Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
Submitted 12 March 2003 ; accepted in final form 24 June 2003
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ABSTRACT |
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gastrointestinal motility; adenosine 5'-triphosphate; voltage-dependent nonselective cation current
There has never been a clear explanation for the rapid repolarization of IJPs, and questions remain about the factors making IJPs so brief (0.5-1.5 s). This is an important question, however, because it directly bears on postjunctional responses and the regulation of excitability of GI smooth muscles by neurotransmitters. An understanding of this repolarization mechanism might be exploited to develop drugs to regulate motility. The repolarization phase of IJPs has been ignored because most investigators consider it to be a passive process that is due to the metabolism and/or dilution by diffusion of neurotransmitters (ATP or NO). For example, as transmitter concentration falls near postjunctional receptors, the K+ channels (SK or NO-dependent K conductances) activated during IJPs may close, producing passive restoration of resting membrane potential. Because the actual spatiotemporal profiles of neurotransmitters concentrations after nerve stimulation are unknown, it is not clear that transmitter depletion can fully explain IJP repolarization. We have considered the possibility that a conductance responsible for an inward current with voltage-dependent properties that render it unavailable at the resting potential might activate at the negative potentials reached during IJPs. Such a conductance could participate in "active repolarization" that would tend to rapidly terminate IJPs.
Our laboratory recently characterized two components of voltage-gated, inward currents in murine colonic myocytes (15). One component had properties of an L-type Ca2+ current and was inhibited by dihydropyridines. The second component did not "run down" during cell dialysis and was resistant to nicardipine (up to 10-6 M). The nicardipine-insensitive current was a low-voltage-activated current, and it was inhibited by Ni2+ (50% inhibitory concentration = 1.4 x 10-5 M), mibefradil (10-6 to 10-5 M), and extracellular Ba2+. Our experiments suggested that the second component of voltage-dependent inward current in murine colonic myocytes appeared to be a novel, voltage-gated nonselective cation current. The properties of this conductance suggest it could contribute to active repolarization of IJPs in GI muscles, and in the present study we designed experiments to test this hypothesis. We have compared the effects of blockers of voltage-gated nonselective cation current on IJPs with responses to locally applied ATP.
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METHODS |
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Muscles were maintained at 37.5 ± 0.5°C by a flowing Krebs-Ringer bicarbonate solution containing (in mM) 120.4 NaCl, 5.9 KCl, 15.5 NaHCO3, 11.5 glucose, 1.2 MgCl2, 1.2 NaH2PO4, and 2.5 CaCl2. The solution was bubbled with 97% O2-3% CO2 and had a pH of 7.3-7.4. Circular muscle cells were impaled with glass microelectrodes filled with 3 M KCl having resistances between 50 and 80 M. Transmembrane potential was measured with a high input impedance amplifier (WPI S-7071, Sarasota, FL). Tissues were equilibrated for
1 h before recordings were begun.
Transmural stimulation and picospritzing of ATP. Two platinum wires were placed parallel to the long axis of the circular muscle layer and were connected to a square wave stimulator (Grass 588, Grass, Quincy, MA) through stimulus isolation unit (Grass SIU 5A). Electrical field stimulation was applied as single and multiple pulses (0.5-ms duration and 150-V intensity at frequencies from 1 to 30 Hz). In some experiments, small puffs of ATP-containing solution were administered directly to the muscles by means of a pressurized application device (Picospritzer; General Valve, East Hanover, NJ). In these experiments, a micropipette (8- to 10-µm diameter) filled with 0.1 M ATP was placed as close as possible to the site of the intracellular microelectrode used to record responses. Pressure pulses at 12 lb./in.2 and 60-ms duration were applied to deliver ATP to the site of recording. Tetrodotoxin, Ba2+, and Ni2+ (as Cl- salts) were purchased from Sigma (St. Louis, MO). Mibefradil was a gift from Dr. Eva-Maria Gutknecht and Dr. Pierre Weber (Hoffmann-La Roche, Basel, Switzerland).
