Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
Submitted 27 August 2004 ; accepted in final form 1 November 2004
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ABSTRACT |
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gastrointestinal motility; calcium ion currents; potassium ion currents; interstitial cells of Cajal; smooth muscle
Using isometric force measurements, carbenoxolone derivatives at concentrations between 104 and 105 M were shown to inhibit the amplitude of phasic contractions of isolated segments of longitudinal muscle, but had no effect on the frequency of spontaneous contractions (3, 8, 10, 31). With the use of intracellular microelectrode recordings, it was also shown that electrical slow waves in the guinea pig small intestine were abolished by octanol (1 mM) (13). Although octanol (0.51 mM) reduced the amplitude and frequency of slow waves in circular muscle in isolated canine ileum, it did not abolish them (10). Octanol caused depolarization of resting membrane potential (RMP) and reduced the nerve-evoked fast-inhibitory junction potentials in this tissue (10). These data suggest that the spread of slow waves between ICC networks and smooth muscle and neurally evoked inhibitory junction potentials may utilize low-resistance gap junctions, but that agents used to study this phenomenon may have indirect effects by directly inhibiting channels involved in slow-wave and inhibitory junction potential generation in ICC networks and smooth muscle cells.
The depolarization in membrane potential in GI muscles observed by using gap junctional uncouplers could be a consequence of the segregation of the ICC networks from neighboring smooth muscles cells. ICCs at the level of the myenteric plexus have been reported to have a more negative membrane potential than smooth muscle cells (21), and, in animal models when ICCs are absent, membrane potential in smooth muscle cells is more positive (17, 37, 41). An alternative explanation is that gap junctional uncouplers have nonspecific side effects on ionic currents on ICC or smooth muscle cells that could lead to membrane depolarization.
The decrease in the contractile activity of GI muscles in the presence of gap junctional uncouplers could also be interpreted in two ways. The inhibition of gap junctions between ICC and smooth muscle could lead to a decrease in the amplitude and coordinated spread of slow waves into the smooth muscle syncytium and subsequently depress phasic contractions. However, this disruption in coupling may cause an effect on the frequency of the phasic contractile activity. This was not the dominant effect observed in contractility experiments (10). A second possibility is that gap junction uncouplers could have direct effects on Ca2+ currents that are essential for contractions of GI smooth muscles.
In the present study, we examined the effects of the commonly used gap junction uncoupler GA using a variety of physiological methods to determine specific or nonspecific effects of this compound on gap junctions and ionic currents in cultured ICC and freshly dispersed smooth muscle cells.
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MATERIALS AND METHODS |
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The animals were maintained and the experiments were performed in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." The Institutional Animal Use and Care Committee at the University of Nevada approved all procedures used.
Isometric force measurements. After animals were killed, jejunal tissues 10 cm from the ileocecal junction or the proximal colon 13 cm distal to the ileocecal junction were removed and placed in Krebs-Ringer bicarbonate solution (KRB). The jejunum was opened along the mesenteric border, and luminal contents were washed away with KRB. Mechanical responses were performed by using standard organ-bath techniques. Strips of muscle (2 x 5 mm) were cut from the tunica muscularis by sharp dissection. Muscles were attached at one end with sutures to a fixed mount within the organ bath and at the other end to an isometric strain gauge (World Precision Instruments, Sarasota, FL). The muscles were immersed in oxygenated KRB and maintained at 37.5 ± 0.5°C. The muscles were set at optimum length by applying 0.1 to 0.3 g of basal tension and then allowed to equilibrate for 12 h with constant perfusion with fresh KRB. Contractions of the muscles were monitored, digitized, and stored with the use of AcqKnowledge software (Biopac Systems, Santa Barbara, CA) running on a personal computer-style computer.
Intracellular microelectrode recordings.
The jejunal tunica muscularis (10 mm x 4 mm) was isolated and placed in a recording chamber with the mucosal aspect of muscle facing upward. In some experiments, proximal colon tissues (13 cm from the ileocecal junction) were used for some experiments. Impalements of cells were made with glass microelectrodes that had resistances of 80120 M
. Transmembrane potentials were recorded with a standard electrometer (Duo 773; World Precision Instruments). Data were recorded on a personal computer running Acknowledge data-acquisition software (Biopac Systems).
