1Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576; and 2Department of Pharmacology, University of Alberta, Edmonton, Canada T6G 2H7
Submitted 3 September 2003 ; accepted in final form 3 September 2003
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ABSTRACT |
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propagation in smooth muscle; PSpice simulation of excitability; slow waves in interstitial cells of Cajal; electric field in junctional cleft
Results demonstrated that transmission of excitation from cell to cell could occur in the complete absence of gap junction channels enabled by the electric field (EF) that develops in the narrow junctional clefts when the prejunctional membrane fires an action potential (AP) (Refs. 8, 13, and unpublished observations). The voltage generated in the junctional cleft (VJC) is negative and serves to depolarize the postjunctional membrane to its threshold by a patch-clamp-like action (Refs. 8, 13, and unpublished observations). Amplitude of VJC depends on several factors, including the values of RJC and the rate of rise of the AP in the prejunctional membrane.
Simulation shows that propagation had a staircase profile, i.e., propagation velocity in each cell was virtually infinite, coupled with a large junctional delay time (e.g., 0.5 ms) (Refs. 9, 12, and unpublished observations). Thus almost all of the propagation time was consumed at the cell junctions. The EF mechanism and evidence supporting it is summarized in two recent review articles (9, 10). This evidence includes the fact that the intercalated disc contains a high density of fast Na+ channels (4). When 10,000 or 1,000 gap junction channels per junction were placed into the model, propagation velocity became nonphysiologically fast (12).
When several chains of cells were placed in parallel with no gap junction channels between them, excitation was capable of jumping from chain to chain (Ref. 6 and unpublished observations). This transverse transmission was affected by the longitudinal resistance of the interstitial fluid (ISF) between the parallel chains (Rol2). The tighter the packing of the chains in the bundle, the faster the transverse propagation velocity. It appeared that the mechanism for the transverse transmission between the parallel chains was the EF that develops in the narrow ISF space. The moderate hyperexcitablility of the basic membrane units composing the cells facilitated successful transfer of excitation by the EF.
In the present study, a two-dimensional planar network of smooth muscle cells (SMCs) consisting of five parallel chains of five cells each (5 x 5 model) was used to investigate activation of the SMC network by a single interstitial cell of Cajal (ICC). It is thought that intramuscular type ICCs (ICC-IM) are the means for physiological activation of the circular smooth muscle layer of the intestine, somewhat analogous to the conduction system of the heart (1-3). Density of ICC-IM varies in the stomach circular muscle but is 1 ICC cell/25 SMCs in the guinea pig pylorus (14). Gap junction connections occur between the intramuscular ICC-IM cell and an SMC cell (1-3). ICC-IM cells in antrum and pylorus do not initiate slow waves but exhibit regenerative APs in response to slow waves from the ICC-MP (myenteric plexus). We have chosen to model only the passive responses they display after the regenerative component is blocked (1-3). This allows our model to represent, in part, slow waves generated by ICC-MP (1-3). The slow waves are undershooting, long-duration potential changes, having an average amplitude of
40 mV and an RP of
60-80 mV (1-3, 14). We have also used an RP of approximately -55 mV for an alternate "depolarized" ICC-IM. In our present PSpice simulation study, it was found that one ICC-IM cell was capable of activating the entire SMC network, regardless of whether gap junctions were present between the ICC-IM cell and the contiguous SMC cell.
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METHODS |
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Because the ICC-IM cell does not exhibit a regenerative AP, the slow wave was simulated by applying a slow-wave current pulse (ISW) to the inside of the ICC cell that would cause its membrane potential to depolarize by 40 mV for a prolonged period (e.g., 4, 8, 16, or 32 ms; standard value, 8 ms). The rate of rise (and rate of fall) of the ISW pulse was 4, 8, 16, or 32 ms (standard value of 4 ms). Amplitude of the slow wave was varied over a wide range (standard value of 40 mV). To produce the standard slow-wave amplitude of 40 mV, the applied ISW was 2.4 nA (ICCA) or 1.8 nA (ICCB). Thus two types of ICC cells, with respect to resting potential (RP), were used 1) RP of -80 mV (ICCA) and 2) RP of -55 mV (ICCB).
In some simulations, a second ICC-IM cell was attached to the SMC network, namely at the last cell of the first chain (A5 cell). Runs were made with stimulations of only this second ICC cell, as well as the first ICC cell.
In some simulations, the ICC-IM cell was made to generate an overshooting AP (by inserting a GTABLE into the basic units) to determine whether this would be equally effective in activating the SMC network. In the case of the ICCA cell (RP of -80 mV), the AP was fast-rising and the overshoot was to approximately +32 mV (resembling a cardiac AP). In the case of the ICCB cell (RP of -55 mV), the AP was slow-rising and its overshoot was to approximately +11 mV (resembling an AP in an SMC).