Measurement of ectonucleotidase activity. 1,N6-etheno (e)modified nucleotides, have been previously used as exogenous substrates for ATPases or AMPases (13, 17, 20, 24). Moreover, e-nucleotides can be detected at fmol concentration range, providing a sensitive approach of measuring small amounts of the degradation products. Stock solution of 50 µM eATP was prepared from ATP as previously described (6). Briefly, 100 µl of a citrate phosphate buffer (pH 4.0) containing 62 parts of 0.1 M citric acid and 38 parts of 0.2 M Na2HPO4, and 10 µl of 2-chloroacetaldehyde were added to 200 µl of the nucleotide solution (final concentration of nucleotide, 50 µM) in a borosilicate glass culture tube (Fisher Scientific), and the samples were heated for 40 min at 80°Cin a dry bath incubator (Fisher Scientific) to produce the fluorescent derivative eATP. Isolated segments of mouse colon (19.2 ± 0.2 mg wet wt; n = 12) were placed in 200-µl BRANDEL superfusion chambers, as described previously (18, 23). Chambers were mounted vertically in a thermostatic block (at 36.5°C), and the tissue segments were superfused from bottom to top with oxygenated Krebs solution containing (in mM) 118.5 NaCl, 4.2 KCl, 1.2 MgCl2, 23.8 NaHCO3, 1.2 KH2PO4, 11.0 dextrose, and 1.8 CaCl2, and was maintained at 36.5°C at a flow rate of 1 ml/min. After 45 min of equilibration, tissues were subjected to perfusion with eATP (50 nM) for 10 min, and samples from the superfusate were collected from the solution before the tissue (S1) and after 10 min of contact with the tissue (S2). All samples were collected in ice-cold test tubes, and the reaction was stopped by immersing the sample vials in liquid nitrogen. The difference of the e-nucleotide content between S1 and S2 was taken as a measure for ectonucleotidase activity. In some experiments, tissues were superfused with 0.2 mM Ni2+ or 1 µM mibefradil for 5 min before the perfusion with the eATP was started. Both Ni2+ and mibefradil were present along with eATP during the 10-min perfusion before S2 was collected.
High-performance liquid chromatography analysis of e-nucleotides and e-ADO. Samples were analyzed for eATP, eADP, eAMP, and eADO content by reverse high-performance liquid chromatography technique in conjunction with fluorescence detection. Thus the e-nucleotides were separated with an ODS-AM C18 column (Waters, Milford, MA) in a gradient high-performance liquid chromotography system (HP1100 LC; Agilent Technologies, Wilmington, DE). The mobile phase comprised 0.1 M KH2PO4 (pH 6.0 adjusted with 1 M KOH) as buffer A and solvent B consisted of 35% methanol and 65% buffer A. Gradient elution was employed according to the following linear program: time 0, 0% solvent B; 18 min, 100% solvent B. Flow rate was 1 ml/min, run time was 20 min, and postrun time was 5 min. Column temperature was ambient, whereas the autosampler temperature was set at 4°C. Fluorescent signals were determined with a programmable fluorescence detector (HP1100) at an excitation wavelength of 230 nm and an emission wavelength of 420 nm at gain 17. The amounts of adenyl nucleotides and nucleosides in each sample were calculated from calibration curves of nucleotide standards run simultaneously with every set of unknown samples. Results were normalized for sample volume and tissue wet weight, and the overflow of nucleotides was expressed in pmol/mg tissue.
Statistics. All values are expressed as means ± SE, and statistical significance was determined by paired t-test.
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RESULTS |
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We tested the effect of external Ba2+ (100 µM to 1 mM) on IJP profiles (Fig. 1, B-D). Application of Ba2+ (100 µM) did not show significant effect on IJP pattern. However, higher concentrations of Ba2+ (500 µM) significantly increased IJP duration (1.28 ± 0.11 s, n = 6, P < 0.01; values from 10-Hz stimulation) and amplitude (30 ± 1.1 mV, n = 6, P < 0.05; Fig. 1, E and F). Ba2+ (500 µM) also significantly depolarized resting membrane potential (i.e., from -55.5 ± 2.1 to -50.3 ± 2.3 mV in the presence of Ba2+, P < 0.001, n = 6).
