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Journal of Physiology (2002), 541.3, pp. 797-810
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.018796

Voltage-dependent inward currents of interstitial cells of Cajal from murine colon and small intestine

Young Chul Kim, Sang Don Koh and Kenton M. Sanders

	ABSTRACT

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Electrical slow waves in gastrointestinal (GI) muscles are generated by pacemaker cells, known as interstitial cells of Cajal (ICC). The pacemaker conductance is regulated by periodic release of Ca²⁺ from inositol 1,4,5-trisphosphate (IP₃) receptor-operated stores, but little is known about how slow waves are actively propagated. We investigated voltage-dependent Ca²⁺ currents in cultured ICC from the murine colon and small intestine. ICC, identified by kit immunohistochemistry, were spontaneously active under current clamp and generated transient inward (pacemaker) currents under voltage clamp. Depolarization activated inward currents due to entry of Ca²⁺. Nicardipine (1 µM) blocked only half of the voltage-dependent inward current. After nicardipine, there was a shift in the potential at which peak current was obtained (-15 mV), and negative shifts in the voltage dependence of activation and inactivation of the remaining voltage-dependent inward current. The current that was resistant to dihydropyridine (I_VDDR) displayed kinetics, ion selectivity and pharmacology that differed from dihydropyridine-sensitive Ca²⁺ currents. I_VDDR was increased by elevating extracellular Ca²⁺ from 2 to 10 mM, and this caused a +30 mV shift in reversal potential. I_VDDR was blocked by Ni²⁺ (100 µM) or mebefradil (1 µM) but was not affected by blockers of N-, P- or Q-type Ca²⁺ channels. Equimolar replacement of Ca²⁺ with Ba²⁺ reduced I_VDDR without effects on inactivation kinetics. BayK8644 had significantly less effect on I_VDDR than on I_VDIC. In summary, two components of inward Ca²⁺ current were resolved in ICC of murine small intestine and colon. Since slow waves persist in the presence of dihydropyridines, the dyhydropyridine-resistant component of inward current may contribute to slow wave propagation.

(Resubmitted 11 February 2002; accepted after revision 2 April 2002)
Corresponding author K. M. Sanders: Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA. Email: kent{at}physio.unr.edu

	INTRODUCTION

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Spontaneous electrical depolarizations, termed slow waves, are generated within the tunica muscularis in many regions of the gastrointestinal (GI) tract (see Szurszewski, 1987). These events time the phasic contractions of the gut (Tomita, 1981; Sanders, 1992). Many studies indicate that interstitial cells of Cajal (ICC) are the pacemaker cells that generate slow waves (Langton et al. 1989; Ward et al. 1994, 2000; Huizinga et al. 1995; Torihashi et al. 1995; Sanders, 1996; Dickens et al. 1999). From ICC, slow waves spread passively via gap junctions to neighbouring smooth muscle cells where depolarization activates voltage-dependent, dihydropyridine-sensitive Ca²⁺ channels (see model in Horowitz et al. 2000). The smooth muscle response to slow waves can be manifest either as Ca²⁺ action potentials or a sustained 'plateau' depolarization depending upon the voltage-dependent K⁺ channels available in smooth muscle cells. Influx of Ca²⁺ during slow waves activates phasic contractions (Ozaki et al. 1991). Recent work has suggested that the pacemaker current that generates slow waves is due to a voltage-independent, Ca²⁺-inhibited, non-selective cationic conductance in ICC (Thomsen et al. 1998; Koh et al. 1998, 2002). This conductance is activated by release of Ca²⁺ from intracellular stores via IP₃ receptors followed by Ca²⁺-stimulated uptake of Ca²⁺ by mitochondria (Suzuki et al. 2000; van Helden et al. 2000; Ward et al. 2000).

Besides initiating slow waves, ICC networks are a pathway for non-decremental propagation of slow waves in GI muscles (Horowitz et al. 1999). Slow waves are initiated from discrete points within sheets of GI muscles, and these 'initiation sites' may vary as a function of time (see Publicover & Sanders, 1984). Impalement of smooth muscle cells in any direction along the surface of a sheet reveals slow waves of relatively constant amplitude. Slow waves decay in amplitude, as predicted by cable equations, in regions of smooth muscle from which pacemaker ICC have been removed (e.g. Sanders et al. 1990). In other experiments in which the continuity of ICC networks was disrupted, slow waves were generated in regions where ICC networks remained but did not actively propagate to adjacent regions lacking ICC (Ördög et al. 1999).

The mechanism of slow wave propagation is poorly understood. Activation of the voltage-independent pacemaker current in successive cells appears to require cycling of Ca²⁺ from stores to mitochondria, but the rate of slow wave propagation (in excess of 5 mm s^-1; Christensen & Hauser, 1971; Bauer et al. 1985) is too fast to be explained on the basis of Ca²⁺ waves or cell-to-cell diffusion of second messengers. One hypothesis is that a voltage-dependent Ca²⁺ entry entrains pacemaker activity in networks of ICC. In such a mechanism, depolarization, caused by activation of the pacemaker conductance in one cell, might initiate Ca²⁺ entry in neighbouring cells. A localized rise in Ca²⁺ due to influx may increase the probability of Ca²⁺ release from IP₃ receptors in coupled cells (Iino, 1990; Hirose et al. 1998). Such a mechanism must be capable of functioning via Ca²⁺ channels that are resistant to dihydropyridines, because slow waves persist in the presence of micromolar dihydropyridines in many GI muscles (e.g. Ward et al. 1994; Malysz et al. 1995). In the present study we have characterized voltage-dependent Ca²⁺ currents in ICC from the murine colon and small intestine. We identified a dihydropyridine-resistant, voltage-dependent Ca²⁺ conductance that may provide entrainment of pacemaker activity in networks of ICC.

	METHODS

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The Institutional Animal Use and Care Committee at the University of Nevada approved the use and treatment of animals.

BALB/C mice (7-12 days old) of either sex were anaesthetized with chloroform and killed by cervical dislocation. Tissues of the small intestine, from 1 cm below the pyloric ring to the caecum, and proximal colon were removed and opened along the myenteric border. Luminal contents were removed with Krebs-Ringer bicarbonate (KRB) solution. Tissues were pinned to the base of a Sylgard dish and the mucosa was removed by sharp dissection.

