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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Objective:

Accelerated gastric emptying that precipitates hunger and frequent eating could be a potential factor in the development of obesity. The aim of this study was to study gastric emptying in diet-induced obese-prone (DIO-P) and DIO-resistant (DIO-R) rats and explore possible differences in electrical properties of calcium (Ca2+) and potassium (K+) channels of antral circular smooth muscle cells (SMCs).

Design and Methods:

Whole-cell patch-clamp technique was used to measure Ca2+ and K+ currents in single SMCs. Gastric emptying was evaluated 90 min after the ingestion of a solid meal.

Results:

Solid gastric emptying in the DIO-P rats was significantly faster compared with that in the DIO-R rats. The peak amplitude of L-type Ca2+ current (IBa,L) at 10 mV in DIO-P rats was greater than that in DIO-R rats without alternation of the current–voltage curve and voltage-dependent activation and inactivation. The half-maximal inactivation voltage of transient outward K+ current (IKto) was more depolarized (∼4 mV) in DIO-P rats compared with that in DIO-R rats. No difference was found in the current density or recovery kinetics of IKto between two groups. The current density of delayed rectifier K+ current (IKdr), which was sensitive to tetraethylammonium chloride but not 4-aminopyridine, was lower in DIO-P rats than that in DIO-R rats.

Conclusion:

The accelerated gastric emptying in DIO-P rats might be attributed to a higher density of IBa,L, depolarizing shift of inactivation curve of IKto and lower density of IKdr observed in the antral SMCs of DIO-P rats.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Obesity, a recognized risk factor for many medical illnesses and premature death, is one of the most prevalent public health problems in the United States, and the prevalence of this disorder has increased dramatically throughout the world (1, 2). In general, human obesity is multifactorial and ultimately results from inadequate energy expenditure or overconsumption of food. In addition to regulating the rates at which nutrients are being processed, gastrointestinal motility also participates in the control of appetite and satiety. The changes in gastric motor functions in obesity could be a potential factor in the maintenance of weight or the development of obesity. There are reports suggesting that gastric emptying of solid food is accelerated in obese subjects, which precipitates hunger and frequent eating (3, 4). And the percentage of gastric emptying during the initial 30 min after a solid meal was normalized after a major weight reduction (5). Others have reported normal (6) or even slow gastric emptying in obesity (7).

Electrical rhythmicity of gastrointestinal smooth muscle is the basis for smooth muscle contractile activity (8). Action potentials are generated by the influx of Ca2+ through voltage-gated L-type Ca2+ channels, and membrane repolarization is mediated by K+ efflux through both voltage- and Ca2+-activated K+ channels (9). The L-type Ca2+ channels are widely expressed throughout the gastrointestinal tract. The influx of extracellular Ca2+ through the L-type Ca2+ channels is a prerequisite for smooth muscle contraction. The L-type Ca2+ channels play a central role in generation of action potentials and activation of contraction in gastrointestinal smooth muscle cells (SMCs). K+ channels are important physiological regulators of membrane potential in excitable tissues, including gastrointestinal smooth muscles. K+ channels shape the action potential by controlling its repolarization phase and determining the membrane potential and duration of the interspike interval (10).

Consumption of a meal relatively high in fat and energy content (HF diet) is correlated with passive overconsumption and the onset of obesity in both rats and humans (11). In specific subsets of human populations, some individuals are resistant to the onset of obesity caused by a HF diet, whereas others are prone to becoming obese (12). The rat model of diet-induced obesity (DIO) is widely used for studying factors underlying the development and maintenance of obesity from exposure to HF diet. When outbred Sprague–Dawley rats are fed with HF diet, about half-develop obesity (DIO-prone, DIO-P) with higher weight gain and accumulated body fat, whereas the remainders are resistant to DIO (DIO-resistant, DIO-R), gaining the same amount of weight and carcass fat as chow-fed controls (13). In humans, accelerated gastric emptying in obese objects may be one of the contributing factors to overconsumption on a HF diet and subsequent obesity (3, 4, 14). And accelerated gastric emptying was reported in lean to slightly overweight Mexican Americans known to be susceptible to obesity compared to age-, gender-, and body-mass-index-matched non-Hispanic Whites (15). Accordingly, we have hypothesized that DIO-P rats have accelerated gastric emptying in comparison with DIO-R rats. Further, we hypothesize that in the cellular level certain electrophysiological characteristics of L-type Ca2+ channels and potassium channels of the antral SMCs that are associated with cell excitability are altered in DIO-P rats, and these alterations may be associated with accelerated gastric emptying.