In the presence of Ba2+, there was significant rebound excitation after repolarization of the IJP. The rebound excitation was characterized by depolarization and a burst of action potentials (see Fig. 1, C and D). The large rebound excitation may somewhat obscure the repolarization phase of the IJP, so additional experiments were performed in the presence of nicardipine (1 µM), which totally abolished both the action potentials and the rebound excitation depolarization. In the presence of nicardipine, Ba2+ (500 µM) significantly increased IJP duration (from 1.03 ± 0.06 to 1.36 ± 0.23 s, n = 6, P < 0.05; values from 10-Hz stimulation; Fig. 2) and amplitude (from 27 ± 1.8 to 31 ± 1.5 mV, n = 6, P < 0.05). Ba2+ (500 µM) also significantly depolarized resting membrane potential (i.e., from -53.4 ± 2.2 to -48.0 ± 2.9 mV in the presence of Ba2+, P < 0.05, n = 6).
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Two additional blockers of the voltage-dependent NSCC channel in colonic myocytes, Ni2+ and mibefradil, were also tested, but these compounds have generalized blocking effects on a variety of Ca2+ channels. Application of these compounds, therefore, may affect neurotransmitter release, and we observed indications of this because both compounds decreased IJP amplitudes. For example, in experiments in which Ni2+ was tested, the mean peak amplitudes of fast IJPs at 1, 10, and 30 Hz were 24.3 ± 2.0, 25.4 ± 1.9, and 26.2 ± 2.1 mV, respectively (n = 6). Ni2+ (500 µM) dramatically decreased IJP amplitude (to 20.7 ± 2.2, 20.3 ± 1.9, and 19.3 ± 1.9mV at 1, 10, and 30 Hz, respectively). In tests with mibefradil, the mean peak amplitudes of fast IJPs at 1, 10, and 30 Hz under control conditions were 28.7 ± 4.7, 27.7 ± 4.6, and 27.5 ± 4.9 mV, respectively. After mibefradil (5 µM), amplitudes of IJPs significantly decreased (to 22.0 ± 1.5, 21.5 ± 1.76, and 22.3 ± 2.19 mV at 1, 10, and 30 Hz, respectively). Thus it is likely that Ni2+ and mibefradil are not useful in assessing the kinetics of the IJP because of possible prejunctional effects.
Effects of NSCC blockers on responses to locally applied ATP. To avoid problems with NSCC blockers on transmitter release, we performed experiments in which ATP, the putative transmitter responsible for the fast IJP, was directly applied to the muscle by pressure ejection. ATP (0.1 M loaded in a micropipette) was picospritzed with a single 60-ms pulse. These experiments were performed in the presence of tetrodotoxin (0.5-1 µM), L-NA (100 µM), and atropine (1 µM). In these tests, resting membrane potential averaged -46.4 ± 0.9 mV (n = 7). The amplitude and duration of ATP-induced hyperpolarizations were 21.8 ± 1.2 mV and 49.4 ± 5.2 s (n = 5), respectively (Fig. 3). While maintaining the same impalement and without moving the pressure ejection pipette, we tested the effects of Ba2+ effects on ATP-induced hyperpolarizations. Ba2+ (500 µM) did not significantly affect the resting membrane potential or the amplitude of ATP-induced hyperpolarization (i.e., -44.2 ± 0.97 and 24.6 ± 1.57 mV, respectively; Fig. 3, C and D). These values were not significantly different from control value. The durations of ATP-induced hyperpolarizations were significantly prolonged by Ba2+ (to 65.0 ± 4.97 s; Fig. 3, A and B).
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Using the same technique, we tested the effects of Ni2+ on ATP-induced hyperpolarizations (Fig. 4). In these tests, the resting membrane potential was -48.3 ± 0.9 mV (n = 6), and the amplitude and duration of ATP-induced hyperpolarizations were 20.8 ± 0.9 mV and 69.5 ± 12.3 s (n = 6), respectively. While the same impalement was maintained and without moving the pressure ejection pipette, addition of Ni2+ (500 µM) did not affect resting membrane potential or the maximum amplitude of ATP-induced hyperpolarizations (-49.0 ± 0.7 and 22.2 ± 1.0 mV, P > 0.05; Fig. 4, C and D). However, the duration of ATP-induced hyperpolarization was significantly increased after Ni2+ (to 111.7 ± 21.6 s; Fig. 4B).
