Shear Stress Induces Change in Extracellular Signal-Regulated Kinase 5 Levels with Sustained Activation under Disturbed and Continuous Laminar Flow.

Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction under laminar flow. Previously reported research indicates that under laminar flow ERK5 is activated with production of atheroprotective molecules. However, the characterization of ERK5 activation and levels under different flow patterns has not been investigated. Confluent HUVECs were serum-starved then seeded on glass slides. HUVECs incubated in 1% FBS were exposed to continuous laminar flow (CLF), to-and-fro flow (TFF), or pulsatile forward flow (PFF) in a parallel plate flow chamber. At the end of experimentation, cell lysates were immunoblotted with antibodies to phospho-ERK5 and total ERK5. ERK5 activation was assessed by the levels of phosphorylated ERK5. The densitometric mean ± SEM is calculated and analyzed by ANOVA. p < 0.05 is considered significant. Levels of ERK5 decreased with all flow conditions with the largest decrease in TFF flow condition. TFF and CLF exhibited sustained ERK5 phosphorylation in HUVECs stimulated for up to 4 hours. PFF had transient phosphorylation of ERK5 at 2 hours, which then became undetectable at 4 hours of exposure to flow. Also, TFF and CLF both showed decreased levels at 4 hours, suggesting a decrease in activation for these flow conditions. Exposure of HUVEC to different types of shear stress results in varying patterns of activation of ERK5. Activation of ERK5 with TFF suggests a role in the pathogenesis of atherosclerosis and vascular remodeling under disturbed flow conditions.

The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion.

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

Olive oil polyphenol oleuropein inhibits smooth muscle cell proliferation.

OBJECTIVES: The Mediterranean diet, with a high content of olive oil, is associated with a reduced risk of coronary artery disease. The aim of this study was to determine the effect of oleuropein, one of the polyphenols in olive oil, on vascular smooth muscle cell (SMC) proliferation in vitro. DESIGN: This was an experimental study. MATERIALS AND METHODS: Bovine vascular SMCs were cultured in the presence of 100 µM of oleuropein. On days 1, 3 and 5, cell number was counted. Cell cycle analysis was performed by flow cytometry. Cell cycle regulators were assessed by immunoblotting. RESULTS: Cell proliferation in the presence of oleuropein was significantly inhibited by 92%. Cell cycle analysis revealed that oleuropein treatment blocked cells in the G1-S phase compared with the non-treated group. Among G1 phase regulators, retinoblastoma protein, cyclinD, p21 and p27 were not affected by oleuropein, but extracellular signal-regulated kinase 1/2 (ERK1/2) activation was inhibited. Growth of SMC treated with 100 µM of the mitogen-activated protein (MAP) kinase/ERK kinase 1 (MEK1) inhibitor PD98059 was also significantly inhibited by 70%. CONCLUSIONS: Oleuropein inhibited SMC proliferation through a cell cycle block between the G1 and the S phases, which may be regulated by ERK1/2. These results suggest a mechanism by which olive oil consumption may have beneficial effects on cardiovascular mortality by inhibiting SMC proliferation.

Review of prevalence and outcome of vascular disease in patients with diabetes mellitus.

OBJECTIVES: Review the literature to determine the prevalence and outcome in patients with diabetes that undergo surgery to correct carotid artery stenosis, lower extremity arterial disease, and abdominal aortic aneurysm (AAA). DESIGN AND MATERIALS: Studies were obtained from searches over the past 15 years on the National Library of Medicine's online search engine. RESULTS: The review demonstrated an equivalent prevalence of carotid artery stenosis requiring surgery in patients with diabetes, it favored no increase risk of post-CEA stroke, and it was split on perioperative morbidity and mortality risk. There was an increase prevalence of lower extremity arterial disease requiring surgery in patients with diabetes, it favored equivalent patency and limb salvage rates, and it was split on the morbidity and mortality risk. The review demonstrated a decrease in AAA prevalence among patients with diabetes, it found an increase in the morbidity risk, and equivalent mortality risk. CONCLUSIONS: Stroke, graft patency, and limb salvage rates in patients with diabetes after surgery are similar to patients without diabetes; however, their risk of complications is increased after surgery and the mortality risk may be higher after CEA.

Vascular smooth muscle cell migration: current research and clinical implications.