Preparation of dispersed cells and cell cultures

Small strips of intestinal and colonic muscles were equilibrated in Ca²⁺-free Hanks' solution for 30 min and cells were dispersed, as previously described (Koh et al. 1998), with an enzyme solution containing: collagenase (Worthington Type II), 1.3 mg ml^-1; bovine serum albumin (Sigma, St Louis, MO, USA), 2 mg ml^-1; trypsin inhibitor (Sigma), 2 mg ml^-1; and ATP, 0.27 mg ml^-1. The tissues were placed in a 37 °C water bath for 25 min without agitation. After five washes with Ca²⁺-free phosphate-buffered saline (PBS) to remove the enzyme, the tissues were triturated with blunt pipettes. The resulting cell suspension was plated on murine collagen-coated (2.5 µg ml^-1, Falcon/BD) sterile glass coverslips in 35 mm culture dishes. The cultures were incubated at 37 °C in a 95 % O₂-5 % CO₂ incubator. The medium was changed after 24 h to smooth muscle growth medium (SMGM; Clonetics Corp., San Diego, CA, USA) containing stem cell factor (SCF; 5 ng ml^-1; Sigma) without antibiotic/antimyocotic. All experiments on single cells were performed on cells cultured for 1 day. In some experiments recordings were made from small networks of up to three cells, and these cells were cultured for 2-3 days to allow networks to form.

Before experiments, the glass coverslips containing ICC cultures were broken into several pieces with forceps. The small pieces of coverslips with adherent cells were transferred to the recording chamber (1.3 0.7 cm). The bath solution was warmed just prior to entering the experimental bath, and the bath volume (0.1 ml) was exchanged at a rate of 0.11 ml s^-1.

Immunohistochemical labelling of ICC

Cultured colonic cells were prepared for immunohistochemistry by fixation in acetone (4 °C; 10 min). After fixation, the cells were incubated in normal goat serum for 1 h (10 % in PBS). The cells were incubated overnight at 4 °C with a rat monoclonal antibody specific for Kit protein (ACK2, 5 µg µl^-1) in PBS. Immunoreactivity was detected using fluorescein isothiocyanate (FITC)-conjugated secondary antibody (FITC-anti-rat: Vector Laboratories, Burlingame, CA, USA; diluted 1:100 in PBS, 1 h, room temperature). Control cultures were prepared in a similar manner, but omitting ACK2 from the incubation solution. The cells were examined with a Bio-Rad MRC 600 (Hercules, CA, USA) confocal microscope with an excitation wavelength appropriate for FITC (488 nm). Final images were constructed with Comos software (Bio-Rad).

Electrophysiological experiments

Membrane currents were measured with the patch-clamp technique in the whole-cell configuration and using dialysing or perforated-patch conditions (cf. Horn & Marty, 1988). Glass pipettes with a resistance of 3~4 M were used to form gigaseals. Membrane currents were measured with an Axopatch-1D patch-clamp amplifier (Axon Instrument, USA) and filtered at 5 kHz. Uncompensated series resistance was between 2 and 4 M. The cell capacitance was measured by a ramp pulse (1 V s^-1) at a holding potential of -60 mV in every tested single cell and the value was used. Application of command pulses and acquisition of data were accomplished using pCLAMP v.5.5.1 (Axon Instruments, Union City, CA, USA) and a 12-bit A/D converter (TL-1, DMA interface, all from Axon Instruments). The data were analysed with pClamp (version 6.0.4, Axon Instruments) and Origin software (OriginLab Corp., Northampton, MA, USA). Linear leak currents were subtracted digitally.

For studies using the perforated-patch technique, nystatin was dissolved in dimethyl sulphoxide (DMSO) as a stock solution (0.15 mg/2 µl) and added to the pipette solution (0.15 mg ml^-1). Membrane potential was held at -60 mV during the formation of pores.

The temperature of the solution bathing the cells was monitored and maintained at 32 ± 0.4 °C (for the recording of pacemaking currents, n = 196) and 35 ± 0.1 °C (for the recording of I_Ca, n = 809). Data were tabulated and presented as means ± S.E.M. The n values given represent the number of cells on which specific protocols were performed. Differences between data sets were determined with Student's unpaired t test and considered significant when P < 0.05.

Solutions and drugs

The physiological salt solution used to bathe cells (Na⁺-Tyrode) contained (mM): 140 NaCl, 4.5 KCl, 2 CaCl₂, 1 MgCl₂, 5 glucose, 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (Hepes), and pH was adjusted to 7.4 by Tris. Na⁺ was replaced in some experiments by N-methyl-D-glucamine (NMDG) while keeping [Ca²⁺]_o constant at 2 mM. In other experiments external NaCl was totally replaced by CsCl.

For experiments to test the role of Ca²⁺ as a charge carrier the following solutions were used: (1) CaCl₂ was replaced by 2 mM MnCl₂ or omitted to produce nominally 0 mM CaCl₂; (2) CaCl₂ (2 mM) was replaced by equimolar BaCl₂; (3) external Ca²⁺ was increased to 10 mM by addition of excess CaCl₂. This solution was used in the absence or presence of nicardipine (1 µM). For recordings of pacemaking currents, the pipette solution contained (mM): 140 KCl, 5 MgCl₂, 2.7 K₂ATP, 0.1 Na₂GTP, 2.5 creatine phosphate (disodium salt), 5 Hepes and 0.1 ethyleneglycol-bis-(-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA), adjusted to pH 7.2 with Tris. This is referred to as the 'K⁺-rich pipette solution' in the text. Cs⁺-rich pipette solution for the recording of pacemaker and voltage-dependent inward currents contained (mM): 120 CsCl, 20 TEACl, 0.1 EGTA, 10 Hepes, 4 MgATP, 1 Na₂GTP, 2 creatine phosphate (disodium salt), adjusted to pH 7.2 with Tris.

