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Ryanodine or ischemic preconditioning attenuate postischemic leukocyte adhesion and microvascular barrier disruption in rat mesentery
G A S T R O E N T E R O L O G Y Vol. 114, No. 4
A380 AGA A B S T R A C T S
• G1549 CL-HCO 3 AND CL-OH EXCHANGES OF BASOLATERAL MEMBRANES (BLM) REGULATE INTRACELLULAR pH (pHi) IN CRYPT CELLS OF RAT DISTAL COLON. M. Ikum~, J. Geibel, H. J. Binder and V. M. Rajendran. Departments of Internal Medicine and Surgery, Yale University, New Haven, CT. Intracellular pH (pHi) is regulated by both apical and basolateral membrane transporters. Evidence of colonocyte pHi regulation by apical ion exchanges has recently been established, but a role for basolateral ion exchanges has as yet not been presented. This study examined the possible importance of Cl-anion (i.e. C1-HCO3 and CI-OH) exchanges on colonocyte pHi by performing 36C1 uptake in BLM vesicles and bath Cl-dependent pHi regulation using microfluorometry techniques in isolated crypts. Imposition of outward gradients of either HCO 3 or OH stimulated a saturable, DIDSsensitive 36C1 uptake in BLMV isolated from scraped mucosa of distal colon. Initial rate of HCO~-gradient driven 36C1 uptake was 30% higher than that of OH-gradient. Simultaneous presence of outward HCO~- and OH-gradients stimulated 36C1 uptake with an additive effect. These results establish that HCO~- and OH-gradient stimulated 36C1uptake occur via distinct and separate transport processes. Bath Cl-dependent phi recovery from an alkaline load (induced by bath Cl-removal) was observed in both the presence and nominal absence of HCO~. Cl-dependent phi recovery in both the presence and nominal absence of HCO 3 was completely inhibited by 2 mM DIDS. These results establish that bath Cl-dependent pHi regulation in the presence and the nominal absence of HCO 3 occurs via CI-HCO3 and C1-OH exchanges, respectively. Conclusion: Distinct CI-HCO, and CI-OH exchanges are present in BLM of rat distal colon and regulate pHi. • G1550 CROSSTALK BETWEEN APICAL H, K-ATPase AND BASOLATERAL K CHANNEL REGULATES INTRACELLULAR (pHi) IN RAT DISTAL COLON. M. Ikuma, H.J. Binder; J.P. Geibel. Departments of Internal Medicine and Surgery, Yale University, New Haven, CT The mammalian colon has multiple mechanisms for potassium (K) transport including active K absorption and active and potential-dependent K secretion. K movement across both apical (AP) and basolateral (BL) membranes may involve ATPases, co-transporters and channels, We have recently identified in colonic crypt cells a K-dependent mechanism for phi regulation that is mediated by an AP ouahain-sensitive H, K-ATPase (AJP 273, in press, 1998), while barium-sensitive K channels have been well described in colonocyte BL membranes. These present studies were designed to examine the possible crosstalk between AP membrane H, K-ATPase and BL membrane K channels in rat distal colon in the regulation of pHi. pHi in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF. We examined whether changes in intracellular [K] produced by increased bath [K] would alter pHi by mechanisms mediated by AP H, K-ATPase. After loading with BCECF, crypts were superfused with a Na-free solution which caused a rapid and reversible fall in pHi (7.38 ± 0.03 to 6.97 ± 0.07, n=8). When extracellular [K] was increased (5 to 20 mM), in the continued absence of bath Na, there was a further, reversible decrease in pHi (0,22 ± 0.09, n=8). This K-dependent acidification was blocked by 2 mM bath barium, a K channel blocker. In a separate series of experiments we perfused isolated crypts with 1 mM luminal ouabain and found that increasing extracellular bath [K] (5 to 20 mM) had no effect on phi: 6.93 ± 0.02 to 6.92 ± 0.02 (n=5). We propose that increased K movement via BL K channels increases intracellular [K] that then results in a decrease in pHi that is mediated by AP H, K-ATPase. Thus, BL K channels indirectly regulate pHi via AP H, K-ATPase modulated by intracellular [K]. • G1551 RYANODINE OR ISCItEMIC PRECONDITIONING ATTENUATE POSTISCHEMIC LEUKOCYTE ADHESION AND MICROVASCULAR BARRIER DISRUPTION IN RAT MESENTERY. T. Ishida. K. Yarimizu, D.C. Gute, and R,J. Korthuis. Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA. Background: It is generally believed that prolonged reductions in blood flow induce a marked calcium influx. The rise in intracellular calcium promotes endothelial cell adhesion molecule expression, which in turn plays a critical role in the pathogenesis of ischemia/reperfusion (I/R) injury by promoting leukocyte adhesion, activation, and emigration. Brief periods of ischemia have been shown to render the intestine's other tissues resistant to the deleterious effects of subsequent prolonged ischemia and reperfusion (I/R), a phenomenon referred to as ischemic preconditioning (IPC). Although the subject of intensive investigation for the last decade, the mechanisms underlying IPC remain uncertain. However, one possible mechanism may be that IPC prevents the release of calcium from the endoplasmic reticulum during a subsequent exposure to prolonged I/R. The aim of this study was to determine whether the small intestine could be pharmacologically preconditioned with ryanodine, an agent that blocks the release of calcium from the endoplasmic reticulum. Methods: To address this postulate, intravital video microscopic techniques were to quantitate leukocyte (WBC) adhesion (LA), leukocyte emigration
(LE), and venular protein leakage (VPL) in single, unbranched venules (20-35 pm in diameter, 150 lam in length) in rat mesentery subjected to I/R alone (20 min ischemia followed by 1 hour reperfusion), IPC (5 min ischemia, 10 min reperfusion) + I/R, or ryanodine preconditioning (RYPC, superfusion of the mesentery with ryanodine, 5 nM, for 5 rain beginning 15 min prior to I/R) + I/R. All variables were measured during baseline conditions and during min 50-60 of reperfusion. Results: I/R increased LA (19.2+1.4 vs 2.0+0.3 WBC/venule, respectively), LE (8.5+0.8 vs 1.0+0.3 WBC/field, respectively), and VPL (57.1+8.6 vs 8.6+4.6%, respectively) compared to baseline values. No change in LA, LE, or VPL were noted in time control experiments in which the mesentery was continuously perfused for a time period equivalent to that used in the I/R protocol. The increases in LA, LE, and VPL induced by I/R were largely prevented by IPC (2.7+0.5 WBC/venule, 1.7+0.2 WBC/field, and 11.7+4.2%, respectively) or RYPC (3.0+0.7 WBC/venule, 2.1+0.7 WBC/fleld, and 21.0+5.7 %, respectively). Conclusions: I/R promotes leukocyte adhesion and emigration and disrupts microvascular barrier function. These deleterious effects of prolonged I/R are prevented by IPC and ryanodine preconditioning. The latter result suggests that agents which block calcium release from the endoplasmic reticulum may be used to pharmacologically precondition the small intestine and prevent postischemic LA, LE, and VPL. This study was supported by NIH grant DK-43750. G1552 IMBALANCE OF BCL-2 FAMILY PROTEIN EXPRESSION BY MUCOSAL T-CELLS FAVORS RESISTANCE TO APOPTOSIS IN CROHN'S DISEASE (CD), J. Itoh, A.D. Levine, C. Fiocchi. Division of Gastroenterology, Case Western Reserve University, Cleveland, OH. A hallmark of CD is a persistent infiltration of T-cells in the mucosa. This may be due to an abnormally prolonged T-eeU survival which could contribute to chronicity of inflammation. Cell survival is regulated by antiand pro-apoptotic gene products, such as bcl-2 and bcl-XL or bax proteins, respectively. Therefore, we investigated potential differences in the expression of bcl-2-related proteins between peripheral blood and mucosal T-cells, and control and CD mucosal T-cells. T-ceUs were purified from normal peripheral blood (PBT) and normal or CD-involved intestinal lamina propria (LPT), stained intracellularly for bcl-2, bcl-xL and bax, and analyzed by flow cytometry, with results being expressed as mean fluorescence intensity (MFI). The expression of bcl-2 and bax, but not bcl-xL, was significantly (*p < 0.01) greater in LPT compared to that of PBT. Cell source control PBT (n = 7) control LPT (n = 8)
bcl-2 30.6 ± 1.7 45.3 ± 4.0*
MFI _+SE bcl-xL 38.6 ± 1.8 42.2 ± 2.1
bax 90.6 ± 4.7 134.1 ± 11.7"
A significant positive correlation between bcl-2 and hax was found in PBT (r=0.982; p < 0.0001) but not control or CD LPT. In contrast, a significant correlation between bcl-XL and bax was found in both PBT (r=0.933; p < 0.0005) and normal LPT (r=0.942; p < 0.0001). Interestingly, although a positive correlation between bcl-XL and bax was also found in CD LPT (r=0.933; p < 0.05), this occurred at a significantly (p < 0.01) lower bax/bcl-XL ratio than normal LPT, as indicated by a lower slope of the correlating curve. 20O
.~ 160
i)
7" 3D
36
40
4S
~0
55
Ud-x expre~m
These results show that regulation of bcl-2 family of proteins varies between circulating and mucosal T-ceils, perhaps reflecting different survival potentials. Furthermore, the reduction in bax/bcl-XL ratio in CD LPT suggests that regulation of cell survival is skewed towards resistance to apoptosis in this condition. This could result in an exceedingly long T-cell survival and explain chronic inflammatory infiltrates in CD.