The aims of this study were, therefore, to investigate a possible difference in solid gastric emptying between DIO-P and DIO-R rats and to study electrical properties of Ca2+ channels and K+ channels of antral SMCs in DIO-P that might be associated with gastric emptying in comparison with DIO-R rats.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Animals

The experimental study was approved by the Institutional Animal Care and Use Committee at the VA Medical Center, Oklahoma City, OK, and the International Guiding Principles for Biomedical Research Involving Animals were followed. A total of 74 male Sprague–Dawley rats, purchased from the Charles River Laboratories, were used in two experiments (30 for the gastric emptying test and 44 for the patch-clamp study). In the gastric emptying experiment, 30 rats (10 weeks old) were fed ad libitum with HF diet (with 45% kcal as fat, D12451 from Research Diet, USA) for 8 weeks by the Charles River Laboratories before they were shipped. In the patch-clamp experiment, 44 rats were purchased from the Charles River Laboratories at the age of 9 weeks. Upon arrival at our facility, 39 rats were fed ad libitum on the HF diet for 8 more weeks during which body weight was measured weekly. Based on the DIO-P and DIO-R phenotyping approach developed by Levin (13), the rats in the top-third of weight gainers after undergoing prolonged exposure to a HF diet were defined as DIO-P rats, and the rats in the bottom-third of weight gainers were defined as DIO-R rats. The rats in both groups were used for the patch-clamp experiment in the next 7 weeks in an age-matched manner. These remaining five rats were fed with normal chow and were used as control at the age of 18-20 weeks.

Solid gastric emptying study

The method for assessing solid gastric emptying was modified from a method previously described (16). In brief, after being fasted in cages with metal-wired mesh for 24 h with free access to water, both DIO-P (n = 10) and DIO-R (n = 10) rats were given access to 2.5 g of HF pellets for 10 min. All rats finished the food and were killed by overdosing pentobarbital sodium 90 min after the end of feeding. The abdomen was opened, and the stomach was surgically isolated and removed. The gastric content was recovered from the stomach that was air dried for 48 h and then weighed. Solid gastric emptying was calculated according to the following formula:

Gastric emptying (%) = [1 − (dried gastric content in g)/2.5 g] × 100

Isolation of antral SMCs

SMCs were enzymatically isolated using the procedures previously described (17). Briefly, rats were killed by overdose anesthesia. The abdomen was opened by a midline incision, and the stomach was quickly removed and placed in Ca2+-free physiological solution (in mM: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 10; NaCl 135; KCl 5; MgCl2 1.2, and glucose 10) with the pH adjusted to 7.4 with NaOH. The antrum was cut open longitudinally along the lesser curvature and pinned out in a dissecting plate with the mucosal side down. Antral circular muscle strips were removed by peeling off the longitudinal muscle layer under an anatomical microscope. The circular muscle strips were cut into small pieces and incubated in a Ca2+-free physiological solution for 30 min at 4°C. The muscle pieces were then transferred to a fresh Ca2+-free physiological solution containing enzymes (in mg/ml: Collagenase IA 1, papain 1, bovine serum albumin 2, and soybean trypsin inhibitor 1) and incubated for 20 min at 35°C. Then, the muscle pieces were washed several times in fresh Ca2+-free physiological solution and gently agitated to create a cell suspension. Isolated cells were stored at 4°C for use within 8 h. All chemicals used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA).

Whole-cell patch-clamp recording

The conventional whole-cell mode of the voltage- and current-clamp technique was used to measure changes in membrane potentials and currents from antral circular SMCs at room temperature (22-24°C) (18). One drop of cell suspension was added onto the acid-washed glass coverslip in a recording chamber (300 μl, Warner Instruments, Hamden, CT, USA) and allowed 10 min for the adhesion to the coverslip. Patch-clamp micropipettes were pulled with a programmable puller (P-97, Sutter Instruments, Novato, CA, USA), and their tips were fire polished. Pipette resistances ranged from 3 to 5 MΩ. The liquid junction potential was nulled while the pipette tip was immersed in the bath for recording, and the correction for liquid junction potential was done after the experiment. To minimize offsets due to large voltage-clamp errors, up to 80% of the series resistance were electronically compensated. The membrane currents were recorded under the voltage-clamp mode, and the resting membrane potentials were recorded under the current-clamp mode using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) and digitized online at 2 kHz. Data acquisition and analysis were performed with the pClamp software (version 9.0, Molecular Devices, Sunnyvale, CA, USA).