Mibefradil dihydrochloride was a gift from Dr Eva-Maria Gutknecht and Dr Pierre Weber (Hoffmann-La Roche Ltd, Basel, Switzerland) and dissolved in distilled water at 5 mM and stored at -20 °C. -Agatoxin (-Aga-IV A) was purchased from Tocris Cookson (Bristol, UK) and Calbiochem (USA). All other drugs were purchased from Sigma (USA). -Aga-IV A, -conotoxin (-CTx-GVIA) and -conotoxin MVIIC (-CTx-MVIIC) were diluted in Na⁺-Tyrode solution to a final concentration of 100 nM, 1 µM, 1 µM, respectively. Nicardipine and BayK8644 were dissolved in ethanol at 1 mM and were tightly sealed in vials with protection from light and stored at -20 °C and 4 °C, respectively.

	RESULTS

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Identification of interstitial cells in cell cultures

ICC have been previously identified in cultures of small intestinal cells (see Thomsen et al. 1998; Koh et al. 1998), and similar techniques for cell dispersion, culturing and cell selection as previously described were used to select small intestinal ICC in the present study. This is the first report using cells cultured from dispersions of murine colonic smooth muscle, so we performed immunohistochemical analysis to determine the morphology of cells and small networks that displayed Kit-like immunoreactivity (Kit-LI). After 1 day in culture, cells with fusiform cell bodies, large, prominent nuclei, and multiple, thin processes extending from the nuclear region were observed with phase-contrast microscopy (Fig. 1A). Cells with this morphology were easily distinguishable from smooth muscle cells and were found to express Kit-LI (Fig. 1B).

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Figure 1. Phase contrast and fluorescence micrographs of cultured ICC from the murine colon A, a phase contrast image of a single multi-process ICC grown for 1 day in culture from a dispersion of cells from the tunica muscularis of the proximal colon. Note the fusiform cell body, prominent nucleus, and multiple thin processes extending from the nuclear region. Cells with this morphology expressed Kit-like immunoreactivity, as shown in the fluorescence micrograph, B. Scale bars in A and B, 25 µm.

Spontaneous rhythmic electrical activity of murine colonic ICC

Using the conventional whole cell patch-clamp technique (at 32 ± 0.4 °C with K⁺-rich pipette solution) we observed spontaneous electrical rhythmicity in cells cultured for 1 day (n = 196). Recordings were made from single cells and from small networks of cells (typically three cells) that had structural characteristics like those with Kit-LI (see Fig. 1). Single ICC had resting potentials averaging -39 ± 1.4 mV (n = 52 cells evaluated in current clamp; Fig. 2A) and capacitance of 25 ± 0.6 pF (n = 125). The cells displayed spontaneous inward currents ranging in amplitude from -19 pA to -257 pA and averaging -50 ± 2.4 pA (n = 125 single cells). Approximately 75 % of the single cells from which recordings were made had an irregular frequency or long periods of quiescence between spontaneous inward currents. Cells with regular inward currents averaged 6 ± 0.5 cycles min^-1 (n = 32) at a holding potential (V_h) of -60 mV (Fig. 2B). Cells in small networks had much more robust and regular spontaneous inward currents (Fig. 2C). We have previously shown that the spontaneous inward currents in small intestinal ICC are not blocked by dihydropyridines (Koh et al. 1998), and similar findings were made in colonic ICC.

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Figure 2. Spontaneous inward currents from cultured single and networked ICC in murine colon A, spontaneous membrane potential oscillations were recorded from a single ICC under current clamp (I = 0) in the presence of nicardipine (1 µM). The pipette solution contained 140 mM K+. B, spontaneous inward currents were recorded from a single ICC under voltage clamp in the presence of nicardipine (1 µM). The cell was held at -80 mV, and the pipette solution contained 140 mM K+. C, spontaneous inward currents were more robust and regular when recorded from ICC in networks. Cell was held at -80 mV with a K+-rich pipette solution in the pipette. D, Cs+-rich pipette solution used to enhance resolution of voltage-dependent Ca2+ currents did not inhibit spontaneous inward currents.

After confirmation that ICC generated spontaneous inward currents, we focused our study on the characterization of voltage-dependent inward currents (I_VDIC) expressed by these cells. For these studies we used only single ICC to avoid problems with space clamp. The Cs⁺-rich pipette solution (see Methods for details) used to block outward currents and enhance resolution of I_VDIC did not block spontaneous inward currents or corresponding voltage transients under current clamp in these cells (e.g. Fig. 2D). The frequency of spontaneous inward currents in single ICC was such that voltage-clamp protocols could be used to study I_VDIC with pulses applied between spontaneous currents. Using Cs⁺-rich pipette solution, spontaneous currents in colonic ICC averaged -51 ± 5.8 pA (n = 237) and -55 ± 4.3 pA (n = 367) in amplitude in the presence and absence of nicardipine (1 µM), respectively. Similar observations were made in the small intestinal ICC (e.g. spontaneous currents in these cells were -49 ± 3.5 pA (n = 98) and -48 ± 6.5 pA (n = 107) in amplitude in the presence and absence of nicardipine (1 µM), respectively.

Characterization of voltage-dependent inward currents in ICC

Voltage clamp experiments to characterize I_VDIC in colonic and small intestinal ICC were performed with the perforated patch, whole-cell configuration at 35 ± 0.1 °C (n = 809), before and after nicardipine (1 µM). Single ICC used in this portion of the study had average capacitances of 27 ± 0.4 pF (n = 604, colon) and 26 ± 0.5 pF (n = 205, small intestine). Capacitance and spontaneous inward currents were recorded in each case prior to administration of voltage-clamp protocols to elicit I_VDIC to verify that the cells were ICC.

For the characterization of I_VDIC the cells were bathed in a solution containing 2 mM Ca²⁺. The cells were held at -80 mV and stepped for 420 ms every 12 s from -70 to +50 mV in 10 mV increments. Depolarization induced transient currents that consisted of a rapid activation phase followed by partial inactivation (Fig. 3A). Peak currents were observed near 0 mV in colonic ICC (Fig. 3B), and the current reversed at +33 ± 1.3 mV (n = 37). The dihydropyridine Ca²⁺ channel blocker, nicardipine (1 µM), reduced the peak of I_VDIC to 56 ± 4.6 % (n = 15, Fig. 3A and B). The inactivation time constant was not significantly changed from control (67 ± 3.0 ms, n = 289) by nicardipine treatment (86 ± 6.2 ms, n = 68) at 0 mV (P > 0.05). Peak current, however, was observed between -20 and -10 mV after nicardipine, and the current remaining after nicardipine reversed at +21 ± 4.1 mV (n = 9; P < 0.05 compared with reversal of I_VDIC).

