RESUMEN
Post-exercise leucocytosis has been found in children and adults in response to exercise. When normalized to the work performed, children demonstrate a lower lactate response to exercise than in adults. This study examines the association between leukocyte and lactate response in children following exercise. 148 healthy children participated in this study. Each subject performed a ramp exercise on a cycle ergometer followed by ten 2-minute bouts of constant work rate separated by 1-minute rest intervals. Lactate, leukocyte and leukocyte subtype levels were taken pre- and post-exercise. Older children showed a significantly higher pre-to post-exercise leukocyte compared to younger children (3599 ± 165 cells/mL, 2544 ± 163, p<0.0001). Compared to older children, younger children demonstrated a smaller median fold change in lactate (2.7 (1.4-8.1), 4.1 (1.7-29.6)) and leukocyte levels (1.4 (1.0-2.1), 1.6 (1.2-2.8)) after exercise, with a larger leukocyte to lactate fold change ratio. CONCLUSION: Younger children have a greater leukocyte to lactate fold change ratio compared to older children. This finding may be due to the lower anaerobic dependence that is found in younger children.
RESUMEN
The epithelial brush border membrane (BBM) Na(+)-H(+) exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na(+) absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na(+) absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent-soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent-resistant membranes. The size of BBM NHE3 complexes increased in carbachol-exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent-soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent-resistant fraction of ileal BBM. NHERF2, alpha-actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na(+) absorption was completely abolished by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with alpha-actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.
Asunto(s)
Actinina/metabolismo , Carbacol/farmacología , Íleon/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Familia-src Quinasas/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Western Blotting , Proteína Tirosina Quinasa CSK , Endosomas/química , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Íleon/efectos de los fármacos , Íleon/metabolismo , Inmunoprecipitación , Masculino , Microscopía Fluorescente , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Fosforilación , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Conejos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Agua/análisis , Agua/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND AND AIMS: In intestinal Na absorptive cells, phosphatidylinositol 3-kinase (PI 3-K) is involved in rapid epidermal growth factor (EGF) stimulation of Na absorption by the brush border membrane (BBM) Na(+)/H(+) exchanger NHE3. However, how NHE3 is regulated by the PI 3-K pathway and the role of Akt2 are poorly defined. METHODS: The localization of Akt, PI 3-K, and NHE3 was determined by either immunocytochemistry and/or membrane fractionation using OptiPrep density gradient centrifugation. RESULTS: In ileum, active total Akt was present most in the villi and basal layer of the crypts, and Akt2 was mostly in villi. In villus cells, PI 3-K and Akt2 were mostly at the apical surface at which they were present partially in lipid rafts (LR). EGF increased PI 3-K and active Akt2 in ileal BBM at the same time that it increased PI 3-K-dependent trafficking of NHE3 to BBM and stimulation of Na absorption. However, Akt2 was only active in the detergent soluble (DS) pool and not LR of ileal BBM, which correlated with the presence of PTEN in LR. In Caco-2 cells, while EGF stimulated BB NHE3, Akt2 was active in both LR and DS pools. This correlated with the lack of PTEN in the LR of Caco-2 membranes. Akt2 also correlated with epithelial cell differentiation. Akt2 amount and activity were greater in differentiated than undifferentiated Caco-2 cells. CONCLUSIONS: These results suggest that LR may play an important role in determining the function of PI 3-K/Akt2 signaling, including stimulation of intestinal Na absorption. These results also suggest that LR-associated Akt2 may be involved in enterocyte differentiation.