Dynamic recycling of extracellular ATP in human epithelial intestinal cells.

Intestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes and food digestion. The function of these cells is strongly influenced by purinergic signalling activated by extracellular ATP (eATP) and other nucleotides. The activity of several ecto-enzymes determines the dynamic regulation of eATP. In pathological contexts, eATP may act as a danger signal controlling a variety of purinergic responses aimed at defending the organism from pathogens present in the intestinal lumen. In this study, we characterized the dynamics of eATP on polarized and non-polarized Caco-2 cells. eATP was quantified by luminometry using the luciferin-luciferase reaction. Results show that non-polarized Caco-2 cells triggered a strong but transient release of intracellular ATP after hypotonic stimuli, leading to low micromolar eATP accumulation. Subsequent eATP hydrolysis mainly determined eATP decay, though this effect could be counterbalanced by eATP synthesis by ecto-kinases kinetically characterized in this study. In polarized Caco-2 cells, eATP showed a faster turnover at the apical vs the basolateral side. To quantify the extent to which different processes contribute to eATP regulation, we created a data-driven mathematical model of the metabolism of extracellular nucleotides. Model simulations showed that eATP recycling by ecto-AK is more efficient a low micromolar eADP concentrations and is favored by the low eADPase activity of Caco-2 cells. Simulations also indicated that a transient eATP increase could be observed upon the addition of non-adenine nucleotides due the high ecto-NDPK activity in these cells. Model parameters showed that ecto-kinases are asymmetrically distributed upon polarization, with the apical side having activity levels generally greater in comparison with the basolateral side or the non-polarized cells. Finally, experiments using human intestinal epithelial cells confirmed the presence of functional ecto-kinases promoting eATP synthesis. The adaptive value of eATP regulation and purinergic signalling in the intestine is discussed.

Experimental study to assess the impact of vasopressors administered during maintenance of the brain-dead donation in the quality of the intestinal graft.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3+ counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3+ mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE (p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver (p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3 + counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3 + mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE ( p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver ( p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.