- Email: [email protected]
S2061 Effects of Aging on Interstitial Cells of Cajal in the Human Stomach
tested the hypothesis that PDGF-BB regulates the physiology of cultured CSMC. Methods: Single smooth muscle cells from intestinal CSMC were isolated using enzymatic digestion and plated in cell culture medium (DMEM+5% FBS). The amphotericin-B perforated patch clamp technique was used (23°C) with a model 700B Multiclamp amplifier (Axon Instruments). Patch pipette resistance ranged from 2 to 4 MΩ. CSMC on glass coverslips were loaded with 5 μM Fura-2 at 37°C for 30 min, followed by a 15 min wash-out prior to recording by sequential illumination at 340 and 380 nm, with data expressed as the fluorescence ratio (FR). Results: Application of PDGF-BB or PDGF-AA (50 ng/mL) to freshly isolated CSMC caused no measurable affects, but by 3-5 days In Vitro, PDGF-BB caused the resting membrane potential to depolarize by 13.6±4.1 mV (n=4, p<0.5). No significant depolarization occurred with 50 ng/mL PDGF-AA. There was a matching 30 % reduction in input resistance in response to a 10 pA electrotonic current pulse with PDGF-BB (p<0.05; 50ng/ml), with no significant changes measured in response to PDGF-AA. In addition, PDGF-BB caused a 2fold increase in the outward K+ currents elicited from a ramp protocol (-100 to 100 mV holding to -60 mV). In contrast, CSMC at 3 days In Vitro showed a significant two-fold increase in the intracellular calcium concentration (p<0.05; 2.0±0.5 fold, n=11) in response to PDGF-BB (3 min exposure; maximum response at 2.41±0.4 min with τ=5.0 (y0+A1e^(x/τ) and mean duration of 4.6±0.6 min). There was a similar response to 10 mM caffeine (FR increased by 2.06±.28; n=18). Nifedipine (1 μM) attenuated but did not block the intracellular calcium rise, indicating that PDGF requires calcium from both intracellular and external sources. There was no measurable intracellular calcium change with PDGF-AA. Conclusions: PDGF-BB can cause the depolarization and an increase in intracellular calcium in cultured CSMC but not in freshly isolated cells. This suggests that the onset of proliferation of CSMC involves an early expression of functional PDGF receptors. Further experiments are needed to clarify the intracellular events of receptor activation. (Supported by CIHR, CAG, Astra Zeneca and CONACYT)
Diabetes-Induced Changes in the Expression of Proteins Involved in the Regulation of Sustained Mlc20 Phosphorylation and cGMP Levels in Gastrointestinal Smooth Muscle Sunila Mahavadi, Pandu R. Gangula, Shobha Ghosh, John F. Kuemmerle, John R. Grider, Karnam S. Murthy The gastrointestinal complications of diabetes mellitus include nausea, vomiting, increase in intragastric pressure and delay in gastric emptying. Several studies showed that the changes in gastric emptying in diabetes are associated with the loss of enteric neurons and interstitial cells of Cajal, and dysfunction of neuronal nitric oxide synthase activity. We have previously shown that in gastrointestinal smooth muscle sustained MLC20 phosphorylation and contraction are dependent on inhibition of MLC phosphatase, which is mediated by RhoA-dependent pathway involving phosphorylation of MYPT1 (at Thr696) by Rho kinase and CPI-17 (at Thr38) by PKC. Smooth muscle relaxation is dependent on stimulation of cAMP and cGMP levels and activation of cAMP- and cGMP-dependent protein kinases. The levels of cAMP and cGMP are regulated mainly by phosphodiesterase (PDE) 4D5 and PDE5, respectively. We hypothesized that diabetes induces changes in the expression of proteins involved in the maintenance of smooth muscle tone and regulation of cGMP levels. Aim. To determine the expression of PDE5, CPI-17 and Rho kinase as well as agonist-induced Rho kinase activity in gastric smooth muscle from control and diabetic animal models. Methods. Gastric smooth muscle was isolated from spontaneously type II diabetic ob/ob mice and type I diabetic rats. Type I diabetes was induced in rats by intravenous injection of streptozotocin. The rats were kept for 9 weeks and induction of diabetes was ascertained by the determination of blood glucose concentrations. Expression of CPI-17, Rho kinase and PDE5 was measured by immunoblot analysis and acetylcholine-induced Rho kinase activity was measured by immunokinase assay. Results. Expression of CPI-17, Rho kinase and PDE5 are significantly higher in gastric smooth muscle from both models of diabetic animals than from control animals. Consistent with the increased expression of Rho kinase, acetylcholine-induced Rho kinase activity was also significantly higher in smooth muscle from both models of diabetic animals than from control animals. Conclusion. This study identified mechanisms for increased smooth muscle tone and decreased smooth muscle relaxation in diabetes that are directly attributable to changes in smooth muscle physiology. The mechanisms involved increased expression of proteins involved in the regulation of sustained MLC20 phosphorylation (CPI-17 and Rho kinase) and cGMP levels (PDE5). Upregulation CPI-17 and Rho kinase could contribute to increased intragastric pressure, whereas upregulation of PDE5 could contribute to rapid hydrolysis of cGMP and decreased relaxation leading to delay in gastric emptying.
