Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
Submitted 27 July 2004 ; accepted in final form 23 September 2004
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ABSTRACT |
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pacemaker activity; slow waves; gastrointestinal motility; calcium channel
Pacemaker potentials of in situ ICC-MY were first recorded in the guinea pig gastric antrum (3). Gastric pacemaker potentials have two components: a primary component and a plateau component (5, 13). Pacemaker potentials conduct electrotonically to smooth muscles cells (3, 14) and activate voltage-dependent conductances in these cells. Responses to pacemaker potentials recorded from smooth muscle cells have been termed slow waves. Slow waves have a secondary depolarization (regenerative component) superimposed on the electrotonic remnant of the pacemaker potential (see also Ref. 32). Intramuscular interstitial cells of Cajal (ICC-IM), which are distributed between smooth muscle cells (1), may contribute to the regenerative component (2). Recently, pacemaker potentials also were recorded in situ from ICC-MY in the murine small intestine (15). These potentials have at least two components: a rapidly rising upstroke component and a plateau component. The rate of rise of the upstroke depolarization (dV/dtmax) was greatly reduced by Ni2+, Ca2+-free solution, and membrane depolarization with high-K+ solution. The plateau component was inhibited by DIDS and low-Cl solution (15). Therefore, different mechanisms appear to be responsible for the two components of pacemaker potentials.
Recent studies have suggested that a dihydropyridine-resistant Ca2+ conductance may be important for the active propagation of pacemaker potentials within ICC networks (12). Such a conductance exists in cultured ICC-MY from murine small and large intestine (12) and in freshly dispersed ICC from the canine colon (20). Activation of this conductance was proposed to increase Ca2+ influx that phase advances (or entrains) Ca2+ release from inositol 1,4,5-trisphosphate (IP3) receptors. Mibefradil, an inhibitor of T-type Ca2+ channels, blocked the dihydropyridine-resistant Ca2+ conductance but did not block the primary pacemaker current (12). Thus this agent may be helpful in determining the role of the dihydropyridine-resistant Ca2+ conductance in pacemaker potentials recorded from ICC-MY in situ. We hypothesize that the upstroke transient in pacemaker potentials may be a reflection of activation of the dihydropyridine-resistant Ca2+ conductance. Ca2+ entry during the upstroke depolarization may entrain the activity of multitudes of active pacemaker units in ICC-MY, causing the unitary potentials generated by these active sites to summate and form the plateau component.
In the present study, we investigated the nature and role of the upstroke and plateau components of pacemaker potentials recorded in ICC-MY in situ of the murine small intestine using conventional microelectrode recording techniques. The frequency of pacemaker potentials was decreased when the upstroke component was inhibited by mibefradil. Reducing the upstroke caused resolution of unitary potentials, suggesting reduced entrainment of pacemaker activity. The results suggest that there is a causal relationship between the generation of the upstroke depolarization and the frequency of pacemaker potentials. The possible ionic mechanisms generating the upstroke and plateau components of pacemaker potentials are discussed.
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METHODS |
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Conventional microelectrode techniques were used to record intracellular electrical responses from smooth muscle tissues, and the glass capillary microelectrodes filled with 3 M KCl had tip resistances ranging from 90 to 150 M. Signals were amplified with an Axoprobe amplifier, low-pass filtered (cutoff frequency 1 kHz), digitized, and stored on a computer for later analysis.
The bath chamber was constantly perfused with oxygenated KRB of the following composition (in mM): 118.5 NaCl, 4.5 KCl, 1.2 MgCl2, 23.8 NaHCO3, 1.2 KH2PO4, 11.0 dextrose, and 2.4 CaCl2. The pH of the KRB solution was 7.37.4 when bubbled with 97% O2-3% CO2 at 37 ± 0.5°C. After pinning, the preparations were left to equilibrate for at least 1 h before experiments began.
Pacemaker potentials and slow waves from ICC-MY and smooth muscle cells, respectively, were recorded (15). These events were analyzed off-line by measuring the amplitude, dV/dtmax, half-amplitude duration, and frequency. Mibefradil slowed the frequency of pacemaker potentials and caused an extension in the duration between them. This period of diastolic depolarization was approximately exponential and was fit with single-exponential functions. Mibefradil also appeared to move the threshold for initiation of the pacemaker potentials toward more positive values. Because it was not possible to rigorously evaluate threshold values in these studies, we tabulated the voltage at which there was a sharp transition in dV/dt and termed this voltage the "take-off" potential.
