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Motor dysfunction of gastric antral smooth muscle in diabetic rats: Contribution of ATP-dependent potassium channels
Fatameh Khoshavi Najafabadi1, Hassan Sadraei1, Nasrin Mehranfard2, Maedeh Ghasemi3
1 Department of Pharmacology and Toxicology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
2 Neurophysiology Research Center, Cellular and Molecular Medicine Research Institute, Urmia University of Medical Sciences, Urmia, Iran
3 Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| Date of Submission | 06-Feb-2023 |
| Date of Acceptance | 20-May-2023 |
| Date of Web Publication | 27-Jul-2023 |
Correspondence Address:
Dr. Maedeh Ghasemi
Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_44_23
Background: The goal of the current research was to further elucidate the role of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels in the motility and contractility force of gastric smooth muscle of diabetic rats.
Materials and Methods: Male Wistar rats (190–230 g) were grouped into control and streptozotocin (STZ)-induced diabetes (55 mg/kg) rats. Thirty days later, gastric muscle contractility was measured using a myograph and a force transducer of antral segments immersed in a tissue bath. Gastric emptying response was measured through feeding of standard pellet. Furthermore, the expression of KATP channel subunits in antral smooth muscle was determined by western blot technique.
Results: The amplitude of KCl-evoked twitch contractions of diabetic antral strips was about 25% more than control (P < 0.05). Application of minoxidil, a KATP channel opener, dose dependently decreased the force of twitch contractions in both normal and diabetic antral strips. Application of 10 μM glibenclamide, a KATP channel blocker, did not antagonize the minoxidil-induced relaxation of antral strips. Diabetic gastric emptying was faster than normal, although not significant. Despite the relaxant effect of minoxidil on gastric emptying rate in normal rats (P < 0.05), this effect was not observed in diabetic rats. Also, glibenclamide increased gastric emptying and antagonized minoxidil-induced relaxation in normal rats (P < 0.05). Furthermore, the expression of KATP Kir6.1 and SUR2B subunits was substantially reduced in antral smooth muscle in diabetic condition (P < 0.01).
Conclusion: These results propose that KATP channels may contribute to the development of gastric motility disorders in diabetes.
Keywords: Diabetes mellitus, gastric emptying, glibenclamide, KATP channel, minoxidil
How to cite this article:
Khoshavi Najafabadi F, Sadraei H, Mehranfard N, Ghasemi M. Motor dysfunction of gastric antral smooth muscle in diabetic rats: Contribution of ATP-dependent potassium channels. Adv Biomed Res 2023;12:199 |
How to cite this URL:
Khoshavi Najafabadi F, Sadraei H, Mehranfard N, Ghasemi M. Motor dysfunction of gastric antral smooth muscle in diabetic rats: Contribution of ATP-dependent potassium channels. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:199. Available from: https://www.advbiores.net/text.asp?2023/12/1/199/382401 |
| Introduction | |  |
Abnormal gastric motility patterns are a common gastrointestinal (GI) complaint in patients with diabetes mellitus, presenting as transient slow gastric emptying, transient fast gastric emptying, sustained delayed gastric emptying, and sustained fast gastric emptying.[1] Fast gastric emptying is associated with loss of inhibitory neuromuscular transmission, reduced relaxation, and augmented smooth muscle contractility of the stomach as a result of chronic hyperglycemia-induced oxidative stress and possibly an enhancement in the number of antral interstitial cells of Cajal.[2] While delayed gastric emptying is associated with motor dysfunctions in different components of the stomach, such as deficits in smooth muscles, neurotransmission, and interstitial cells of Cajal, which result in weakness of the smooth muscle and impaired contractility.[3]
