Gastrointestinal muscles are able to maintain negative resting membrane potentials in spite of stretch. We investigated whether stretch-dependent K+ channels might contribute to myogenic regulation of smooth muscle cells from the mouse colon.
Negative pressure applied to on-cell membrane patches activated K+ channels that were voltage independent and had a slope conductance of 95 pS in symmetrical K+ gradients. The effects of negative pressure on open probability were graded as a function of pressure and reversible when atmospheric pressure was restored.
Cell elongation activated K+ channels with the same properties as those activated by negative pressure, suggesting that the channels were stretch-dependent K+ (SDK) channels.
Channels with the same properties were maximally activated by patch excision, suggesting that either an intracellular messenger or interactions with the cytoskeleton regulate open probability.
Internal 4-aminopyridine, Ca2+ (10−8 to 10−6m), and tetraethylammonium (internal or external) were without effect on SDK channels.
Nitric oxide donors (and cell-permeant cGMP analogues) activated SDK channels, suggesting that these channels may mediate a portion of the enteric inhibitory neural response in colonic muscles.
In summary, SDK channels are an important conductance expressed by colonic muscle cells. SDK channels may stabilize membrane potential during dynamic changes in cell length and mediate responses to enteric neurotransmitters.
The tunica muscularis of the gastrointestinal (GI) tract contains continuous sheets of smooth muscle cells. The diameter of GI organs changes dramatically during digestion as food and chyme are passed through the system. As a result of the distension and contractions that occur, individual smooth muscle cells experience dramatic length changes, and cell stretch (or distortion) might affect membrane potential, excitability and responsiveness to agonist stimulation. Although many investigators believe that smooth muscles exhibit stretch-dependent contraction (Burnstock & Prosser, 1960; Himpens & Somlyo, 1988; Kirber et al. 1988; Fay, 2000), stretch of colonic muscles does not initiate an obvious contractile response (K. Keef, personal communication). Thus, it is possible that part of the cellular apparatus includes ionic conductance(s) that stabilize membrane potential and limit excitability during distension of the bowel wall. This may be an important aspect of the ‘myogenic response’ to stretch that facilitates the reservoir function of regions of the GI tract and prevents interference in the coordination of segmental and/or peristaltic movements provided by the enteric nervous system.
Ion channels activated by distortion of the plasma membrane have been observed in numerous cell types and under a variety of experimental conditions. Three types of mechanosensitive ion channels have been described in gastrointestinal smooth muscle cells: swelling-activated chloride channels (Dick et al. 1998), stretch-activated non-selective cation channels (Waniishi et al. 1997) and Ca2+ channels (Farrugia et al. 1999). Activation of these ion channels, under physiological ionic gradients, would result in inward current, depolarization and contractions. Contraction, however, does not appear to be a basic response to stretch in many GI muscles, and this may be an important feature allowing volume expansion of GI organs without significant increases in luminal pressure. This feature may allow some GI organs to provide a reservoir function. Such a mechanism might involve stretch-dependent K+ channels expressed by GI smooth muscle cells, but conductances of this type have not been found in GI muscles to date. If stretch-dependent K+ channels are expressed in smooth muscles, they could provide a negative-feedback pathway by generating outward current in response to stretch and contraction, and, in this way, these channels could regulate contractile behaviour (Brayden & Nelson, 1992). Thus, it is possible that both inhibitory neural reflexes and myogenic mechanisms might contribute to the regulation of bowel wall compliance.
In the present study we have tested whether stretch-dependent K+ channels are expressed in colonic smooth muscle cells. We have characterized the channels that respond to stretch and surveyed some of the means by which this conductance might be regulated. The studies demonstrate an important new class of channels in GI smooth muscles that may participate in the regulation of membrane potential and excitability and may mediate some of the responses of these tissues to neurotransmitters.
Colonic smooth muscle cells were prepared form Balb/C mice of either sex, 1-2 months of age. Mice were anaesthetized with chloroform and killed by cervical dislocation, and the proximal colon was quickly removed, as approved by the Institutional Animal Care and Use Committee. Colons were cut open along the longitudinal axis, pinned out in a Sylgard-lined dish, and washed with Ca2+-free Hanks' solution containing (mm): 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4, 0.44 KH2PO4, 10 glucose, 2.9 sucrose and 11 Hepes, pH 7.4. After removal of the mucosa and submucosa, pieces of muscle were incubated in a Ca2+-free Hanks' solution containing 4 mg ml−1 fatty acid-free bovine serum albumin (Sigma), 14 U ml−1 papain (Sigma), 230 U ml−1 collagenase (Worthington), and 1 mm trypsin inhibitor (Sigma). The muscle pieces were incubated at 37 °C in enzyme solution for 8-12 min, washed with Ca2+-free Hanks' solution, and then gently triturated to create a cell suspension. Dispersed cells were stored at 4 °C in Ca2+-free Hanks' solution supplemented with minimum essential medium and containing (mm): 0.5 CaCl2, 0.5 MgCl2, 4.17 NaHCO3 and 10 Hepes, pH 7.4.