S2060 Ano1 Plays a Role in the Proliferation of ICC Jennifer E. Stanich, Simon J. Gibbons, Jason R. Rock, Brian D. Harfe, Tamas Ordog, Gianrico Farrugia Background: Density of networks of interstitial cells of Cajal (ICC) is dependent on a balance between factors that regulate proliferation, survival and loss of ICC. Ano1 (TMEM16A) is a Ca2+ activated Cl- channel expressed on ICC. Ano1 is also expressed in gastrointestinal stromal tumors and Cl- plays an important role in the advancement of cell cycle raising the possibility that Ano1 is involved in the regulation of the cell cycle of ICC. Aim: Investigate the role of Ano1 in the proliferation of ICC and its effect on ICC network volumes. Methods: For primary cultures, small intestinal muscle strips obtained from 2-3 day old Balb/c mice were enzymatically dissociated. The broad spectrum Cl- channel inhibitors 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and tamoxifen were added every 20h. Cells were fixed at 48 hours and ICC counted. Antibodies to Kit (ACK2) and Ki67 were used to label ICC and proliferating cells respectively. Proliferating ICC were detected in whole mount preparations of the jejunum from post-natal day (PND) 0 Ano1(-/-) mice and Ano1(+/+) littermates. ICC from the myenteric plexus (MY) region were visualized by confocal microscopic imaging and dividing cells counted from 0.5 μm thick optical sections. The volumes of Kit-positive ICC networks were determined by 3D reconstruction and volume rendering of the images using Analyze™. Results: Proliferation of ICC was significantly lower in culture following treatment with inhibitors (vehicle; 10.7, 9.7 - 11.8, 10 μM DIDS; 2.7, 2.1 - 3.7, 1 μM Tamoxifen; 6.4, 3.5 - 6.5, %Ki67 positive ICC, median, quartiles, n = 4-5, p < 0.01, Mann Whitney test). There were also significantly fewer Ki67-positive ICC-MY of whole mounts from the Ano1(-/-) mice compared to tissues from Ano1(+/+) mice (4.1 ± 0.96, 7.9 ± 0.84, %Ki67 positive ICC per stack respectively, mean ± SE, n =5, p < 0.01, t test). However, the Kit-positive ICC-MY volumes were not significantly different in the PND 0 whole mounts (831000 ± 223000 (-/-), 925,000 ± 196,000 (+/+) voxels/stack respectively, mean ± SE, n =6, p > 0.05, t test). Conclusions: Ano1 appears to be involved in ICC cell cycle regulation in both primary cultures and intact tissues. The preservation of ICC network volume at birth, despite the decreased proliferation, indicates that other factors, e.g., reduced cell death, may compensate for loss of Ano1 or that Ano1 may play a more prominent role in ICC network maintenance after birth. Supported by NIH grants DK57061 and DK68055
S2058 Altered Circular Smooth Muscle (CSM) Cell Function in Lower Esophageal Sphincter (LES) of W/Wv Mutant Mice Francisco Bautista-Cruz, William G. Paterson Background: In addition to having evidence of impaired nitrergic neurotransmission, W/Wv mutant mice, which lack intramuscular interstitial cells of Cajal (ICC-IM), also demonstrate a more negative resting membrane potential (RMP) and smaller membrane potential fluctuations or unitary potential (UPs) than control mice. It has therefore been proposed that ICCs generate UPs and thereby contribute to RMP of GI smooth muscle. It remains possible, however, that the more negative RMP and decreased UPs are due to alterations at the level of the smooth muscle itself. Methods: a) Patch Clamp: Single smooth muscle cells from W/ Wv mutant and control mice LES were freshly isolated using enzymatic digestion. The amphotericin-B perforated Patch Clamp technique was performed at room temperature (~23°C) in both current and voltage clamp modes using a model 700 B Multiclamp amplifier and the Clampex software for the data acquisition. Patch pipette resistance ranged from 2 to 4 MΩ. b) Calcium Imaging: Aliquots of freshly isolated LES smooth muscle cells were added to the recording chamber and at the same time loaded with 5 μM Fura 2 for 30 minutes at room temperature in the physiological salt solution, followed by a 15 minute wash-out period prior to recording. Ca2+ changes were obtained by sequential illumination at 340 and 380 nm and expressed as the fluorescence ratio. Results: There was no significant difference in input resistance in response to applied electrotonic current pulses between CSM cells from W/Wv mutants vs controls. RMP was more negative in CSM cells from mutant mice LES as compared to controls (-47.91±2.06 mV, n=9 vs. -41.66±1.43 mV, n= 8, p < 0.03). UPs were also significantly smaller in mutant mice (3.80±0.16 mV, n=9 vs. 4.85±0.41 mV, n=7, p < 0.03). Administration of caffeine (10 mM) induced an inward current in cells from both mutant and control mice, but the current density was significantly larger in cells from W/Wv mutants (8.01±2.32 pA/pF, n=5 vs 2.12±0.81 pA/pF, n=5; p < 0.05; holding voltage of -60 mV). Furthermore, membrane potential hyperpolarization induced by 1 mM sodium nitroprusside was more pronounced in cells from W/Wv mutants (-15.12±2.48 mV, n=8 vs -8.69±1.28 mV, n=9, p<0.05). In addition, Ca2+ mobilization induced by caffeine was increased in cells from mutants (3.1±0.57, n=7 vs 1.76 ±0.06, n= 6, p=0.05). Conclusions: These findings suggest that the more negative RMP and lower amplitude of UPs seen in LES CSM of W/Wv mutant mice in intact tissue relates to dysfunction of the smooth muscle cell itself, rather than to the absence of ICC-IM. (Supported by CIHR, CAG, Astra Zeneca and CONACYT)
S2061 Effects of Aging on Interstitial Cells of Cajal in the Human Stomach Pedro J. Gomez-Pinilla, Simon J. Gibbons, Michael L. Kendrick, Michael G. Sarr, K.Robert Shen, Maria J. Pozo, Tamas Ordog, Gianrico Farrugia Background: Interstitial cells of Cajal (ICC) are required for normal function of the stomach. Several diseases such as gastroparesis are associated with loss of ICC. Due to a relative lack of access to properly collected and stored human tissue, controls for disease specimens are not always closely age-matched, this may be important, because the effect of age on ICC number and network volumes is unknown. Aim: To determine the effects of aging on ICC numbers and network volumes in the stomach of patients with no co-existent motility disorders. Methods: Human gastric tissue from the anterior mid body was obtained under IRB approval from 20 female patients (age range 25-70 yrs) undergoing operation for morbid obesity. 12-μm tissue sections were cut from paraformaldehyde-fixed, full-thickness specimens and labeled with a mouse monoclonal antibody cocktail against Kit (LabVision, Fremont, CA) to identify ICC. The number of ICC in the circular muscle was quantified by counting Kit-positive structures surrounding a DAPI-positive nucleus in 39 fields per patient. Each field was 0.12 mm2. ICC network volumes were determined from confocal microscopic images and reconstructed in 3D. The volume of Kit-positive structures was expressed as percentage of the total volume of the confocal stack. The ratio of total Kitpositive voxel number divided by number of cell bodies was used to determine mean individual ICC size. Effect of age was assessed by testing trends using linear regression analysis. Results: The number of intramuscular ICC bodies declined significantly (P = 0.0005) with age with a decrease of 0.88 ICC bodies/field/10 years and R2 value of 0.5. This represents a 12.9% decrease in ICC number per 10 years. The volume of Kit-positive ICC structures also decreased significantly (P < 0.0001) with age with a slope of 15% per 10 years and R2
S2059 Functional Evidence of PDGF Receptor Activation in Cultured Rat Intestinal Smooth Muscle Cells Francisco Bautista-Cruz, William G. Paterson, Michael G. Blennerhassett Platelet-derived growth factor-BB (PDGF) is a mitogenic factor for vascular smooth muscle but its role in the intestine is unclear. Circular smooth muscle cells (CSMC) from the rat colon initiated expression of PDGF-R and became highly sensitive to PDGF by 10 days in primary culture (eg, 50 ng/mL caused 8-fold increase in 3H-thymidine), suggesting that PDGF could promote smooth muscle proliferation in the inflamed intestine. This study
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value of 0.7. However, mean ICC size did not change (P=0.18). Conclusion: There is a significant decrease in ICC numbers and network volumes with age in the human gastric body. These observations also suggest that studies on gastric tissues and functions must include carefully age-matched controls. Support: BFU-2007-60563, DK57061, DK68055 and DK52766. PJGP is supported by MECYT and FUNDECYT. MJP is supported by RETICEF.
