Electrical slow waves are periodic oscillations in membrane potential that occur in smooth muscle cells in many regions of the gastrointestinal (GI) tract of animals and man (e.g. Tomita, 1981). Slow waves consist of a rapid upstroke depolarization, partial repolarization, a plateau phase lasting several seconds and repolarization to the resting membrane potential. The ionic currents involved in generation of slow waves have been characterized in colonic muscles (see Sanders, 1992, for review); however, the conductances responsible for the maintenance of the resting membrane potential (i.e. the maximum level of polarization between slow waves) have yet to be defined.
Membrane potentials of GI muscles determine the open probability of ion channels involved in rhythmic electrical and contractile activity. At the submucosal surface of the circular muscle layer in the canine colon, resting membrane potential is several millivolts positive to the equilibrium potential for potassium, EK (Smith et al. 1987). The membrane potential at the myenteric surface of the circular muscle layer is substantially less negative (averaging about -45 mV; Smith et al. 1987). The negative resting membrane potential in cells near the submucosal surface and the gradient in membrane potential across the circular layer has been attributed to a significant contribution from an electrogenic Na+-K+-ATPase. This is based on the observations that: (i) ouabain abolishes the membrane potential gradient (Burke et al. 1988); and (ii) differential expression of Na+-K+-ATPase mRNA between the submucosal vs. myenteric regions of the circular muscle layer (Burke et al. 1991). Barajas-Lopez & Huizinga (1992) suggested that the effects of ouabain could be due to inhibition of K+ conductances in response to increased intracellular Na+. However, the nature of such a K+ conductance was not resolved.
An inward rectifier K+ conductance could contribute to the negative resting potential and the membrane potential gradient across the circular muscle layer in the colon. Ba2+-sensitive inward rectifier K+ currents have been identified in vascular smooth muscles (Quayle et al. 1993; Knot et al. 1996). These currents are likely to be due to the expression of Kir2.1 mRNA (Bradley et al. 1999), but this conductance has not been reported in GI muscles to date. Although referred to as ‘inward rectifiers,’ these conductances carry small outward currents at potentials positive to EK, and this current contributes to the negative membrane potentials of several cell types, including cardiac muscle, mast cells and neurones (see Jan & Jan, 1997, for review).
In the present study, we examined the hypothesis that a Ba2+-sensitive inward rectifier potassium conductance contributes to the resting potentials of canine colonic muscles. Regional differences in expression of an inward rectifier conductance may contribute to the gradient in resting membrane potential within the circular muscle layer (Smith et al. 1987).
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In the present study we have demonstrated the presence of a Ba2+-sensitive K+ conductance that is manifest in cells along the submucosal surface of the circular muscle layer in the canine colon. Electrophysiological observations were consistent with the expression of Kir2.1, a class of Ba2+-sensitive inward rectifying K+ channels in colonic muscles. The Ba2+-sensitive conductance in colonic muscles appears to contribute to the negative membrane potentials recorded from cells near the submucosal surface of the circular muscle layer (Smith et al. 1987) for the following reasons: (i) low concentrations of Ba2+ induced depolarization of the circular muscles; and (ii) the gradient in resting membrane potential across the circular muscle layer was abolished by Ba2+. Under basal conditions, the Ba2+-sensitive conductance, which has the properties of an inward rectifier K+ conductance, appeared to be mainly expressed by ICC, which are the pacemaker cells in the colon (Langton et al. 1989). Low doses of Ba2+ depolarized membrane potentials in intact muscle strips; however, there was far less sensitivity to Ba2+ in strips of muscle from which the pacemaker regions (containing ICC) were removed. Stimulation of isolated circular muscle preparations (ICM) with forskolin resulted in enhanced sensitivity to Ba2+, suggesting that the inward rectifier conductance may be regulated by cAMP.
This study identifies a potential role for an inward rectifier K+ conductance in GI smooth muscle. Previous studies have examined the effects of BaCl2 on GI muscles; however, these investigations failed to specifically identify contributions of inward rectifier K+ conductances to membrane potential (Liu & Huizinga, 1994; Murillo et al. 1997). This is because the effects of Ba2+ can be obscured by the activation of spike activity in GI muscles due to the increased current carried by Ba2+ through L-type Ca2+ channels (Hagiwara & Byerly, 1981; Bean, 1985). Prior studies tested the effects of much higher concentrations of Ba2+ (0.5-5 mM) than that used in the present study, and non-specific effects on other K+ conductances may have obscured the effects of this ion on inward rectifiers (Liu & Huizinga, 1994; Murillo et al. 1997).
Benham et al. (1987) described the presence of an inwardly rectifying non-selective current in isolated smooth muscle cells of the rabbit jejunum. This conductance is unlike those of the Kir gene family in that it carries both K+ and Na+ ions, and is significantly inhibited by 1 mM Cs+, and to a lesser extent by 10 mM Ba2+. This ‘anomalous rectifier’ may be similar to the currents described by DiFrancesco in sino-atrial cells and Purkinje cells in the heart (1985) and by Mayer & Westbrook (1988) in murine sensory neurones.
Little is known about the ionic conductance (s) responsible for the negative resting potentials in GI muscles, although it is apparent that the resting conductance for K+ ions is an important determinant of RMP (see Casteels, 1981, for review). In canine colon a membrane potential gradient exists across the circular muscle layer in which cells near the submucosal surface are highly polarized (i.e. -78 mV), and the resting membrane potential of cells near the myenteric surface lie at about -45 mV (Smith et al. 1987). Burke et al. (1988) proposed that a gradient in Na+-K+-ATPase activity might contribute to the gradient in membrane potential. It was suggested that the Na+ pump produced up to -30 mV of membrane potential at the submucosal surface of the circular muscle, and the contribution of the pump to resting potential decreased across the muscle layer. Barajas-Lopez & Huizinga (1992) argued that the depolarization caused by inhibition of the Na+-K+-ATPase was due to block of a K+ conductance by increased intracellular Na+, but the properties of the proposed K+ conductance were not determined.