For preparation of canine myocytes, dogs of either sex were killed with pentobarbital sodium (100 mg kg−1) and the colon was removed. Circular muscle strips were cut from the proximal colon with the aid of a dissecting microscope. Isolation and incubation of canine myocytes have been described previously (Koh et al. 1995).
Dispersed smooth muscle cells were pipetted into a small recording chamber on an inverted microscope and allowed to adhere to the bottom for 5 min prior to commencement of experiments. All experiments were performed at room temperature and cells were used within 6 h after enzymatic dispersion.
Voltage clamp experiments
Single channel voltage clamp experiments were performed using murine and canine colonic smooth muscle cells. Gigaohm seals were obtained using heat-polished borosilicate glass pipettes with resistances of 5-10 MΩ. Estimated patch area was 0.5-1 μm2. Currents were amplified with a List EPC-7 amplifier and/or Axopatch-1A amplifier and digitized with a 12 bit analog to digital converter (Model TL-1, DMA interface, Axon instruments). Data were stored on videotape or directly digitized on-line using pCLAMP software (version 5.5.1 or 6.03, Axon instruments). Data were sampled at 1-5 kHz, and low-pass filtered at 0.2-1 kHz using an eight-pole Bessel filter. Data were displayed and analysed using pCLAMP (version 6, Axon instruments) and Origin software (MicroCal Software) to obtain amplitude histogram and channel activity (NPo, where N is the number of channels in the patch, and Po is the probability of a channel being open). NPo was determined from 30 s or 1 min of channel recording.
Application of negative pressure and mechanical stretch
Membrane stretch was elicited by applying suction (negative pressure) to a side port of the patch pipette holder. The degree of negative pressure was calibrated with a pressure transducer. The negative pressure and volume relation was linear. For experiments involving direct mechanical stretch, a two-microelectrode technique was used. One electrode was gigasealed in the cell-attached patch configuration at one end of the cell, and a second gigaseal was formed with another electrode at the opposite end of the cell (as depicted in Fig. 4). Stretch was applied to the cell by manipulating one pipette with a micromanipulator.
For the recording of K+ channels in cell-attached or excised patches, the bath solution contained (mm): 140 KCl, 1 EGTA, 0.61 CaCl2 and 10 Hepes adjusted to pH 7.4 with Tris. In order to test the Ca2+ sensitivity of channels in the patch, Ca2+ was added to bath solutions buffered by 1 mm EGTA to create Ca2+ activities from 10−8 to 10−6m. Activities were calculated with a program developed by C.-M. Hai (University of Virginia, Charlottesville, VA, USA). The pipette solution for asymmetrical K+ gradients was 5 mm KCl and 135 mm NaCl instead of the 140 mm KCl used in the bath solution and for symmetrical K+ gradient experiments the pipette solution was identical to the bath solution except for the addition of 200 nm charybdotoxin to the pipette solution to inhibit large conductance Ca2+-activated K+ channels. Tetraethylammonium (TEA), 4-aminopyridine (Sigma), sodium nitroprusside and 8-bromo-cGMP (8-Br-cGMP) (Calbiochem, San Diego, CA, USA) were added to the bath solution in some experiments.
Characteristics of stretch-dependent K+ channels
On-cell patches of murine colonic myocytes were held at 0 mV in asymmetrical K+ gradients (i.e. 5 mm K+ in the patch solution/cellular K+ concentration). Under these conditions there were few openings of K+ channels and contributions from non-selective cation channels were minimized. Application of negative pressure to the pipette caused activation of a large number of channels that produced outward unitary currents (Fig. 1A). Immediately after application of negative pressure, NPo increased sharply. Open probability changes every 2 s are shown for a representative patch in Fig. 1B exposed to a negative pressure of -40 cmH2O. After restoring atmospheric pressure to the pipette, open probability returned to the control level (close to a probability density of 0), but restoration of control NPo was always slower (τ1/2= 9 ± 1 s, n = 19) than the increase in open probability when negative pressure was applied. A similar conductance, activated by the same range of negative pressure changes, was observed in on-cell patches of canine colonic myocytes studied under the same conditions (Fig. 1C).