Solutions for isometric force measurements and intracellular recordings. The bath chamber was constantly perfused with oxygenated Krebs solution 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 was 7.37.4 when bubbled with 97% O23% CO2 at 37 ± 0.5°C. Muscles were left to equilibrate for at least 1 h before experiments were begun. For intracellular recordings, nifedipine (Sigma, St. Louis, MO) was dissolved in ethanol at a stock concentration of 10 mM before being added to perfusion solution at a final concentration of 1 µM to reduce muscle contractions. Nifedipine has been shown previously not to affect slow waves (43). All experiments were performed in the presence of tetrodotoxin (1 µM). Drugs were diluted to the desired concentrations and applied to the muscles by switching the perfusion to one containing the drug.
Preparation of isolated myocytes. Colonic and jejunal smooth muscle cells were prepared from colons removed from BALB/c mice, as described above. Colons and jejunums were cut open along the longitudinal axis, pinned out in a Sylgard-lined dish, and washed with Ca2+-free solution containing (in mM) 125 NaCl, 5.36 KCl, 15.5 NaOH, 0.336 Na2HPO4, 0.44 KH2PO4, 10 glucose, 2.9 sucrose, and 11 HEPES, adjusted to pH 7.4 with Tris. Mucosa and submucosa were removed. Most experiments were conducted by using myocytes isolated from bulk circular smooth muscle. Pieces of muscle were incubated in a Ca2+-free solution supplemented with 2 mg fatty acid-free bovine serum albumin, 1.3 mg collagenase, and 2 mg trypsin inhibitor in 1 ml. Tissues were incubated at 37°C in enzyme solution for 812 min and then washed with Ca2+-free solution. Tissue pieces were gently agitated to create a cell suspension. Dispersed cells were stored at 4°C in Ca2+-free solution. Experiments were performed at room temperature within 6 h of dispersing cells.
Preparation of cultured ICC networks. ICC networks were cultured as previously described (21). Briefly, the mucosa was removed from small strips of intestinal muscle that were subsequently equilibrated in Ca2+-free Hanks' solution for 30 min, and cells were dispersed with an enzyme solution containing 1 mg/ml collagenase (Worthington Type II), 2 mg/ml bovine serum albumin (Sigma), 2 mg/ml trypsin inhibitor (Sigma), and 0.7 mg/ml ATP. Cells were plated onto sterile glass coverslips coated with murine collagen (2 µg, Falcon/BD) in 35-mm culture dishes. The cells were cultured at 37°C in a 95% O25% CO2 incubator in smooth muscle growth medium (Clonetics, San Diego, CA) supplemented with 2% antibiotic/antimycotic (Gibco, Grand Island, NY) and murine stem cell factor (5 ng/ml, Sigma).
ICCs were identified immunologically with a monoclonal antibody for kit protein (see Ref. 21) labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR). These studies were performed on small networks of ICCs (<10 cells). The morphologies of ICC and ICC networks were distinct from other cell types in the cultures, so it was possible to identify the cells with phase-contrast microscopy once the cells were verified with ACK2-Alexa Fluor 488 labeling. As previously reported, the pacemaker currents from small clusters of ICCs were more robust and more regular than from isolated ICCs (21).
Voltage patch-clamp experiments.
The whole cell patch-clamp technique was used to record membrane currents from dissociated murine colonic smooth muscle cells. Currents were amplified with a list EPC-7 (List Electronic, Darmstadt, Germany) or Axopatch 200B (Axon Instruments, Foster City, CA). Data were digitized with a 16-bit analog-to-digital converter (Digidata 1322A, Axon instruments). Data were stored directly and digitized online using pClamp software (version 9.2, Axon Instruments). Data were sampled at 4 kHz and filtered at 2 kHz by using an eight-pole Bessel filter and analyzed by using pClamp (version 9.2; Axon Instruments), Graphpad Prism (version 3.0, Graphpad Software, San Diego, CA), and Origin software (version 5.0, MicroCal Software, Northampton, MA). Pipette resistances were 14 M. To measure holding current, cell capacitance, and input resistance simultaneously from ICC networks, we used the built-in membrane capacitance tool in pClamp software (version 9.2; Axon Instrument, see Fig. 2). In most experiments on isolated smooth muscle cells, the uncompensated series resistance was between 2 and 4 M
, and voltage errors during the largest currents approached 12 mV. We compensated for at least 70% of the series resistance. Voltage errors were much smaller during steps to negative potentials.