Determinations of the total propagation time (TPT) were made by measuring the time difference between when the first response (e.g., AP in E5 cell) reached a membrane potential (VM) of -20 mV and when the last response (e.g., AP in a cell of the A chain) crossed -20 mV. In some cases, the latest period was measured from the beginning of the ISW pulse applied to the ICC cell to when the AP response in the attached E5 cell crossed -20 mV.
As illustrated in Fig. 1, each cell (SMC and ICC) was represented by four basic units: two units for the surface membrane (one upward and one downward) and one unit for each of the two junctional membranes. To simplify the recorded traces (APs), voltage markers were placed across only one of the units of each of the 25 cells in the SMC network, namely the upward-facing surface unit. The same was true for the ICC cell. The cleft potential VJC (measured across RJC) was measured only for the ICC-E5 junction and the subsequent E5-E4 junction. In addition, the voltage change was measured across the postjunctional membrane (of the E5 cell) at the ICC-E5 junction.
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RESULTS |
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In some experiments, the ICC-IM cells (ICCA and ICCB) were made inexcitable (by removing the GTABLE), and instead, a slow wave was generated by internal application of depolarizing current (ISW). The standard slow-wave amplitude was 40 mV. Thus in the ICCA cell, VM went from -80 mV (RP) to -40 mV; in the ICCB cell, VM went from -55 mV (RP) to -15 mV. The standard slow-wave rise time was 4 ms, plateau of 8 ms, and fall-time of 4 ms. Because the SMCs in the network were not given pacemaker properties, the slow-wave plateau duration (and fall-time) were not important.
Excitable ICC-IM Cells
When the ICC-IM cells were made excitable, ICCA and ICCB were capable of activating all 25 SMCs in the 5 x 5 network. This, as illustrated in Fig. 3A, shows a typical result with the ICCA cell and Fig. 3B with the ICCB cell. The first trace in A and B is the AP of the ICC cell. Figure 3A, right shows the AP at a faster sweep speed.
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Nonexcitable ICC-IM Cells
Voltage recordings across selected units of cells ICC, E5, E4, and E3. Recordings were made from only a few cells near the ICC-E5 junction to reduce the number of traces and so simplify their identification. This is illustrated in Fig. 4. The circuit block diagram for the ICC cell and SMCs E5, E4, and E3 is illustrated in Fig. 4A. Numbers indicate the points at which voltage was measured corresponding to the traces labeled in B and C. Data from an ICCA cell are illustrated in Fig. 4B. Trace 1 is the slow-wave voltage applied to the ICCA cell. Trace 2 is the VJC recorded at the ICCA-E5 junction. Trace 4 is the voltage recorded across the postjunctional membrane of cell E5 (VJU; facing the ICCA-E5 junction). The three AP traces were recorded from cells E5, E4, and E3. The potential changes of the junctional membranes are superimposed on those for the surface membrane as indicated by the labels. Data from an ICCB cell are illustrated in Fig. 4C. Traces are labeled as in Fig. 4B. The main difference is that VJC (ICCB-E5) is smaller in amplitude and shorter in duration. Hence the VJU trace is similar to other AP traces in cell E5. That is, there is no subtraction from the VJU trace, as occurs in Fig. 4B.
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Effect of gap junctions. In these experiments, and all of the following, voltage recordings were made from all 25 cells of the SMC network and from the ICC-IM cell (from the upward-facing surface membrane unit). In addition, a voltage recording was made across RJC (VJC) of the last three junctions: ICC-E5, E5-E4, and E4-E3. VJU of the E5 cell was facing the ICC-E5 junction.
Control response in the absence of gap junctions at the ICCA-E5 junction is shown in Fig. 5A for standard conditions. Standard conditions included an RJC value of 20 M throughout the SMC network and a slow wave of 40 mV amplitude (from -80 mV RP to -40 mV) in the ICCA cell. The standard slow wave had a rise time of 4 ms, plateau of 8 ms, and fall-time of 4 ms. As can be seen in Fig. 5A, the peak cleft potential (VJC) at the ICCA-E5 junction was -10 mV, and it followed the time course of the slow wave. As predicted, the VJC subtracts from the VJU. VJC brought the E5 cell to threshold, which then propagated an AP throughout the network.