Smooth muscle contractility is responsible for propelling luminal content through the GI tract. Although chronic hyperglycemia influences the molecular signaling of autonomic and enteric neurons, and interstitial cells of Cajal via inflammation and oxidative stress, smooth muscle contraction abnormalities are one of the causes of gastric motility disorders in diabetic patients.[4] Smooth muscle contractility is principally determined by alterations in the concentration of intracellular free Ca2+, which influxes through voltage-activated Ca2+ channels. Intracellular free Ca2+ can be affected by many factors, such as potassium channels' activity.[5] Potassium channels are especially implicated in the regulation of GI smooth muscle contraction as well as vascular contraction. Closure of adenosine triphosphate (ATP)-sensitive potassium channels (KATP channels), which couple cell metabolism to cell membrane potential by ATP, requires opening of voltage-dependent Ca2+ channels and subsequent Ca2+ influx, which then leads to muscle contraction. Research findings indicate that functional KATP channels are obviously found in intestinal smooth muscle and their activity contributes to motor regulation of the GI system.[6],[7] Animal studies on diabetes-induced gastric abnormalities have revealed that hyperglycemia stimulates glucose-sensitive neurons in the vagal afferents by closing KATP channels, possibly reducing antral contractions and gastric emptying.[8] However, there are reports showing that hyperglycemia functions directly on inhibitory neurons and counteracts their stimulatory effect on glucose-sensitive neurons. Changes in KATP channel expression and activation in neural and Cajal interstitial cells or smooth muscle may also be involved in smooth muscle contraction dysfunction, which affects gastric accommodation and peristaltic contractility in diabetic condition. KATP channels may, therefore, form a drug target that directly affects smooth muscle contraction.[1],[9] For example, previous studies have suggested that diminished motor activity of colonic smooth muscle during inflammation is associated with altered transcriptional regulation of KATP channel subunits.[10] Further, an enhancement in the expression of KATP channel subunits in vascular smooth muscle of diabetic rats contributes to impaired smooth muscle contractility.[11] York et al.[12] demonstrated that intestinal contractility is dependent on the normal expression of KATP channels, whose overactivity have been demonstrated to lead to contraction and motor impairment in the small intestine and colon. However, it is unclear whether KATP channels in gastric smooth muscle are involved in gastric contractility as well as emptying abnormalities in diabetes. Understanding the mechanisms that mediate these pathological processes may lead to the identification of novel targets for treatment. Hence, the goal of the current research was to determine the role of KATP channels in in vitro contractile response of the antral smooth muscle as well as the rate of gastric emptying in normal and diabetic rats. Furthermore, KATP channel subunit expression in antral smooth muscle was investigated.
| Materials and Methods | | |
Drugs and solutions
Tyrode's solution consists of the following (mM): NaCl 136.9, KCl 2.7, CaCl2 1.8, MgCl2 1.05, NaHCO3 11.9, NaH2PO4 0.42, and glucose 5.6, and is saturated with oxygen. Streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA) was prepared freshly in 0.1 M citrate-buffered saline (pH 4.5) to induce diabetes.
Acetylcholine (Sigma) and KCl were prepared in distilled water. Glibenclamide (Sigma) and minoxidil sulfate (Merck, Germany) were made up in dimethylsulfoxide (DMSO); further dilution was made in 50% DMSO. Unless stated, all the chemicals were from Merck.
Experimental protocols
Male Wistar rats (190–230 g) were used in this research. All procedures conducted on the animals were approved by the Animal Care and Use Committee at Isfahan University of Medical Sciences (IR.MUI.RESEARCH.REC.1399.132) and were based on the guidelines set by this committee. The rats were housed under standardized conditions at room temperature and allowed free access to food and tap water throughout the experiments. One week after adaption, the animals were randomly grouped into normal (control) and STZ-induced diabetes groups. To establish a model of diabetic gastropathy, the rats were subjected to fasting for 10–12 h and intraperitoneally (IP) injected with STZ (55 mg/kg). Three days after STZ injection, tail blood was taken for glucose level determination using a glucometer (GALA, TD-4277). Rats with glucose >250 mg/dl were considered diabetic and included in the research. Diabetic rats were kept under standard conditions for 30 days to develop a model of diabetes-induced gastropathy.[13] After 30 days, blood glucose and body weight of rats were assessed and rats with hyperglycemic index (blood glucose >250 mg/dl) were divided into two main groups: one group for examining contractility of isolated gastric antral muscle strips and the second group for gastric emptying test.