Drugs used were caffeine, glibenclamide, nifedipine, and pinacidil (all obtained from Sigma, St. Louis, MO). Mibefradil was a gift from Hoffmann-La Roche. Glibenclamide was dissolved in dimethyl sulfoxide (DMSO) to make stock solutions. Nifedipine and pinacidil were dissolved in ethanol. Other drugs tested were dissolved in distilled water. The final concentration of the solvent in KRB did not exceed 1:1,000. Addition of these chemicals to KRB solution did not alter the pH of the solution.
Experimental values were expressed as means ± SD. Statistical significance was tested using Student's t-test, and P < 0.05 was considered significant.
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RESULTS |
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Cells in which slow waves were recorded had peak membrane potentials of 67.4 ± 2.7 mV (n = 12). Slow waves occurred at a frequency of 25.2 ± 1.4 min1 (n = 12) and had peak amplitudes of 30.0 ± 2.3 mV (n = 12), dV/dtmax of 0.52 ± 0.08 V s1 (n = 12), and half-widths of 0.90 ± 0.08 s (n = 12). The peak amplitudes and dV/dtmax measured from pacemaker potentials differed significantly from those of slow waves, while membrane potentials between the spontaneous depolarizations were not significantly different in the two types of cells. Comparison of the electrical properties of the spontaneous depolarizations with those reported previously (15) suggests that pacemaker potentials and slow waves were recorded from ICC-MY and circular smooth muscle cells, respectively.
When circular muscle cells were impaled, the membrane potential dropped sharply to a maximum negative potential of about 65 mV (data not shown). When ICC-MY were impaled, membrane potential dropped to between 20 and 50 mV initially (Fig. 1A) and then increased gradually to nearly 70 mV (Fig. 1B). In more than one-half of the cells tested (63.6%), unitary potentials (see Refs. 4 and 16 for definition and description of these events) were resolved during the intervals between pacemaker potentials just after impalement of ICC-MY (Fig. 1A). However, resolvable unitary potentials decreased as the resting membrane potential stabilized (Fig. 1B).
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The effects of mibefradil on slow waves (i.e., events recorded from smooth muscle cells) were similar to its effects on pacemaker potentials. Mibefradil, at concentrations of 10 and 30 µM but not 1 µM, reduced the amplitude, frequency, and dV/dtmax of slow waves (Fig. 4). Membrane potential between slow waves was depolarized by 3.7 ± 2.2 mV at 10 µM mibefradil (n = 8) and by 8.4 ± 0.7 mV at 30 µM mibefradil (n = 8) (Figs. 4 and 5). As with pacemaker potentials, mibefradil converted slow waves into slowly developing changes in potential, indicating that modulation of pacemaker potentials is closely reflected in slow-wave activity. The transient repolarization at the end of the upstroke, which is distinctive behavior recorded from smooth muscle cells, was abolished in the presence of 10 µM mibefradil (Fig. 4B). The effects of mibefradil on slow waves are summarized in Fig. 5.
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Figure 10 shows a comparison of the configuration of pacemaker potentials with the configuration of slow waves before (Fig. 10, Aa and Ba) and during (Fig. 10, Ab and Bb) exposure to 10 µM pinacidil after normalization of the amplitudes of slow waves to the amplitudes of pacemaker potentials. Pinacidil had no appreciable effect on the waveforms of pacemaker potentials (Fig. 10Ab). In contrast, there was a distinct change in the configuration of slow waves in the presence of pinacidil. The transient repolarization after the upstroke of slow waves was reduced by pinacidil (Fig. 10Bb).
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DISCUSSION |
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It was shown previously that Ni2+ and reduced extracellular Ca2+ decreased the dV/dtmax and frequency of pacemaker potentials recorded in ICC-MY in the murine small intestine (15). The depolarization during the diastolic period was also slowed by Ni2+ and reduced extracellular Ca2+, suggesting the involvement of Ca2+ entry during the initial and upstroke depolarizations. To further explore this possibility, we tested the effects of mibefradil, a blocker of T-type Ca2+ channels (24), on pacemaker potentials, because this compound has been shown to inhibit a dihydropyridine-resistant Ca+ current in ICC-MY of the murine small intestine without affecting the primary pacemaker conductance (12). Mibefradil decreased the dV/dtmax and frequency of pacemaker potentials. The inhibitory effects of mibefradil were strikingly similar to the effects of Ni2+ and reduced extracellular Ca2+, thus strengthening the hypothesis that a dihydropyridine-resistant Ca2+ conductance contributes to the depolarization during the diastolic period and to the upstroke depolarization of pacemaker potentials.