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When a parallel shunt resistance (RGJ) was inserted across the ICCA-E5 junction (from the inside of the ICCA cell to the inside of E5 cell), the effect of gap junctions, in parallel with the EF mechanism (VJC), could be assessed. Figure 5B shows that an RGJ of 1,000 M, equivalent of one gap junction channel (100 pS each) had almost no effect. However, when RGJ was 10 M
(Fig. 5C; equivalent to 1,000 channels) or 1.0 M
(Fig. 5D; 10,000 channels) cell E5 closely followed the VM change of the ICCA cell, namely the slow wave. The postjunctional membrane of the E5 cell, as expected, also followed the slow wave, but at a reduced amplitude due to subtraction of VJC. As can be seen, only 20 cells of the SMC network responded in Fig. 5, C and D (failure of one chain). Although cell E5 is excitable, it does not fire an AP because of the "voltage clamping" effect of the slow wave in the ICC cell.
Similar results were obtained with the ICCB-type cell (Fig. 6). The control response in the absence of gap junction channels at the ICCB-E5 junction is shown in Fig. 6A for standard conditions. The slow wave of the ICCB cell went from -55 mV (RP) to -15 mV. The peak VJC was approximately -2mV and of short duration. Therefore, the shape of VJU was nearly identical to the AP across the surface membrane of the E5 cell. VJC was sufficient to excite E5, and an AP was propagated throughout the network. When an RGJ of 1,000 M (1 channel) was inserted across the ICCB-E5 junction (Fig. 6B), there was almost no effect. However, when RGJ was 10 M
(Fig. 6C; 1,000 channels) or 1.0 M
(Fig. 6D; 10,000 channels), cell E5 closely followed the VM change of the ICCB cell. Only 22 cells of the network responded (failure of part of the D-chain cells).
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Therefore, with both ICCA and ICCB, insertion of gap junction channels did not facilitate activation of the SMC network. However, TPT was slightly reduced, as would be expected on the basis of one less cell (E5) that needed to be excited and propagated to.
Effect of slow-wave amplitude. Amplitude of the slow wave was varied to determine its effect on the amplitude of the junctional cleft potential (VJC) of the ICC-E5 junction. This was done for both the ICCA-type cell (Fig. 7) and the ICCB-type cell (Fig. 8). As can be seen in Fig. 7, VJC was -20 mV when the slow-wave amplitude was 80 mV (Fig. 7A), -10 mV when the slow wave was the standard 40 mV (Fig. 7B), -5 mV when slow wave was 20 mV (Fig. 7C), and approximately -2.5 mV when slow wave was 10 mV (Fig. 7D). Thus there was a linear relationship between VJC amplitude and slowwave amplitude, as shown in Fig. 9A. As can be seen in Fig. 7, the delay time between the beginning of the slow wave and the AP response of the E5 cell (to where VM crossed -20 mV) was slightly shorter the larger the slow-wave amplitude; e.g., the delay was 2.7 ms in Fig. 7A vs. 3.3 ms in Fig. 7D. The small slow wave, and hence small VJC, was capable of activating the E5 cell and propagating throughout the network, because of the high level of excitability (i.e., low threshold) of the SMCs.
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As can be seen in Fig. 8, peak VJC amplitude was greater when slow-wave amplitude was greater. However, the relationship between the two parameters was not linear (Fig. 9B). As in the case of ICCA, delay time between the beginning of the slow wave and the AP response of cell E5 (to -20 mV) was slightly shorter; e.g., the delay was 3.1 ms in Fig. 9A vs. 3.6 ms in Fig. 9D. There was failure of one (Fig. 9C) or two (Fig. 9D) SMCs at the lower slow-wave amplitudes. On the other hand, TPT (to -20 mV) was less with the small slow waves, e.g., 5.3 ms in Fig. 9A vs. 3.8 ms in Fig. 9D.
Effect of RJC amplitude. The effect of variation in the amplitude of the radial RJC on amplitude of VJC (ICC-E5 junction) and effectiveness of the ICCA cell (Fig. 10) and ICCB cell (Fig. 11) in activating the SMC network was determined. When RJC was varied, it was changed globally throughout the network. As can be seen in Fig. 10, VJC amplitude was -25 mV at RJC of 80 M (Fig. 10A), -17 mV at 40 M
(Fig. 10B), -10 mV at the standard 20 M
(Fig. 10C), and -6 mV at 10 M
(Fig. 10D). There was almost a linear relationship between VJC amplitude and RJC amplitude, as shown in Fig. 12A. All 25 cells of the SMC network responded when RJC was 20 M
(Fig. 12C) or 40 M
(Fig. 12D), but one cell failed to respond at 10 M
(Fig. 12D), and many cells failed at 80 M
(Fig. 12A). Therefore, there is an optimum value of RJC.