To record L-type Ca2+ channel currents without contamination of K+ currents, tetraethylammonium chloride (TEA), 6 mM, was added to the bath solution, and K+ was replaced by cesium in the pipette solution to block K+ currents. The bath solution contained (in mM): HEPES 10, NaCl 135, TEA–Cl 6, BaCl2 10, MgCl2 1.2, and glucose 10 (pH 7.4 with NaOH). The intracellular solution contained (in mM): HEPES 10, CsCl 130, ethylene glycol tetraacetic acid (EGTA) 10, TEA 10, Na2ATP 1, Na3GTP 1, phosphocreatine disodium 2, and MgCl2 1; pH was adjusted with CsOH to 7.2. To study the current–voltage curve, Ca2+ channel currents (IBa,L) were evoked using a standard stimulus protocol, that is, the membrane potential was stepped up to test potentials between −80 and +60 mV for 400 ms from a holding potential of −80 mV in a 10-mV increment with an interval of 10 s to allow for full recovery of Ca2+ channels (Figure 2A). To study the effect of nifedipine on IBa,L, cells were depolarized to 10 mV every 5 s for 400 ms till current amplitude was stable. To record voltage-gated K+ channel currents, the bath solution contained (in mM): HEPES 10, NaCl 135, KCl 5.4, MgCl2 1.2, and glucose 10 (pH 7.4 adjusted with NaOH to 7.4). The intracellular solution contained (in mM): HEPES 10, KCl 20, potassium gluconate 110, EGTA 10, Na2ATP 1, Na3GTP 1, and MgCl2 1; pH was adjusted with KOH to 7.2. In some experiments, TEA or 4-aminopyridine (4-AP) was added into the bath solution to record transient K+ (IKto) or delayed rectifier K+ current (IKdr), respectively. When recording the resting membrane potential, 2 mM CaCl2 was added into the bath solution, and the pipette solution was same as that in recording voltage-gated K+ currents.

The activation curves of Ca2+ channel and K+ channel currents were derived from their IV curves, where GBa,L or Gk was calculated by dividing the initial peak current value by the driving force (difference between membrane potential and equilibrium potential) and plotted as a function of membrane potential. The activation curves were fitted to a Boltzmann function G/Gmax = 1/{1 + exp[(VVh)]/k}, where V is membrane potential, Vh is the half-maximal activation voltage, and k is the slope constant (mV).

The steady-state inactivation curves of Ca2+ channel and K+ channel currents were obtained by different double pulse protocols. Ca2+ currents were elicited by a depolarizing pulse of 10 mV for 100 ms with 2-s preconditioning pulses from −80 to +20 mV in an increment of 10 mV. The time interval between two sweeps was set at 20 s. K+ currents were elicited by a depolarizing pulse of +20 mV with 2-s preconditioning pulses from −100 to +20 mV in an increment of 10 mV. The time interval between two sweeps was also set at 20 s. A plot of normalized peak current (I/Imax) as a function of preconditioning potentials was drawn. The steady-state inactivation curves were drawn by fitting these data to a Boltzmann function: I/Imax = 1/{1 + exp[(VhV)]/k}, where V is membrane potential, Vh is the half-maximal inactivation voltage, and k is the slope constant. To measure recovery of Kto, a two-step voltage protocol was used from inactivation by applying a pair of voltage pulses at +20 mV, separated by a variable time interval at −90 mV (Figure 6A). Channel inactivation was initially produced using a 1-s long pulse, and the time course for recovery from inactivation was defined using a second pulse evoked after a variable recovery time, ranging from 25 to 1,000 ms. Recovery was determined by plotting the related peak test current as a function of varied interval.

Statistical analysis

Data are presented as mean ± SEM. Significant differences between two groups of means were assessed by Student's t-test, and P value was accepted at the 0.05 level. Statistical analysis, data fitting, and graph plotting were performed using the Microcal Origin software (OriginLab, Northampton, MA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Solid gastric emptying in DIO-P and DIO-R rats

Among the 30 rats assigned to the gastric emptying test, the top-third and bottom-third weight gainers were defined as the DIO-P (600 ± 13 g, n = 10) and DIO-R rats (487 ± 9 g, n = 10, P < 0.05), respectively (Figure 1A) and were used in the solid gastric emptying test. Solid gastric emptying in the DIO-P rats was found to be significantly faster than that in the DIO-R rats (73 ± 3% vs. 62 ± 4%, P < 0.05; see Figure 1B).

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Figure 1. Gastric emptying in DIO-P and DIO-R rats. (A) DIO-P rats (n = 10) were significantly heavier than DIO-R rats (n = 10) after feeding with HF diet. (B) The percentage of gastric emptying in DIO-P (n = 10) rats was significantly faster than that of in DIO-R rats (n = 10). (C) Body weight gain in DIO-P and DIO-R rats that were used for the patch-clamp study during the exposure to a HF (HF) diet. Male Sprague–Dawley rats (n = 39) were fed with HF diet at the age of 10 weeks for 8 weeks. The top-third highest weight gainers were designated as DIO-P rats (n = 13) and the bottom-third weight gainers as DIO-R rats (n = 12).