Preparation of gastric antral muscle strips
After euthanizing by CO2, the rat stomachs were immediately removed and washed with Tyrode's solution. The dissected stomach was cut into fundus and antrum sections. The antral region was cut open longitudinally and then further cut into four strips (3–6 mm) along the greater curvature. Each strip was cleaned and then suspended in an organ bath containing 20 ml of oxygenated Tyrode's solution at 37°C. One end of the strip was fixed with a thread to a hook. The tissue holder was then moved into the bath and the other end of the tissue was tied up to an external isotonic force transducer (Harvard) under 1 g tension. Gassing with O2 was continuously maintained throughout the experiment. Following several washes with Tyrode's solution, the muscle strips were allowed to relax to their baseline tonicity. Tissue contractions were induced by the addition of KCl or acetylcholine (ACh) into the organ bath. Initially, the tissues were primed by adding three successive concentrations of ACh (50 μM) to obtain reproducible responses. ACh was in contact with the tissue for 30 s before being washed out with fresh Tyrode's solution. Once a stable baseline was established, KCl or ACh was added to the organ bath at 5 min intervals to induce tissue contractions. Concentration response curves were constructed for both KCl and ACh in the diabetic and nondiabetic gastric strips. Furthermore, the inhibitory effect of minoxidil (0.069, 0.27, 1.1, and 4.4 mM) was examined on the KCl (20 mM)- and ACh (50 μM)-induced contractions. The effect of minoxidil was examined in a cumulative way for KCl response and in a noncumulative manner for ACh. The effect of minoxidil on gastric strip contractions was also examined in the presence of glibenclamide (10 μM). Before examining their impacts, minoxidil and glibenclamide were in contact with the tissue for 10 min. Time-matched, vehicle-treated control experiments were also considered by adding equivolume amount of the vehicle. The effect of each drug was examined on six different tissues.
Gastric emptying
Normal and diabetic rats were fasted for 16–18 h and were divided into the following subgroups: vehicle control, pretreated minoxidil (50 mg/kg), and pretreated glibenclamide (50 mg/kg) + minoxidil (50 mg/kg) (n = 6 for each group). Glibenclamide, minoxidil, or saline was administered orally and the rats were fed 30 min later. Rats had access to preweighed food pellets (1.6 g) in separate cages for 15 min, as mentioned earlier.[14] The rats that did not consume food within 15 min were excluded from the research. Sixty minutes after the feeding time, the animals were killed by CO2 and the stomach was removed. The gastric content was recovered from the stomach, dried, and weighed.
Western blotting
Frozen antral smooth muscle tissues were homogenized in lysis buffer containing protease inhibitor cocktail to extract protein (Tris-HCl, ethylenediaminetetraacetic acid [EDTA], NaCl, sodium deoxycholate, sodium dodecyl sulfate, and Triton [NP40] [1%]). The protein concentration was evaluated using Bradford assay (Bio-Rad, Hercules, CA, USA). The protein samples were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were probed with mouse anti-SUR2B monoclonal antibodies (S323A-31; Novus Biologicals, Centennial, USA) and goat anti-Kir6.1 polyclonal antibodies (sc-11224, Santa Cruz Biotechnology) at 4°C overnight. Mouse anti-β actin monoclonal antibodies (sc-47778, Santa Cruz Biotechnology) were used as an internal reference. After washing with Tris-buffered saline and Tween 20, the membranes were probed with corresponding horseradish peroxidase–conjugated secondary antibodies. Immunoreactive protein bands were detected by enhanced chemiluminescence western blotting reagents and analyzed using ImageJ (NIH, Bethesda, MD, USA).
Statistical analysis
Muscle contractions were assessed as the amplitude of the recorded contraction. The results were expressed as the percentage of initial contraction produced by ACh (50 μM) at the beginning of the experiment. Solid gastric emptying was determined based on the following formula, as mentioned earlier:[14],[15] % gastric emptying = [1 – (dried weight of food recovered from stomach/weight of food intake)]×100. The results are given as means ± standard error of mean (SEM). Data analysis was conducted by using Student's t-test or analysis of variance (ANOVA) followed by a Tukey–Kramer multiple comparisons test. P value < 0.05 was regarded as significant.