Atherosclerosis and intimal hyperplasia are major causes of morbidity and mortality. These processes develop secondary to endothelial injury due to multiple stimuli, including smoking, diabetes mellitus, hypertension, and hyperlipidemia. Once this injury occurs, an essential element in the development of both these processes is vascular smooth muscle cell (VSMC) migration. Understanding the mechanisms involved in VSMC migration and ultimately the development of strategies by which this process can be inhibited, has been a major focus of research. The authors present a review of the extracellular proteins (growth factors, extracellular matrix components, and cell surface receptors) and intracellular signaling pathways involved in VSMC migration, as well as potential therapeutic approaches to inhibit this process.

The multidisciplinary approach to limb salvage.

There is considerable morbidity and mortality associated with ulcerations of the lower limbs in patients with diabetes as well as non-diabetic patients. The role of the primary care physician and podiatrist in the evaluation, diagnosis, and management of lower extremity wounds is critical. Careful assessment and management of vascular disease by vascular surgeons and interventionalists, biomechanical foot abnormalities by the podiatrist, wound coverage by the plastic surgeons and aggressive treatment of infections and metabolic derangements by the physicians are required. The multidisciplinary approach provides a comprehensive treatment protocol and significantly increases the chances of successfully healing the ulcer and prevents recurrence. It also provides for increased academic output through publications, participation in clinical trials and educational venues.

An appraisal of different cardiac risk reduction strategies in vascular surgery patients.

OBJECTIVES: to summarize existing evidence regarding the benefits and the risks of all available interventional and medical means aimed at cardiac risk reduction in patients undergoing vascular surgery. DESIGN: review of the literature. MATERIALS AND METHODS: a critical review of all studies examining the impact of various prophylactic cardiac maneuvers on perioperative outcome following vascular surgery was performed. Overall mortality, cardiac mortality and myocardial infarction rate were used as the outcome measures. RESULTS: coronary artery bypass grafting is associated with a 60% decrease in perioperative mortality in patients undergoing vascular surgery, but in most of the cases this decrease does not outweigh the combined risk of the cardiac and the subsequent noncardiac vascular procedure. Data supporting the cardioprotective effect of percutaneous transluminal angioplasty in the perioperative setting are insufficient. beta-blockade has been shown to decrease perioperative mortality and cardiac morbidity in both high-risk (strong evidence) and low-risk (weak evidence) patients. CONCLUSIONS: coronary revascularization is rarely indicated to simply get the patient through vascular surgery and should be reserved for patients who would need it irrespective of the scheduled vascular procedure. Among all available pharmacological agents, including beta-blockers, alpha-agonists, calcium channel blockers and nitrates, only beta-blockers have been proven to reduce the cardiac risk of vascular surgery.

Immunobiologic analysis of arterial tissue in Buerger's disease.

INTRODUCTION: the cause of thromboangiitis obliterans (TAO) still remains unknown. We have reported that immunologic injury associated with T lymphocytes infiltration might be the initial etiologic mechanism in TAO. The present study was undertaken to examine further the mechanism of immune injury. METHODS: arterial walls affected by TAO were obtained from eight patients with eight non-pulsatile arteries and one patent artery. Immunohistochemical and TUNEL studies were performed for phenotyping of the infiltrating cells with CD4 (helper T cell), CD8 (cytotoxic T cell), CD56 (natural killer cell), and CD68 (macrophage), for identification of cell activation with VCAM-1 and i -NOS, for the presence of cell death with TUNEL analysis, and for inflammatory cytokine detection with RT-PCR. RESULTS: the characteristic features were luminal obliteration, together with a varying degree of recanalization. T cells infiltrated mainly in thrombus, intima, and adventita. Among infiltrating cells, CD4 T cells greatly outnumbered CD8 cells. VCAM-1 and i -NOS were expressed in endothelial cells around the intima (patent segment) or vaso vasorum (occluded segment). Endothelial cells in vaso vasorum stained positive with TUNEL. Interferon-gamma mRNA was detected in two specimens. CONCLUSIONS: our results suggest that T cell mediated immune inflammation is a significant event in the development of TAO.

Cyclic strain activates the pro-survival Akt protein kinase in bovine aortic smooth muscle cells.