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Body weight gain during the exposure to HF diet in DIO-P and DIO-R rats used in the patch-clamp study

Among the 39 rats fed with HF diet assigned for the patch-clamp study, the 13 highest and 12 lowest weight gainers were retrospectively identified as DIO-P and DIO-R rats, respectively. Before starting the HF diet, no significant difference in body weight existed between the two groups (DIO-P vs. DIO-R: 312 ± 8 vs. 311 ± 5 g, P > 0.05). After 8 weeks of exposure to the HF diet, the DIO-P rats gained 32% more weight compared to their initial body weight than the DIO-R rats. By then, DIO-P rats (620 ± 8 g) were about 19% heavier than DIO-R rats (521 ± 6 g, P < 0.001; see Figure 1). The rats in both groups were used for the patch-clamp experiment in an age-matched manner (DIO-P: n = 13, 22.0 ± 2.1 weeks vs. DIO-R: vs. 22.1 ± 2.1 weeks, n = 12, P > 0.05). These rats were kept on the HF diet before the study, and the mean body weight on the day of the study was 712 ± 14 g for the DIO-P rats and 620 ± 13 g for the DIO-R rats (P < 0.001).

Similar passive electrical properties of antral SMCs between DIO-P and DIO-R rats

The cell capacitances in DIO-P and DIO-R rats were 46.8 ± 2.2 pF (n = 56) and 45.3 ± 1.8 pF (n = 45, P > 0.05), respectively. These values may be considered to represent a rough measure of cell surface area (1 μF/cm2) and to indicate no differences in mean cell size between DIO-P and DIO-R rats. No difference in the resting membrane potentials of SMCs was noted between the DIO-P (−46.8 ± 1.2 mV, n = 22) and DIO-R (−45.8 ± 0.8 mV, n = 18, P > 0.05) rats.

Biophysical properties of L-type Ca2+ channels in antral SMCs

Current–voltage relationship of L-type Ca2+ channel currents

In rat gastric circular SMCs, depolarizing pulses activated inward currents (Figure 2A), and the amplitude of these currents displayed U-shaped dependence on membrane potentials between −80 and +60 mV. The inward currents were activated around −30 mV, peaked around +10 mV, and reversed at around +60 mV (Figure 2C). A higher current density was noted in the DIO-P rats compared with the DIO-R rats. The peak current density of IBa,L at 10 mV in DIO-P SMCs (−17.7 ± 1.7 pA/pF, n = 19) was greater than that in DIO-R SMCs (−12.3 ± 1.7 pA/pF, n = 16, P < 0.05). No significant difference was observed in cell capacitance between the DIO-P (47.8 ± 1.7pF, n = 19 out of 56 cells) and DIO-R rats (44.5 ± 1.3pF, n = 16 out of 45 cells, P > 0.05, not shown in the figure). To study the effect of the duration of HF diet exposure, the current density of IBa,L were compared between cells from rats of 18-21 weeks (average ∼ 10weeks exposure to HF diet) and cells from rats of 22-25 weeks (average ∼ 14weeks exposure to HF diet). No significant difference of IBa,L was observed in cells from both DIO-P (10 weeks, −17.5 ± 2.8 pA/pF, n = 10 vs. 14 weeks HF diet exposure, −18.0 ± 1.5 pA/pF, n = 9, P > 0.05) and DIO-R rats (10 weeks, −12.9 ± 2.8 pA/pF, n = 8 vs. 14 weeks HF diet exposure −11.7 ± 2.0 pA/pF, n = 8, P > 0.05). In the normal chow-fed rats (18-20 weeks age), the peak current density of IBa,L was −14.6 ± 1.7 pA/pF (n = 16), which was not significantly different from that of the age-matched (18-21 weeks) DIO-P (P > 0.05) or DIO-R (P > 0.05) rats. These inward currents were thought to be L-type Ca2+ channel currents, as they were sensitive to nifedipine (Figure 2B). No significant difference in the sensitivities to nifedipine (10 nM) was noted between DIO-P (45.3 ± 6.2% of the control, n = 4) and DIO-R rats (43.9 ± 3.0% of the control, n = 4, P > 0.05, not shown in the figure). Nifedipine (1 μM) blocked all the inward current in both groups (n = 4 in each group, not shown in the figure).

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Figure 2. Current–voltage (IV) relationship of L-type Ca2+ channel currents in antral circular SMCs. Ba2+ (10 mM) in bath solution was used to amplify current amplitudes. (A) Superimposed current traces recorded every 10 s at test potentials from −80 to 60 mV with holding potential at −80 mV in DIO-P, DIO-R, and normal chow-fed rats. (B) Sensitivity to nifidipine. L-type calcium current was elicited by depolarizeing cell membrane potential to 10 mV at 5 s interval. The original tract is from a cell of DIO-P rat. (C) IV curve of peak current density of L-type Ca2+ channel currents in antral circular SMCs from DIO-P, DIO-R, and normal chow-fed rats.