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Figure 3. Voltage-dependent inward currents (IVDIC and IVDDR) in ICC under whole-cell configuration IVDIC was generated by depolarizing pulses from -70 to +50 mV. Data were obtained using the perforated patch technique (A-C from colonic ICC and D-F from small intestinal ICC). A and D, currents were elicited by voltage steps from a holding potential of -80 mV (currents displayed resulted from test potentials from 0 to +40 mV, voltage protocol shown as inset in A). Currents were generated before (top current traces) and after nicardipine (1 µM; bottom traces in A and D). B and E, summaries of current-voltage relationships of IVDIC () and current remaining after nicardipine (IVDDR; ). C and F, voltage dependences of activation and inactivation of IVDIC () and IVDDR () are shown as plots of normalized peak currents. For steady-state activation, maximum conductance was determined from the peak inward current and corrected for the change in driving force at each test potential. Half-activation and inactivation potentials were determined from Boltzmann functions fitted to the data. Data are plotted as means ± S.E.M.

Protocols to analyse steady-state activation and inactivation properties of the I_VDIC were used in additional studies. To evaluate steady-state activation relationships, the peak conductance at each test potential was calculated using the equation: I_Ca = g_Ca (V - E_rev) where g_Ca, V and E_rev are peak conductance, test potential and reversal potential, respectively. The results were plotted and fitted with a Boltzmann equation. In colon, the half-activation voltage of I_VDIC was -12 ± 0.7 mV (n = 37) with a slope factor (k) of 6 ± 0.3 (n = 37; Fig. 3C). After nicardipine treatment, half-activation voltage (I_VDDR) was shifted to the left (-19 ± 3.7 mV, n = 10; P < 0.05). The slope factor was increased to 7 ± 0.6 (Fig. 3C).

A modified double-pulse protocol was used to measure steady-state inactivation of the I_VDIC as a function of membrane potential. Conditioning steps from -120 to +20 mV were applied for 3.75 s. After a 7 ms step to -60 mV, the membrane potential was stepped to 0 mV for 1 s. Resulting currents were normalized to the maximum current obtained after a conditioning potential of -120 mV (I/I_max) and plotted as a function of the conditioning potential. The data were fitted by a Boltzmann equation. ICC of colon had a half-inactivation voltage (V_0.5) averaging -55 ± 3.0 mV with slope factor (k) of 6 ± 1.6 (n = 5; Fig. 3C). After nicardipine treatment, the half-inactivation voltage (I_VDDR) was shifted to the left (-70 ± 5.2 mV, n = 6; P < 0.01) and the slope factor increased to 7 ± 0.2 (Fig. 3C).

Cells from the small intestine demonstrated similar currents in response to depolarization (n = 15, Fig. 3D). Peak currents were observed between -10 and 0 mV in small intestinal ICC (Fig. 3E), and the current reversed at +35 ± 2.0 mV (n = 15 small intestine cells). The inactivation time constant did not change before and after nicardipine treatment. Peak current, however, was observed between -20 and -10 mV after nicardipine, and the current remaining after nicardipine reversed at +24 ± 2.6 mV (n = 5). The half-activation voltage of the current from the small intestinal ICC occurred at -15 ± 0.9 mV with a slope factor (k) of 6 ± 0.8 (n = 10; Fig. 3F). After nicardipine treatment, the half-activation voltage (I_VDDR) was shifted to the left (-26 ± 4.1 mV, n = 7, P < 0.01) and the slope factor was 6 ± 0.6 (Fig. 3F). The half-inactivation voltage of I_VDIC from small intestinal ICC was -54 ± 1.4 mV with a slope factor (k) of 7 ± 0.4 (n = 6; Fig. 3F). After nicardipine treatment, the half-inactivation voltage (I_VDDR) was shifted to the left (-66 ± 1.9 mV, n = 5, P < 0.01) and the slope factor decreased to 6 ± 1.2 (Fig. 3F). The data demonstrate that the current remaining after nicardipine treatment had different kinetics from the net voltage-dependent inward current under control conditions.

Reversal potentials (E_rev) of the voltage-dependent inward currents for colon cells were further evaluated using protocols to determine the reversal of instantaneous tail currents. Membrane currents were elicited in cells under perforated patch conditions by a double-pulse protocol in which membrane potential was depolarized from -80 mV to 0 or -10 mV for 5-7 ms and then stepped to potentials ranging from -30 to +60 mV. Membrane currents were measured after capacitance transients relaxed. Using these protocols, instantaneous tail currents of I_VDIC reversed at +31 ± 2.6 mV (n = 6 colon cells, Fig. 4A and C). After treatment with nicardipine, E_rev of I_VDDR shifted to an average of +17 ± 2.4 mV (n = 17) in normal Na⁺ gradients (Fig. 4B and C).

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Figure 4. Reversal potential of IVDIC and IVDDR using instantaneous tail current analysis A, cells were held at -80 mV, stepped to 0 mV for 7 ms to activate IVDIC, and then stepped to various potentials (-10 to +50 mV; data displayed were obtained with test potentials of 0 to +50 mV using voltage protocol shown in the inset). B, the same protocols were applied after nicardipine (1 µM) to determine the reversal of IVDDR tail currents. Representative currents in panels A and B are from different cells. C, a summary plot of the amplitude of tail currents as a function of test potential. , reversal of IVDIC (n = 6); , the reversal of the current remaining after nicardipine (IVDDR; n = 17). Note that IVDDR reverses at more negative potentials.

Ion selectivity of I_VDDR

The ionic nature of the voltage-dependent inward currents was investigated in a further series of experiments. V_h was held at -80 mV and 420 ms depolarizing steps were applied to 0 mV every 12 s to record maximal voltage-dependent inward current. When [Ca²⁺]_o was increased from 2 to 10 mM, peak I_VDIC in colonic ICC was increase d by 240 ± 31 % of control (n = 7; Fig. 5A). Data from test protocols in 2 and 10 mM Ca²⁺ were plotted as I-V curves (Fig. 5B). The reversal of I_VDIC shifted significantly in the positive direction by 10 ± 2 mV after switching to the external solution from 2 to 10 mM Ca²⁺ (n = 5, P < 0.05).