BACKGROUND: Pulsatile pressure induced by the beating heart causes cyclic strain on arterial endothelial cells and smooth muscle cells (SMCs). This study examined whether Akt, a serine/threonine protein kinase known to promote cell survival by inhibiting apoptosis, is activated by cyclic strain in bovine aortic SMCs. METHODS: Bovine aortic SMCs were cultured on flexible-bottomed membranes and then serum-starved for 24 to 36 hours. The cells were then exposed to 150-mm Hg repetitive deformations, which created an average of 10% strain on the monolayer SMCs at a frequency of 60 cycles/minute for 0 (negative control) and 30 minutes. Platelet-derived growth factor (PDGF)--stimulated SMCs were used as positive controls. Phosphorylation of Akt was determined by means of Western blot analysis. An apoptosis assay (TUNEL) was also performed on SMCs exposed to cyclic strain. RESULTS: Akt phosphorylation was significantly increased over that of the negative control after 30 minutes of cyclic strain and in the PDGF group. Cyclic strain did not increase the prevalence of apoptosis in SMCs over the control. CONCLUSIONS: Cyclic strain activated the pro-survival Akt kinase. The pro-survival function was supported by the fact that cyclic strain did not increase apoptosis in bovine aortic SMCs. This experiment suggests that cyclic strain may induce arterial wall thickening by tipping the balance toward arterial SMC proliferation through the inhibition of apoptosis.

The integrin-mediated cyclic strain-induced signaling pathway in vascular endothelial cells.

The irregular distribution of plaque in the vasculature results from the interaction of local hemodynamic forces with the vessel wall. One well-characterized force is cyclic circumferential strain, the repetitive pulsatile pressure distention on the arterial wall. This review summarizes current research, which has aimed to elicit the signal transduction pathway by which cyclic strain elicits functional and structural responses in endothelial cells; specifically, it summarizes the signaling pathway that begins with the reorganization of integrins. One method by which these extracellular matrix receptors affect signal transduction is through their ability to initiate the process of phosphorylation on tyrosine residues of cytoplasmic protein kinases, including focal adhesion kinase. The strain-induced pathway appears to also involve ras and the mitogen-activated protein kinase family of enzymes, and preliminary data suggests a role for src as well. Ultimately, it is the regulation of gene expression through the modulation of transcription factors that allows endothelial cells to respond to changes in local hemodynamics.

Cyclic stretch induces the expression of vascular endothelial growth factor in vascular smooth muscle cells.

OBJECTIVE: Accumulating evidence links the release of vascular endothelial growth factor (VEGF) by vascular smooth muscle cells (VSMC) to normal endothelial cell (EC) function, repair and maintenance. Using an in vitro model we investigate the role of cyclic stretch on both the release of VEGF by VSMC and the phosphorylation of a VEGF receptor on EC. METHODS: Bovine VSMC and EC were exposed to 10% cyclic strain for 4 hours. VEGF mRNA steady-state levels of VSMC were analysed by northern blot hybridisation. The presence of secreted VEGF from VSMC was determined by assaying the migration of EC. VEGF receptor phosphorylation on stretched EC was assayed by immunoblotting. RESULTS: The steady-state level of VEGF mRNA in stretched VSMC increased 3.3 (+/- 0.6) fold above that of unstretched VSMC (p < 0.005). Migration of EC was stimulated 8.3 (+/- 1.1) and 14.6 (+/- 1.3) fold by media from unstretched and stretched VSMC respectively, demonstrating a 1.8 fold increase due to stretch alone (p < 0.05). Cyclic stretch resulted in phosphorylation of the VEGF receptor KDR. CONCLUSION: Exposure of VSMC to physiological levels of stretch induces a biologically significant increase in VEGF secretion and may provide an arterial stimulus for maintenance of steady state levels of VEGF essential for EC survival.

Intermittent pneumatic compression devices -- physiological mechanisms of action.

There are many reports of how IPC is used effectively in the clinical setting; including the prevention of deep venous thrombosis, improvement of circulation in patients with lower extremity arterial diseases, reduction of lymphoedema, and the healing of venous ulcers. However, despite the widely accepted use of IPC, it is still unclear how IPC actually exerts its beneficial effects. The exact physiological mechanisms of action are unknown. The clinical utility of IPC and the putative mechanisms by which IPC could exert its therapeutic effect will be reviewed. The paper will examine the mechanical effects of IPC exerted on the lower extremity, and the subsequent biochemical changes in the circulation. In vitro studies of the effects of mechanical stress such as compressive strain and shear on cultured endothelial cells, and their clinical relevance to IPC will also be reviewed.

Basic science curriculum in vascular surgery residency.