A380 AGA A B S T R A C T S
• G1549 CL-HCO 3 AND CL-OH EXCHANGES OF BASOLATERAL MEMBRANES (BLM) REGULATE INTRACELLULAR pH (pHi) IN CRYPT CELLS OF RAT DISTAL COLON. M. Ikum~, J. Geibel, H. J. Binder and V. M. Rajendran. Departments of Internal Medicine and Surgery, Yale University, New Haven, CT. Intracellular pH (pHi) is regulated by both apical and basolateral membrane transporters. Evidence of colonocyte pHi regulation by apical ion exchanges has recently been established, but a role for basolateral ion exchanges has as yet not been presented. This study examined the possible importance of Cl-anion (i.e. C1-HCO3 and CI-OH) exchanges on colonocyte pHi by performing 36C1 uptake in BLM vesicles and bath Cl-dependent pHi regulation using microfluorometry techniques in isolated crypts. Imposition of outward gradients of either HCO 3 or OH stimulated a saturable, DIDSsensitive 36C1 uptake in BLMV isolated from scraped mucosa of distal colon. Initial rate of HCO~-gradient driven 36C1 uptake was 30% higher than that of OH-gradient. Simultaneous presence of outward HCO~- and OH-gradients stimulated 36C1 uptake with an additive effect. These results establish that HCO~- and OH-gradient stimulated 36C1uptake occur via distinct and separate transport processes. Bath Cl-dependent phi recovery from an alkaline load (induced by bath Cl-removal) was observed in both the presence and nominal absence of HCO~. Cl-dependent phi recovery in both the presence and nominal absence of HCO 3 was completely inhibited by 2 mM DIDS. These results establish that bath Cl-dependent pHi regulation in the presence and the nominal absence of HCO 3 occurs via CI-HCO3 and C1-OH exchanges, respectively. Conclusion: Distinct CI-HCO, and CI-OH exchanges are present in BLM of rat distal colon and regulate pHi. • G1550 CROSSTALK BETWEEN APICAL H, K-ATPase AND BASOLATERAL K CHANNEL REGULATES INTRACELLULAR (pHi) IN RAT DISTAL COLON. M. Ikuma, H.J. Binder; J.P. Geibel. Departments of Internal Medicine and Surgery, Yale University, New Haven, CT The mammalian colon has multiple mechanisms for potassium (K) transport including active K absorption and active and potential-dependent K secretion. K movement across both apical (AP) and basolateral (BL) membranes may involve ATPases, co-transporters and channels, We have recently identified in colonic crypt cells a K-dependent mechanism for phi regulation that is mediated by an AP ouahain-sensitive H, K-ATPase (AJP 273, in press, 1998), while barium-sensitive K channels have been well described in colonocyte BL membranes. These present studies were designed to examine the possible crosstalk between AP membrane H, K-ATPase and BL membrane K channels in rat distal colon in the regulation of pHi. pHi in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF. We examined whether changes in intracellular [K] produced by increased bath [K] would alter pHi by mechanisms mediated by AP H, K-ATPase. After loading with BCECF, crypts were superfused with a Na-free solution which caused a rapid and reversible fall in pHi (7.38 ± 0.03 to 6.97 ± 0.07, n=8). When extracellular [K] was increased (5 to 20 mM), in the continued absence of bath Na, there was a further, reversible decrease in pHi (0,22 ± 0.09, n=8). This K-dependent acidification was blocked by 2 mM bath barium, a K channel blocker. In a separate series of experiments we perfused isolated crypts with 1 mM luminal ouabain and found that increasing extracellular bath [K] (5 to 20 mM) had no effect on phi: 6.93 ± 0.02 to 6.92 ± 0.02 (n=5). We propose that increased K movement via BL K channels increases intracellular [K] that then results in a decrease in pHi that is mediated by AP H, K-ATPase. Thus, BL K channels indirectly regulate pHi via AP H, K-ATPase modulated by intracellular [K]. • G1551 RYANODINE OR ISCItEMIC PRECONDITIONING ATTENUATE POSTISCHEMIC LEUKOCYTE ADHESION AND MICROVASCULAR BARRIER DISRUPTION IN RAT MESENTERY. T. Ishida. K. Yarimizu, D.C. Gute, and R,J. Korthuis. Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA. Background: It is generally believed that prolonged reductions in blood flow induce a marked calcium influx. The rise in intracellular calcium promotes endothelial cell adhesion molecule expression, which in turn plays a critical role in the pathogenesis of ischemia/reperfusion (I/R) injury by promoting leukocyte adhesion, activation, and emigration. Brief periods of ischemia have been shown to render the intestine's other tissues resistant to the deleterious effects of subsequent prolonged ischemia and reperfusion (I/R), a phenomenon referred to as ischemic preconditioning (IPC). Although the subject of intensive investigation for the last decade, the mechanisms underlying IPC remain uncertain. However, one possible mechanism may be that IPC prevents the release of calcium from the endoplasmic reticulum during a subsequent exposure to prolonged I/R. The aim of this study was to determine whether the small intestine could be pharmacologically preconditioned with ryanodine, an agent that blocks the release of calcium from the endoplasmic reticulum. Methods: To address this postulate, intravital video microscopic techniques were to quantitate leukocyte (WBC) adhesion (LA), leukocyte emigration
(LE), and venular protein leakage (VPL) in single, unbranched venules (20-35 pm in diameter, 150 lam in length) in rat mesentery subjected to I/R alone (20 min ischemia followed by 1 hour reperfusion), IPC (5 min ischemia, 10 min reperfusion) + I/R, or ryanodine preconditioning (RYPC, superfusion of the mesentery with ryanodine, 5 nM, for 5 rain beginning 15 min prior to I/R) + I/R. All variables were measured during baseline conditions and during min 50-60 of reperfusion. Results: I/R increased LA (19.2+1.4 vs 2.0+0.3 WBC/venule, respectively), LE (8.5+0.8 vs 1.0+0.3 WBC/field, respectively), and VPL (57.1+8.6 vs 8.6+4.6%, respectively) compared to baseline values. No change in LA, LE, or VPL were noted in time control experiments in which the mesentery was continuously perfused for a time period equivalent to that used in the I/R protocol. The increases in LA, LE, and VPL induced by I/R were largely prevented by IPC (2.7+0.5 WBC/venule, 1.7+0.2 WBC/field, and 11.7+4.2%, respectively) or RYPC (3.0+0.7 WBC/venule, 2.1+0.7 WBC/fleld, and 21.0+5.7 %, respectively). Conclusions: I/R promotes leukocyte adhesion and emigration and disrupts microvascular barrier function. These deleterious effects of prolonged I/R are prevented by IPC and ryanodine preconditioning. The latter result suggests that agents which block calcium release from the endoplasmic reticulum may be used to pharmacologically precondition the small intestine and prevent postischemic LA, LE, and VPL. This study was supported by NIH grant DK-43750. G1552 IMBALANCE OF BCL-2 FAMILY PROTEIN EXPRESSION BY MUCOSAL T-CELLS FAVORS RESISTANCE TO APOPTOSIS IN CROHN'S DISEASE (CD), J. Itoh, A.D. Levine, C. Fiocchi. Division of Gastroenterology, Case Western Reserve University, Cleveland, OH. A hallmark of CD is a persistent infiltration of T-cells in the mucosa. This may be due to an abnormally prolonged T-eeU survival which could contribute to chronicity of inflammation. Cell survival is regulated by antiand pro-apoptotic gene products, such as bcl-2 and bcl-XL or bax proteins, respectively. Therefore, we investigated potential differences in the expression of bcl-2-related proteins between peripheral blood and mucosal T-cells, and control and CD mucosal T-cells. T-ceUs were purified from normal peripheral blood (PBT) and normal or CD-involved intestinal lamina propria (LPT), stained intracellularly for bcl-2, bcl-xL and bax, and analyzed by flow cytometry, with results being expressed as mean fluorescence intensity (MFI). The expression of bcl-2 and bax, but not bcl-xL, was significantly (*p < 0.01) greater in LPT compared to that of PBT. Cell source control PBT (n = 7) control LPT (n = 8)
bcl-2 30.6 ± 1.7 45.3 ± 4.0*
MFI _+SE bcl-xL 38.6 ± 1.8 42.2 ± 2.1
bax 90.6 ± 4.7 134.1 ± 11.7"
A significant positive correlation between bcl-2 and hax was found in PBT (r=0.982; p < 0.0001) but not control or CD LPT. In contrast, a significant correlation between bcl-XL and bax was found in both PBT (r=0.933; p < 0.0005) and normal LPT (r=0.942; p < 0.0001). Interestingly, although a positive correlation between bcl-XL and bax was also found in CD LPT (r=0.933; p < 0.05), this occurred at a significantly (p < 0.01) lower bax/bcl-XL ratio than normal LPT, as indicated by a lower slope of the correlating curve. 20O
.~ 160
i)
7" 3D
36
40
4S
~0
55
Ud-x expre~m
These results show that regulation of bcl-2 family of proteins varies between circulating and mucosal T-ceils, perhaps reflecting different survival potentials. Furthermore, the reduction in bax/bcl-XL ratio in CD LPT suggests that regulation of cell survival is skewed towards resistance to apoptosis in this condition. This could result in an exceedingly long T-cell survival and explain chronic inflammatory infiltrates in CD.