| Results | | |
Evaluation of the animal model
Three days after modeling, all the animals in the diabetic group had blood glucose concentrations >250 mg/dl and were considered diabetic. At the end of the 30-day diabetic period, blood glucose was measured again. The diabetic rats had higher blood glucose values than the controls (P < 0.001) [Figure 1]. | Figure 1: Changes in blood glucose concentration in the STZ (55 mg/kg)-induced diabetic rats relative to normal rats. Data are represented as mean ± SEM, ***P < 0.001 versus normal. SEM = standard error of mean, STZ = streptozotocin
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Evaluation of gastric muscle contractility
Addition of KCl (10, 20, 40, and 80 mM) to the organ bath induced fast contraction of gastric strips in both diabetic and nondiabetic rats. However, maximum induced response was substantially higher in the diabetic rats relative to the nondiabetic rats (P < 0.05) [Figure 2]. As a bath concentration of 20 mM KCl almost produced a maximum response, this concentration was selected for further studies. Minoxidil from 20 μg/ml (0.069 mM) to 1.28 mg/ml (4.4 mM) inhibited KCl responses in gastric strip in both diabetic and nondiabetic rats in a concentration-dependent manner [Figure 3]a and [Figure 3]b (two-way ANOVA; for non-diabetic rats: concentration [F {2.201, 33.01} =212.3, P < 0.0001], concentration × column factor [F {6, 45} =28.99, P < 0.0001] and for diabetic rats: concentration [F {2.326, 34.89} =180.8, P < 0.0001], concentration × column factor [F {6, 45} =27.83, P < 0.0001]). Glibenclamide had no marked impact on the inhibitory action of minoxidil, except at a bath concentration of 320 μg/ml (1.1 mM) in nondiabetic rats. Glibenclamide seems to potentiate the inhibitory effect of minoxidil (P < 0.05) [Figure 3]a. There was no statistically substantial difference in the inhibitory action of minoxidil between the diabetic and nondiabetic rats (two-way ANOVA). Equivolume of minoxidil vehicle had no significant effect on the contractile activity of KCl on gastric strips in diabetic or nondiabetic rats (two-way ANOVA). | Figure 2: Concentration response curve of gastric strips for KCl in diabetic and nondiabetic rats. KCl was added at 10 min intervals in a cumulative manner. Tissue response was expressed as the percentage of initial control response for each tissue. Data are represented as mean ± SEM (n = 6). There was statistically significant difference between the responses of KCl in diabetic and nondiabetic rats (two-way ANOVA; concentration: P <0.0001, column factor: P <0.05, concentration × column factor: P <0.01). ANOVA = analysis of variance, SEM = standard error of mean
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 | Figure 3: Inhibitory effect of minoxidil and glibenclamide on KCl-induced contraction of gastric strip in the nondiabetic (a) and diabetic (b) rats. Minoxidil was added at 10 min intervals in a cumulative manner. Tissue response was expressed as the percentage of initial control response for each tissue. Data are represented as mean ± SEM (n = 6). There was no significant statistical difference between the KCl responses in the control tissues (two-way ANOVA). Stars show statistically significant differences between the test groups and the corresponding control groups (*P < 0.05, **P < 0.01, ***P < 0.001). Reduction in gastric strip response in the presence of glibenclamide (10 μM) with minoxidil (1.1 mM) was statistically significant in nondiabetic rats (#P < 0.05). ANOVA = analysis of variance, SEM = standard error of mean
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ACh contracted gastric strips of the antrum section in a concentration-dependent manner. These findings were observed in the antral muscle strips of both diabetic and nondiabetic rats. However, on comparing the potency of Ach, no substantial difference was detected in the contractile responses of isolated antral strips in the diabetic and nondiabetic rats [Figure 4]. The effect of minoxidil in higher concentrations was also examined on Ach-induced contraction of gastric strips. In the nondiabetic rats, minoxidil significantly reduced the contractile response to ACh (50 μM) only at the highest concentration used (4.4 mM) (P < 0.001, two-way ANOVA; concentration [F {1.944, 29.16} =69.73, P < 0.0001] and concentration × column factor [F {6, 45} =24.71, P < 0.0001]) [Figure 5]a. However, in the diabetic rats, the inhibitory impact of minoxidil was seen at a lower concentration of minoxidil (1.1 mM, P < 0.05) [Figure 5]b. Furthermore, glibenclamide potentiated the inhibitory effect of minoxidil (1.1 mM) in nondiabetic rats compared to control (P < 0.05) [Figure 5]a and [Figure 5]b. | Figure 4: Concentration response curve of gastric strips for ACh in diabetic and nondiabetic rats. ACh was added at 5 min intervals with 30 s contact times. Tissue response was expressed as the percentage of initial control response for each tissue. Data are represented as mean ± SEM (n = 6). There was no significant statistical difference in ACh responses between diabetic and nondiabetic rats (two-way ANOVA). ANOVA = analysis of variance, SEM = standard error of mean
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 | Figure 5: Inhibitory effect of minoxidil and glibenclamide on Ach-induced contraction of gastric strip in nondiabetic (a) and diabetic (b) rats. ACh was added at 5 min intervals with 30 s contact times. Tissue response was expressed as the percentage of initial control response for each tissue. Data are represented as mean ± SEM (n = 6). There was no significant statistical difference in ACh responses between the control tissues (two-way ANOVA). Stars show statistically significant differences between the test groups and the corresponding control groups (*P < 0.05, **P < 0.01, ***P < 0.001). Reduction in gastric strip response in the presence of minoxidil (1.1 and 4.4 mM) was statistically significant between the nondiabetic and diabetic rats. ANOVA = analysis of variance, SEM = standard error of mean
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In the vehicle-treated (DMSO), time-matched control tissues, no substantial difference was detected in the contractile responses of gastric strips between diabetic and nondiabetic rats (two-way ANOVA).