presents an attractive strategy for virtual hypothesis testing, and can be used to predict the functional consequences of structural defects such as ICC loss. AIMS: (i) To adapt a biophysically-based ICC model so that it could be applied to simulate slow wave entrainment in multi-scale tissue models; (ii) To simulate slow wave entrainment in realistic wild-type (WT) and 5-HT2B knockout (degraded) ICC network structures, in order to predict the effects of ICC loss. METHODS: (i) IMAGING: Jejunal tissue was sourced from WT and 5-HT2B (serotonin receptor) knockout mice. 5-HT2B mice have decreased volumes of ICC networks (30% for myenteric). ICC networks were imaged with immunofluorescence labeling against Kit, and the myenteric ICC network geometries were imaged using confocal microscopy. (ii) SIMULATION: A biophysically-based mathematical ICC model was adapted to enable entrainment simulations by incorporation of a voltage-dependent IP3-related mechanism, and embedded into geometric models of the WT and 5-HT2B knockout ICC myenteric networks. Slow wave propagation was simulated over an activation time of 400 ms in each case. Current density and [Ca2+]i density were quantified and compared between the WT and 5-HT2B knockout models. RESULTS: Virtual slow wave phase-locking confirmed that ICC-entrainment was successfully simulated in realistic tissue ICC myenteric networks. The total activation time to depolarize the 5-HT2B knockout model (260 ms) was approximately 40 ms longer than in the WT model (220 ms). The peak current density in the WT model was 0.49 μA mm-2 higher than the 5-HT2B knockout model. The [Ca2+]i density after 400 ms in the WT model was 0.26 mM mm-2 higher than in the 5-HT2B knockout model. CONCLUSIONS: Tissue-specific models of slow waves quantitatively demonstrated physiological differences between WT and 5-HT2B knockout models. The findings of the relationship between structural loss and slow waves are of relevance to clinical conditions such as diabetic gastroparesis, in which ICC network depletion and degradation is a central pathogenic feature. ACKNOLEDGEMENTS: This work is supported by the NZ HRC, the NZ Society of Gastroenterology, NIH grants DK57061, DK68055 and DK64775.
S2062 Age-Related Loss of Interstitial Cells of Cajal in the Human Colon Pedro J. Gomez-Pinilla, Simon J. Gibbons, Robert R. Cima, Eric J. Dozois, David W. Larson, Maria J. Pozo, Tamas Ordog, Gianrico Farrugia Background: Changes in bowel function are more common in the elderly and are likely due to several factors including diet, exercise, medications and cellular changes at the level of the gastrointestinal tract. Interstitial cells of Cajal (ICC) play a key role in the control of gastrointestinal motility. The impact of aging on ICC numbers is unknown. Aim: Determine if the number of ICC and network volume in human colon is influenced by age or gender. Methods: Colonic tissue was obtained from patients undergoing colectomies for non-obstructive colon cancer. No patients had co-existing motility disorders. Tissues from 23 patients ranging in age from 36-92 were studied (14 male and 9 female). 12-μm sections were incubated with an anti-Kit antibody to identify ICC. For each patient, we counted the number of ICC bodies present in 39 fields each of 0.12 mm2 for circular muscle and 0.036 mm2 for the myenteric plexus region. ICC network volumes were determined from confocal microscopic images reconstructed in 3D. The volume of Kit-positive structures was expressed as percentage of the total volume of the confocal stack. Effect of age was assessed by testing trends using linear regression analysis. Results: The number of ICC bodies declined significantly (P < 0.0001) with age with slopes of -0.69 (r2 = 0.55) and -0.9 (r2 = 0.77) ICC bodies/field per 10 years for the circular muscle and myenteric plexus region respectively. These translated into a 12.6% and 11.5% decrease in ICC number per 10 years. ICC network volumes were also decreased by 16.8% (r2 = 0.42) and 18.3% (r2 = 0.43) per 10 years for the circular muscle and myenteric plexus region respectively. Gender had no influence on the age-associated changes in ICC numbers. Mean ICC size, obtained by dividing the number of Kit-positive voxels by the number of ICC bodies did not change in the circular muscle with age (P=0.27) while in the myenteric plexus region there was a drop in average ICC size that just reached statistical significance (P= 0.048). Conclusion: ICC numbers and network volumes decrease significantly with age in a gender-independent fashion. Agerelated loss of ICC may increase susceptibility to colonic dysfunction in the elderly. Support: RETICEF, BFU-2007-60563, DK57061, DK68055 and DK52766. PJGP is supported by MECYT and FUNDECYT.