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Steady-state activation and inactivation curves of L-type Ca2+ channel currents

No difference was noted in voltage dependence of steady-state activation and inactivation of L-type Ca2+ channel currents between the DIO-P and DIO-R rats. Voltage dependence of activation and inactivation of L-type Ca2+ channels in the two groups of rats were of the same order of magnitude. To examine the voltage dependence of activation, peak currents were converted into relative conductance by dividing the initial peak current value by the driving force (difference between membrane potential and equilibrium potential). The conductance was plotted as a function of membrane potential (Figure 3A). Fitting these data with a Boltzmann function gave a voltage of half-activation (Vh) of −5.8 − 0.8 mV with a slope constant of 6.0 ± 0.2 mV in the DIO-P rats (n = 19), both of which were similar to Vh of −8.0 ± 1.6 mV (P > 0.05) and the slope constant of 6.2 ± 0.3 mV (P > 0.05) in DIO-R rats (n = 16). Voltage dependences of inactivation were determined as described previously, and the stimulus protocol and current trace are shown in Figure 3C. Steady-state inactivation curve was used to describe the probability of L-type Ca2+ channels being open at certain membrane potentials, that is, how many ion channels might be activated (opened) at a given voltage. As shown in Figure 3B, the mean value of Vh was −23.2 ± 1.8 and −20.8 ± 1.9 mV (P > 0.05), respectively, in DIO-P (n = 11) and DIO-R rats (n = 9). The slope constant k was −6.1 ± 0.5 mV in the DIO-P rats (n = 11) and −6.2 ± 0.4 mV in the DIO-R rats (n = 9, P > 0.05).

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Figure 3. No significant difference in steady-state activation and inactivation of L-type Ca2+ channels between DIO-P and DIO-R gastric circular SMCs. (A) Activation curves were derived from current–voltage curves, where GBa,L was calculated by dividing the initial peak current value by the driving force and plotted as a function of membrane potential. (B). Inactivation curves of IBa,L in DIO-P and DIO-R groups. Currents were activated by voltage steps to 10 mV after a 2-s preconditioning pulses between −80 and +20 mV in a 10-mV increment. Inactivation is shown as a plot of normalized peak current as a function of conditional potential from −80 to +20 mV. Smooth curves were fitted with Boltzmann equation that yields values of half-maximally inactivated voltage (Vh) and slope constant (k). (C) Double pulses protocol for inactivation curve of L-type calcium current and actual traces of currents measured following 2-s preconditioning pulses from −80 to +20 mV with an increment of 10 mV.

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Biophysical properties of voltage-gated K+ channels in SMCs

Pharmacological separation of different voltage-gated K+ channel currents

Outward K+ currents are mainly composed of voltage-gated K+ channel currents and Ca2+-dependent K+ channel currents. Under the Ca2+-free bath solution and the pipette solution with 10 mM EGTA, contamination of currents by Ca2+-activated K+ currents was minimized. Voltage-gated K+ currents were evoked using a standard stimulus protocol, that is, the membrane potential was stepped up from a holding potential of −90 mV to test potentials between −80 and +50 mV in 10 mV increments for 400 ms. The time interval was set at 10 s. In this study, depolarization of membrane potentials above −60 mV activated nonlinear, time- and voltage-dependent outward currents in both groups (Figure 4G). Previous studies in guinea pig gastric antrum and colon SMCs have shown that the voltage-gated K+ channel current that consists of IKto and IKdr can be separated by voltage protocols or pharmacological agents (9, 19, 20).

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Figure 4. Current–voltage (IV) relationship of transient outward K+ currents of antral circular SMCs in DIO-P and DIO-R. The original traces of A–E are from an antral cell of DIO-P rat. (A) Whole-cell outward K+ channel current evoked when the membrane potential was stepped up in 400 ms from a holding potential of −90 mV to test potentials between −80 and +50 mV in a 10-mV increment. (B) Outward K+ currents recorded in the presence of 10 mM TEA. (C) TEA-sensitive current. (D) Outward K+ currents recorded in the presence of 5 mM 4-AP. (E) 4-AP-sensitive IKto current. (F) TEA-sensitive currents (IKdr) in DIO-P and DIO-R antral circular SMC were plotted as a function of the membrane potential. (G) Peak current density of transient outward K+ channel currents, as a function of the membrane potential, was measured during the first 20 ms of test pulses in DIO-P and DIO-R antral circular SMCs. (H) End pulse (350-400 ms of test pulse) of 4-AP-insensitive IKdr currents in DIO-P and DIO-R antral circular SMC were plotted as a function of the membrane potential.

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In this study, IKto and IKdr were pharmacologically separated by their different sensitivities to TEA and 4-AP. IKto but not IKdr was insensitive to 10 mM TEA. As shown in Figure 4B, after IKdr was blocked by externally applied 10 mM TEA, the peak of TEA-insensitive IKto at 50 mV in the DIO-P rats was 41.5 ± 4.8 pA/pF (78.4 ± 4.0% of the control, n = 14), not different from that in the DIO-R rats (47.8 ± 9.7 pA/pF, 73.2 ± 5.4% of the control, n = 10, P > 0.05, not shown in the figure). The TEA-sensitive IKdr (Figure 4C) was isolated by digitally subtracting the TEA-insensitive components from the control, and its current–voltage relationship was constructed (Figure 4F). In Figure 4F, the end pulse of the TEA-sensitive IKdr (average current from 350 to 400 ms after test pulse onset) at 50 mV in the DIO-P rats (16.0 ± 2.5pA/pF, n = 14) was lower than that in the DIO-R rats (25.7 ± 3.3pA/pF, n = 10, P < 0.05).

