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Metabolic acidosis stimulates intestinal arginine absorption
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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
untreated control grafts (P ⬍ 0.05). In addition, neointimal cell proliferation assayed by bromodeoxyuridine (BrdU) incorporation was reduced in both proximal (2.03 ⫾ 0.81%) and distal (3.12 ⫾ 0.14%) anastomoses of heparin-coated grafts compared with proximal (3.54 ⫾ 1.23%) and distal (5.58 ⫾1.15%) anastomoses of untreated control grafts (P ⬍ 0.05). Conclusions: Heparin-coated ePTFE graft significantly reduces anastomotic intimal hyperplasia and cell proliferation without measurable side effects in a baboon model. This approach supports a useful strategy for improving prosthetic bypass graft patency.
CRITICAL CARE ORAL POSTER SESSION I
solution (n ⫽ 6), and 2) by measuring L p after 25 g/ml of the type 2 (AT2) receptor blocker PD-123319 (PD) in HK, and after PD plus AngII in HK (n ⫽ 6). Results: HK prevented the increase in L p seen with AngII (Fig. 1). AT1 activation with both Sar 1-AngII in HK (p ⫽ 0.001) (Fig 2) and AngII⫹PD in HK (p ⫽ 0.008) (Fig 3) slightly increased L p, however, this effect was less than that observed with AT1 activation alone. Units for L p are ⫻10 –7 cm sec –1 䡠 cm H 2O –1. Conclusions: Inhibition of endothelial cell calcium influx prevented the 5-fold increase in L p due to AngII alone and attenuated the 5-fold increase in L p due to AT1 activation. This suggests that AngII activation of the AT1 receptor increases L p primarily, but not exclusively, via modulation of intracellular calcium and raises the question of notable cross-talk between the AT1 and AT2 receptors.
P41. Metabolic Acidosis Stimulates Intestinal Arginine Absorption. M. J. Epler, M.D., W. W. Souba, M.D., Sc.D., Q. Meng, M.D., A. M. Karinch, Ph.D., C. Lin, Ph.D., T. C. Vary, Ph.D., M. Pan, M.D., Ph.D. Penn State College of Medicine. Introduction: L-Arginine is the exclusive precursor for nitric oxide (NO) biosynthesis and an essential nutrient for intestinal integrity during stress states. However, the effect of extracellular pH on intestinal arginine uptake is poorly understood. The purpose of this in vitro study was to investigate the regulation of intestinal arginine transport during acidosis. Methods: 3H-L-arginine transport activity was measured in intestinal epithelial Caco-2 cells at various ambient pHs (pH 6.6-7.6) ⫾ actinomycin-D (1 M), cycloheximide (20 M), phosphatidylinositide-3 kinase (PI-3) inhibitor wortmannin (1 uM), and the NO synthetase inhibitor N--Nitro-LArginine (L-NA, 100 M). Data were analyzed by ANOVA. Results: Decreasing the extracellular pH stimulated arginine transport activity in a time and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a 4-fold increase in arginine transport activity (0.94 ⫾ 0.10 acidosis vs. 0.23 ⫾ 0.05 control, nmole/mg/min, p ⬍ 0.01) and a 3-fold increase of arginine transporter mCAT-1 mRNA levels (p ⬍ 0.01). This acidosis-induced increase in arginine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 1.53 ⫾ 0.08 acidosis vs. 0.56 ⫾ 0.05 control, nmole/mg /min, p ⬍ 0.01) rather than a change in transporter affinity (Km ⫽ 54 ⫾ 10 acidosis vs. 43 ⫾ 6 control, M arginine, p ⫽ NS). This acidosis-stimulated arginine transport activity was individually blocked by actinomycin-D, cycloheximide, and L-NA. The acidosisinduced arginine transport activity was further stimulated by wortmannin (1.4 ⫾ 0.25 acidosis ⫹ wortmannin vs. 0.94 ⫾ 0.10 acidosis vs. 0.21 ⫾ 0.03 wortmannin vs. 0.23 ⫾ 0.05 control, nmole/mg /min). Conclusion: Acidosis stimulates arginine transport in Caco-2 cells via a mechanism that leads to transcription of the gene and translation of arginine transporter. Phosphatidylinositide-3 kinase is a key intracellular regulator involved in this signal transduction cascade. The nitric oxide