S2065 Primary Cultures of Colonic Smooth Muscle Cells Retain Contractile Phenotype With Specific Modifications of Gene Expression by Epigenetic Mechanisms Xuan-Zheng P. Shi, Kuicheon Choi, Sushil K. Sarna Background and aims: Cell cultures are an integral part of molecular and genetic investigations. During in situ proliferation or development, the epigenetic information is inherited from the progenitor cells to impart/retain the profiles of gene expression in daughter cells. The epigenetic regulation is affected by the microenvironment, which is different in cell cultures from that in situ. Therefore, we investigated whether the expression of specific genes of the excitation-contraction coupling changes in primary cell cultures of smooth muscle cells resulting in a partial change in phenotype. Methods: The experiments were performed on freshly obtained muscularis externa, freshly dispersed single cells and in resuspended cells of primary cultures of rat colonic circular smooth muscle cells (RCCSMCs). Results: RCCSMCs retained their contractile phenotype. However, the cell shortening in response to 10-9 to 10-5 M ACh decreased in these cells vs. that in freshly dissociated cells (19±3% vs. 35±3% with 10-5 M ACh, p<0.05). We investigated the expression of 10 key cell-signaling proteins of the excitation-contraction coupling in the three types of tissues, using β-actin as the housekeeping protein. The expression of all investigated proteins did not differ between muscularis externa tissues and freshly dissociated cells (p>0.05). The expression of Gαq , 20 kD regulatory light chain (RLC20 ) and muscarinic M3 receptor did not differ between fresh cells and RCCSMCs. The expression of PP1c, Gαi3 , and ROK increased in RCCSMCs vs. those in fresh cells (470±50%, 134±12%, and 144±18%, p<0.05). By contrast,, the expressions of MLCK (68+5%), Ca v 1.2 α1C subunit (58±16%) , smooth muscle α-actin (47±8%) , MYPT1 (60±14%), and CPI-17 (19±7%) decreased in RCCSMCs vs. those in fresh cells (p<0.05). The over-expression of CPI-17 in RCCSMCs partially reversed the suppression of contractility in re-suspended cells. Methylation-specific PCR showed that the methylation of CPI-17 gene promoter increased in cell cultures (208±17%), vs. that in muscularis externa tissues. Conclusions: The colonic circular smooth muscle cells retain their contractile phenotype in culture. However, the reactivity to ACh is suppressed due to alterations in the expressions of specific cell-signaling proteins. DNA methylation of the promoters of the suppressed genes may account for gene suppression. Selective genetic changes are likely to occur in primary cell cultures of all cell-types due to their restricted microenvironment. An understanding of these changes with reference to the goals of investigation would enhance the translational relevance of the findings.
S2063 Impaired Heme Oxygenase-1 Induction in the Gastric Antrum of Streptozotocin-Induced Diabetic Rats Sachiko Suzuki, Hidekazu Suzuki, Hitoshi Tsugawa, Juntaro Matsuzaki, Yoshio Kase, Toshifumi Hibi Background. Diabetic gastroparesis and loss of interstitial cells of Cajal (ICC) has been reported to be observed in some patients and in animal models with diabetes. Moreover, failure to up-regulate heme oxygenase-1 (HO-1) in response to oxidative stress is reported to induce the loss of ICC in non-obese diabetic mice (Gastroenterology, 2008, 135(6):205564). Streptozotocin (STZ) is known to induce type I diabetes with the loss of ICC (Nerurogastroenterol. Motil., 2009, 21(10):1095-e92), however, the regulation of HO-1 expression in the stomach has not yet been elucidated in this rat model. The aim of the present study was to investigate the alteration of HO-1 expression and ICC in the stomach of the rat model of STZ-induced diabetes. Materials and Methods. 8-week-old female Wistar rats were assigned to the control and the STZ groups. STZ was intraperitoneally administered to STZ group (65 mg/Kg). The expression of HO-1 mRNA and c-kit (a marker for ICC) mRNA were assessed by quantitative RT-PCR. The expressions of HO-1 and c-Kit protein were investigated by western-blot analysis and immunofluorescent staining. Serum malondialdehyde (MDA) levels were also measured using a commercial kit. Results. Significant increase of mRNA and protein expression levels of HO-1 in the stomach were observed in the STZ group at 8 weeks after the injection (mRNA; 378 ± 59.2 %, protein; 480 ± 309 % of the control), but not at 1 week, as compared with the levels in the control group. Immunofluorescent staining revealed HO-1 immunoreactivity in the gastric corpus at 8 weeks in the STZ rats, but not in the control group. HO-1 immunoreactivity was not observed in the antrum of either the control or STZ rats. c-kit mRNA and c-Kit protein expressions in the stomach were significantly decreased at 8 weeks after STZ injection (mRNA; 38.2 ± 59.2%, protein; 75.0 ± 13.7% of control), but not at 1 week, as compared with the levels in the control group. c-Kit immunoreactivity was decreased in the gastric antrum, but not in the corpus of STZ group at 8 weeks after STZ injection. Significant increase of the serum MDA levels was observed in the STZ group, both at 1 week and 8 weeks after the injection, as compared with the levels at the corresponding time-points in the controls. Conclusion. Up-regulation of HO-1 was limited to the gastric corpus of the STZ group in response to 8 week-hyperglycemia. HO-1 in the corpus might have protected ICC in the stomach of STZ group. However, ICC decreased in the antrum probably due to impaired HO-1 enhancement.