When externally applied with 5 mM 4-AP, the peak of potassium channel current was significantly reduced. The peak of the 4-AP-sensitive IKto (Figure 4E), which was isolated by digitally subtracting the 4-AP-insensitive IKdr components (Figure 4) from the control (Figure 4A), was 72.8 ± 3.6% of the control in the DIO-P rats (45.6 ± 8.2 pA/pF, n = 12) and 71.6 ± 3.1% of the control in the DIO-R rats (49.2 ± 7.6pA/pF, n = 11, P > 0.05, not shown in the figure). In Figure 4H, significant difference was observed in the end pulse of 4-AP-insensitive IKdr at 50 mV between the DIO-P (23.7 ± 1.7pA/pF, n = 12) and DIO-R rats (29.8 ± 1.7pA/pF, n = 11, P < 0.05).

Current–voltage relationship of IKto in SMCs

No difference was observed in current–voltage relationship of IKto in SMCs between the DIO-P and DIO-R rats. In both DIO-P and DIO-R rats, depolarizing pulses were used to activate outward currents at membrane potentials between −80 and +50 mV. In Figure 4G, the outward currents were activated around −60 mV. The peak amplitude of IKto at +50 mV in DIO-P SMCs (46.6 ± 5.5 pA/pF, n = 22) was not significantly different from that in DIO-R SMCs (52.2 ± 5.6 pA/pF, n = 15, P > 0.05).

Steady-state activation and inactivation curves of IKto

In Figure 5A, the parameters for Boltzmann distribution of voltage-dependent activation were as follows: DIO-P rats (n = 22), Vh at −1.3 ± 1.0 mV and k at 14.4 ± 0.9 mV; DIO-R rats (n = 15), Vh at −4.2 ± 1.0 mV (P > 0.05) and k at 13.0 ± 0.8 mV (P > 0.05). A classic double pulse protocol was used to determine the voltage dependence of inactivation of IKto (Figure 5C). As shown in Figure 5B, comparing to the inactivation curve in DIO-R rats with the half-maximal inactivation voltage of inactivation Vh at −73.9 ± 1.2 mV (n = 13), the inactivation curve in DIO-P rats was more depolarized with Vh at −69.1 ± 1.9 mV (n = 17, P < 0.05). The slope constant k was 6.6 ± 0.6 mV in DIO-P rats (n = 17) and 6.5 ± 0.7 mV in DIO-R rats (n = 13; P > 0.05).

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Figure 5. Steady-state activation and inactivation of transient outward K+ channel current of antral SMCs in DIO-P and DIO-R rats. (A) Activation curves were derived from current–voltage curves where GKto was calculated by dividing the initial peak current value by the driving force and plotted as a function of membrane potential. (B) The steady-state inactivation is shown as a plot of normalized peak current (I/Imax) as a function of conditioning potential from −100 to +20 mV and fitted with a Boltzmann function. (C) Upper panel: double pulses protocol for inactivation curve of transient outward K+ channel current. Bottom panel: membrane currents were measured for 200 ms following 2-s preconditioning pulses from −100 to +20 mV by increments of 10 mV.

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Recovery kinetics of transient outward K+ channel currents

In Figure 6B, the time constant of recovery from inactivation was well fitted with a single exponential of 227 ± 50 ms in DIO-P (n = 10) and 239 ± 20 ms in DIO-R rats (n = 11, P > 0.05), respectively. No difference was observed between the two groups of rats.

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Figure 6. Recovery kinetics from inactivation of transient outward K+ channels in DIO-P and DIO-R rat gastric circular SMCs. (A) Stimulus protocol for recovery kinetics. Membrane potential was stepped for 1 s from −90 to 20 mV followed by a repolarization to −90 mV; The membrane potential was stepped back to 20 mV following variable recovery intervals at −90 mV. Representing traces from a cell of DIO-P rat showed transient outward K+ channels recovered from inactivation at longer intervals. (B) Peak currents were normalized and were plotted as a function of recovery interval and fitted with a single exponential function. No significant difference was found in the time constant of recovery from inactivation between DIO-P and DIO-R rats.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

In this study, we investigated the electrical properties of Ca2+ channels and K+ channels in antral circular SMCs in DIO-P and DIO-R rats. The results showed that: (a) The DIO-P rats showed accelerated gastric emptying in comparison with the DIO-R rats; (b). The current density of L-type Ca2+ current in DIO-P antral SMCs was greater than that in DIO-R antral SMCs, whereas no difference was noted in the current–voltage curves and steady-state inactivation between the two groups. (c) The half-maximal inactivation voltage of transient outward K+ current was more depolarized in DIO-P rats comparing to that in DIO-R rats. No difference was found in the current density and recovery kinetics of IKto between the DIO-P and DIO-R rats. (d) The TEA-sensitive and 4-AP-insensitive IKdr current in DIO-P was lower than that of in DIO-R rats.