Recognizing the importance of basic science teaching in surgical education, the leadership of the Association of Program Directors in Vascular Surgery (APDVS) appointed a panel to gather information and to present its findings at the 1999 annual fall meeting of the Apdvs. A questionnaire was distributed to the program directors present. In addition, information was gathered from the American Board of Surgery regarding the basic science content in the vascular surgery item pool on the vascular surgery qualifying examination (VQE). The vascular surgery unit of the surgical resident curriculum was also analyzed. Fifty-three program directors (64%) completed the questionnaire. Although only two program directors felt that their residents were better prepared to answer basic science questions, the results of the Vqe showed that the examinees do not, as a group, perform differently on basic science items than on clinical management questions. In addition, only a minority of program directors (15%) use a specific method to monitor the learning process of their residents. The majority of the program directors responding (75%) felt that they were capable of teaching basic science to residents. Interestingly, almost half the 53 respondents (47%) said that a basic science curriculum should be comprehensive, not exclusively relevant to the clinical setting. Vqe content outline and the vascular surgery unit of the surgical resident curriculum revealed great emphasis on clinically relevant basic science information. The Apdvs panel recommends that a basic science curriculum should be comprehensive, yet clinically pertinent, and completely integrated with the clinical curriculum. In terms of how to teach basic science in vascular residencies, the panel supports teaching conferences that are problem-based with a faculty member acting as the "resource person" and with specific goals set for the conferences. The panel also suggested establishing a Web site that provides a series of questions, the answers of which could be readily available to trainees and program directors. such immediate feedback could be of great help to program directors to focus the learning process of their residents and monitor its progress.

The C-terminal domain of thrombospondin-1 induces vascular smooth muscle cell chemotaxis.

OBJECTIVE: Thrombospondin-1 (TSP-1), an acute-phase reactant implicated in vascular disease, is a 420-kd multifunctional glycoprotein chemotactic for vascular smooth muscle cells (VSMCs). TSP-1 has six domains of repeating homologous amino acid sequences: N-terminal, procollagen homology, type 1 repeat, type 2 repeat, type 3 repeat/RGD (T3), and C-terminal (COOH). The purpose of this experiment was to determine which domains of TSP-1 induce VSMC chemotaxis. METHODS: A modified Boyden Chamber chemotaxis assay was used to assess VSMC migration. Serum-free medium, TSP-1, or each of the fusion proteins (10 and 20 microg/mL) synthesized for the different domains were placed in the bottom wells. Quiescent bovine aortic VSMCs (50,000) were placed in the top wells. After 4 hours at 37 degrees C, migrated VSMCs were recorded as cells per five fields (400x) and analyzed with the paired t test. To verify the fusion protein data, we performed chemotaxis assays with antibodies to each of the domains (25 microg/mL) combined with TSP-1 (20 microg/mL) in the bottom wells and VSMCs in the top wells. RESULTS: The COOH domain significantly stimulated VSMC chemotaxis (P = <.001). To a lesser extent, the N-terminal and T3 domains also induced chemotaxis (P <.05). However, only the anti-COOH antibody (C6.7) and the anti-integrin-associated protein portion of COOH antibody (D4.6) significantly inhibited TSP-1-induced VSMC chemotaxis (by 85% and 92%, respectively). CONCLUSIONS: These results implicate the COOH domain as the portion of the TSP-1 molecule primarily responsible for VSMC chemotaxis. This experiment suggests that future strategies in the prevention of VSMC migration, an initial step in the development of vascular lesions, may involve selective inhibition of the COOH domain of TSP-1.

Translocation of PKC isoforms in bovine aortic smooth muscle cells exposed to strain.

Our laboratory has previously reported that the exposure of smooth muscle cells (SMC) to the cyclic strain results in significant stimulation of protein kinase C (PKC) activity by translocating the enzyme from the cytosol to the particulate fraction. We now sought to examine the strain-induced translocation of individual PKC isoforms in SMC. Confluent bovine aortic SMC grown on collagen type I-coated plates were exposed to cyclic strain for up to 100 s at average 10% strain with 60 cycles/min. Immunoblotting analysis demonstrates that SMC express PKC-alpha, -beta and -zeta in both cytosolic and particulate fractions. Especially, PKC-alpha and -zeta were predominantly expressed in the cytosolic fraction. However, cyclic strain significantly (P < 0.05) increased PKC-alpha and -zeta in the particulate fraction and decreased in the cytosolic fraction. Thus, the cyclic strain-mediated stimulation of PKC activity in SMC may be due to the translocation of PKC-alpha and -zeta from the cytosolic to the particulate fraction. These results demonstrate that mechanical deformation causes rapid translocation of PKC isoforms, which may initiate a cascade of proliferation responses of SMC since NF-kappaB, which is involved in the cellular proliferation has been known to be activated by these PKC isoforms.

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells.

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 microM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 microM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH(2)-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the alpha2-, alpha3-, alpha6-, or beta1-integrin subunits but not on a substrate of functional antibody to the alpha5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.

Role of p38 MAP kinase in endothelial cell alignment induced by fluid shear stress.