synthetase activity plays a pivotal role in this stimulation. An increased availability of arginine to cells during acidosis provides substrate for NO biosynthesis and may help in maintaining cellular integrity. P42. Intracellular Calcium Modulates Angiotensin II Type 1 Receptor Mediated Increases in Hydraulic Permeability. C. R. Newton, M.D., B. Curran, B.S., G. P. Victorino, M.D. Department of Surgery, UCSF–East Bay. Background: Previous work has documented a 5-fold increase in L p due to AngII. We hypothesized that the increase in L p due to activation of the AT1 receptor is modulated by intracellular calcium [Ca 2⫹]. Methods: L p was measured in rat mesenteric venules using the Landis micro-occlusion model. A 100 mM KCl Ringer’s solution (HK) was used to abolish the electrochemical driving force for [Ca 2⫹] influx. Measures of L p were obtained at baseline (BL), after HK, and after 20 ng/ml AngII plus HK (n ⫽ 6). Modulation of [Ca 2⫹] via selective AT1 activation was evaluated by: 1) measuring L p at BL, after HK, and after 100 M of the AT1 agonist Sar 1-AngII in HK
P43. Addition of Positive Charge Augments the AntiEndotoxin Properties of LALF-Based Peptides. P. S. Vietzen, M.D., D. B. Leslie, M.D., K. R. Wasiluk, Ph.D., V. Lazaron, Ph.D., D. L. Dunn, M.D., Ph.D. University of Minnesota. Introduction: The endotoxin-binding domain of Limulus antilipopolysaccharide factor (LALF 31-52) contains eight positivelycharged amino acids. We hypothesized that adding more positivelycharged residues to this domain would further enhance its antiendotoxin properties. Methods: A series of peptides (PV11 to PV42) was created based upon the endotoxin-binding region of LALF (PV00). Neutral or negatively-charged amino acids were sequentially replaced with one to four positively-charged (lysine ⫹1) residues at various positions. The peptides were then tested for LPS neutralization by two distinct assays and for bactericidal activity against P. aeruginosa. Results for each peptide were compared to the native sequence using Student’s t-test. Results: Most peptides with at least two additional lysine residues displayed significantly increased bactericidal activity versus the native peptide sequence (p ⬍ 0.05; left graph). Many lysine-addition peptides also demonstrated significantly increased activity versus the native peptide in a Limulus
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
untreated control grafts (P ⬍ 0.05). In addition, neointimal cell proliferation assayed by bromodeoxyuridine (BrdU) incorporation was reduced in both proximal (2.03 ⫾ 0.81%) and distal (3.12 ⫾ 0.14%) anastomoses of heparin-coated grafts compared with proximal (3.54 ⫾ 1.23%) and distal (5.58 ⫾1.15%) anastomoses of untreated control grafts (P ⬍ 0.05). Conclusions: Heparin-coated ePTFE graft significantly reduces anastomotic intimal hyperplasia and cell proliferation without measurable side effects in a baboon model. This approach supports a useful strategy for improving prosthetic bypass graft patency.
CRITICAL CARE ORAL POSTER SESSION I
solution (n ⫽ 6), and 2) by measuring L p after 25 g/ml of the type 2 (AT2) receptor blocker PD-123319 (PD) in HK, and after PD plus AngII in HK (n ⫽ 6). Results: HK prevented the increase in L p seen with AngII (Fig. 1). AT1 activation with both Sar 1-AngII in HK (p ⫽ 0.001) (Fig 2) and AngII⫹PD in HK (p ⫽ 0.008) (Fig 3) slightly increased L p, however, this effect was less than that observed with AT1 activation alone. Units for L p are ⫻10 –7 cm sec –1 䡠 cm H 2O –1. Conclusions: Inhibition of endothelial cell calcium influx prevented the 5-fold increase in L p due to AngII alone and attenuated the 5-fold increase in L p due to AT1 activation. This suggests that AngII activation of the AT1 receptor increases L p primarily, but not exclusively, via modulation of intracellular calcium and raises the question of notable cross-talk between the AT1 and AT2 receptors.