S2066 A Computational Pipeline for the Registration, 3D Visualization and Interpretation of High-Resolution Slow Wave Maps Leo K. Cheng, Gregory O'Grady, Jonathan Erickson, Peng Du, John A. Windsor, Andrew J. Pullan BACKGROUND: The advent of high resolution (HR) multi-electrode mapping has enabled the accurate assessment of the propagation patterns, velocities and amplitudes of GI slow wave activity. However, unlike in the neurological and cardiac electrical imaging fields, there are currently no established methods for the anatomical registration and anatomically realistic visualization of GI electrical maps. Anatomical registration and visualization are critical for accurately describing regional activity differences, for localizing the anatomical site of functional problems, and for intuitively presenting mapped data. Novel methods were developed and validated for this purpose. METHODS: Human gastric serosal slow wave activity was mapped in human volunteers undergoing surgery with printed circuit board arrays (interelectrode spacing 7.6 mm; 192 electrodes). Novel automated analysis methods were employed for slow wave event detection, wave clustering, and spatial mapping of activation times and velocity fields. Pre-operative CT images were used to construct subject-specific anatomically realistic 3D stomach models. The CT images were digitized and a non-linear fitting method was used to reconstruct the stomach surface. As the pre-operative stomach geometries were found to be distended compared with the intraoperative state, measurements from defined landmarks and photogrammetry were used to scale the stomach geometry, position and correlate the data from multiple mapped sites and to correlate inter-subject variation in PCB
S2064 Tissue-Specific Mathematical Models of Slow Wave Entrainment in Wild-Type and 5-HT2B Knockout Mice With Altered Interstitial Cells of Cajal Networks Peng Du, Gregory O'Grady, Simon J. Gibbons, Rita Yassi, Rachel Lees-Green, Gianrico Farrugia, Leo K. Cheng, Andrew J. Pullan BACKGROUND: Slow waves propagate via the entrainment of ICCs. Degradation of ICC networks is associated with several motility disorders such as gastroparesis, however the pathophysiological mechanisms of this relationship is uncertain. Mathematical modeling
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Diabetes-Induced Changes in the Expression of Proteins Involved in the Regulation of Sustained Mlc20 Phosphorylation and cGMP Levels in Gastrointestinal Smooth Muscle Sunila Mahavadi, Pandu R. Gangula, Shobha Ghosh, John F. Kuemmerle, John R. Grider, Karnam S. Murthy The gastrointestinal complications of diabetes mellitus include nausea, vomiting, increase in intragastric pressure and delay in gastric emptying. Several studies showed that the changes in gastric emptying in diabetes are associated with the loss of enteric neurons and interstitial cells of Cajal, and dysfunction of neuronal nitric oxide synthase activity. We have previously shown that in gastrointestinal smooth muscle sustained MLC20 phosphorylation and contraction are dependent on inhibition of MLC phosphatase, which is mediated by RhoA-dependent pathway involving phosphorylation of MYPT1 (at Thr696) by Rho kinase and CPI-17 (at Thr38) by PKC. Smooth muscle relaxation is dependent on stimulation of cAMP and cGMP levels and activation of cAMP- and cGMP-dependent protein kinases. The levels of cAMP and cGMP are regulated mainly by phosphodiesterase (PDE) 4D5 and PDE5, respectively. We hypothesized that diabetes induces changes in the expression of proteins involved in the maintenance of smooth muscle tone and regulation of cGMP levels. Aim. To determine the expression of PDE5, CPI-17 and Rho kinase as well as agonist-induced Rho kinase activity in gastric smooth muscle from control and diabetic animal models. Methods. Gastric smooth muscle was isolated from spontaneously type II diabetic ob/ob mice and type I diabetic rats. Type I diabetes was induced in rats by intravenous injection of streptozotocin. The rats were kept for 9 weeks and induction of diabetes was ascertained by the determination of blood glucose concentrations. Expression of CPI-17, Rho kinase and PDE5 was measured by immunoblot analysis and acetylcholine-induced Rho kinase activity was measured by immunokinase assay. Results. Expression of CPI-17, Rho kinase and PDE5 are significantly higher in gastric smooth muscle from both models of diabetic animals than from control animals. Consistent with the increased expression of Rho kinase, acetylcholine-induced Rho kinase activity was also significantly higher in smooth muscle from both models of diabetic animals than from control animals. Conclusion. This study identified mechanisms for increased smooth muscle tone and decreased smooth muscle relaxation in diabetes that are directly attributable to changes in smooth muscle physiology. The mechanisms involved increased expression of proteins involved in the regulation of sustained MLC20 phosphorylation (CPI-17 and Rho kinase) and cGMP levels (PDE5). Upregulation CPI-17 and Rho kinase could contribute to increased intragastric pressure, whereas upregulation of PDE5 could contribute to rapid hydrolysis of cGMP and decreased relaxation leading to delay in gastric emptying.
S2060 Ano1 Plays a Role in the Proliferation of ICC Jennifer E. Stanich, Simon J. Gibbons, Jason R. Rock, Brian D. Harfe, Tamas Ordog, Gianrico Farrugia Background: Density of networks of interstitial cells of Cajal (ICC) is dependent on a balance between factors that regulate proliferation, survival and loss of ICC. Ano1 (TMEM16A) is a Ca2+ activated Cl- channel expressed on ICC. Ano1 is also expressed in gastrointestinal stromal tumors and Cl- plays an important role in the advancement of cell cycle raising the possibility that Ano1 is involved in the regulation of the cell cycle of ICC. Aim: Investigate the role of Ano1 in the proliferation of ICC and its effect on ICC network volumes. Methods: For primary cultures, small intestinal muscle strips obtained from 2-3 day old Balb/c mice were enzymatically dissociated. The broad spectrum Cl- channel inhibitors 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and tamoxifen were added every 20h. Cells were fixed at 48 hours and ICC counted. Antibodies to Kit (ACK2) and Ki67 were used to label ICC and proliferating cells respectively. Proliferating ICC were detected in whole mount preparations of the jejunum from post-natal day (PND) 0 Ano1(-/-) mice and Ano1(+/+) littermates. ICC from the myenteric plexus (MY) region were visualized by confocal microscopic imaging and dividing cells counted from 0.5 μm thick optical sections. The volumes of Kit-positive ICC networks were determined by 3D reconstruction and volume rendering of the images using Analyze™. Results: Proliferation of ICC was significantly lower in culture following treatment with inhibitors (vehicle; 10.7, 9.7 - 11.8, 10 μM DIDS; 2.7, 2.1 - 3.7, 1 μM Tamoxifen; 6.4, 3.5 - 6.5, %Ki67 positive ICC, median, quartiles, n = 4-5, p < 0.01, Mann Whitney test). There were also significantly fewer Ki67-positive ICC-MY of whole mounts from the Ano1(-/-) mice compared to tissues from Ano1(+/+) mice (4.1 ± 0.96, 7.9 ± 0.84, %Ki67 positive ICC per stack respectively, mean ± SE, n =5, p < 0.01, t test). However, the Kit-positive ICC-MY volumes were not significantly different in the PND 0 whole mounts (831000 ± 223000 (-/-), 925,000 ± 196,000 (+/+) voxels/stack respectively, mean ± SE, n =6, p > 0.05, t test). Conclusions: Ano1 appears to be involved in ICC cell cycle regulation in both primary cultures and intact tissues. The preservation of ICC network volume at birth, despite the decreased proliferation, indicates that other factors, e.g., reduced cell death, may compensate for loss of Ano1 or that Ano1 may play a more prominent role in ICC network maintenance after birth. Supported by NIH grants DK57061 and DK68055