Gastric emptying in normal and diabetic rats
As shown in [Figure 6], 75% of the gastric contents of the normal or nondiabetic rats was emptied within 1 h after feeding. The gastric emptying response (% of emptying) was slightly increased in the diabetic rats relative to the controls [Figure 6]. Following minoxidil gavage (50 mg/kg), the gastric emptying response was considerably decreased in nondiabetic rats (one-way ANOVA, P < 0.001), while it was unchanged in diabetic rats relative to the controls. The effect of minoxidil on the gastric emptying response was reversed by pretreatment with glibenclamide (50 mg/kg) in the nondiabetic rats (P < 0.05) [Figure 6]. | >Figure 6: Effect of minoxidil and glibenclamide on gastric emptying in normal and diabetes rats. Drugs were administered orally in fasted rats and 30 min later, the rats had access to food for 15 min. An hour later, the stomach was dissected out for determination of gastric emptying. Data are represented as mean ± SEM (n = 6). The symbol * shows the statistical difference between the treated groups and their corresponding control groups (***P < 0.001, #P < 0.05 glibenclamide [50 mg/kg]–minoxidil vs. minoxidil in nondiabetic rats, one-way ANOVA). ANOVA = analysis of variance, DMSO = dimethylsulfoxide, SEM = standard error of mean
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Evaluation of the expression of Kir6.1 and SUR2B subunits in the antral muscle portion of stomach
Western blotting of the antral muscle layer showed a substantial reduction in the levels of Kir6.1 and SUR2B subunits in the diabetic rats relative to the normal rats (both P < 0.01) [Figure 7]a and [Figure 7]b. The reduced expression of Kir6.1 (as pore-forming subunit) shows that gastric antral KATP channel expression was decreased in the diabetic rats relative to the controls. | Figure 7: The Protein expression of pore and regulatory subunits of KATP channels in gastric antrum of normal and diabetic rats. (a) The protein expression level of the KATP channel subunit Kir6.1 in normal and diabetic rats (n=5). (b) The protein expression level of the KATP channel subunit SUR2B in normal and diabetic rats (n=5). Data are represented as mean ± SEM, **P<0.01 versus normal. SEM=Standard error of mean
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| Discussion | | |
Gastric motility patterns arise from the coordinated activity of autonomic and enteric circuits, gastric smooth muscle cells, and interstitial cells of Cajal. The current research focused on the role of KATP channels in the contractile responses of the gastric antral muscle and gastric motility in healthy and diabetic rats. Our findings confirmed that KATP channels (Kir6.1/SUR2B) are found in intestinal smooth muscle and are functionally related to antral smooth muscle contraction, and demonstrated that KATP channel impairment is involved in type 1 diabetes.
