The capacity of the intestine to secrete fluid is dependent on the basolateral Na + -K + -2Cl − co-transporter (NKCC1). Given that cAMP and Ca2+ signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na + -K + -ATPase, M3 muscarinic acetylcholine receptor (M 3 AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive NH 4 + uptake), intracellular Ca 2+ and cell volume were assessed by 2 ,7 -bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), Fura-2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca2+ signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca 2+ -dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re-expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin-AG1478, and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re-expression were accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)-stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalization and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca 2+ -dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.

Intracellular pH (pHi) in the perfused rat mandibular gland was determined from the distribution of DMO (5,5-dimethyl-2,4-oxazolidinedione). In unstimulated glands, pHi averaged 7.12 +/- 0.02. Stimulation with a "standard" (submaximal) concentration (0.3 mumol/l) of acetylcholine (ACh) caused a fall in pHi to 6.81 +/- 0.06 over 60 min, but a maximal concentration (1.0 mumol/l) caused an initial rise in pHi to 7.60 +/- 0.02, followed by a fall to 7.45 +/- 0.02 over 60 min. After replacement of perfusate Cl with gluconate, the standard ACh concentration caused a rise in pHi to 7.50 +/- 0.02 followed by a fall to 7.27 +/- 0.04 after 60 min, concomitant with a 76% fall in secretory rate and a rise in salivary HCO3 concentration from 14 +/- 0.9 to 67 +/- 1.5 mmol/l. Furosemide (1 mmol/l) had a similar effect to gluconate replacement except that secretory rate fell only by 60%. Bumetanide (1 mmol/l), which inhibited secretion by 67%, did not cause pHi to rise following ACh stimulation but prevented the fall seen with ACh alone. Acetazolamide and methazolamide (1 mmol/l) had no effect on the salivary secretory response to ACh but they caused pHi to rise, respectively, to 7.20 +/- 0.03 and 7.43 +/- 0.02. Bumetanide and methazolamide together caused pHi to rise to 7.58 +/- 0.02 and reduced the secretory response to ACh by 91%. The disulfonic stilbene, SITS, caused pHi to rise to 7.26 +/- 0.03. Ouabain and amiloride both caused resting pHi to fall closer to equilibrium and largely abolished the gland's responsiveness to ACh.(ABSTRACT TRUNCATED AT 250 WORDS)

Critical Care 2017, 21(Suppl 1):P349 Introduction Imbalance in cellular energetics has been suggested to be an important mechanism for organ failure in sepsis and septic shock. We hypothesized that such energy imbalance would either be caused by metabolic changes leading to decreased energy production or by increased energy consumption. Thus, we set out to investigate if mitochondrial dysfunction or decreased energy consumption alters cellular metabolism in muscle tissue in experimental sepsis. Methods We submitted anesthetized piglets to sepsis (n = 12) or placebo (n = 4) and monitored them for 3 hours. Plasma lactate and markers of organ failure were measured hourly, as was muscle metabolism by microdialysis. Energy consumption was intervened locally by infusing ouabain through one microdialysis catheter to block major energy expenditure of the cells, by inhibiting the major energy consuming enzyme, N+/K + -ATPase. Similarly, energy production was blocked infusing sodium cyanide (NaCN), in a different region, to block the cytochrome oxidase in muscle tissue mitochondria. Results All animals submitted to sepsis fulfilled sepsis criteria as defined in Sepsis-3, whereas no animals in the placebo group did. Muscle glucose decreased during sepsis independently of N+/K + -ATPase or cytochrome oxidase blockade. Muscle lactate did not increase during sepsis in naïve metabolism. However, during cytochrome oxidase blockade, there was an increase in muscle lactate that was further accentuated during sepsis. Muscle pyruvate did not decrease during sepsis in naïve metabolism. During cytochrome oxidase blockade, there was a decrease in muscle pyruvate, independently of sepsis. Lactate to pyruvate ratio increased during sepsis and was further accentuated during cytochrome oxidase blockade. Muscle glycerol increased during sepsis and decreased slightly without sepsis regardless of N+/K + -ATPase or cytochrome oxidase blocking. There were no significant changes in muscle glutamate or urea during sepsis in absence/presence of N+/K + -ATPase or cytochrome oxidase blockade. ConclusionsThese results indicate increased metabolism of energy substrates in muscle tissue in experimental sepsis. Our results do not indicate presence of energy depletion or mitochondrial dysfunction in muscle and should similar physiologic situation be present in other tissues, other mechanisms of organ failure must be considered. , and long-term follow up has shown increased fracture risk [2]. It is unclear if these changes are a consequence of acute critical illness, or reduced activity afterwards. Bone health assessment during critical illness is challenging, and direct bone strength measurement is not possible. We used a rodent sepsis model to test the hypothesis that critical illness causes early reduction in bone strength and changes in bone architecture. Methods 20 Sprague-Dawley rats (350 ± 15.8g) were anesthetised and randomised to receive cecal ligation and puncture (CLP) (50% cecum length, 18G needle single pass through anterior and posterior wa...

Gallstone ileus is an uncommon cause of intestinal obstruction and occurs following the formation of a cholecystoenteric fistula, permitting passage of gallstones into the gastrointestinal tract. Impaction of a gallstone in the sigmoid colon is rare and is usually at sites of previous colonic disease. Definitive management can be challenging due to the advanced age and co-morbidity usually seen in this group of patients. We describe a patient successfully managed with on-table endoscopy and, under local anaesthetic, the formation of a left iliac fossa trephine loop colostomy, permitting an enterolithotomy to deliver the stone whilst accommodating for severe pre-existing distal sigmoid diverticular disease. A review of the literature identified various endoscopic and surgical treatments that, depending on local expertise and patient characteristics, can be considered on a case-by-case basis. We advocate the management described in this case for patients presenting with large bowel obstruction due to gallstone ileus, with a background of diverticular disease and who are not fit for general anaesthetic or formal bowel resection, as an alternative to medical palliation alone.