The increase in open probability was dependent on the degree of negative pressure applied to the pipette. Patches from five cells were exposed to pressures ranging from -20 to -80 cmH2O. In order be sure that the effects of negative pressure were reversible and lacked desensitization, different levels of negative pressure were applied to the same patch and each pressure was tested twice (e.g. Fig. 2A). A negative pressure of -20 cmH2O had little effect on channel activity (NPo= 0.5 ± 0.4), but application of negative pressure of -40 cmH2O to the same patches caused NPo of the K+ channels to increase from 0.0 to 6.2 ± 0.7 (n = 4). Application of -60 cmH2O or -80 cmH2O to the pipette, caused a maximal increase in NPo. After restoration of atmospheric pressure each time, the open probability returned to near 0 (Fig. 2A). Data summarizing the effects of negative pressure on NPo are shown in Fig. 2B.
In 19 patches we found an average of seven K+ channels per patch activated by negative pressure. We estimate, based on patch size and calculations of membrane surface area from whole capacitance measurements (i.e. 30 pF), that a maximal cell conductance of 150-300 nS at -20 mV would be obtained if all channels of this type were activated.
Patch excision also caused an increase in the open probability of channels with similar characteristics to the channels activated by negative pressure. Figure 2A shows activation of channels with negative pressure, restoration of control activity several times by restoring atmospheric pressure, and then patch excision. Channels with the same unitary current amplitude were activated by both manoeuvres.
The channels activated by negative pressure were K+ channels. Figure 3A shows changes in unitary currents as a function of patch potential. Figure 3B shows the current-voltage relationship for the channels activated by negative pressure. The data were fitted with the Goldmann-Hodgkin-Katz (GHK) equation and the fit confirmed that the channels showed high selectivity for potassium. The channels activated by negative pressure had a conductance of 52 ± 1 pS at 0 mV in asymmetrical K+ gradients (n = 5). There was no change in NPo as a function of voltage. The effects of potential on the single channels observed in excised patches were also characterized and the current-voltage relationship was fitted with the GHK equation. Although typical patches had several channels, for display purposes we selected an example of a patch with only a single channel that was activated by patch excision to demonstrate that this channel had the same current amplitude as the channels activated by negative pressure (Fig. 3C). In asymmetrical K+ gradients (Fig. 3C and D) the amplitude of unitary currents activated by patch excision was 2.2 ± 0.2 pA at 0 mV (n = 5). This current amplitude, corresponding to a conductance of 53 ± 1 pS in excised patches, was the same current amplitude as the channels activated by negative pressure (i.e. 2.0 ± 0.4 pA; P < 0.001). There was no effect of voltage on the open probability of the channels activated by patch excision (data not shown). In studies using symmetrical K+ gradients (140/140 mm), we found the conductance of these channels was 95 ± 1 pS (n = 4, Fig. 3D). These data suggest that the same K+ channels were activated by cell excision and stretch. It should be noted that in symmetrical K+ gradients it was often hard to isolate the stretch- and excision-activated channels from a non-selective cation channel that was also present in the patches (data not shown). Therefore, asymmetrical K+ gradients (5/140 mm K+) were used in the remaining experiments.
It is possible that stretch of the cell membrane resulting from application of negative pressure to the patch pipette was the main stimulus for channel activation in the experiments above. In order to test this hypothesis, we performed experiments in which two patch pipettes were used to form gigaseals on the same cell (Fig. 4A). Similar channel activity was recorded in both patches. After confirming that SDK channels were activated by negative pressure (Fig. 4C), one of the pipettes was used to stretch the cell, and the 2nd pipette was used to record single channel activity. Cell elongation (4-7 μm; Fig. 4B) caused a significant increase in the open probability of channels of similar characteristics to those activated by negative pressure (unitary currents were 2.1 ± 0.3 pA at 0 mV and resulted from openings of 53 ± 1 pS channels; n = 4). These observations suggest that activation of the 52 pS K+ channels by negative pressure was due to stretch of the cell membrane. Since these channels were activated by stretch, we have referred to them as stretch-dependent K+ (SDK) channels throughout the remainder of the paper.