KATP channels belong to the family of inward rectifier channels with the pore-forming subunit Kir6.x and the regulatory subunit SUR, both of which are needed for functional expression. Although, KATP channels have been shown to couple cell metabolism to membrane potential, direct assessments of KATP channel activity indicate that brief openings of these channels can occur in the absence of the channel openers.[16] This proposes that KATP channels contribute to resting membrane potential and basal tone of smooth muscles. Currently, genetic manipulation of the KATP channel subunits has revealed not only the physiological, but also the pathophysiological roles of KATP channels. Studies using KATP channel knockout and transgenic mice models suggest that opening of KATP channels, as metabolic sensors, is required for adaptation to stress and is critical in the protective mechanisms against a variety of different acute metabolic stresses such as hypoxia and hyperglycemia. However, a number of open issues remain to be resolved.[17]
Consistent with our results, previous studies also reported the presence of KATP channels in gastric tissue.[18] Moreover, a dose-dependent inhibition of smooth muscle contraction by KATP channel activators such as minoxidil, cromakalim, and diazoxide has been demonstrated in the stomach and other regions of the GI tract, including the esophagus, ileum, and colon.[19],[20] The activation of KATP channel increases potassium conductance, leading to cell membrane hyperpolarization and subsequent inhibition of extracellular Ca2+ influx and Ca2+ release from intracellular storage organelles.[21] Studies have also demonstrated that KATP channel opener-induced hyperpolarization is responsible for its relaxing action on ACh- and KCl-induced contractions in the smooth muscle of rat and the gastric antrum of guinea pig.[22] We demonstrated that the agonist minoxidil, which can open KATP channels, inhibited antral smooth muscle contractions induced by ACh or 20 mM K+, while glibenclamide, a KATP channel blocker, did not antagonize the minoxidil-induced relaxations in isolated antral strips. Many studies have shown a great potency of glibenclamide in inhibiting KATP channels in the smooth muscle cells and reversing the effect of the KATP channel openers on GI muscle. However, in our study, glibenclamide was unable to induce contraction or antagonize the relaxing effect of minoxidil on antral strips.
In agreement with our results, Saponara et al.[18] reported a similar effect of glibenclamide in fundus strips, even at doses much higher than those needed to suppress KATP channels in smooth muscle cells. This observation suggests that the resistance of isolated antral muscle to channel inhibition by glibenclamide may occur by different mechanisms, which may related to the dose used or direct inhibition of Ca2+ influx by minoxidil. For example, Horii et al.[19] showed a dose-dependent effect of glibenclamide on esophagus muscle contractions in rat. In this study, glibenclamide increased vagally induced contractions of the rat esophagus at an effective dose of more than 500 μM, while glibenclamide at a dose of 50 μM did not affect esophageal muscle motility. It seems that high concentrations of glibenclamide are required to reverse minoxidil-induced relaxation of antral smooth muscle. In addition to its impact on KATP channels, minoxidil may also inhibit voltage-sensitive calcium channels and thereby exert a relaxing effect on the smooth muscle, although this hypothesis needs further support through docking experiments. Moreover, there are several important lines of evidence indicating that sulfonylurea receptor subunits, which play a principal role in regulating KATP gating kinetics, determine significant differences in the pharmacological characteristics of KATP channels.[20],[21],[22],[23],[24],[25] For example, the SUR2/Kir6.2 channel has a low sensitivity to both ATP and glibenclamide and can be activated by KATP channel openers of cromakalim and pinacidil, but not by diazoxide. Also, glibenclamide binds to SUR2 subunit with lower affinity.[26] Functional KATP channels are composed of four Kir6.x (Kir6.1 or Kir6.2) pore-forming subunits, involving in ATP suppression, and four regulatory SURx (SUR2A and B) subunits, involving in Mg-ADP stimulation. SUR subunits are also sensitive to the KATP channel openers diazoxide and pinacidil and the blocker glibenclamide.[27] Our molecular assay detected the expression of Kir6.1 and SUR2B subunit proteins of KATP channels in the rat antral muscle. This subunit profile has been suggested for the KATP channel in gastric smooth muscle cells of guinea pig.[28] This may account for the low affinity and partial sensitivity to glibenclamide, an impact that is particularly evident in isolated antral strips. In vivo studies showed that glibenclamide (10 μM) partially antagonized minoxidil-induced relaxation of stomach and increased gastric motility in the gastric emptying test of normal rats. The physiological role of KATP channels in gastric smooth muscle is not clear. In guinea pig gastric myocytes, a modulatory effect of KATP channels has been documented by substance P and ACh.[28] KATP channels may participate in regulation and/or changes in contractility induced by neurotransmitters in the stomach.