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Figure 5. The effects of external Ca2+on IVDIC and IVDDRin colonic ICC A, representative traces shown in 2 (a), 10 (b) and 0 (c) mM Ca2+. B, changes in IVDIC caused by switching external solution from 2 to 10 to 0 mM Ca2+. Data were obtained from steps from -80 to 0 mV every 12 s. C, current-voltage relationship for IVDIC recorded from seven cells in 2 () and 10 () mM Ca2+. D, IVDDR in 2 (a) and 10 (b) mM Ca2+ in the presence of nicardipine (1 µM). E, changes in peak caused by switching external solution from 2 to 10 mM Ca2+. Data were obtained from steps from -80 to 0 mV every 10 s. F, current-voltage relationship for IVDDR recorded from five cells in 2 () and 10 () mM Ca2+.

I_VDIC was reduced in nominally Ca²⁺-free solution (to 0.3 ± 0.1 % of control; n = 3) or by equimolar replacement of Ca²⁺ with Mn²⁺ (to 0.2 ± 0.1 % of control; n = 4). The effects of changing [Ca²⁺]_o on the I_VDIC were plotted as a function of time in Fig. 5A.

The current remaining after treatment with nicardipine (i.e. I_VDDR) was also affected by changes in extracellular Ca²⁺ concentration. After nicardipine, when [Ca²⁺]_o was increased from 2 to 10 mM, peak I_VDDR in colonic ICC was increased by 355 ± 70 % (n = 5; Fig. 5C, P < 0.05). Data from test protocols in 2 and 10 mM Ca²⁺ were plotted as I-V curves (Fig. 5D). I_VDDR was reduced in nominally Ca²⁺-free solution (to 0.3 ± 0.1 % of control; n = 3). I_VDDR reversed at +20 ± 3.5 mV in the presence of nicardipine (n = 4), and the reversal potential shifted to more positive potentials with 10 mM [Ca²⁺]_o (+56 ± 1.5 mV, n = 5; P < 0.01). I_VDDR in small intestinal ICC was increased by 419 ± 83 % and reversed at 27 ± 3.9 mV (n = 5) in the presence of nicardipine and normal Ca²⁺ (2 mM), and the reversal potential shifted to more positive potentials with 10 mM [Ca²⁺]_o (+54 ± 2.1 mV, n = 5; P < 0.01, not shown).

Equimolar replacement of Ca²⁺ with Ba²⁺ did not significantly change the amplitude of peak current (81 ± 6 % of control peak current, n = 12, P > 0.1), but slowed the kinetics of inactivation (Fig. 6A). Figure 6B shows the time constants of inactivation of I_VIDC in the presence of Ca²⁺-PSS (82 ± 16 ms) and Ba²⁺-PSS (121 ± 16 ms, n = 12, P < 0.01). After addition of nicardipine, switching the external solution to Ba²⁺-PSS reduced I_VDDR to 39 ± 8 % (colon; n = 14; P < 0.01) and 49 ± 15 % (small intestine; n = 9; P < 0.05) of control (Fig. 6C-F shows data for colon). In the presence of nicardipine, replacement of Ca²⁺-PSS with Ba²⁺-PSS did not affect the inactivation kinetics of I_VDDR (see Fig. 6C and D). The lack of enhancement of the magnitude of I_VDIC (which is composed of L-type Ca²⁺ current and I_VDDR) by switching from Ca²⁺-PSS to Ba²⁺-PSS may have been due to the offsetting effects of inhibition of I_VDDR by Ba²⁺-PSS.

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Figure 6. The effect of external Ba2+on IVDIC and IVDDR in murine colon A, representative traces showing IVDIC from a colonic ICC before () and after () equimolar Ba2+ replacement for Ca2+. Voltage steps from -80 to 0 mV were applied repetitively. B, the summary of inactivation time constants of IVDIC at 0 mV before () and after () equimolar Ba2+ replacement for Ca2+. C, representative traces of IVDDR from a colonic ICC before () and after () equimolar Ba2+ replacement for Ca2+. Voltage steps from -80 to 0 mV were applied in the presence of nicardipine (1 µM). D, the summary of inactivation time constants of IVDDR at 0 mV before () and after () equimolar Ba2+ replacement for Ca2+. Experiments were performed in the presence of nicardipine (1 µM) to isolate IVDDR. E, change in peak current due to equimolar Ba2+ replacement for Ca2+ on IVDDR as a function of time. Data were obtained for steps from -80 to 0 mV every 12 s. F, current-voltage relationship for peak IVDDR in Ca2+-PSS () and Ba2+-PSS ().

Replacement of Na⁺ with NMDG decreased I_VDIC to 51 ± 3 % of control (n = 11; Fig. 7A and B; P < 0.01). After Na⁺ replacement, addition of nicardipine (1 µM) further decreased current to 2 ± 1.4 % (n = 3) of peak I_VDIC. This experiment was also performed in the reverse order. Addition of nicardipine first reduced I_VDIC to 55 ± 3.5 %, and then replacement of Na⁺ with NMDG⁺ reduced I_VDDR to 13 ± 7.4 % (n = 4) of peak I_VDIC in colonic ICC (Fig. 7C and D) without significant change in the E_rev of I_VDDR (i.e. 17 ± 3.4 mV to 16 ± 3.2 mV; P > 0.05; n = 4; data not shown).

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Figure 7. The effect of external Na2+ replacement on IVDIC and IVDDR in colonic ICC A, IVDIC elicited from a colonic ICC by voltage steps from -80 to 0 mV (a), after replacement of external Na+ with NMDG (b), after addition of nicardipine (1 µM) to isolate IVDDR (c) and after washout of nicardipine (d). Traces a-d were taken at the time points noted in B. B, peak currents elicited by stepping from -80 to 0 mV every 12 s during equimolar replacement of Na+ with NMDG and after addition of nicardipine. C, currents elicited from a colonic ICC by voltage steps from -80 to 0 mV (a), after addition of nicardipine (1 µM) to isolate IVDDR (b) and after replacement of external Na+ with NMDG (c). Traces a-c were taken at the time points noted in D. D, peak currents elicited by steps from -80 to 0 mV every 12 s during exposure to nicardipine and after equimolar replacement of Na+ with NMDG. NMDG markedly reduced IVDDR in a reversible manner.

