Hydrogen peroxide activates L-type Ca2+ channels in colonic smooth muscle
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FAK itself may in turn initiate regulate intestinal epithelial motility in an ‘inside-out’ fashion. Hydrogen peroxide activates L-type Ca 21 channels in colonic smooth muscle Madhu Prasad MD, Jeffrey B Matthews, MD, FACS*. Department of Surgery, Portland VA Medical Center and Oregon Health Sciences University. 3710 SW US Veterans Hospital Road, Portland, OR, 97207 USA. *Department of Surgery Beth Israel Deaconess Medical Center, Harvard Medical School. Phone: (503)-273-5221. Introduction: Abnormal motor activity contributes significantly to the morbidity of colitis resulting from ischemia, infection, and inflammatory bowel disease. During colitis, activated polymorphonuclear neutro- phils (PMN) infiltrate the bowel wall and release a variety of reactive oxygen metabolites (ROM). The ROM hydrogen peroxide (H 2 O 2 ) is present in high concentrations at sites of inflammation. In gastrointes- tinal smooth muscle, the action potential upstroke is principally medi- ated by Ca 21 influx through voltage-gated L-type Ca 21 channels. To test our hypothesis that ROM produced during colitis contribute to motor dysfunction, we examined the effects of H 2 O 2 on Ca 21 channels in colonic smooth muscle. Methods: Cells were isolated by enzymatic dispersal of freshly har- vested smooth muscle from rabbit distal colon and placed in the recording chamber of an inverted, phase-contrast microscope. Whole cell patch clamp recording techniques ware used to measure ionic currents in voltage clamp. Ca 21 channel activity was measured in conventional whole-cell configuration using 2mM Ba 21 as the charge carrier to minimize Ca 21 channel inactivation. Standard voltage proto- cols and high Cs 1 (130 mM) pipette solutions were used to elicit Ca 21 currents in voltage clamp. Results: H 2 O 2 (0.1–100 mM) enhanced Ca 21 currents in a dose- dependent manner. At a holding potential of 110 mV, currents were enhanced from 2.1 6 0.2 pA/pF to 5.4 6 0.3 pA/pF (control vs 1 mM H 2 O 2 , n 5 6). The currents elicited by H 2 O 2 were blocked by nifedipine(1mM), suggesting that they represented activation of L-type Ca 21 channels. The structurally unrelated tyrosine kinase inhibitors genistein (50mM) and tyrphostin A48 (50mM) inhibited H 2 O 2 -induced Ca 21 currents and decreased basal Ca 21 currents. Dialysis of cells with anti-c-src and anti-FAK antibodies reduced basal Ca 21 currents by 65% and 40% respectively and inhibited the H 2 O 2 -induced in- crease in Ca 21 currents. Nonspecific anti-IgG antibodies were without effect. Basal and H 2 O 2 -induced Ca 21 currents were unaffected by the MEK inhibitor PD98059 or anti-Ras antibodies. Conclusions: L-type Ca 21 channels in colonic smooth muscle are enhanced in a dose-dependent manner by H 2 O 2 . This effect of H 2 O 2 appears to result from specific activation of the intracellular tyrosine kinase FAK with resulting downstream enhancement of c-src activity. This action of H 2 O 2 on colonic myocytes is likely to modify excitation- contraction coupling and may partly explain abnormal motility ob- served during colitis. Cross-talk between calcium and cyclic-amp signaling at serosal potassium channels in human colonic mucosae. DC Winter, M.D., B McNamara, M.D., GC O’Sullivan, M.Ch., F.R.C.S.I., F.A.C.S., BJ Harvey, Ph.D., D.Sc. Departments of Cellular Physiology and Surgery, National University of Ireland, Cork, Ireland.; Mailing Address: Prof. G. O’Sullivan, Department of Surgery, Mercy Hospital, Cork, Ireland. Telephone: 353-21-271971 Fax: 353- 21-875660 Introduction: The regulatory mechanisms of intestinal chloride (Cl 2 ) secretion are not fully characterized. The secretory synergism of cal- cium and cAMP (adenosine 39 59-monophosphate) signals is thought to occur by differential stimulation of serosal or apical membrane ion channels. Since serosal potassium (K 1 ) channels play a key role in maintaining charge balance during secretion the effects of cAMP and calcium signaling on these channels was investigated. Methods: Cl 2 secretory responses of mucosal epithelium dissected from surgical specimens were measured as short circuit current changes (DI sc ) in Ussing chambers. Serosal K 1 currents (I K ) were quantified after permeabilization of the apical membrane with an iono- phore (nystatin 100 mg/ml) DI sc and DI K are expressed in mA/cm 2 . Intracellular calcium measurements were recorded from isolated co- lonic crypts by spectrofluorescence imaging. Drug doses were carba- chol 100mM, forskolin 15mM, and tetrapentylammonium (TPeA) 100 mM. Results are mean 6 S.E.M., for n 5 6. A Mann-Whitney U test was performed for statistical analysis. Results: Carbachol and forskolin generated Cl 2 secretion (DI sc 5 57.2 6 10.6 and 71.2 6 16.9 respectively) and synergism was ob- served (cumulative DI SC 5 186.3 6 29.3; P , 0.001). Blocking serosal K 1 channels with TPeA inhibited secretion (carbachol DI SC 5 4.2 6 1.8; forskolin DI SC 5 5.9 6 2.3; P , 0.001). Carbachol, but not forskolin activated K 1 channels (DI K 5 59.8 6 13.3). However, pretreatment with forskolin augmented the K 1 channel response to carbachol (DI K 5 163.7 6 20.1; P , 0.001). Moreover, priming with forskolin (after inhibiting I K with TPeA) restored the activation of K 1 channels by carbachol (P , 0.01). Neither basal nor carbachol-stimulated increases in intracellular calcium were altered by cAMP (P . 0.3). Conclusions: An intersection of cAMP and calcium-mediated secre- tory pathways occurs at the serosal membrane of human colonic mucosa. This cross-talk between second messenger systems con- verges at the level of the potassium channels independent of intracel- lular calcium. Alimentary Tract II: Stomach and Pancreas Chronic bilateral vagal nerve stimulation (VNS) changes eating behavior resulting in weight loss in a canine model Ramesh Reddy MD, Joel Horovitz MD, Mitchell Roslin MD. Maimonides Medical Center, Brooklyn, NY, USA; For Correspondence: Mitchell S. Roslin, M.D., F.A.C.S., 953 49 th Street, Brooklyn, NY 11219, USA 718-283-8461 Telephone 718-283-6860 Fax Email [email protected] Introduction: Obesity is the most prevalent chronic disease in the Western world. However, treatment options remain limited. Previous reports suggested the importance of Vagal afferents in transmission of satiety signals from the gut to the brain. The aim of this study was to determine whether VNS could alter eating behavior and result in weight loss in a canine model. Permanent VNS was provided by nerve stim- ulator (NCP) (Cyberonics, TX). Methods: 8 canines (wt 55– 60 lbs) underwent placement of bilateral NCP’s in supradiaphragmatic position via thoracotomy. Stimulation was adjusted (0 – 4 mAmps) with frequency (30Hz), pulsewidth (500mi- crosec) and on/off cycle (1 on:1.8 off) Dogs were given 400 gm of lab diet at 9AM and 5PM. Six dogs were placed in the chronic VNS group with round the clock stimulation and two in the acute group 5 meal time only (activation 20 minutes prior to feeding). In the chronic group, chronic dogs 1 and 2, NCP’s were cycled for a period on, turned off and then back on to provide additional information Results: Acute dogs (1&2): no effect seen, including bilateral VNS and unilateral (left or right only). Chronic dogs showed marked changes in eating behavior that correlated with weight loss. The following table shows the results: Dog VNS OFF VNS ON Weight Reduction, Baseline to Last Visit Food Consumption Time, minutes Average Meal Consumption, grams Food Consumption Time, minutes Average Meal Consumption, grams Acute 1 (1899) ,5 400 ,5 400 None Acute 2 (2764) ,5 400 ,5 400 None Chronic 1 (B1024) ,5 400 20 400 None Chronic 2 (1964) ,5 400 20 200–400 24% Chronic 3 (2041) ,5 400 20 60–350 35% Chronic 4 (2082) ,5 400 20 100–400 15% Chronic 5 (2578) ,5 400 20 110–400 24% Chronic 6 (2091) ,5 400 20 105–400 None S27 Vol. 191, No. 4S, October 2000 Surgical Forum Abstracts
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Transcript of Hydrogen peroxide activates L-type Ca2+ channels in colonic smooth muscle
FAK itself may in turn initiate regulate intestinal epithelial motility in an‘inside-out’ fashion.