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For studies of inward currents, we performed the perforated-patch technique. Amphotericin B (Sigma) was dissolved in dimethyl sulphoxide (Sigma) as a stock solution (0.06 mg/µl) and added to the pipette solution (0.3 mg/ml). Membrane potential was held at 60 mV during the formation of membrane pores. External solution was CaPSS. Internal solution for voltage-dependent inward currents contained (in mM) 120 CsCl, 20 TEA-Cl, 0.1 EGTA, and 10 HEPES, adjusted to pH 7.2 with Tris.
Analysis of electrophysiological data. Data are expressed as means ± SE. The Student's t-test was used where appropriate to evaluate differences in the data. P values < 0.05 were taken as statistically significant differences. For intracellular recordings, several electrical parameters were analyzed: 1) RMP, 2) slow-wave amplitude, and 3) slow-wave frequency. For patch-clamp experiments, we analyzed the peak currents before and after drug treatments.
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RESULTS |
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Effects of GA on Lucifer dye spread and passive electrical properties of ICC cultures.
To determine the effects of GA on ICC networks, we used 3- to 4-day primary cultures of ICC networks (21). Connected ICCs had low-input resistances (62 M) and high capacitances (13.5 pF). ICC networks possessed spontaneous inward currents averaging 316 pA at a frequency of 17 cycles/min. The slow-wave activity of these networks was similar to that previously reported (21). After treatment of
-GA (3 x 105 M), input resistance increased to 348 M
, and membrane capacitance decreased to 6.4 pF. GA also abolished the spontaneous inward currents that were observed under control conditions (Fig. 2, AC). We also performed current-clamp (I = 0) experiments to characterize the effects of GA on freshly dispersed jejunal myocytes (Fig. 2D). The membrane potential under normal physiological condition averaged 54 ± 7 mV. The application of
-GA (105 M) did not affect the membrane potentials (55 ± 7 mV, n = 5; P > 0.05). This data suggest that depolarization observed in tissue experiments and ICC networks was caused by the uncoupling effects of GA between ICCs and ICCs to smooth muscle cells.
ICC networks were also dialyzed by using Lucifer yellow dye using patch pipettes (2 mg/ml added to the internal solution in the pipette) to examine the spread of dye through junctions that formed between individual ICCs. Under control conditions, Lucifer yellow dye normally spread to four or five connected ICCs (Fig. 3, A and B, n = 5). After preincubation of -GA (105 M) for 15 min, Lucifer yellow dye was never observed to spread to adjacent ICCs when dialyzed (Fig. 3, C and D). The increase in input resistance and decrease in cell capacitance with the decrease in the spread of Lucifer yellow dye between cells suggest that GA uncoupled the cell junction between neighboring ICCs.
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To isolate KDR currents, cells were bathed in MnPSS (see MATERIALS AND METHODS), and patch pipettes contained BAPTA (10 mM) to minimize any contamination of BK channels. The application of -GA (105 M) significantly decreased KDR currents of jejunal myocytes under these conditions. Peak outward currents were 614 ± 53 and 1,581 ± 110 pA under control conditions at 0 and +30 mV, respectively (Fig. 8, AC). The treatment of
-GA significantly decreased peak outward currents to 519 ± 43 and 1,277 ± 104 pA, respectively (P < 0.05, n = 5). To examine the voltage dependence of
-GA, we used a double-pulse protocol with long 3- to 5-s prepulses. The treatment of 4-AP (5 mM) revealed a slowly activating and inactivating current (KDR) in jejunal myocytes. The application of
-GA (105 M) significantly decreased this current from an average of 745 ± 120 to 527 ± 70 pA at 0 mV (P < 0.05, n = 5, Fig. 8, DF). The application of TEA (10 mM) revealed a rapidly activating and inactivating current (IA).
-GA (105 M) significantly decreased this outward current in the presence of TEA from 1,103 ± 137 pA under control conditions to 871 ± 88 pA after
-GA at +30 mV (n = 4, P < 0.05, Fig. 8, GI).