Previous studies have shown that elevation in extracellular K+ increases the slope of the relationship between external K+ concentration and membrane potential in GI muscles, suggesting an increase in K+ conductance (Bolton, 1973; Bauer & Sanders, 1985; Hirst & van Helden, 1985; Hara & Szurszewski, 1986). The increase in Nernst-like behaviour was attributed simply to an enhancement in outward rectification when cells are depolarized by elevated K+. In the present study, we showed that membrane potential approached EK at potentials much more negative than the range in which voltage-dependent outward rectifier conductances are activated in canine colonic myocytes (Thornbury et al. 1992). We believe that the increase in apparent K+ conductance at these potentials may be due to an increase in the inward rectifier K+ conductance, which is activated at negative potentials by enhanced extracellular K+ (Kubo, 1996; Lopatin & Nichols, 1996).
In some studies of vascular smooth muscles, small increases in external K+ have been shown to produce hyperpolarization of RMP (Edwards et al. 1988; Knot et al. 1996). This apparently paradoxical behaviour has been attributed to the presence of an inward rectifier K+ conductance that is activated by elevated external K+. In the present study small elevations in external K+ caused depolarization. The difference between the vascular muscles tested and colonic muscles may be explained by the following: cerebral and coronary arteries have resting potentials of approximately -45 mV, which is at least 30 mV positive to EK (Edwards et al. 1988; Knot et al. 1996). Therefore, an increase in K+ conductance results in hyperpolarization as RMP approaches EK. In colonic myocytes we found that RMP approached EK when external K+ was enhanced. Colonic muscles have negative resting potentials, only a few millivolts positive to EK. Therefore, colonic muscles depolarized and membrane potential followed the estimated changes in EK (see Fig. 5).
There appears to be an important heterogeneity in the expression of the Ba2+-sensitive inward rectifier conductance in colonic muscles. ICC had significant inward rectifier currents under resting conditions, and this may contribute to the very negative potentials recorded in the submucosal pacemaker region (Smith et al. 1987), which is populated by ICC (Berezin et al. 1988). Previous studies have suggested that conductances in ICC may participate in setting the membrane potentials of GI muscles, because removal of ICC results in less polarized smooth muscle tissues (Liu & Huizinga, 1993; Ward et al. 1994; Torihashi et al. 1995). In contrast, smooth muscle cells may either have a much lower expression of these channels, or the conductance may be less available under basal conditions. Molecular studies support our electrophysiological findings with regard to heterogeneous expression of inward rectifier currents. Transcriptional expression of Kir2.1 mRNA (but not Kir2.2 or Kir2.3) was found in the circular muscle layer of the canine proximal colon. The expression of Kir2.1 was significantly higher in SCM preparations (which contained the ICC from which inward rectifier currents were measured) than in either ICM or MyCM preparations.
Our study suggests that a significant portion of the membrane potential gradient across the thickness of the circular muscle layer (see Smith et al. 1987) is due to the heterogeneous expression of Kir2.1 in colonic muscles. In the present study we reproduced the large depolarization caused by ouabain (see Burke et al. 1988), although the effects of ouabain were significantly reduced after prior application of Ba2+. This suggests that ouabain may be acting on the same conductance blocked by Ba2+.
We observed an increase in the sensitivity of isolated smooth muscle strips to Ba2+ in response to forskolin. This suggests that inward rectifier channels may be present in smooth muscle cells (also supported by molecular studies showing expression of Kir2.1 in ICM), but these channels may require activation by cAMP-dependent mechanisms before appreciable current is generated by this conductance. Regulation of the inward rectifier K+ conductance may provide a novel mechanism by which hormones and neurotransmitters could regulate membrane potential and excitability of GI muscles. Several endogenous compounds (including noradrenaline, vasoactive intestinal polypeptide, pituitary adenylyl cyclase-activating protein, prostaglandins and secretin) hyperpolarize and relax GI smooth muscles via cAMP-dependent mechanisms (see Makhlouf & Murthy, 1997, for review). cAMP-dependent mechanisms have been linked to activation of delayed rectifier currents (via 20 and 80 pS channels; Koh et al. 1996) and BKCa channels (Carl et al. 1991) in colonic myocytes. Activation of these conductances could result in membrane hyperpolarization or decreased membrane excitability, but these channels have low open probabilities at the resting potentials of colonic cells and they are insensitive to inhibition by Ba2+. Therefore, the hyperpolarization caused by forskolin was unlikely to be due to activation of delayed rectifier and BK channels.
In summary, membrane potential was sensitive to low concentrations of Ba2+ in the circular muscle of the canine colon. Depolarization caused by Ba2+ may be due to the expression of Kir2.1. This conductance may be predominantly located in interstitial cells of Cajal. We hypothesize that increased expression of Kir2.1 channels is responsible for the negative resting potentials in and near the submucosal surface of the circular muscle layer. The negative membrane potentials of ICC may influence the resting potential of electrically coupled circular muscle cells, creating the gradient in membrane potential across the circular muscle layer. Hormones and neurotransmitters coupled to effects via cAMP may enhance the open probabilities of Kir2.1 channels, leading to hyperpolarization and reduction in excitability.