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We also tested the effects of mibefradil on ATP-induced hyperpolarizations (Fig. 5). In these experiments, the resting membrane potential was -49.0 ± 0.7 mV (n = 4). The amplitude and duration of ATP-induced hyperpolarization were 21.3 ± 0.8 mV and 41.5 ± 2.2 s (n = 4), respectively. While the same impalements were maintained, mibefradil (1 µM) did not affect resting membrane potential or the amplitudes of ATP-induced hyperpolarizations (-48.0 ± 0.7 and 21.3 ± 0.5 mV, respectively, P > 0.1). However, the duration of ATP-induced hyperpolarizations was significantly increased after mibefradil (to 72 ± 4.2 s, P < 0.001; Fig. 5, A and B).
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Ni2+ and mibefradil effect on ATPase activity. One possible explanation for the increase in the duration of ATP-induced hyperpolarization could be that the compounds used might block tissue ATPase activity and thus prolong the actions of ATP. Therefore, we tested the effects of Ni2+ and mibefradil on colonic tissue ATPase activity.
A typical chromatogram of eATP consists of a major eATP peak with a retention time of 10.5 ± 0.4 min and a small eADP peak (6% of the eATP peak), with no signals corresponding to eAMP or eADO (S1; Fig. 6A). eATP was not degraded for a period of 30 min at 36.5°C in the absence of tissue (data not shown). With the tissue present, however, a 10-min incubation with the eATP resulted in decreased eATP, increased eADP, and appearance of eAMP and eADO (collectively called e-product) (Fig. 6, A and B), suggesting that eATP undergoes degradation during a 10-min contact period with colonic muscles, presumably due to the action of membrane-bound nucleotidases (25). No significant reduction of eATP or enhancement of the e-products occurred in the mouse colon treated with Ni2+ (0.2 mM) (Fig. 6C), suggesting that Ni2+ inhibited ATPase activity. In the presence of mibefradil (1 µM), however, eATP was degraded the same as in control tissues (Fig. 6D).
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DISCUSSION |
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None of the other known inward current conductance(s) in colonic myocytes is likely to significantly contribute to the active repolarization mechanism. Neuronal stimulation in the presence of dihydropyridine to block L-type Ca2+ channels did not change the profiles of IJPs in the present study or in previous investigations of IJPs (7). This observation tends to exclude the involvement of L-type Ca2+channels or Ca2+-activated Cl- channels, which depend, in part, on Ca2+ entry for activation in the active repolarization of IJPs. Additionally, the voltage range for activation of resolvable L-type Ca2+ channels (i.e., positive to -50 mV) is too positive for these channels to be a factor in IJP repolarization (16, 27).
An active repolarization mechanism would require a conductance that activates at negative potentials (greater than -60 mV) and exhibits voltage dependence. One class of channels that satisfies this criterion is the T-type Ca2+ channels, which are low-voltage-activated Ca2+ channels. There have been reports in the literature about T-type Ca2+ conductance in GI smooth muscle cells (30, 31, 33, 34). The well-described characteristics of T-type Ca2+ currents are 1) low threshold activation, 2) rapid inactivation, 3) negative potentials for half-inactivation, 4) slow deactivation, 5) resistance to dihydropyridine, and 6) equal Ca2+ and Ba2+ permeability. Our previous voltage-clamp studies of murine colonic muscles showed that the voltage-dependent NSCC is a discrete (non-T-type) conductance. The properties of the voltage-dependent NSCC include 1) low threshold activation at negative potentials, 2) current reversal at 0 mV, 3) fast recovery from inactivation, and 4) block by Ni2+, Ba2+, and mibefradil. The low threshold activation of the NSCC suggests that this conductance could provide a mechanism for active repolarization of IJPs.
Having identified several blockers of the NSCC in our laboratory's previous study of the voltage-dependent NSCC (15), we used these agents to study the involvement of this conductance in enteric inhibitory responses and responses to ATP. Ba2+ was the most useful agent to study the role of the NSCC in IJPs elicited by electrical field stimulation, reducing the rate of repolarization without any significant effect on amplitude. We found that other blockers of the NSCC (i.e., Ni2+ and mibefradil) reduced the amplitude of EJPs. Because these compounds are nonspecific Ca2+-channel blocking agents, it is possible that they interfered with transmitter release, which is Ca2+ dependent. Ba2+ can block some of the conductance(s) present in smooth muscle cells (e.g., inward rectifier K+ conductance), and application of 500 µM Ba2+ induced depolarization of resting membrane potential by a few millivolts.
