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.
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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.