S2058 Altered Circular Smooth Muscle (CSM) Cell Function in Lower Esophageal Sphincter (LES) of W/Wv Mutant Mice Francisco Bautista-Cruz, William G. Paterson Background: In addition to having evidence of impaired nitrergic neurotransmission, W/Wv mutant mice, which lack intramuscular interstitial cells of Cajal (ICC-IM), also demonstrate a more negative resting membrane potential (RMP) and smaller membrane potential fluctuations or unitary potential (UPs) than control mice. It has therefore been proposed that ICCs generate UPs and thereby contribute to RMP of GI smooth muscle. It remains possible, however, that the more negative RMP and decreased UPs are due to alterations at the level of the smooth muscle itself. Methods: a) Patch Clamp: Single smooth muscle cells from W/ Wv mutant and control mice LES were freshly isolated using enzymatic digestion. The amphotericin-B perforated Patch Clamp technique was performed at room temperature (~23°C) in both current and voltage clamp modes using a model 700 B Multiclamp amplifier and the Clampex software for the data acquisition. Patch pipette resistance ranged from 2 to 4 MΩ. b) Calcium Imaging: Aliquots of freshly isolated LES smooth muscle cells were added to the recording chamber and at the same time loaded with 5 μM Fura 2 for 30 minutes at room temperature in the physiological salt solution, followed by a 15 minute wash-out period prior to recording. Ca2+ changes were obtained by sequential illumination at 340 and 380 nm and expressed as the fluorescence ratio. Results: There was no significant difference in input resistance in response to applied electrotonic current pulses between CSM cells from W/Wv mutants vs controls. RMP was more negative in CSM cells from mutant mice LES as compared to controls (-47.91±2.06 mV, n=9 vs. -41.66±1.43 mV, n= 8, p < 0.03). UPs were also significantly smaller in mutant mice (3.80±0.16 mV, n=9 vs. 4.85±0.41 mV, n=7, p < 0.03). Administration of caffeine (10 mM) induced an inward current in cells from both mutant and control mice, but the current density was significantly larger in cells from W/Wv mutants (8.01±2.32 pA/pF, n=5 vs 2.12±0.81 pA/pF, n=5; p < 0.05; holding voltage of -60 mV). Furthermore, membrane potential hyperpolarization induced by 1 mM sodium nitroprusside was more pronounced in cells from W/Wv mutants (-15.12±2.48 mV, n=8 vs -8.69±1.28 mV, n=9, p<0.05). In addition, Ca2+ mobilization induced by caffeine was increased in cells from mutants (3.1±0.57, n=7 vs 1.76 ±0.06, n= 6, p=0.05). Conclusions: These findings suggest that the more negative RMP and lower amplitude of UPs seen in LES CSM of W/Wv mutant mice in intact tissue relates to dysfunction of the smooth muscle cell itself, rather than to the absence of ICC-IM. (Supported by CIHR, CAG, Astra Zeneca and CONACYT)
S2061 Effects of Aging on Interstitial Cells of Cajal in the Human Stomach Pedro J. Gomez-Pinilla, Simon J. Gibbons, Michael L. Kendrick, Michael G. Sarr, K.Robert Shen, Maria J. Pozo, Tamas Ordog, Gianrico Farrugia Background: Interstitial cells of Cajal (ICC) are required for normal function of the stomach. Several diseases such as gastroparesis are associated with loss of ICC. Due to a relative lack of access to properly collected and stored human tissue, controls for disease specimens are not always closely age-matched, this may be important, because the effect of age on ICC number and network volumes is unknown. Aim: To determine the effects of aging on ICC numbers and network volumes in the stomach of patients with no co-existent motility disorders. Methods: Human gastric tissue from the anterior mid body was obtained under IRB approval from 20 female patients (age range 25-70 yrs) undergoing operation for morbid obesity. 12-μm tissue sections were cut from paraformaldehyde-fixed, full-thickness specimens and labeled with a mouse monoclonal antibody cocktail against Kit (LabVision, Fremont, CA) to identify ICC. The number of ICC in the circular muscle was quantified by counting Kit-positive structures surrounding a DAPI-positive nucleus in 39 fields per patient. Each field was 0.12 mm2. ICC network volumes were determined from confocal microscopic images and reconstructed in 3D. The volume of Kit-positive structures was expressed as percentage of the total volume of the confocal stack. The ratio of total Kitpositive voxel number divided by number of cell bodies was used to determine mean individual ICC size. Effect of age was assessed by testing trends using linear regression analysis. Results: The number of intramuscular ICC bodies declined significantly (P = 0.0005) with age with a decrease of 0.88 ICC bodies/field/10 years and R2 value of 0.5. This represents a 12.9% decrease in ICC number per 10 years. The volume of Kit-positive ICC structures also decreased significantly (P < 0.0001) with age with a slope of 15% per 10 years and R2
S2059 Functional Evidence of PDGF Receptor Activation in Cultured Rat Intestinal Smooth Muscle Cells Francisco Bautista-Cruz, William G. Paterson, Michael G. Blennerhassett Platelet-derived growth factor-BB (PDGF) is a mitogenic factor for vascular smooth muscle but its role in the intestine is unclear. Circular smooth muscle cells (CSMC) from the rat colon initiated expression of PDGF-R and became highly sensitive to PDGF by 10 days in primary culture (eg, 50 ng/mL caused 8-fold increase in 3H-thymidine), suggesting that PDGF could promote smooth muscle proliferation in the inflamed intestine. This study
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value of 0.7. However, mean ICC size did not change (P=0.18). Conclusion: There is a significant decrease in ICC numbers and network volumes with age in the human gastric body. These observations also suggest that studies on gastric tissues and functions must include carefully age-matched controls. Support: BFU-2007-60563, DK57061, DK68055 and DK52766. PJGP is supported by MECYT and FUNDECYT. MJP is supported by RETICEF.