At 30 µM, mibefradil dramatically increased the resolution of unitary potentials during the intervals between pacemaker potentials (see Figs. 2 and 7). Unitary potentials may represent the basic pacemaker events of ICC-MY. Unitary potentials are also a common feature of small bundles of antral circular muscle (containing ICC-IM), and these events can summate to produce events termed regenerative potentials (22, 26). Unitary potentials also occur in ICC-MY of the gastric antrum (5, 16), and summation of these events has been suggested as the basis for the plateau component of pacemaker potentials (i.e., "driving potentials" in the terminology of Edwards and colleagues). In the present study, the shape of pacemaker potentials in the presence of 30 µM mibefradil was similar to that of the regenerative potentials recorded from single bundles of circular muscle from the guinea pig gastric antrum (22, 26). Thus it is reasonable to suggest that pacemaker potentials in the presence of mibefradil (or low concentrations of Ni2+ or extracellular Ca2+; see Ref. 15) may similarly result from the summation of unitary potentials. We also noted a buildup of unitary potentials before the discharge of pacemaker potentials in the presence of mibefradil (see Figs. 2 and 7). A basic difference between ICC-MY (which generate regular pacemaker potentials with fast upstroke depolarizations) and ICC-IM (which also have an intrinsic capacity for generation of unitary potentials, but such events are typically uncoordinated and entrained by the pacemaker activity of ICC-MY in situ) may be the channel density of voltage-dependent, mibefradil-sensitive Ca2+ channels.
ICC-MY contain an abundance of intracellular structures (e.g., sarcoplasmic reticulum and mitochondria) that are arranged in close apposition to the plasma membrane (25). The plasma membrane contains ionic conductances necessary for generation and propagation of pacemaker potentials. We have referred to these complexes (i.e., intracellular organelles and plasma membrane conductances) as "pacemaker units" and have suggested that these complexes are responsible for the generation of pacemaker currents by regulating submembrane Ca2+ concentration (12). Each pacemaker unit is capable of spontaneous generation of inward current, and random activation of these currents from a multitude of pacemaker units is likely to be the basis for unitary potentials. Thus a mechanism must exist to entrain the activity of pacemaker units throughout ICC-MY networks. Our data suggest that a dihydropyridine-resistant Ca2+ conductance is required for entrainment of the activity of pacemaker units and active regeneration of pacemaker potentials (see Refs. 12 and 15). Thus voltage-dependent, dihydropyridine-resistant Ca2+ channels appear to be a critical participant in the pacemaker unit complex. On the basis of this realization, we interpret our results in the following manner. Under control conditions, activation of dihydropyridine-resistant Ca2+ channels facilitates entrainment of unitary potentials, which provides the basis for regeneration of pacemaker potentials. When the availability of dihydropyridine-resistant Ca2+ channels (as occurs in the presence of mibefradil, Ni2+, or elevated external K+; see Ref. 15) or the driving force for Ca2+ entry is reduced (as occurs when extracellular Ca2+ is depleted), the probability of entrainment is reduced. Under these conditions, the activity of pacemaker units is effectively uncoupled, and the tendency to resolve unitary potentials is therefore increased. A further observation consistent with previous studies (12) is that manipulations that reduce current through dihydropyridine-resistant Ca2+ channels do not appear to affect the intrinsic pacemaker activity of ICC-MY (at least for a period of many minutes), and thus the generation of unitary potentials persists.
Previous studies of pacemaker activity in ICC-MY of the gastric antrum have shown that shortly after repolarization of pacemaker potentials, the occurrence of unitary potentials is low but the probability of these events increases steadily during the diastolic period (5). It is likely that the net depolarization caused by the summation of unitary potentials is responsible for bringing membrane potential to the point of threshold. Threshold in ICC-MY appears to be due to the inward current generated by activation of voltage-dependent, dihydropyridine-resistant Ca2+ channels. Reducing the availability of these Ca2+ channels would tend to shift threshold away from the maximum diastolic (i.e., resting) potential between slow waves and increase the time required for sufficient recovery of unitary potentials from refractoriness and summation of these events to reach threshold. This would result in a slowing of the frequency of pacemaker potentials, which is what we observed in response to mibefradil. This concept is also in agreement with our findings that mibefradil slowed the rate of the diastolic depolarization and caused a positive shift in the potential at which the upstroke depolarization developed (take-off potential). Reduction in the current from dihydropyridine-resistant Ca2+ channels also resulted in slower upstroke depolarizations, suggesting that this current contributes significantly to the charge movement responsible for this phase of pacemaker potentials. This conclusion is consistent with findings in a long history of experiments on slow waves and pacemaker activity in gastrointestinal muscles in which several authors have concluded that the upstroke potential fundamentally depends on voltage-dependent, dihydropyridine-resistant Ca2+ entry (4, 7, 15, 36).