The p38/mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MAPKAP kinase 2)/heat shock protein (HSP)25/27 pathway is thought to play a critical role in actin dynamics. In the present study, we examined whether p38 was involved in the morphological changes seen in endothelial cells (EC) exposed to shear stress. Cultured bovine aortic EC were subjected to 14 dyn/cm(2) laminar steady shear stress. Peak activation of p38, MAPKAP kinase 2, and HSP25 were sixfold at 5 min, sixfold at 5 min, and threefold at 30 min compared with static control, respectively. SB-203580 (1 microM), a specific inhibitor of p38, abolished the activation of MAPKAP kinase 2 and HSP25 as well as EC elongation and alignment in the direction of flow elicited by shear stress. The mean orientation angle of cells subjected to shear without SB-203580, with SB-203580, or static control were 17, 50, and 43 degrees, respectively (P < 0. 05). EC transfected with the dominant negative mutant of p38-alpha aligned randomly with no stress fiber formation despite exposure to shear stress. These data suggests that the pathway of p38/MAPKAP kinase 2/HSP25/27 is activated in response to shear stress, and this pathway plays an important role in morphological changes induced by shear stress.

Role of mitogen-activated protein kinases in pulmonary endothelial cells exposed to cyclic strain.

The aim of this study was to examine the role of mitogen-activated protein kinases (MAPKs) activation in bovine pulmonary arterial endothelial cells (EC) exposed to cyclic strain. EC were subjected to 10% average strain at 60 cycles/min. Cyclic strain induced activation of extracellular signal-regulated kinase (ERK; 1.5-fold), c-Jun NH(2)-terminal protein kinase (JNK; 1.9-fold), and p38 (1. 5-fold) with a peak at 30 min. To investigate the functional role of the activated MAPKs, we analyzed cells after treatment with PD-98059, a specific ERK kinase inhibitor, or SB-203580, a catalytic inhibitor for p38, and after transient transfection with JNK(K-R), and MEKK(K-M) the respective catalytically inactive mutants of JNK1 and MAPK kinase kinase-1. Cyclic strain increased activator protein-1 (AP-1) binding activity, which was blocked by PD-98059 and SB-203580. Activity of AP-1-dependent luciferase reporter driven by 12-O-tetradecanoyl-phorbol-13-acetate-responsive element (TRE) was induced by cyclic strain, and this was attenuated by PD-98059, MEKK(K-M), JNK(K-R), and SB-203580. PD-98059 and SB-203850 did not inhibit cell alignment and migration induced by cyclic strain. MEKK(K-M) and JNK(K-R) transfection did not block cyclic strain-induced cell alignment. In conclusion, cyclic strain activates ERK, JNK, and p38, and their activation plays a role in transcriptional activation of AP-1/TRE but not in cell alignment and migration changes in bovine pulmonary arterial EC.

Effects of glutamine isomers on human (Caco-2) intestinal epithelial proliferation, strain-responsiveness, and differentiation.

Enteral feeding with small amounts to stimulate bowel motility, and glutamine supplementation, which provides nutrients selectively used by intestinal epithelial cells, might preserve the gut mucosa during fasting. We evaluated the effects of the interaction between mechanical strain and glutamine supplementation in human Caco-2 intestinal epithelial cells, and pursued the finding of equivalent effects of L- and D-glutamine in Caco-2, HT-29, and primary malignant human colonocytes. Caco-2 cells were subjected to repetitive strain in media containing 2 mmol/L of L-glutamine and media supplemented with L- or D-glutamine. Proliferation was determined by automated cell counting. Differentiation and cellular production of L-glutamine were determined spectroscopically. Rhythmic deformation stimulated Caco-2 proliferation in a frequency-dependent manner. Maximal stimulation occurred at 10 cpm, consistent with in vivo frequencies of peristalsis and villous motility. Deformation at 10 cpm and L-glutamine supplementation from 2 to 5 mmol/L concentrations independently stimulated Caco-2 proliferation; the combination further increased proliferation. D-Glutamine supplementation yielded similar results, although with lesser potency. Furthermore, both L- and D-glutamine equivalently reduced Caco-2 dipeptidyl dipeptidase activity. The effects of each isoform were blocked by 1 to 3 mmol/L acivicin, a selective antagonist of glutamine metabolism. Indeed Caco-2 and HT-29 cells and primary malignant colonocytes each metabolized D-glutamine to L-glutamine. Glutamine supplementation in fasting patients might prove synergistic with stimulation of bowel motility by non-nutritive feeding, whereas tissue-specific variations in D-glutamine metabolism might facilitate selective nutripharmaceutical targeting of the gut mucosa.