Relatively little has been reported on the ion channels in rodent antral SMCs in contrast to the large body of information available on ion channels in guinea pig and canine antral SMCs. The predominant voltage-dependent Ca2+ channels in smooth muscle are dihydropyridine-sensitive L-type channels. L-type Ca2+ channels seem to be ubiquitously expressed in the SMCs of the gastrointestinal tract (8). The Ca2+ channel current may not be a homogeneous current, because low-threshold transient (T-type) Ca2+ channels have been described in some gastrointestinal SMCs such as rat colon and guinea pig taenia coli (21, 22). However, a previous patch-clamp experiment in our laboratory detected only the presence of a single component of high-threshold, dihydropyridine-sensitive inward current in rat gastric circular SMC (17). This finding was in agreement with previous reports in canine gastric (23) and colon (24) SMCs in which only L-type Ca2+ channel currents were observed. In this study, the inward currents were activated around −30 mV, peaked around +10 mV, and reversed around +60 mV. Their sensitivity to nifedipine indicated that these currents were L-type Ca2+ channel currents. The current density of L-type Ca2+ currents of antral SMCs in rats was higher than that in guinea pigs (25). HF diet was reported to increase blood pressure via a process involving the elevation of Ca2+ current density and an alteration of channel kinetics in the cerebrovascular SMC of male Osborne-Mendel rats (26). Whether this difference is attributed to species differences or to the impact of HF diet needs further investigation. Other mechanisms, such as hormones, neurotransmitters, intracellular messengers, and phosphorylation pathways, have also been suggested (27). For example, insulin is known to increase the L-type calcium channel current (28). In this study, we did not measure blood pressure; neither did we assess possible compounding effects of certain comorbidities, such as insulin resistance.

The larger currents recorded in the DIO-P rats were not the result of differences in cell size, as these currents were normalized to cell capacitance, and actually there was no difference in cell capacitances between DIO-P and DIO-R rats. It indicated that the differences in Ca2+ current densities resulted from a larger number of channels or a higher opening probability of the L-type Ca2+ channels. Ca2+ influx is essential to generate gastric smooth muscle tone and contractile activity. The main pathway for Ca2+ entry into SMCs is through dihydropyridine-sensitive L-type Ca2+ channels (8). Blockade of Ca2+ channels reduces the duration and amplitude of electrical slow waves in SMCs and blocks generation of action potentials (8). The enhancement or down-regulation of Ca2+ channel function and expression in cell adaptation and disease have been reported in mesenteric vascular SMCs of spontaneously hypertensive rats (29) and inflamed circular SMCs of the canine colon with smooth muscle dysfunction (24). In this study, the increased Ca2+ current densities may confer a higher level of Ca2+-dependent myogenic tone to gastric antrum. In this study, there are no differences in the voltage dependence of activation and inactivation such as half-maximal activation/inactivation voltages and slope constants between the DIO-P rats and the DIO-R rats. There are also reports on the enhancement or down-regulation of Ca2+ channel functions and expressions without modification of its voltage-dependent activation/inactivation (24, 30).

In SMCs, K+ channels play a crucial role in the regulation of contraction via the control of membrane potential and excitability. Kto currents have been identified in several gastrointestinal smooth muscles, including guinea pig colon (9) and mouse colon (31) and antrum (19). Inhibition of IKto with 4-AP (a Kto channel blocker) was reported to shift the resting membrane potential to more positive potentials (9, 19, 31), increased the velocity of the action potential upstroke and abolished the quiescent periods between slow waves and induced a slight depolarization (32). In this study, the half-inactivation voltages for transient outward K+ channel currents in DIO-P and DIO-R rats were around −70 mV, close to the resting membrane potential recorded in muscle strips, indicating that a substantial portion of this current contributes to the establishment of the rat antrum SMC resting membrane potential. The −4 mV hyperpolarizing shift of the inactivation curve of IKto in DIO-R rats may render the availability of more Kto channels to hyperpolarize the membrane excitation induced by slow wave to favor relaxation. The resting membrane potentials are more negative (−55 to −60 mV) when recorded in intact rat gastric smooth muscle strips incubated with warmed solution (19, 33). Abolition of electrogenic Na+–K+-ATPase activity in isolated cells at room temperature in this study might contribute to the discrepancy in the resting membrane potentials between isolated cells and intact muscle strips (34). Compared with the transient outward K+ channel current in murine antral SMCs, the current density in rat was lower with the similar recovery kinetics from inactivation (252 ms in murine) and a more negative half-maximal inactivation voltage of steady-state inactivation curve (−65 mV in murine) (19). The discrepancy may be due to species and organ differences. K+ channel subunits with transient outward K+ channel properties are found in several K+ channel families including “Shaker” (Kv1.4), “Shaw” (Kv3.4), and “Shal” (Kv4.1, Kv4.2, and Kv4.3). Different heteromeric subunit combinations of A-type potassium channel in SMCs have profound effects on cell excitability and increase channel functional diversity (35). Further study is needed to understand the underlying mechanism and physiological role of Kto in modulating gastric motility in DIO-P and DIO-R rats.

