ABSTRACT
Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3- ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.
ABSTRACT
AIM: To study non-cardiac chest pain (NCCP) in relation to ineffective esophageal motility (IEM) and rapid food intake. METHODS: NCCP patients with a self-reported habit of fast eating underwent esophageal manometry for the diagnosis of IEM. Telephone interviews identified eating habits of additional IEM patients. Comparison of manometric features was done among IEM patients with and without the habit of rapid food intake and healthy controls. A case study investigated the effect of 6-mo gum chewing on restoration of esophageal motility in an IEM patient. The Valsalva maneuver was performed in IEM patients and healthy controls to assess the compliance of the esophagus in response to abdominal pressure increase. RESULTS: Although most patients diagnosed with NCCP do not exhibit IEM, remarkably, all 12 NCCP patients who were self-reporting fast eaters with a main complaint of chest pain (75.0%) had contraction amplitudes in the mid and distal esophagus that were significantly lower compared with healthy controls [(23.45 mmHg (95%CI: 14.06-32.85) vs 58.80 mmHg (95%CI: 42.56-75.04), P < 0.01 and 28.29 mmHg (95%CI: 21.77-34.81) vs 50.75 mmHg (95%CI: 38.44-63.05), P < 0.01, respectively)]. In 7 normal-eating IEM patients with a main complaint of sensation of obstruction (42.9%), the mid amplitude was smaller than in the controls [30.09 mmHg (95%CI: 19.48-40.70) vs 58.80 mmHg (95%CI: 42.56-75.04), P < 0.05]. There was no statistically significant difference in manometric features between the fast-eating and normal-eating groups. One NCCP patient who self-reported fast eating and was subsequently diagnosed with IEM did not improve with proton-pump inhibition but restored swallow-induced contractions upon 6-mo gum-chewing. The Valsalva maneuver caused a markedly reduced pressure rise in the mid and proximal esophagus in the IEM patients. CONCLUSION: Habitual rapid food intake may lead to IEM. A prospective study is needed to validate this hypothesis. Gum-chewing might strengthen weakened esophageal muscles.