Previous studies have demonstrated that gastric relaxation is mediated by depolarization of glucose-activated afferent neurons via suppressing KATP channels.[8] Therefore, hyperglycemia causes a decrease in peristaltic contractions in the corpus and antrum, resulting in slow gastric emptying. However, hyperglycemia has also been reported to cause fast gastric emptying.[2] A recent observation showed that hyperglycemia results in inhibition of purinergic and nitrergic transmission on end cholinergic motor neurons and depolarization of smooth muscles, resulting in their increased excitability.[29] Concordant with these results, it seems that oral administration of glibenclamide could increase the depolarization and excitability of corpus and antral smooth muscles and cause fast gastric emptying. These data propose that KATP channels may be involved in the regulation of smooth muscle contractility in the antrum.
GI motor dysfunctions frequently occur in patients with long-standing diabetes. Aye-Mon et al.[30] assessed diabetic gastropathy by measuring gastric motility and sensitivity for 10 weeks after diabetic induction. They observed that the rate of 2-h solid gastric emptying was substantially enhanced in diabetic rats relative to controls, 2 weeks after diabetic induction. This response was also faster after 4 weeks in diabetic rats than controls, although not significant. From 6 to 10 weeks, fast gastric emptying was no longer seen in diabetic rats. Consistent with these results, after 30 days of diabetes induction, we also observed a nonsignificant enhancement in the rate of gastric emptying. Furthermore, in spite of the relaxant impact of minoxidil on the gastric emptying rate in normal rats, this KATP channel-modulating agent could not induce this effect in diabetic rats.
Our molecular results indicated that diabetes induced about 50% downregulation of both Kir6.1 and SUR2B subunits compared to normal. Intracellular ATP and Mg-ADP have been shown to inhibit and activate KATP channels, respectively. Four Kir6.x pore-forming subunits (Kir6.1 or Kir6.2) are responsible for ATP inhibition and four regulatory SURx subunits (SUR1, SUR2A, or SUR2B) are responsible for Mg-ADP. SUR subunits are also sensitive to agonists (diazoxide/pinacidil) and antagonists (glibenclamide).[26] Studies of Kir6.1−/− and SUR2−/− mice have revealed that the Kir6.1/SUR2 subunits play an important role in the modulation of vascular tone.[17],[31] York et al.[12] also identified KATP channels (Kir6.1/SUR2) as a direct regulator of intestinal smooth muscle contraction, and mice carrying mutations in Kir6.1 or SUR2, as KATP gain-of-function, as well as patients who suffer from Cantú syndrome had attenuated intrinsic contractility throughout the intestine. Interestingly, we observed a reduction in both Kir6.1 or SUR2B expression and fasting gastric emptying in the diabetic rats relative to the controls. These results suggest that gastric motility depends on the normal expression of Kir6.1 and SUR2B subunits in stomach smooth muscle, and pathogenic defects in the subunits result in impaired motility of smooth muscle in some disease such as diabetes. Diabetes can also influence gastric motility via alterations in neurotransmission, enteric neuron number and size, interstitial cells of Cajal, and neurodegenerative alterations. Multiple studies have also indicated a loss of neuronal nitric oxides synthase (nNOS) function in diabetes, leading to delayed gastric emptying or alterations in gastric accommodation.[32],[33] A decrease in interstitial cells of Cajal and nNOS neurons in type 2 diabetes has been demonstrated to be partly due to an enhancement of apoptosis.[34] A reduction in substance P in the stomach and ileum of diabetic rats has also been shown in previous studies. Generally, diabetes can cause an excitatory/inhibitory neuropeptide imbalance, resulting in GI motility dysregulation. Moreover, hyperglycemia through inducing oxidative stress can lead to nerve damage and thereby cause altered GI motility.[32]
In conclusion, we found that KATP channel acts as a regulator of contractility via its effect on antral smooth muscle and suggest that pathogenic impairment in the subunits of Kir6.1 and SUR2 may contribute to in vitro and in vivo gastric contractile and pharmacological dysfunction. This may provide a potential new target for the treatment of time-dependent gastric symptoms of diabetes and broader GI abnormalities.
Ethics approval
All procedures conducted on the animals were approved by the Animal Care and Use Committee at Isfahan University of Medical Sciences (IR.MUI.RESEARCH.REC.1399.132) and were based on the guidelines set by this committee.
Financial support and sponsorship
This work was supported by a grant from the Isfahan University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
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