potential, the inhibitory response to purinergic neurotransmission (1, 18), and the development of excitatory responses to muscarinic stimulation.

There are several mechanisms by which elevated Ca2+ or Ca2+ binding proteins might decrease the tendency of IP3 to release Ca2+. IP3 receptors have intrinsic Ca2+ binding sites (23), and it is possible that direct binding of Ca2+ could decrease the sensitivity of IP3 receptors for IP3. Two of the sites for Ca2+ binding lie within the IP3 binding domain, and thus Ca2+ could exert antagonism on IP3 binding to its receptor (30). It is also possible that interactions with calmodulin could be involved in regulation or IP3 sensitivity. IP3 receptor subunits bind one calmodulin molecule in the absence of Ca2+ and two when Ca2+ is present (10). The effectiveness of IP3 in releasing Ca2+ from IP3 receptor-operated stores is reduced by calmodulin binding (28). Ca2+-dependent enzymes may also regulate the interactions of IP3 and its receptor. IP3 receptors are tightly associated with the immunophilin, FKBP12 (8), and calcineurin (which is expressed by colonic smooth muscle cells; G. Amberg, B. Perrino, and K. M. Sanders, unpublished observations) is physiologically associated with the IP3 receptor-FKBP12 complex. The immunosuppressant drug FK-506 binds to FKBP12 and inhibits its association with IP3 receptors (8). It is thought that anchoring calcineurin to IP3 receptors with FKBP12 controls the level of phosphorylation of the receptor and contributes to the Ca2+-dependent regulation of Ca2+ release from IP3 receptor-operated stores (9, 34). Our data showing that FK-506 decreased the inhibition of spontaneous Ca2+ transients associated with the rise in basal Ca2+ due to ACh or elevated external Ca2+ suggest that IP3 receptor-FKBP12 association, possibly via tethering of calcineurin, might be important in regulating Ca2+ sensitivity of IP3 receptors. Additionally, we found that cyclosporin A, which disrupts calcineurin activity by interacting with cyclophilin to form a calcineurin-inhibitory complex [see Perrino and Soderling (32)], also inhibited the tendency of elevated external Ca2+ to inhibit Ca2+ transients. Together, these data suggest that cytoplasmic Ca2+ regulation of IP3 receptors, possibly via the actions of calcineurin, might be an important aspect of muscarinic responses in colonic myocytes.

In summary, the response of colonic myocytes to muscarinic stimulation includes enhanced IP3 receptor-operated Ca2+ release that is potentially coupled to activation of Ca2+-dependent K+ channels. This tendency is suppressed in colonic cells by Ca2+ entry and a rise in basal Ca2+ that inhibits spontaneous Ca2+ transients. Ca2+-dependent negative feedback of Ca2+ release from IP3 receptor-operated channels may be an important step in defining whether muscarinic stimulation is an excitatory or inhibitory signal in GI muscles.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant DK-41315. The Noran confocal microscope was purchased by National Heart, Lung, and Blood Institute shared equipment Grant HL-44455.


    FOOTNOTES

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (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. Section 1734 solely to indicate this fact.

Received 28 June 2000; accepted in final form 4 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bayguinov, O, Hagen B, Bonev AD, Nelson MT, and Sanders KM. Intracellular calcium events activated by ATP in murine colonic myocytes. Am J Physiol Cell Physiol 279: C126-C135, 2000[Abstract/Free Full Text].

2.   Benham, CD, and Bolton TB. Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J Physiol (Lond) 381: 385-406, 1986[Abstract].

3.   Benham, CD, Bolton TB, and Lang RJ. Acetylcholine activates an inward current in single mammalian smooth muscle cells. Nature 316: 345-347, 1985[ISI][Medline].

4.   Boittin, FX, Coussin F, Macrez N, Mironneau C, and Mironneau J. Inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channel-dependent Ca2+ signalling in rat portal vein myocytes. Cell Calcium 23: 303-311, 1998[ISI][Medline].

5.   Bolton, TB, Prestwich SA, Zholos AV, and Gordienko DV. Excitation-contraction coupling in gastrointestinal and other smooth muscles. Annu Rev Physiol 61: 85-115, 1999[ISI][Medline].

6.   Bolton, TB, and Zholos AV. Activation of M2 muscarinic receptors in guinea-pig ileum opens cationic channels modulated by M3 muscarinic receptors. Life Sci 60: 1121-1128, 1997[ISI][Medline].

7.   Bonev, AD, Jaggar JH, Rubart M, and Nelson MT. Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries. Am J Physiol Cell Physiol 273: C2090-C2095, 1997[Abstract/Free Full Text].

8.   Bootman, MD, Berridge MJ, and Lipp P. Cooking with calcium: the recipes for composing global signals from elementary events. Cell 91: 367-373, 1997[ISI][Medline].

9.   Cameron, AM, Steiner JP, Roskams AJ, Ali SM, Ronnett GV, and Snyder SH. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux. Cell 83: 463-472, 1995[ISI][Medline].

10.   Cameron, AM, Steiner JP, Sabatini DM, Kaplin AI, Walensky LD, and Snyder SH. Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. Proc Natl Acad Sci USA 92: 1784-1788, 1995[Abstract].

11.   Cardy, TJ, and Taylor CW. A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors. Biochem J 334: 447-455, 1998[ISI][Medline].

12.   Cole, WC, Carl A, and Sanders KM. Muscarinic suppression of Ca2+-dependent K+ current in colonic smooth muscle. Am J Physiol Cell Physiol 257: C481-C487, 1989[Abstract/Free Full Text].

13.   Gordienko, DV, Bolton TB, and Cannell MB. Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells. J Physiol (Lond) 507: 707-720, 1998[Abstract/Free Full Text].