Pharmacology of SDK channels in excised patches
Using inside-out patches held at 0 mV, we tested classical K+ channel blockers on SDK channels. Intracellular applications of 4-aminopyridine (up to 5 mm, n = 5) and tetraethylammonium (TEA, up to 10 mm, n = 5) had no effect on the open probability or the amplitude of unitary currents due to SDK channels (data not shown). Putting TEA in the patch pipette (up to 20 mm) also appeared to have no effect on SDK channels (data not shown), further confirming that SDK channels are distinct from large conductance Ca2+-activated K+ channels. At the present time, we have not identified a specific inhibitor of SDK channels. SDK channels were also not affected by changes in Ca2+ concentrations from 10−8 to 10−6m applied to the cytoplasmic surface of the patch (n = 5, data not shown).
Mechanisms regulating open probability of SDK channels
Besides the obvious importance of regulation by stretch in gastrointestinal muscles, it is also possible that SDK channels are regulated by other physical parameters and by biologically active substances such as cell lipids and neurotransmitters. Previous studies have demonstrated that K+ channels of the same conductance in colonic muscle cells are activated by NO and the cGMP-dependent pathway (Koh et al. 1995). We performed experiments to survey some of the means by which the open probability of SDK channels might be regulated in GI muscles.
We tested the effects of sodium nitroprusside (SNP) and 8-Br-cGMP on the open probability of SDK channels. In on-cell patches at rest, openings of SDK channels were minimal. Application of SNP (10−6m) caused an increase in the openings of channels with the same amplitude as SDK channels (Fig. 5). Increasing the concentration of SNP (10−5m) led to a significant increase in NPo similar to SDK channels (n = 4). All-points amplitude histograms for a representative experiment are shown in Fig. 5B and C. These data show the activation of SDK-like channels in response to SNP. It should also be noted that SNP activated small conductance K+ channels (< 4 pS) in patches from murine colonic myocytes as previously demonstrated in studies of canine colonic myocytes (Koh et al. 1995). Activation of these channels was regularly observed, as in Fig. 5, but these channels were not further characterized in the present study.
We also tested the effects of SNP on SDK channels pre-activated by negative pressure. At a negative pressure of -30 mmH2O the NPo was 1.5 ± 0.3. After addition of SNP, NPo increased to 4.2 ± 0.7 (n = 3; Fig. 5D). When levels of negative pressure were great enough to produce maximal activation of SDK channels, addition of SNP had no further effect on channel activation (data not shown).
NO increases intracellular cyclic GMP via activation of guanylyl cyclase, and this pathway has been proposed as the major means by which NO acts in murine colonic myocytes (Shuttleworth et al. 1997). Therefore we also tested the effects of the membrane-permeant cGMP analogue 8-Br-cGMP on the open probability of SDK channels. Application of 8-Br-cGMP (10−5m) in the bathing solution dramatically increased the NPo of SDK channels (n = 4, Fig. 6).
In the present study, we have characterized a stretch-dependent K+ conductance (SDK channels) that is expressed in murine and canine colonic myocytes. The conductance of SDK channels is 95 pS in symmetrical K+ gradients, and the open probability of these channels is highly dependent upon cell length, apparently via interactions with the cytoskeleton. Neurotransmitters and second messenger signalling pathways also regulate SDK channels. An exciting result is that SDK channels appear to be the same channels that were previously shown to be a major target for NO-dependent effects in colonic muscle cells (Koh et al. 1995). Thus it is possible that besides contributions to resting potential and regulation of excitability during dynamic changes in cell length, SDK channels may also be an important conductance in mediating responses to enteric inhibitory neurotransmission. NO-dependent regulation of SDK channels appears to occur, at least in part, via cGMP-dependent pathways, which are the main mechanisms responsible for the electrical and mechanical effects of nitrergic nerve regulation of colonic muscles (Shuttleworth et al. 1997).
Application of negative pressure to on-cell patches pulls the plasma membrane into the patch pipette, stretching the membrane from which single channel currents are recorded. Although many stretch-activated conductances have been studied by this technique, there are possible artifacts associated with this approach to stretching the plasma membrane (e.g. Morris & Horn, 1991). Therefore, we attempted to compare the effects of negative pipette pressure and actual cell elongation on SDK channels in colonic myocytes. Both means of stretching the cell membrane activated single channel currents of the same amplitude suggesting that either technique is suitable for activating this class of ion channels in smooth muscle cells. Changes in cell length occur during contraction, relaxation and distension of the colon in vivo, and therefore changes in cell length might be considered to be one of the physiological stimuli for SDK channels.