presents an attractive strategy for virtual hypothesis testing, and can be used to predict the functional consequences of structural defects such as ICC loss. AIMS: (i) To adapt a biophysically-based ICC model so that it could be applied to simulate slow wave entrainment in multi-scale tissue models; (ii) To simulate slow wave entrainment in realistic wild-type (WT) and 5-HT2B knockout (degraded) ICC network structures, in order to predict the effects of ICC loss. METHODS: (i) IMAGING: Jejunal tissue was sourced from WT and 5-HT2B (serotonin receptor) knockout mice. 5-HT2B mice have decreased volumes of ICC networks (30% for myenteric). ICC networks were imaged with immunofluorescence labeling against Kit, and the myenteric ICC network geometries were imaged using confocal microscopy. (ii) SIMULATION: A biophysically-based mathematical ICC model was adapted to enable entrainment simulations by incorporation of a voltage-dependent IP3-related mechanism, and embedded into geometric models of the WT and 5-HT2B knockout ICC myenteric networks. Slow wave propagation was simulated over an activation time of 400 ms in each case. Current density and [Ca2+]i density were quantified and compared between the WT and 5-HT2B knockout models. RESULTS: Virtual slow wave phase-locking confirmed that ICC-entrainment was successfully simulated in realistic tissue ICC myenteric networks. The total activation time to depolarize the 5-HT2B knockout model (260 ms) was approximately 40 ms longer than in the WT model (220 ms). The peak current density in the WT model was 0.49 μA mm-2 higher than the 5-HT2B knockout model. The [Ca2+]i density after 400 ms in the WT model was 0.26 mM mm-2 higher than in the 5-HT2B knockout model. CONCLUSIONS: Tissue-specific models of slow waves quantitatively demonstrated physiological differences between WT and 5-HT2B knockout models. The findings of the relationship between structural loss and slow waves are of relevance to clinical conditions such as diabetic gastroparesis, in which ICC network depletion and degradation is a central pathogenic feature. ACKNOLEDGEMENTS: This work is supported by the NZ HRC, the NZ Society of Gastroenterology, NIH grants DK57061, DK68055 and DK64775.
S2062 Age-Related Loss of Interstitial Cells of Cajal in the Human Colon Pedro J. Gomez-Pinilla, Simon J. Gibbons, Robert R. Cima, Eric J. Dozois, David W. Larson, Maria J. Pozo, Tamas Ordog, Gianrico Farrugia Background: Changes in bowel function are more common in the elderly and are likely due to several factors including diet, exercise, medications and cellular changes at the level of the gastrointestinal tract. Interstitial cells of Cajal (ICC) play a key role in the control of gastrointestinal motility. The impact of aging on ICC numbers is unknown. Aim: Determine if the number of ICC and network volume in human colon is influenced by age or gender. Methods: Colonic tissue was obtained from patients undergoing colectomies for non-obstructive colon cancer. No patients had co-existing motility disorders. Tissues from 23 patients ranging in age from 36-92 were studied (14 male and 9 female). 12-μm sections were incubated with an anti-Kit antibody to identify ICC. For each patient, we counted the number of ICC bodies present in 39 fields each of 0.12 mm2 for circular muscle and 0.036 mm2 for the myenteric plexus region. ICC network volumes were determined from confocal microscopic images reconstructed in 3D. The volume of Kit-positive structures was expressed as percentage of the total volume of the confocal stack. Effect of age was assessed by testing trends using linear regression analysis. Results: The number of ICC bodies declined significantly (P < 0.0001) with age with slopes of -0.69 (r2 = 0.55) and -0.9 (r2 = 0.77) ICC bodies/field per 10 years for the circular muscle and myenteric plexus region respectively. These translated into a 12.6% and 11.5% decrease in ICC number per 10 years. ICC network volumes were also decreased by 16.8% (r2 = 0.42) and 18.3% (r2 = 0.43) per 10 years for the circular muscle and myenteric plexus region respectively. Gender had no influence on the age-associated changes in ICC numbers. Mean ICC size, obtained by dividing the number of Kit-positive voxels by the number of ICC bodies did not change in the circular muscle with age (P=0.27) while in the myenteric plexus region there was a drop in average ICC size that just reached statistical significance (P= 0.048). Conclusion: ICC numbers and network volumes decrease significantly with age in a gender-independent fashion. Agerelated loss of ICC may increase susceptibility to colonic dysfunction in the elderly. Support: RETICEF, BFU-2007-60563, DK57061, DK68055 and DK52766. PJGP is supported by MECYT and FUNDECYT.