Caffeine is thought to have multiple effects on intracellular Ca2+ mobilization, such as enhancing Ca2+ release from ryanodine receptors (10), blocking IP3 receptors (23), and inhibiting IP3 production (30). These effects are related to Ca2+ release from internal Ca2+ stores. Pinacidil inhibits the production of IP3 (11). A previous study showed that BAPTA-AM abolished pacemaker potentials and unitary potentials recorded from ICC-MY in murine small intestine (15). In the present study, pinacidil reduced the duration of the plateau potential. It seems reasonable to hypothesize that the generation of unitary potentials and the plateau component of pacemaker potentials are related to Ca2+ release from internal Ca2+ stores. Indeed, it is known that intracellular Ca2+ handling mechanisms mediated by IP3-induced Ca2+ release from internal stores (27, 35) and Ca2+ uptake into mitochondria (35) play a key role in generating spontaneous activity in gastrointestinal muscles and ICC. Furthermore, our results support the idea that pacemaker potentials result from at least two conductances: an initial Ca2+ influx (upstroke component) and a second conductance activated via Ca2+ release from IP3 receptor-operated stores and mitochondrial Ca2+ uptake (plateau component) (see Ref. 15).
Pinacidil increased the dV/dtmax of the upstroke of pacemaker potentials. This is likely caused by the increase in driving force for Ca2+ ions caused by hyperpolarization due to activation of KATP channels. While significant hyperpolarization would be expected to stabilize membrane potentials and decrease the likelihood of activation of voltage-dependent Ca2+ channels, it appears that the 10-mV hyperpolarization caused by pinacidil was not sufficient to block pacemaker potentials. Hyperpolarization would also be likely to increase the amplitude of unitary potentials, because these are also due to inward currents with equilibrium potentials positive to the resting potential. In fact, previous studies of unitary potentials in gastric tissues confirms the increase in amplitude in response to hyperpolarization (4, 16). The dihydropyridine-resistant (mibefradil sensitive) Ca2+ current activates and inactivates at potentials more negative than those of dihydropyridine-sensitive current (12). In our previous characterization of the dihydropyridine-resistant Ca2+ current in ICC-MY, we found that only 50% of channels were available at the resting potentials recorded from ICC-MY in situ in the present study. Hyperpolarization by 10-mV increased the availability of these channels to
80%. Thus, as these cells hyperpolarize, there may be accommodation to the hyperpolarization that results in a negative shift in threshold. The increase in dihydropyridine-resistant Ca2+ channel availability would help to preserve the frequency and would tend to increase the amplitude and dV/dtmax. While the pacemaker current underlying unitary potentials and the plateau component of pacemaker potentials are apparently independent of membrane potential (17, 29), the contributions of the voltage-dependent, dihydropyridine-resistant Ca2+ conductance to the process of entrainment and generation of the upstroke potential lend voltage dependency to the integrated phenomenon of pacemaker activity in ICC.
The recordings in the present study show a contrast in the manifestations of slow waves in pacemaker cells (ICC-MY) and smooth muscle cells. Previous studies performed in gastric muscle cells suggest that slow waves passively conduct in smooth muscle cells (3). The results of the present study are consistent with this conclusion. Slow waves recorded from smooth muscle cells were always smaller in amplitude than the pacemaker potentials recorded in ICC-MY, and the rate of rise of the upstroke phase of the slow waves was slower in smooth muscle cells. Because slow waves are driven by the pacemaker potentials in ICC-MY, any treatment that affected specific parameters of events in ICC-MY would be expected to have analogous effects in smooth muscle cells. Our observations demonstrate that mibefradil reduced the dV/dtmax of the upstroke component in ICC-MY and had equivalent effects in smooth muscle cells and that caffeine reduced the plateau phase and had equivalent effects in smooth muscle cell recordings. Pinacidil, which did not block pacemaker potentials in ICC-MY (see previous paragraph), would be expected to increase K+ conductance (via activation of KATP channels) of the smooth muscle syncytium. Reduction in input resistance would tend to reduce the amplitude of voltage signals that conduct passively through smooth muscle cells. Consistent with this hypothesis, pinacidil greatly reduced slow-wave amplitude.
In consideration of previous results published by several authors, our observations in the present study support the following concept (see also Fig. 11). Pacemaker potentials recorded from ICC-MY in the murine small intestine are composed of a rapidly rising upstroke component due to Ca2+ influx via a dihydropyridine-resistant Ca2+ conductance. The upstroke triggers a sustained plateau component that represents a summation of primary pacemaker currents. These events are initiated by Ca2+ release from internal stores. The primary pacemaker currents (which result in unitary potentials) persist when the availability of the dihydropyridine-resistant Ca2+ channels decreases, but under these circumstances, entrainment of unitary potentials is reduced. Thus the upstroke component due to the entry of Ca2+ is critical for 1) entrainment of pacemaker activity in the multitudes of active pacemaker units in ICC-MY networks and 2) active regeneration of pacemaker potentials.
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GRANTS |
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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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