The pharmacology of stretch-dependent K+ channels is ambiguous at the present time. Amiloride, TEA and quinine (all non-specific ion channel blockers) were found to block stretch-dependent K+ channels of Lymnaea neurons from the outside but not from the cytoplasmic surface of the membrane (Small & Morris, 1995). Others have found that stretch-dependent K+ channels in cultured chick ventricular myocytes are sensitive to intracellular Ca2+ and ATP (Kawakubo et al. 1999). The channels we identified in colonic myocytes were not sensitive to 4-aminopyridine, TEA, or intracellular Ca2+. Until a distinctive and selective pharmacology can be identified for SDK channels, it will be difficult to assess their physiological function in intact muscles.
Ca2+-activated K+ channels modulated by membrane stretch have been observed in apical membranes of cultured medullary thick ascending limb cells (Taniguchi & Guggino, 1989), embryonic rat neuroepithelial cells (Mienville et al. 1996) and apical membranes of rat and rabbit cortical collecting tubules (Pacha et al. 1991). The conductance of these channels varies from cell to cell and ranges from 20 to 200 pS in symmetrical K+ gradients. In endocardial endothelium, large conductance Ca2+-activated K+ channels are activated by stretch (Hoyer et al. 1994). These channels display both voltage dependence and Ca2+ sensitivity. SDK channels in canine colonic myocytes are distinct from these conductances in that we were unable to detect voltage dependence or Ca2+ sensitivity (from 10−6 to 10−8m). In addition SDK channels were not blocked by either internal or external TEA or charybdotoxin.
We previously described K+ channels of approximately 90 pS in inside-out patches from circular smooth muscle cells of the canine proximal colon that were activated by NO donors and cGMP-dependent pathways (Koh et al. 1995). Openings of these channels were abundant in excised patches, but we rarely observed currents in cell-attached patches unless the cells were stimulated with NO or membrane-permeant cGMP analogues. At that time, it was unclear how the low open probability of these channels was maintained in the cell-attached configuration. From our current observations we believe that the 90 pS K+ channels in canine colonic muscles are SDK channels. In the present study we found that the open probability of SDK channels was increased dramatically upon patch excision, and the conductance of SDK channels is approximately the same as the 90 pS K+ channels previously described (Koh et al. 1995). The data suggest that SDK channels may be held in a closed state by the cytoskeleton in resting cells and activation may occur when the cytoskeleton is disrupted during patch excision.
Muscle tension affects intraluminal pressure in the hollow organs of the GI tract. If the cells in the wall of these organs are elastic, then intraluminal pressure would not tend to rise significantly as filling occurs. However, previous studies have suggested that stretch of GI muscle cells activates inward currents carried by chloride (Dick et al. 1998), non-selective cation conductances (Waniishi et al. 1997) and calcium channels (Farrugia et al. 1999). Many regions of the GI tract, including the proximal colon, serve a reservoir function in normal GI motility, holding contents until it is appropriate to move food or chyme to the next region. In order for GI muscles to remain in the relaxed state during filling of the organs and elongation of smooth muscle cells, activation of outward currents may be necessary for stabilizing resting potential. This mechanism could also be important in other visceral organs that expand dramatically without initiation of contraction (e.g. uterus and bladder). Part of suppressing contraction might come from neural reflexes that actively inhibit electrical excitability and contractile processes, but the present study demonstrates a novel myogenic mechanism that might participate in the inhibition of contractions during elongation of smooth muscle cells. The fact that SDK channels are also activated by NO, the primary inhibitory neurotransmitter in GI muscles, suggests that these channels are an important point of convergence for myogenic and neurogenic control of motility.
In summary, SDK channels are abundant in colonic myocytes of mouse and dog. These channels are regulated by stretch, possibly by involving interactions with the cytoskeleton. Localized stretch of membrane patches or cell elongation activated channels with similar properties. Patch excision maximally activated SDK channels. These channels are likely to be important physiologically by maintaining membrane potential during cell elongation (e.g. during organ filling) and participating in enteric inhibitory neural responses mediated by NO via cGMP-dependent pathways. The pharmacology of SDK channels is ambiguous at the present time, but new blockers of these channels may be potentially useful in controlling GI motility, particularly in disorders involving organ distention.
This study was supported by a Program Project Grant from NIDDK: DK40569. The authors thank Nancy Horowitz for preparation of the colonic smooth muscle cells.
S. D. Koh: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.