14.   Hurley, BR, Preiksaitis HG, and Sims SM. Characterization and regulation of Ca2+-dependent K+ channels in human esophageal smooth muscle. Am J Physiol Gastrointest Liver Physiol 276: G843-G852, 1999[Abstract/Free Full Text].

15.   Iino, M. Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. J Gen Physiol 95: 1103-1122, 1990[Abstract].

16.   Inoue, R, and Isenberg G. Acetylcholine activates nonselective cation channels in guinea pig ileum through a G protein. Am J Physiol Cell Physiol 258: C1173-C1178, 1990[Abstract/Free Full Text].

17.   Inoue, R, and Isenberg G. Effect of membrane potential on acetylcholine-induced inward current in guinea-pig ileum. J Physiol (Lond) 424: 57-71, 1990[Abstract].

18.   Inoue, R, and Isenberg G. Intracellular calcium ions modulate acetylcholine-induced inward current in guinea-pig ileum. J Physiol (Lond) 424: 73-92, 1990[Abstract].

19.   Jaggar, JH, Porter VA, Lederer WJ, and Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278: C235-C256, 2000[Abstract/Free Full Text].

20.   Koh, SD, Dick GM, and Sanders KM. Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle. Am J Physiol Cell Physiol 273: C2010-C2021, 1997[Abstract/Free Full Text].

21.   Kong, ID, Koh SD, and Sanders KM. Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes. Am J Physiol Cell Physiol 278: C352-C362, 2000[Abstract/Free Full Text].

22.   Kunze, WA, and Furness JB. The enteric nervous system and regulation of intestinal motility. Annu Rev Physiol 61: 117-142, 1999[ISI][Medline].

23.   Mak, DO, McBride S, and Foskett JK. Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition. Proc Natl Acad Sci USA 95: 15821-15825, 1998[Abstract/Free Full Text].

24.   Merritt, JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh BK, McCarthy SA, Moores KE, and Rink TJ. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J 271: 515-522, 1990[ISI][Medline].

25.   Mignery, GA, Johnston PA, and Sudhof TC. Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor. J Biol Chem 267: 7450-7455, 1992[Abstract/Free Full Text].

26.   Mironneau, J, Arnaudeau S, Macrez-Lepretre N, and Boittin FX. Ca2+ sparks and Ca2+ waves activate different Ca2+-dependent ion channels in single myocytes from rat portal vein. Cell Calcium 20: 153-160, 1996[ISI][Medline].

27.   Miyakawa, T, Maeda A, Yamazawa T, Hirose K, Kurosaki T, and Iino M. Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. EMBO J 18: 1303-1308, 1999[Abstract/Free Full Text].

28.   Nelson, MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

29.   Pacaud, P, and Bolton TB. Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells. J Physiol (Lond) 441: 477-499, 1991[Abstract].

30.   Patel, S, Morris SA, Adkins CE, O'Beirne G, and Taylor CW. Ca2+ -independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization. Proc Natl Acad Sci USA 94: 11627-11632, 1997[Abstract/Free Full Text].

31.   Perez, GJ, Bonev AD, Patlak JB, and Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol 113: 229-238, 1999[Abstract/Free Full Text].

32.   Perrino, BA, and Soderling TR. Biochemistry and pharmacology of calmodulin regulated phosphatase calcineurin. In: Calmodulin and Signal Transduction. New York: Academic, 1998, p. 170-236.

33.   Pietri, F, Hilly M, and Mauger JP. Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor. J Biol Chem 265: 17478-17485, 1990[Abstract/Free Full Text].

34.   Saunders, HH, and Farley JM. Pharmacological properties of potassium currents in swine tracheal smooth muscle. J Pharmacol Exp Ther 260: 1038-1044, 1992[Abstract].

35.   Sieck, GC, Kannan MS, and Prakash YS. Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells. Can J Physiol Pharmacol 75: 878-888, 1997[ISI][Medline].

36.   Vogalis, F, and Sanders KM. Cholinergic stimulation activates a non-selective cation current in canine pyloric circular muscle cells. J Physiol (Lond) 429: 223-236, 1990[Abstract].

37.   Wade, SM, and Sims GR. Muscarinic stimulation of tracheal smooth muscle cells activates large-conductance Ca2+-dependent K+ channel. Am J Physiol Cell Physiol 265: C658-C665, 1993[Abstract/Free Full Text].

38.   Wang, YX, and Kotlikoff MI. Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes. J Physiol (Lond) 523: 131-138, 2000[Abstract/Free Full Text].

39.   Zhang, BX, Zhao H, and Muallem S. Ca2+-dependent kinase and phosphatase control inositol 1,4,5-trisphosphate-mediated Ca2+ release. Modification by agonist stimulation. J Biol Chem 268: 10997-11001, 1993[Abstract/Free Full Text].

40.   Zhang, LB, and Buxton IL. Muscarinic receptors in canine colonic circular smooth muscle. II. Signal transduction pathways. Mol Pharmacol 40: 952-959, 1991[Abstract].

41.   Zhang, LB, Horowitz B, and Buxton IL. Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes. Mol Pharmacol 40: 943-951, 1991[Abstract].

42.   Zholos, AV, and Bolton TB. Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle. Br J Pharmacol 122: 885-893, 1997[Abstract].

43.   ZhuGe, R, Sims SM, Tuft RA, Fogarty KE, and Walsh JV, Jr. Ca2+ sparks activate K+ and Cl- channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J Physiol (Lond). 513: 711-718, 1998[Abstract/Free Full Text].


Am J Physiol Cell Physiol 280(3):C689-C700
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society