Pharmacology of I_VDIC in ICC

BayK8644 (0.4 µM) increased I_VDIC to 161 ± 19.6 % of control in colonic ICC (n = 13), and Fig. 8A and B shows an example of the effects of BayK8644 as a function of time. We also tested the effects of BayK8644 after replacing Na⁺ with NMDG⁺. The current remaining after NMDG⁺ was enhanced by BayK8644 (0.4 µM) by 170 ± 10 % (n = 5) compared with NMDG⁺-resistant current (Fig. 8C and D). After nicardipine, the effects of BayK8644 (0.4 µM) on I_VDDR were significantly less than the effects of BayK8644 on I_VDIC in colonic ICC (P < 0.05). BayK8644 increased the current remaining after nicardipine to 128 ± 8 % of maximal I_VDDR (n = 15, P < 0.01). It should also be noted that the time course of the effects of BayK8644 on I_VDDR were significantly slower than the effects of this compound on I_VDIC (Fig. 8E and F). These data suggest that there is a highly sensitive component of current to BayK8644 in I_VDIC (i.e. the component blocked by nicardipine), and I_VDDR is significantly less sensitive to BayK8644.

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Figure 8. The effect of BayK8644 on IVDIC and IVDDR in colonic ICC A, IVDIC elicited from a colonic ICC by voltage steps from -80 to 0 mV at various times after addition of BayK8644 (0.4 µM). Traces a-c were taken at the time points noted in B. B, change in peak current after addition of BayK8644 (0.4 µM) as a function of time. Data obtained from steps from -80 to 0 mV every 12 s. C, IVDIC elicited from a colonic ICC by voltage steps from -80 to 0 mV after replacement of external Na+ with NMDG and after subsequent addition of BayK8644 (0.4 µM). Traces a-c were taken at the time points noted in D. D, peak currents elicited by steps from -80 to 0 mV every 12 s after replacement of Na+ with NMDG and after addition of BayK8644. E, effects of BayK8644 (0.4 µM) on IVDDR elicited from a colonic ICC (i.e. in the presence of nicardipine) by voltage steps from -80 to 0 mV. Traces a-c were taken at the time points noted by the same letters in F. F, peak currents elicited by steps from -80 to 0 mV every 12 s during exposure to BayK8644 in the presence of nicardipine. The effects of BayK8644 on IVDDR were significantly less than the effects of this compound on IVDIC (see text for details).

The inorganic Ca²⁺ channel blocker, Ni²⁺ (40, 50, 100 and 200 µM), reduced peak I_VDIC to 86 ± 2.5 %, 78 ± 4.9 %, 26 ± 3.1 % and 23 ± 6.4 % of control in ICC of colon (n = 7, 4, 8, 6, respectively). In the presence of nicardipine (which reduced I_VDIC to 56 ± 4.6 % of control), nickel (100 µM) produced a further reduction of I_VDDR to 30 ± 9.2 % of I_VDIC at 0 mV (Fig. 9A and B; n = 3). In small intestinal ICC, addition of 100 µM nickel in the presence of 1 µM nicardipine reduced the peak of the I_VDIC to 39 ± 1.7 % of I_VDIC (n = 8, data not shown). The dihydropyridine-insensitive current was partially inhibited by Ni²⁺, but showed less sensitivity than typical low voltage-activated Ca²⁺ currents (Ganitkevich & Isenberg, 1990).

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Figure 9. Inhibition of IVDDR in small intestinal ICC by nickel and mibefradil A, representative currents elicited by test potentials from -80 to of 0 mV in control (IVDIC; ), and after subsequent addition of nicardipine (1 µM) and nickel (100 µM). Traces displayed were obtained by steps to 0 mV. B, current-voltage relationship showing IVDIC (), IVDDR (current after nicardipine, 1 µM; ), and the effects of nickel on IVDDR (100 µM; ). C, typical currents elicited from a small intestinal ICC by voltage steps from -80 to 0 mV after application of nicardipine (1 µM) and mibefradil (1 µM). Traces displayed (a-d) were taken at the time points noted in D. D, peak current elicited by voltage step from -80 to 0 mV applied every 12 s after addition of nicardipine (1 µM) and mibefradil (0.3 µM). Nicardipine blocked approximately half of IVDIC and mibefradil blocked the remaining current (IVDDR). The effects of mibefradil were difficult to wash out. E, typical currents elicited from a colonic ICC by voltage steps from -80 to 0 mV after application of mibefradil (0.3 µM) and nicardipine (1 µM) Traces displayed (a-d) were taken at the time points noted in D. F, peak current elicited by voltage step from -80 to 0 mV applied every 12 s after addition of mibefradil (0.3 µM) and nicardipine (1 µM). At this concentration, mibefradil blocked approximately half of IVDlC and nicardipine blocked the remaining current. G and H, the concentration-response relationship for the effects of mibefradil on IVIDC and IVDDR (i.e. mibefradil concentrations added in the presence of nicardipine, 1 µM), respectively. Data in G were fitted with a two-site competition equation, and dotted lines show IC50 values for the fit. In H the block by mibefradil was fitted with a one-site competition equation, and the IC50 is denoted by the dotted lines.

We also tested the effects of mibefradil on I_VDIC and I_VDDR in colonic ICC. As shown in Fig. 9C and D, in the presence of 1 µM nicardipine, mibefradil (0.3 µM) completely blocked I_VDDR. In experiments conducted in the opposite order, mibefradil decreased I_VDIC to 49 ± 8 % (n = 8). Application of nicardipine (1 µM) after mibefradil blocked most of the remaining current (Fig. 9E and F). We also tested the concentration-response relationship of the block of I_VIDC and I_VDDR by mibefradil (Fig. 9G and H). The concentration-response relationship for the effects of mibefradil on I_VIDC is shown in Fig. 9G. These data were fitted with a two-site competition equation with IC₅₀ at 23 nM and 1.8 µM (n = 9). This observation suggests that mibefradil has at least two binding sites affecting I_VDIC. After nicardipine, the concentration-response relationship was fitted with a single site (Fig. 9H, IC₅₀ = 73 nM; n = 7). Thus, the lower affinity block by mibefradil was blocked by nicardipine, and I_VDDR was more sensitive to mibefradil than the dihydropyridine-sensitive component of I_VIDC.