Hydrogen peroxide activates L-type Ca21 channels incolonic smooth muscleMadhu Prasad MD, Jeffrey B Matthews, MD, FACS*. Department ofSurgery, Portland VA Medical Center and Oregon Health SciencesUniversity. 3710 SW US Veterans Hospital Road, Portland, OR,97207 USA. *Department of Surgery Beth Israel Deaconess MedicalCenter, Harvard Medical School. Phone: (503)-273-5221.
Introduction: Abnormal motor activity contributes significantly to themorbidity of colitis resulting from ischemia, infection, and inflammatorybowel disease. During colitis, activated polymorphonuclear neutro-phils (PMN) infiltrate the bowel wall and release a variety of reactiveoxygen metabolites (ROM). The ROM hydrogen peroxide (H2O2) ispresent in high concentrations at sites of inflammation. In gastrointes-tinal smooth muscle, the action potential upstroke is principally medi-ated by Ca21 influx through voltage-gated L-type Ca21 channels. Totest our hypothesis that ROM produced during colitis contribute tomotor dysfunction, we examined the effects of H2O2 on Ca21 channelsin colonic smooth muscle.
Methods: Cells were isolated by enzymatic dispersal of freshly har-vested smooth muscle from rabbit distal colon and placed in therecording chamber of an inverted, phase-contrast microscope. Wholecell patch clamp recording techniques ware used to measure ioniccurrents in voltage clamp. Ca21 channel activity was measured inconventional whole-cell configuration using 2mM Ba21 as the chargecarrier to minimize Ca21 channel inactivation. Standard voltage proto-cols and high Cs1 (130 mM) pipette solutions were used to elicit Ca21
currents in voltage clamp.
Results: H2O2 (0.1–100 mM) enhanced Ca21 currents in a dose-dependent manner. At a holding potential of 110 mV, currents wereenhanced from 2.1 6 0.2 pA/pF to 5.4 6 0.3 pA/pF (control vs 1 mMH2O2, n 5 6). The currents elicited by H2O2 were blocked bynifedipine(1mM), suggesting that they represented activation of L-typeCa21 channels. The structurally unrelated tyrosine kinase inhibitorsgenistein (50mM) and tyrphostin A48 (50mM) inhibited H2O2-inducedCa21 currents and decreased basal Ca21 currents. Dialysis of cellswith anti-c-src and anti-FAK antibodies reduced basal Ca21 currentsby 65% and 40% respectively and inhibited the H2O2-induced in-crease in Ca21 currents. Nonspecific anti-IgG antibodies were withouteffect. Basal and H2O2-induced Ca21 currents were unaffected by theMEK inhibitor PD98059 or anti-Ras antibodies.
Conclusions: L-type Ca21 channels in colonic smooth muscle areenhanced in a dose-dependent manner by H2O2. This effect of H2O2
appears to result from specific activation of the intracellular tyrosinekinase FAK with resulting downstream enhancement of c-src activity.This action of H2O2 on colonic myocytes is likely to modify excitation-contraction coupling and may partly explain abnormal motility ob-served during colitis.
Cross-talk between calcium and cyclic-amp signaling atserosal potassium channels in human colonic mucosae.DC Winter, M.D., B McNamara, M.D., GC O’Sullivan, M.Ch.,F.R.C.S.I., F.A.C.S., BJ Harvey, Ph.D., D.Sc. Departments ofCellular Physiology and Surgery, National University of Ireland, Cork,Ireland.; Mailing Address: Prof. G. O’Sullivan, Department of Surgery,Mercy Hospital, Cork, Ireland. Telephone: 353-21-271971 Fax: 353-21-875660
Introduction: The regulatory mechanisms of intestinal chloride (Cl2)secretion are not fully characterized. The secretory synergism of cal-cium and cAMP (adenosine 39 59-monophosphate) signals is thoughtto occur by differential stimulation of serosal or apical membrane ionchannels. Since serosal potassium (K1) channels play a key role inmaintaining charge balance during secretion the effects of cAMP andcalcium signaling on these channels was investigated.