-GA have no effect on the voltage dependence of activation and inactivation (e.g., 3 ± 1 mV in control vs. 4 ± 1 mV after
-GA for activation; 44 ± 1 mV in control vs. 45 ± 1 mV after
-GA for inactivation; n = 4). These data suggest that the decrease of net outward currents by GA is caused by direct inhibition of IA.
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DISCUSSION |
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Slow waves coordinate the phasic contractile activity of the smooth muscle cells by causing membrane depolarization via L-type calcium channels that leads to an increase in intracellular calcium and the subsequent activation of the contractile apparatus of the cells (16). The exact role of how ICCs coordinate the spread of electrical excitability within GI muscles is controversial. Ultrastructural analysis (9, 23) and immunohistochemical (33) studies have provided morphological evidence for the existence of gap junctions between ICCs, between ICCs and smooth muscle cells, and between smooth muscle cells of the circular muscle layer. Little ultrastructural or immunohistochemical evidence has been provided to demonstrate the presence of gap junctions within the longitudinal muscle layers throughout the GI tract. However, one immunohistochemical report has shown the presence of low levels of connexins that were localized to the longitudinal muscle layer of the murine stomach but not small intestine (14). The identity of the connexin proteins that make up the formation of gap junctions has also been investigated by using immunohistochemical techniques (23, 39). It has been demonstrated that connexin 40, 43, and 45 were differentially distributed in the muscle layers of the GI tract of several species (14, 32, 39). Connexin 43 was the highest expressed connexin protein in GI tissues and was expressed in all regions where gap junctions occur, and connexin 40 is also widely distributed in the circular muscle of the lower esophageal sphincter, stomach, and ileum.
Other investigations examining the coupling between smooth muscle cells in the GI tract have utilized dye spread with Lucifer yellow (19). Although gap junctions have been observed in the circular but not the longitudinal muscle layer of the small intestine, dye coupling between smooth muscle cells was observed in both muscle layers. The spread of dye between muscle cells in the circular and longitudinal layers suggests the presence of gap junctions in both muscle layers and is inconsistent with previous ultrastructural studies that failed to demonstrate the presence of gap junctions in the longitudinal muscle layer (45).
Numerous functional studies have been performed to examine the role of gap junctional communication between smooth muscle cells in the GI tract (3, 10, 31) and other visceral and vascular systems (5, 6, 24, 25, 30, 38). GA in either the - or
-isoform has been widely used to examine the functional role of gap junctions in smooth muscles using several assays, including electrical measurements, dye coupling, or intracellular Ca2+ measurements. 18
-GA was shown to abolish coupling through gap junctions using these assays in a variety of smooth muscle preparations (15, 27, 35, 36, 44).
In the GI tract, mechanical experiments using GA have been performed to examine the role of gap junctions in coupling of smooth muscle cells (8, 10). In these experiments, GA was reported to decrease both the amplitude and frequency of spontaneous contractions in canine ileum and colon. These findings were consistent with the hypothesis that GA uncouples gap junctions between ICCs and between ICCs and smooth muscle, which leads to a reduction in the mechanical activity of this tissue (10, 23).
To confirm the uncoupling effects of GA on gap junctions, we performed a series of patch-clamp and dye-spread experiments using this compound on ICC networks that were maintained in primary culture for 34 days. In these experiments, the application of GA increased input cellular resistance and decreased capacitance. Furthermore, the spread of Lucifer yellow within ICC networks was greatly reduced after application of GA. Taken together, these observations suggested that the uncoupling effects of GA on gap junctions are consistent with previous reports.
In the present study, we also examined the nonspecificity of GA. Using isometric force measurements, intracellular microelectrode recordings and the patch-clamp technique examined the effects of GA on mechanical activity, membrane potential, and voltage-dependent ionic currents from jejunal and colonic tissues and cells. In mechanical experiments, GA decreased the amplitude and frequency of spontaneous contractions from jejunal tissues. This finding is consistent with a previous report in canine ileal muscle (10, 23) and could be interpreted as an uncoupling effect of GA on gap junctions between ICCs and smooth muscle cells, leading to a reduction in mechanical activity. However, microelectrode recordings revealed that GA also induced a significant membrane depolarization with an associated decrease in slow-wave amplitude. In contrast to the contractility experiments, these findings could not be explained by the uncoupling effects of GA alone. It has been previously reported that gap junction blockers may have nonspecific effects in addition to the actions of uncoupling gap junctions (4, 8, 18, 34). Although 18-GA is considered more specific than 18
-GA and heptanol or octanol (11, 12), a systematic study on the nonspecific effects of GA on ionic channels in the GI tract has not been performed.