Finally, I_VDDR was not affected by TTX (2.5 µM). I_VDDR was also insensitive to N-, P- and Q-type Ca²⁺ channel blocking toxins (i.e. -Aga-IV A (500 nM), -conotoxin (-CTx-GVIA; 1 µM) and -conotoxin MVIIC (-CTx-MVIIC; 1 µM) (n = 2, respectively, in ICC of colon, data not shown).

In order to determine the role of the two components of I_VDIC in pacemaker activity, it would be extremely useful to identify concentrations of blockers that would inhibit either component of inward current without blocking the spontaneous inward (pacemaker) currents in ICC. Therefore, we tested the effects of 1 µM nicardipine and 0.3 µM mibefradil (which blocked over 90 % of the I_VDDR; see Fig. 9G and H) on spontaneous inward currents in single ICC. With Cs⁺-rich pipette solution and at a holding potential of -80 mV, the spontaneous inward current small intestinal ICC (average amplitude -44 ± 4.7 pA) were not blocked by either nicardipine (1 µM; amplitude -43 ± 3.9 pA) or by subsequent addition of mibefradil (0.3 µM; amplitude -44 ± 5.5 pA) in the continued presence of nicardipine (n = 16; see Fig. 10).

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Figure 10. The effects of nicardipine and mibefradil on spontaneous inward currents in small intestinal ICC A, representative traces elicited by test potentials from -80 to 0 mV in the presence of nicardipine (1 µM) and nicardipine plus mibefradil (0.3 µM). B and C, neither nicardipine nor nicardipine and mibefradil together blocked spontaneous inward currents in isolated ICC. The holding potential was -80 mV.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

We have investigated voltage-dependent inward currents (I_VDIC) in cultured ICC from the murine colon and small intestine. The spontaneous rhythmic properties of small intestinal ICC have been described previously in detail. This is the first report of activity from murine colonic ICC. Like small intestinal ICC, these cells generated spontaneous inward currents. As with small intestinal ICC, these currents were more robust and regular when colonic ICC were coupled into small networks (Koh et al. 1998). However, since the purpose of the present study was to investigate voltage-dependent currents, we chose cells that were isolated, and the isolated cells had smaller and more irregular spontaneous inward currents. The characteristics of the spontaneous inward currents in colonic ICC and how they compare with similar currents recorded from small intestinal ICC were not studied. Instead we sought to determine the mechanisms for voltage-dependent Ca²⁺ entry in ICC.

The voltage-dependent inward current generated by depolarization was composed of at least two components: a typical, dihydropyridine-sensitive (L-type) Ca²⁺ current, and a dihydropyridine-resistant current (I_VDDR). The two components of currents were separated on the basis of kinetic behaviour, ion permeability and pharmacology. The following are differences between I_VDDR and the dihydropyridine-sensitive current (see Table I): (i) addition of nicardipine shifted the voltage-dependence of activation and inactivation to more negative potentials; (ii) nicardipine caused a shift in reversal potential to less positive potentials; (iii) Ba²⁺, which has greater permeability than Ca²⁺ through L-type channels and equal permeability to Ca²⁺ through cardiac T-type channels (Ertel & Ertel, 1997), inhibited I_VDDR; (iv) replacement of Na⁺ with NMDG⁺ inhibited I_VDDR, but this occurred without shifting the reversal potential, suggesting that NMDG⁺ may be a blocker of the conductance; (v) BayK8644 only slightly increased I_VDDR; (vi) mibefradil was more potent as a blocker of I_VDDR than the dihydropyridine-sensitive current. At least a portion of I_VDDR is carried by Ca²⁺ since increasing extracellular Ca²⁺ increased the current and shifted the reversal potential towards more positive potentials. Removing extracellular Ca²⁺ inhibited I_VDDR. Ni²⁺ and mibefradil reduced I_VDDR, but the sensitivity of the current towards Ni²⁺ was lower than for T-type Ca²⁺ channels (e.g. Arnoult et al. 1998; Hagiwara et al. 1988). Blockers of N-, P- and Q-type Ca²⁺ channels had no effect on I_VDDR. Thus, we believe that I_VDDR is due to a Ca²⁺-selective conductance, but its properties are different from low voltage-activated (T-type) Ca²⁺ currents and other high voltage-activated currents. Molecular and single-channel studies will undoubtedly be necessary to identify the channels responsible for I_VDDR.

We have now identified three voltage-dependent inward currents in murine GI smooth muscle cells and ICC. In interstitial cells the voltage-dependent inward current is composed of dihydropyrine-sensitive and dihydropyridine-resistant components, as described in this study. In murine colonic smooth muscle cells we have previously reported that the voltage-dependent inward current is composed of dihydropyrine-sensitive (L-type) Ca²⁺ current and a voltage-dependent non-selective cation current (Koh et al. 2001). A previous study has also reported a dihydropyridine-insensitive conductance in mesenteric arterial smooth muscle cells (see Morita et al. 1999). Table 1 compares the similarities and differences between these conductances. This comparison suggests that the dihydropyridine-resistant current in ICC is characteristically different from the voltage-dependent non-selective cation current expressed by smooth muscle cells of the murine colon.

A previous study reported two components to the Ca²⁺ current in freshly isolated ICC from the canine colon (Lee & Sanders, 1993). The portion of the Ca²⁺ current that was not blocked by dihydropyridines in colonic ICC differed from I_VDDR recorded in murine ICC. The activation of the current in colonic ICC was shifted to more negative potentials, and the current was typically apparent in recordings with the permeabilized patch technique as a pronounced hump at potentials negative to the potential where L-type Ca²⁺ current could be resolved. The current in colonic ICC was enhanced by negative holding potentials and inactivated by more positive holding potentials, and the current was blocked by relatively low concentrations of Ni²⁺ and insensitive to nifedipine. Thus the characteristics of the colonic current were more consistent with a T-type Ca²⁺ conductance. However, until both channels can be identified and expressed in the absence of contaminating inward currents, it will be difficult to assess the degree of similarity between the dihydropyridine-resistant Ca²⁺ conductances in canine and murine ICC. The results of this study, however, are consistent with previous work (Lee & Sanders, 1993) in showing that dihydropyridine-resistant Ca²⁺ currents are an intrinsic property of ICC.