P41. Metabolic Acidosis Stimulates Intestinal Arginine Absorption. M. J. Epler, M.D., W. W. Souba, M.D., Sc.D., Q. Meng, M.D., A. M. Karinch, Ph.D., C. Lin, Ph.D., T. C. Vary, Ph.D., M. Pan, M.D., Ph.D. Penn State College of Medicine. Introduction: L-Arginine is the exclusive precursor for nitric oxide (NO) biosynthesis and an essential nutrient for intestinal integrity during stress states. However, the effect of extracellular pH on intestinal arginine uptake is poorly understood. The purpose of this in vitro study was to investigate the regulation of intestinal arginine transport during acidosis. Methods: 3H-L-arginine transport activity was measured in intestinal epithelial Caco-2 cells at various ambient pHs (pH 6.6-7.6) ⫾ actinomycin-D (1 M), cycloheximide (20 M), phosphatidylinositide-3 kinase (PI-3) inhibitor wortmannin (1 uM), and the NO synthetase inhibitor N--Nitro-LArginine (L-NA, 100 M). Data were analyzed by ANOVA. Results: Decreasing the extracellular pH stimulated arginine transport activity in a time and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a 4-fold increase in arginine transport activity (0.94 ⫾ 0.10 acidosis vs. 0.23 ⫾ 0.05 control, nmole/mg/min, p ⬍ 0.01) and a 3-fold increase of arginine transporter mCAT-1 mRNA levels (p ⬍ 0.01). This acidosis-induced increase in arginine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 1.53 ⫾ 0.08 acidosis vs. 0.56 ⫾ 0.05 control, nmole/mg /min, p ⬍ 0.01) rather than a change in transporter affinity (Km ⫽ 54 ⫾ 10 acidosis vs. 43 ⫾ 6 control, M arginine, p ⫽ NS). This acidosis-stimulated arginine transport activity was individually blocked by actinomycin-D, cycloheximide, and L-NA. The acidosisinduced arginine transport activity was further stimulated by wortmannin (1.4 ⫾ 0.25 acidosis ⫹ wortmannin vs. 0.94 ⫾ 0.10 acidosis vs. 0.21 ⫾ 0.03 wortmannin vs. 0.23 ⫾ 0.05 control, nmole/mg /min). Conclusion: Acidosis stimulates arginine transport in Caco-2 cells via a mechanism that leads to transcription of the gene and translation of arginine transporter. Phosphatidylinositide-3 kinase is a key intracellular regulator involved in this signal transduction cascade. The nitric oxide synthetase activity plays a pivotal role in this stimulation. An increased availability of arginine to cells during acidosis provides substrate for NO biosynthesis and may help in maintaining cellular integrity. P42. Intracellular Calcium Modulates Angiotensin II Type 1 Receptor Mediated Increases in Hydraulic Permeability. C. R. Newton, M.D., B. Curran, B.S., G. P. Victorino, M.D. Department of Surgery, UCSF–East Bay. Background: Previous work has documented a 5-fold increase in L p due to AngII. We hypothesized that the increase in L p due to activation of the AT1 receptor is modulated by intracellular calcium [Ca 2⫹]. Methods: L p was measured in rat mesenteric venules using the Landis micro-occlusion model. A 100 mM KCl Ringer’s solution (HK) was used to abolish the electrochemical driving force for [Ca 2⫹] influx. Measures of L p were obtained at baseline (BL), after HK, and after 20 ng/ml AngII plus HK (n ⫽ 6). Modulation of [Ca 2⫹] via selective AT1 activation was evaluated by: 1) measuring L p at BL, after HK, and after 100 M of the AT1 agonist Sar 1-AngII in HK
P43. Addition of Positive Charge Augments the AntiEndotoxin Properties of LALF-Based Peptides. P. S. Vietzen, M.D., D. B. Leslie, M.D., K. R. Wasiluk, Ph.D., V. Lazaron, Ph.D., D. L. Dunn, M.D., Ph.D. University of Minnesota. Introduction: The endotoxin-binding domain of Limulus antilipopolysaccharide factor (LALF 31-52) contains eight positivelycharged amino acids. We hypothesized that adding more positivelycharged residues to this domain would further enhance its antiendotoxin properties. Methods: A series of peptides (PV11 to PV42) was created based upon the endotoxin-binding region of LALF (PV00). Neutral or negatively-charged amino acids were sequentially replaced with one to four positively-charged (lysine ⫹1) residues at various positions. The peptides were then tested for LPS neutralization by two distinct assays and for bactericidal activity against P. aeruginosa. Results for each peptide were compared to the native sequence using Student’s t-test. Results: Most peptides with at least two additional lysine residues displayed significantly increased bactericidal activity versus the native peptide sequence (p ⬍ 0.05; left graph). Many lysine-addition peptides also demonstrated significantly increased activity versus the native peptide in a Limulus