There are two inward rectifier-type conductance(s) that could be involved. First, activation of an "IF-like" current (an inwardly rectifying cation conductance) could participate in repolarization of IJPs. However, we have never observed an IF-like conductance in colonic cells, thus activation of such a conductance during the IJP is unlikely. Second, it is possible that inactivation of a K+ conductance could participate in repolarization of the IJP. However, how an inwardly rectifying K+ conductance could increase the rate of repolarization is unclear. At the peak of the fast IJP, these channels would produce outward current that would summate with the influence of SK channels (main conductance activated during the fast IJP), and, during repolarization of the IJP, inwardly rectifying K+ channels would tend to impede (not enhance) repolarization. A pharmacological inactivation of inwardly rectifying K+ channels might contribute, but there is no evidence that ATP or other inhibitory transmitters affect inwardly rectifying K+ channels. Thus the mechanism for inactivation of a K+ conductance and the rationale for considering the involvement of such channels is vague. Therefore, the prolongation of IJPs would have to be due to activation of an outward current or inhibition of an inward current component. The only conductance we are aware of in murine colonic myocytes with these characteristics is the voltage-dependent NSCC.
Stimulation of enteric inhibitory nerves releases at least two neurotransmitters, ATP and NO. Thus a potential prejunctional effect of inhibitors of NSCC makes it difficult to directly assess the role of the NSCC in postjunctional neural responses. Therefore, we applied ATP directly, via picospritzing, in the presence of tetrodotoxin to block release of other neurotransmitters from neural activation by ATP. In these studies, we were able to compare several disparate blockers of NSCC. Spritzing ATP on colonic muscles resulted in transient hyperpolarizations that varied somewhat in amplitude and duration from experiment to experiment due to control resting potential, spritz pipette characteristics, and placement of the tip of the pipette. To avoid problems in interpreting our data, we conducted these experiments by comparing responses before and after application of NSCC blockers without moving the spritz pipette and without changing impalements. Under these conditions, we observed consistent significant increases in the duration of the hyperpolarization responses after application of the three NSCC blockers tested. Ni2+ and mibefradil are known to also be blockers of T-type Ca2+ channels, but we have never found evidence of expression of T-type Ca2+ channels in colonic myocytes (e.g., see Ref. 15). In our laboratory's previous report (15), we found that a low concentration (300 µM) of Ni2+ inhibited voltage-dependent NSCC. These data confirm the observations made with nerve-evoked IJPs and suggest that this conductance is activated during ATP-dependent hyperpolarization responses. Thus the data suggest that the voltage-dependent NSCC participates in active repolarization of IJPs in colonic muscles.
We also tested the effect of the NSCC conductance blockers on the breakdown of ATP. A variety of extracellular enzymes participate in degrading extracellular ATP and its metabolites, including ecto-ATPase and ecto-ATP diphosphohydrolases or ADPase. These enzymes are activated by Ca2+ or Mg2+ and are inhibited by millimolar concentrations of Cu2+, Zn2+, or La3+ (35). Therefore, we tested the idea that part of the prolongation of IJPs or ATP responses due to NSCC blockers could be due to inhibition of ATP metabolism. Mouse colon tissues demonstrated detectable ecto-ATPase activity, and Ni2+ significantly inhibited this activity. Mibefradil, however, did not significantly affect ATP metabolism. Because Ni2+ and mibefradil had the same effect on hyperpolarizations due to locally applied ATP, it is likely that ATP metabolism was not the primary factor in ending these responses. It is possible that, after the hyperpolarization response to ATP develops, the NSCC is activated and tends to counteract the hyperpolarization and restore membrane potential. In the presence of blockers of the NSCC, the ATP responses were prolonged and may have lasted until the ATP concentration fell due to diffusion from the site of application.
In summary, the fast IJP component of the enteric inhibitory response in murine colonic muscles recovers too quickly to be simply a passive result of shutting off of the K+ conductance(s) responsible for the hyperpolarization. We have described a voltage-dependent NSCC that, although unavailable at the resting membrane potential, can reset during the IJP. Activation of the conductance during the repolarization response hastens recovery of resting membrane potential. This is an interesting conductance because, at present, it is unique to GI muscles. Thus it is potentially a target for reducing smooth muscle excitability in colonic muscles.
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DISCLOSURES |
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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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