Methods: Cl2 secretory responses of mucosal epithelium dissectedfrom surgical specimens were measured as short circuit currentchanges (DIsc) in Ussing chambers. Serosal K1 currents (IK) werequantified after permeabilization of the apical membrane with an iono-
phore (nystatin 100 mg/ml) DIsc and DIK are expressed in mA/cm2.Intracellular calcium measurements were recorded from isolated co-lonic crypts by spectrofluorescence imaging. Drug doses were carba-chol 100mM, forskolin 15mM, and tetrapentylammonium (TPeA) 100mM. Results are mean 6 S.E.M., for n 5 6. A Mann-Whitney U test wasperformed for statistical analysis.
Results: Carbachol and forskolin generated Cl2 secretion (DIsc 557.2 6 10.6 and 71.2 6 16.9 respectively) and synergism was ob-served (cumulative DISC 5 186.3 6 29.3; P , 0.001). Blocking serosalK1 channels with TPeA inhibited secretion (carbachol DISC 5 4.2 61.8; forskolin DISC 5 5.9 6 2.3; P , 0.001). Carbachol, but not forskolinactivated K1 channels (DIK 5 59.8 6 13.3). However, pretreatment withforskolin augmented the K1 channel response to carbachol (DIK 5163.7 6 20.1; P , 0.001). Moreover, priming with forskolin (afterinhibiting IK with TPeA) restored the activation of K1 channels bycarbachol (P , 0.01). Neither basal nor carbachol-stimulated increasesin intracellular calcium were altered by cAMP (P . 0.3).
Conclusions: An intersection of cAMP and calcium-mediated secre-tory pathways occurs at the serosal membrane of human colonicmucosa. This cross-talk between second messenger systems con-verges at the level of the potassium channels independent of intracel-lular calcium.
Alimentary Tract II:Stomach and PancreasChronic bilateral vagal nerve stimulation (VNS)changes eating behavior resulting in weight loss in acanine modelRamesh Reddy MD, Joel Horovitz MD, Mitchell Roslin MD.Maimonides Medical Center, Brooklyn, NY, USA; ForCorrespondence: Mitchell S. Roslin, M.D., F.A.C.S., 953 49th Street,Brooklyn, NY 11219, USA 718-283-8461 Telephone 718-283-6860Fax Email [email protected]
Introduction: Obesity is the most prevalent chronic disease in theWestern world. However, treatment options remain limited. Previousreports suggested the importance of Vagal afferents in transmission ofsatiety signals from the gut to the brain. The aim of this study was todetermine whether VNS could alter eating behavior and result in weightloss in a canine model. Permanent VNS was provided by nerve stim-ulator (NCP) (Cyberonics, TX).
Methods: 8 canines (wt 55–60 lbs) underwent placement of bilateralNCP’s in supradiaphragmatic position via thoracotomy. Stimulationwas adjusted (0–4 mAmps) with frequency (30Hz), pulsewidth (500mi-crosec) and on/off cycle (1 on:1.8 off) Dogs were given 400 gm of labdiet at 9AM and 5PM. Six dogs were placed in the chronic VNS groupwith round the clock stimulation and two in the acute group 5 mealtime only (activation 20 minutes prior to feeding). In the chronic group,chronic dogs 1 and 2, NCP’s were cycled for a period on, turned offand then back on to provide additional information
Results: Acute dogs (1&2): no effect seen, including bilateral VNS andunilateral (left or right only). Chronic dogs showed marked changes ineating behavior that correlated with weight loss. The following tableshows the results:
Dog
VNS OFF VNS ONWeight
Reduction,Baseline toLast Visit
FoodConsumptionTime, minutes
Average MealConsumption,
grams
FoodConsumptionTime, minutes
Average MealConsumption,
grams
Acute 1 (1899) ,5 400 ,5 400 None
Acute 2 (2764) ,5 400 ,5 400 None
Chronic 1 (B1024) ,5 400 20 400 None
Chronic 2 (1964) ,5 400 20 200–400 24%
Chronic 3 (2041) ,5 400 20 60–350 35%
Chronic 4 (2082) ,5 400 20 100–400 15%
Chronic 5 (2578) ,5 400 20 110–400 24%
Chronic 6 (2091) ,5 400 20 105–400 None
S27Vol. 191, No. 4S, October 2000 Surgical Forum Abstracts