The decrease in mechanical activity observed in the presence of GA could have resulted from the inhibition of an inward calcium conductance in smooth muscle cells. Therefore, we examined net inward currents using perforated patches in murine jejunal and colonic myocytes. The IC50 of -GA was 1.9 µM. This concentration of GA was significantly lower than the concentrations that have been used to uncouple gap junctions, which often range between 20 and 100 µM. At a concentration of 30 µM, GA inhibited 90% of Ca2+ currents in murine jejunal myocytes. Although the inhibitory mechanism of GA on Ca2+ currents is not clear, it was observed to decrease net calcium currents at all potentials tested without a change in the voltage-dependent activation and inactivation of these currents. The inhibitory potency of
-GA on inward currents was also not different than that observed with the
-isoform of GA. These data suggest that decrease in the spontaneous mechanical activity resulted from inhibition of Ca2+ currents in murine jejunum and colon.
The membrane depolarization produced by GA could have resulted from the inhibition of an outward, or activation of an inward, ionic conductance in smooth muscle cells. GA has been shown to inhibit a chloride membrane conductance in confluent primary cultures of rat hepatocytes (2). Inhibition of a chloride conductance is unlikely in the present study, as this would have caused membrane hyperpolarization rather than the observed depolarization. We, therefore, examined the effects of GA on outward potassium currents.
To examine the effect of GA on outward K+ currents, we performed conventional dialyzed whole-cell experiments. Under these conditions, net outward currents were decreased by external application of both isoforms of GA. To examine the effects of GA on specific K+ currents, we isolated three types of voltage-dependent K+ currents using voltage protocols and pharmacological approaches. First, we isolated BK currents using a holding potential of 30 mV to induce full inactivation of voltage-dependent delayed rectifier K+ currents (22). Under these conditions, GA increased BK currents. In vascular smooth muscles, BK channel is one of major conductances that regulate RMP. The activation of BK channels by GA may induce hyperpolarization of vascular tissues. Indeed, a previous report suggested that gap junction blockers attenuate myogenic tone through membrane hyperpolarization (2, 24). In the circular muscle layer of the murine small intestine and colon, BK channels do not appear to act as an active conductance for the regulation of RMPs in these tissues (1, 2, 22, 24). The expression of SK channels in murine colonic myocytes has been reported, and these channels appear to be involved in setting RMP in this tissue (20). Using the same voltage protocol described above, incubation of TEA reveals a small amplitude of outward currents. -GA had no effect on the remaining outward current in the presence of TEA. These data would suggest that the observed depolarization produced by GA was not through the inhibition of SK channels.
To examine the effects of GA on voltage-dependent IAs, we isolated these currents using MnPSS (see MATERIALS AND METHODS) and dialyzed smooth muscle cells with BAPTA (10 mM) to reduce intracellular concentration to 10 nM. Under these conditions, when smooth muscle cells were exposed with 4-AP (5 mM), a slow-activating and inactivating delayed rectifier current remained (KDR) (22). GA significantly inhibited the KDR currents, suggesting that decrease in net outward currents resulted from inhibition of KDR currents. Therefore, the membrane depolarization produced by GA could be a consequence of its blocking effects on delayed rectifier K+ currents in smooth muscle cells.
Finally, using TEA (10 mM), we isolated a fast-activating and inactivating delayed rectifier K+ current (IA) (22). GA (30 µM) decreased IA current without a change in the voltage dependence of activation and inactivation.
In conclusion, we have performed a systematic investigation of the effects of GA on networks of ICC cultures and ionic currents in smooth muscle cells. The effects of GA on membrane properties and dye spread are consistent with this compound inhibiting coupling between individual ICCs. However, GA also has several nonspecific side effects on calcium and potassium currents that make the use of this agent to specifically inhibit gap junctional coupling in GI tissues unreliable.
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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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