The presence of voltage-dependent, dihydropyridine-resistant Ca²⁺ channels in ICC of mouse and dog suggests a role for these conductances in the functions of these cells. Evidence suggests that ICC are pacemaker cells in GI muscles (see Introduction), and the network formed by pacemaker ICC is necessary for active propagation of slow waves around and along the gut (for review see Horowitz et al. 1999). In many GI muscles generation of slow waves and propagation of these events is unaffected by concentrations of dihydropyridines that quantitatively block L-type Ca²⁺ channels in smooth muscle cells. Several studies have shown that the pacemaker mechanism and the conductance responsible for pacemaker currents are voltage-independent (Ohba et al. 1975; Thomsen et al. 1998; Koh et al. 1998, 2002; Ward et al. 2000; i.e. the frequency of pacemaker currents and the openings of pacemaker channels are not significantly affected by changing voltage over the physiological range). The pacemaker current in ICC is due to a voltage-independent non-selective cation conductance (Thomsen et al. 1998; Koh et al. 1998). This conductance is controlled via Ca²⁺ cycling from the sarcoplasmic reticulum (through IP₃ receptors) to mitochondria (Ward et al. 2000). Mitochondrial uptake of Ca²⁺, perhaps by reducing local Ca²⁺ in a restricted volume between the mitochondria and the plasma membrane, appears to be the stimulus for activation of pacemaker current (Koh et al. 2002). The primary event, however, that initiates the pacemaker mechanism is release of Ca²⁺ from IP₃ receptors (Ward et al. 2000; van Helden et al. 2000), and animals lacking IP₃R1 receptors fail to generate slow waves (Suzuki et al. 2000). Blocking this mechanism at any stage (IP₃ receptor anatagonism, mitochondrial inhibitors, etc.) blocks propagation of slow waves (S. M. Ward and K. M. Sanders, unpublished observation). At the present time it is unclear how such a mechanism can propagate slow waves without decrement over many centimetres at rates in excess of 5 mm s^-1 (see Publicover & Sanders, 1984; Sanders et al. 1990).

We suggest that the dihydropyridine-resistant Ca²⁺ current observed in the present study might be a necessary component of slow wave propagation. Propagation of slow waves (which is really entrainment of an array of spontaneously active pacemaker units in networks of ICC) might be accomplished by a stimulus that increases the probability of Ca²⁺ release from IP₃ receptors in coupled cells. We propose the following mechanism (see Fig. 11): Each individual ICC contains the basic mechanism for initiation of pacemaker current. Spontaneous activation of pacemaker (inward) current in one cell within a network causes depolarization and activation of voltage-dependent Ca²⁺ entry in surrounding, coupled cells. Both dihydropyridine-sensitive and -resistant Ca²⁺ conductances may contribute to Ca²⁺ entry, but the fact that dihydropyridines do not block slow wave propagation suggests that the dihydropyridine-resistant current is sufficient to sustain propagation. Local increases in Ca²⁺ near IP₃ receptors can increase the probability of channel opening and Ca²⁺ release (Iino, 1990; Hirose et al. 1998). Thus, local Ca²⁺ entry might serve as the means to entrain (i.e. phase-advance) the pacemaker mechanism in the cells around the cell where the initial pacemaker current occurs. This mechanism would provide a means of pacemaker entrainment at rates compatible with physiological measurements from several species. Leak through this conductance may also enhance the rate of firing of the pacemaker mechanism in the primary (initial) pacemaker cell. Recent data suggest that dihydropyridine-resistant Ca²⁺ entry may provide a similar regulation of pacemaker activity in the heart. Entry of Ca²⁺ via T-type channels increases spontaneous Ca²⁺ release events from ryanodine receptors (i.e. Ca²⁺ sparks), and this stimulates an inward current during the late diastolic phase in atrial pacemakers (Huser et al. 2000). Further studies of ICC networks and intact GI muscles using agents identified in the present study to block I_VDDR without blocking the pacemaker conductance must be performed on intact muscles to test the role of I_VDDR in slow wave propagation.

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Figure 11. Role of voltage-dependent Ca2+ conductances in propagation of slow waves Pacemaker activity is initiated in ICC by Ca2+ release from IP3 receptor-operated stores (Suzuki et al. 2000; Ward et al. 2000) in the sarcoplasmic reticulum (SR). The site where this first occurs in an ICC network is the primary pacemaker (ICC1). Leak of Ca2+ through a dihydropyridine-resistant conductance may increase the rate of firing of the pacemaker mechanism in the primary pacemaker since reduced extracellular Ca2+ slows the pacemaker frequency (cf. Ward & Sanders, 1992). Ca2+ released from IP3 receptors, which are in close physical proximity to mitochondria, stimulates Ca2+ uptake into mitochondria (M) via a Ca2+-dependent transporter (CU). Mitochondrial Ca2+ uptake transiently reduces Ca2+ activity in the space close to the plasma membrane (PM; and see Ward et al. 2000 ). Reducing Ca2+ in this space activates a Ca2+-inhibited, non-selective cation conductance (INSCC). Activation of these channels generates the pacemaker current (see Koh et al. 2002). Inward current depolarizes adjacent ICC within the network (ICCn) that are coupled to the primary pacemaker via gap junctions. The extent of depolarization depends upon the cable properties of the network, which depend upon parameters such as internal resistance (ri), junctional resistance due to gap junctions (rj), and membrane resistance (rm) and capacitance (cm). Depolarization of neighbouring ICC activates voltage-dependent Ca2+ channels. The present study demonstrates that both dihydropyridine-sensitive and dihydropyridine-resistant conductances are expressed by ICC (IVDIC) and might contribute to Ca2+ entry, but slow waves are propagated in the presence of dihydropyridines, suggesting that the dihydropyridine-resistant conductance (IVDDR) is sufficient. Ca2+ entry into the space near IP3 receptors promotes (i.e. phase advances) release of Ca2+ and regenerates the pacemaker mechanism. This process also depends upon re-uptake of Ca2+ into stores by the SR Ca2+-ATPase (SERCA) to reset the mechanism.

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Acknowledgements

This work was supported by a Program Project Grant from the NIDDK, PO1 DK41315.

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