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As can be seen in Fig. 11, VJC amplitude was greater when slow-wave amplitude was greatest. These data are plotted in Fig. 12B. As in the case of ICCA, there was an optimum RJC value for maximum effectiveness of the ICCB cell in activating the network. All 25 cells responded at RJC of 40 M (Fig. 12B) and the standard 20 M
(Fig. 12C), but several cells failed at 10 M
(Fig. 12D), and many cells failed at 80 M
(Fig. 12A).
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DISCUSSION |
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Two types of ICC-IM cells with respect to the RP were modeled: ICCA (RP of -80 mV) and ICCB (RP of -55 mV). The latter had an RP similar to that of the SMC in the network. Both types were capable of activating the SMC network, but the first type (ICCA) worked more readily. This may be due to the fact that the VJC produced at the ICC-E5 junction was greater with the ICCA cell type (see Fig. 4). Therefore, successful activation of the SMC network should occur whether the RP of the ICC-IM cell is low (e.g., -55 mV) or high (e.g., -80 mV).
Even when the ICC-IM cell was made excitable to intrinsically generate an overshooting AP (to +32 mV in the case of ICCA and to +11 mV in the case of ICCB) by introducing positive feedback between Na+ or Ca2+ conductance and Vm, there was successful activation of the SMC network. This occurred despite the fact that the AP was very fast rising in the case of ICCA (e.g., dV/dtmax of 200 V/s). In the case of ICCB, the AP rate of rise was
10 V/s, i.e., much closer to the rate of rise of the physiological slow wave. Therefore, successful activation of the SMC network also occurred when the ICC-IM cell was made intrinsically excitable. Thus the present results with the special modeling of slow waves are valid.
Special modeling of the slow wave for the ICC-IM cell was done by making it inexcitable (i.e., no positive feedback between Ca2+ conductance and Vm) and applying an intracellular stimulating current shaped like a slow wave. This had a number of advantages, including being able to readily vary characteristics of the slow wave such as amplitude, duration, rate of rise, and rate of fall. Because the SMC modeled were not given pacemaker properties (i.e., repetitive firing during prolonged depolarizing pulses), long-duration slow waves were no more effective than those of short duration. Hence, the standard slow wave used had a plateau duration of 8 ms and a rise time of 4 ms. The standard current (ISW) applied to the ICC-IM was 2.4 nA in the case of the ICCA type and 1.8 nA in the ICCB type, because those were the current amplitudes required to produce a slow-wave amplitude of 40 mV (about the physiological value) in both types. The slow wave in both types of ICC-IM cell produced substantial negative cleft potentials at the ICC-E5 junction (see Fig. 4).
Circuit analysis of the ICC cell and associated cell junction (ICC-E5) demonstrated that a large negative cleft potential should be generated in the junction, whose magnitude should be nearly directly proportional to the magnitude of the slow wave and ISW. This was actually found for the ICCA cell. (There was deviation from linearity in the case of the ICCB cell). As previously reported (6, 13) VJC amplitude was determined by the amplitude of RJC (see Figs. 10 and 11). As shown, there was almost a linear relationship between RJC and the amplitude of VJC in the ICC-E5 junction.
In the present study, there were no gap junction channels between the 25 SMCs of the planar network (5 parallel chains of 5 cells each). It was previously shown (5, 6, 8) that transmission from cell to cell occurs by the EF (negative VJC) generated in the junctional cleft when the prejunctional membrane fires an AP. This accounts for longitudinal propagation. Transverse propagation between parallel chains may occur by a similar EF mechanism, because the longitudinal resistance of the ISF space between the chains (Rol2) was a key factor in facilitating the transverse propagation (unpublished observations) (the higher the Rol2, reflecting tighter packing of the chains, the better and faster the transverse transmission). Moderate hyperexcitability of the SMCs in the network facilitates the longitudinal and transfer propagation throughout the network by the EF mechanism. The excitability of the basic membrane units was originally adjusted to give a propagation velocity (longitudinal) in the average physiological range with the cells having AP characteristics also in the physiological range.
This hyperexcitability may account for why slow waves of smaller amplitude were almost as effective in activating the network in both the ICCA and ICCB cases (see Figs. 7 and 8). The same may be true as to why longer rise times for the slow waves were also very effective
In summary, one ICC-IM cell displaying a simulated slow wave (40 mV amplitude) was able to activate a planar network of 25 SMCs. The ICC-IM cell could be either type A (RP of -80 mV) or type B (RP of -55 mV), but type A was slightly more effective. Successful transmission occurred without any gap junction channels at the ICC-E5 junction and was mediated by the negative junctional cleft potential (VJC) developed at that junction. Therefore, gap junctions are not required at this junction. Successful transmission also occurred when the ICC-IM cell was made intrinsically excitable to produce regenerative APs; this was true for both ICCA and ICCB types.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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