S2065 Primary Cultures of Colonic Smooth Muscle Cells Retain Contractile Phenotype With Specific Modifications of Gene Expression by Epigenetic Mechanisms Xuan-Zheng P. Shi, Kuicheon Choi, Sushil K. Sarna Background and aims: Cell cultures are an integral part of molecular and genetic investigations. During in situ proliferation or development, the epigenetic information is inherited from the progenitor cells to impart/retain the profiles of gene expression in daughter cells. The epigenetic regulation is affected by the microenvironment, which is different in cell cultures from that in situ. Therefore, we investigated whether the expression of specific genes of the excitation-contraction coupling changes in primary cell cultures of smooth muscle cells resulting in a partial change in phenotype. Methods: The experiments were performed on freshly obtained muscularis externa, freshly dispersed single cells and in resuspended cells of primary cultures of rat colonic circular smooth muscle cells (RCCSMCs). Results: RCCSMCs retained their contractile phenotype. However, the cell shortening in response to 10-9 to 10-5 M ACh decreased in these cells vs. that in freshly dissociated cells (19±3% vs. 35±3% with 10-5 M ACh, p<0.05). We investigated the expression of 10 key cell-signaling proteins of the excitation-contraction coupling in the three types of tissues, using β-actin as the housekeeping protein. The expression of all investigated proteins did not differ between muscularis externa tissues and freshly dissociated cells (p>0.05). The expression of Gαq , 20 kD regulatory light chain (RLC20 ) and muscarinic M3 receptor did not differ between fresh cells and RCCSMCs. The expression of PP1c, Gαi3 , and ROK increased in RCCSMCs vs. those in fresh cells (470±50%, 134±12%, and 144±18%, p<0.05). By contrast,, the expressions of MLCK (68+5%), Ca v 1.2 α1C subunit (58±16%) , smooth muscle α-actin (47±8%) , MYPT1 (60±14%), and CPI-17 (19±7%) decreased in RCCSMCs vs. those in fresh cells (p<0.05). The over-expression of CPI-17 in RCCSMCs partially reversed the suppression of contractility in re-suspended cells. Methylation-specific PCR showed that the methylation of CPI-17 gene promoter increased in cell cultures (208±17%), vs. that in muscularis externa tissues. Conclusions: The colonic circular smooth muscle cells retain their contractile phenotype in culture. However, the reactivity to ACh is suppressed due to alterations in the expressions of specific cell-signaling proteins. DNA methylation of the promoters of the suppressed genes may account for gene suppression. Selective genetic changes are likely to occur in primary cell cultures of all cell-types due to their restricted microenvironment. An understanding of these changes with reference to the goals of investigation would enhance the translational relevance of the findings.
S2063 Impaired Heme Oxygenase-1 Induction in the Gastric Antrum of Streptozotocin-Induced Diabetic Rats Sachiko Suzuki, Hidekazu Suzuki, Hitoshi Tsugawa, Juntaro Matsuzaki, Yoshio Kase, Toshifumi Hibi Background. Diabetic gastroparesis and loss of interstitial cells of Cajal (ICC) has been reported to be observed in some patients and in animal models with diabetes. Moreover, failure to up-regulate heme oxygenase-1 (HO-1) in response to oxidative stress is reported to induce the loss of ICC in non-obese diabetic mice (Gastroenterology, 2008, 135(6):205564). Streptozotocin (STZ) is known to induce type I diabetes with the loss of ICC (Nerurogastroenterol. Motil., 2009, 21(10):1095-e92), however, the regulation of HO-1 expression in the stomach has not yet been elucidated in this rat model. The aim of the present study was to investigate the alteration of HO-1 expression and ICC in the stomach of the rat model of STZ-induced diabetes. Materials and Methods. 8-week-old female Wistar rats were assigned to the control and the STZ groups. STZ was intraperitoneally administered to STZ group (65 mg/Kg). The expression of HO-1 mRNA and c-kit (a marker for ICC) mRNA were assessed by quantitative RT-PCR. The expressions of HO-1 and c-Kit protein were investigated by western-blot analysis and immunofluorescent staining. Serum malondialdehyde (MDA) levels were also measured using a commercial kit. Results. Significant increase of mRNA and protein expression levels of HO-1 in the stomach were observed in the STZ group at 8 weeks after the injection (mRNA; 378 ± 59.2 %, protein; 480 ± 309 % of the control), but not at 1 week, as compared with the levels in the control group. Immunofluorescent staining revealed HO-1 immunoreactivity in the gastric corpus at 8 weeks in the STZ rats, but not in the control group. HO-1 immunoreactivity was not observed in the antrum of either the control or STZ rats. c-kit mRNA and c-Kit protein expressions in the stomach were significantly decreased at 8 weeks after STZ injection (mRNA; 38.2 ± 59.2%, protein; 75.0 ± 13.7% of control), but not at 1 week, as compared with the levels in the control group. c-Kit immunoreactivity was decreased in the gastric antrum, but not in the corpus of STZ group at 8 weeks after STZ injection. Significant increase of the serum MDA levels was observed in the STZ group, both at 1 week and 8 weeks after the injection, as compared with the levels at the corresponding time-points in the controls. Conclusion. Up-regulation of HO-1 was limited to the gastric corpus of the STZ group in response to 8 week-hyperglycemia. HO-1 in the corpus might have protected ICC in the stomach of STZ group. However, ICC decreased in the antrum probably due to impaired HO-1 enhancement.
S2066 A Computational Pipeline for the Registration, 3D Visualization and Interpretation of High-Resolution Slow Wave Maps Leo K. Cheng, Gregory O'Grady, Jonathan Erickson, Peng Du, John A. Windsor, Andrew J. Pullan BACKGROUND: The advent of high resolution (HR) multi-electrode mapping has enabled the accurate assessment of the propagation patterns, velocities and amplitudes of GI slow wave activity. However, unlike in the neurological and cardiac electrical imaging fields, there are currently no established methods for the anatomical registration and anatomically realistic visualization of GI electrical maps. Anatomical registration and visualization are critical for accurately describing regional activity differences, for localizing the anatomical site of functional problems, and for intuitively presenting mapped data. Novel methods were developed and validated for this purpose. METHODS: Human gastric serosal slow wave activity was mapped in human volunteers undergoing surgery with printed circuit board arrays (interelectrode spacing 7.6 mm; 192 electrodes). Novel automated analysis methods were employed for slow wave event detection, wave clustering, and spatial mapping of activation times and velocity fields. Pre-operative CT images were used to construct subject-specific anatomically realistic 3D stomach models. The CT images were digitized and a non-linear fitting method was used to reconstruct the stomach surface. As the pre-operative stomach geometries were found to be distended compared with the intraoperative state, measurements from defined landmarks and photogrammetry were used to scale the stomach geometry, position and correlate the data from multiple mapped sites and to correlate inter-subject variation in PCB
S2064 Tissue-Specific Mathematical Models of Slow Wave Entrainment in Wild-Type and 5-HT2B Knockout Mice With Altered Interstitial Cells of Cajal Networks Peng Du, Gregory O'Grady, Simon J. Gibbons, Rita Yassi, Rachel Lees-Green, Gianrico Farrugia, Leo K. Cheng, Andrew J. Pullan BACKGROUND: Slow waves propagate via the entrainment of ICCs. Degradation of ICC networks is associated with several motility disorders such as gastroparesis, however the pathophysiological mechanisms of this relationship is uncertain. Mathematical modeling
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