IKto, a slow activating and inactivating one, was also identified in every cell studied using high K+ pipette solution. In contrast to IKto, IKdr was sensitive to millimolar concentration of TEA in both DIO-P and DIO-R rats in this study. In contrast to the ability of 4-AP to depolarize cell membrane potential potentials (9, 19, 31), resting membrane potentials were not altered by externally applied TEA, indicating that IKdr is not involved in maintenance of the resting membrane potential (17). But TEA can increase the amplitude of action potentials and decrease the threshold to evoke an action potential in rat antral SMCs (17). A reduction of IKdr in DIO-P rats was supported in this study by comparing the current density of the TEA-sensitive current in two groups. It may lead to cell membrane depolarization and enhanced contractility of antral SMC in DIO-P rats.

Alterations in gastrointestinal motility have been observed in obese patients, and these alterations could be contributing factors to the development and maintenance of obesity and altered eating behaviors. In this study, solid gastric emptying was faster in the DIO-P rats than that in the DIO-R rats. It is generally accepted that liquid gastric emptying is dependent principally on the fundic tone, because the pressure gradient between the stomach and the duodenum is the major factor controlling liquid gastric emptying. In contrast, gastric emptying of a solid meal is mainly determined by antral contractions, which play a pivotal role in the pumping or emptying effect of the stomach (36), and that was why we studied the electrical characteristics of channels that influence antral contractility. In this study, the DIO-P rats exhibited electrical activities of a higher density of Ca2+ current and lower TEA-sensitive potassium current that favor enhanced antral contraction, whereas the DIO-R rats exhibited a hyperpolarizing shift of the inactivation curve of Kto that favors muscle relaxation. Alteration of ion channel will result in alteration of excitability and contraction in gastrointestinal smooth muscle. The spontaneous contractile activity was almost diminished in the mice under the smooth muscle-specific disruption of the gene encoding the L-type Cav1.2 Ca2+ channel that lead to paralytic ileus. The balance of these modulated electrical activities may render the enhanced excitability and contraction in antral SMCs of the DIO-P rats compared with that of DIO-R rats, which is associated with the accelerated gastric emptying in the DIO-P rats. Rats with ventromedial hypothalamic lesions have accelerated gastric emptying that is related to overeating and obesity (37). A rapid rate of gastric emptying would precipitate a feeling of hunger as a result of decreased gastric distension that reduces the negative feedback satiety signal produced by the presence of nutrients inside the distal stomach. The subsequent increased rate of energy intake would favor the development of obesity.

Whether the differences observed in the Ca2+ and K+ channels of antral SMCs between the DIO-P and DIO-R rats are the causes or the consequences of HF diet induced obesity is of great interest. Hypertension induced by dietary fat was reported to be associated with the elevation of current density and an alteration of channel kinetics of Ca2+ channel in the cerebrovascular SMC of male Osborne-Mendel rats (26). Ion channel remodeling may be involved in DIO rats under HF diet. In this study, the DIO-P rats exhibited a higher density of Ca2+ current and lower TEA-sensitive potassium current as well as accelerated gastric emptying. In normal chow-fed rats, the current density of Ca2+ channel was between that of DIO-P and DIO-R rats. It is reasonable as normal chow-fed rats consist of both the DIO-P and the DIO-R rats. It has been reported that the weight gain and body composition change persist even with the regular chow diet, once the DIO-P and DIO-R phenotypes are established on a HF diet (38). The difference in the ion channel properties between DIO-P and DIO-R may be genetically different, though the possibility of HF diet involved in the ion channel remodeling as well as gastric emptying cannot be totally excluded as DIO-P rats consumed more HF diet to gain more weight than DIO-R rats. Acceleration of gastric emptying was reported in healthy male subjects after the consumption of a high-fat diet for a short duration of 2 weeks (39). It will be of great interest to study the gastric emptying as well as the ion channel activities in both humans that fed a high-fat diet without develop obesity and selectively bred DIO-P and DIO-R rats with normal chow diet.

In conclusion, gastric emptying is accelerated in DIO-P rats, compared with DIO-R rats, and this may be attributed to an increased density of L-type Ca2+ channels, down regulated delayed rectifier K+ channel current and depolarizing shift of inactivation curve of transient outward potassium channel observed in the antral smooth muscles in the DIO-P rats.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References