Muscarinic cholinergic stimulation of phosphatidylinositol turnover in the longitudinal smooth muscle of guinea-pig ileum

1. The metabolism of phosphatidylinositol and phosphatidate was investigated in fragments of longitudinal smooth muscle from guinea-pig ileum incubated with cholinergic and anticholinergic drugs. 2. Incorporation of Pi into these lipids was enhanced by acetylcholine and carbamoylcholine. 3. The receptor responsible for triggering this response was of the muscarinic type, since (a) the response was also produced by the muscarinic agonists acetyl-beta-methylcholine, carbamoyl-beta-methylcholine and pilocarpine, and (b) the response was prevented by atropine and prophylbenzilylcholine mustard, but not by tubocurarine. 4. Increased phosphatidylinositol labellin was clearly observed within 5 min in tissue treated with a high concentration of carbamoylcholine. 5. Halfmaximal stimulation of phosphatidylinositol labelling occurred at approx. 10 muM-muM-carbamoylcholine. 6. Incubation of muscle fragments with carbamoylcholine provoked a decrease in phosphatidylinositol concentration, as would be expected if phosphatidyl-inositol breakdown is the reaction controlled by agonists. 7. This information all appears consistent with the proposal that phosphatidylinositol breakdown may be a reaction intrinsic to the mechanisms of muscarinic cholinergic receptor systems

Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts.

AIMS: Cardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav 1.5 currents in homozygous RyR2-P2328S (RyR2(S/S) ) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav 1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2(S/S) mice and their wild-type (WT) littermates. METHODS: We explored effects of 1 μm flecainide on WT and RyR2(S/S) atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na(+) currents (INa ) were recorded using loose patch clamping of superfused atria. RESULTS: RyR2(S/S) showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2(S/S) but pro-arrhythmic in WT. It increased INa in RyR2(S/S) atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2(S/S) , but reduced CV while sparing AERP in WT. Thus, RyR2(S/S) hearts have low λ relative to WT; flecainide then increases λ in RyR2(S/S) but decreases λ in WT. CONCLUSIONS: Flecainide (1 μm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca(2+) release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, UK) under a David Phillips Fellowship held by JAF (BB/FO23863/1) and by the Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grants Scheme.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/apha.1250

Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G

Background: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. Methodology/Principal Findings: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. Conclusions/Significance: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality

Contemporary review of risk-stratified management in acute uncomplicated and complicated diverticulitis

BACKGROUND: Acute colonic diverticulitis is a common clinical condition. Severity of the disease is based on clinical, laboratory, and radiological investigations and dictates the need for medical or surgical intervention. Recent clinical trials have improved the understanding of the natural history of the disease resulting in new approaches to and better evidence for the management of acute diverticulitis. METHODS: We searched the Cochrane Library (years 2004-2015), MEDLINE (years 2004-2015), and EMBASE (years 2004-2015) databases. We used the search terms "diverticulitis, colonic" or "acute diverticulitis" or "divertic*" in combination with the terms "management," "antibiotics," "non-operative," or "surgery." Registers for clinical trials (such as the WHO registry and the https://clinicaltrials.gov/) were searched for ongoing, recruiting, or closed trials not yet published. RESULTS: Antibiotic treatment can be avoided in simple, non-complicated diverticulitis and outpatient management is safe. The management of complicated disease, ranging from a localized abscess to perforation with diffuse peritonitis, has changed towards either percutaneous or minimally invasive approaches in selected cases. The role of laparoscopic lavage without resection in perforated non-fecal diverticulitis is still debated; however, recent evidence from two randomised controlled trials has found a higher re-intervention in this group of patients. CONCLUSIONS: A shift in management has occurred towards conservative management in acute uncomplicated disease. Those with uncomplicated acute diverticulitis may be treated without antibiotics. For complicated diverticulitis with purulent peritonitis, the use of peritoneal lavage appears to be non-superior to resection

Investigation of the relationship between cell-surface calcium-ion gating and phosphatidylinositol turnover by comparison of the effects of elevated extracellular potassium ion concentration on ileium smooth muscle and pancreas.

Incubation of fragments of guinea-pig ileum smooth muscle in the presence of an elevated extracellular K+ concentration, which causes an increase in cell-surface Ca2+ permeability and thus leads to contraction, caused a marked increase in phosphatidylinositol turnover, as assessed by incorporation of 32Pi. This response was not diminished by atropine or propylbenzilycholine mustard, two muscarinic cholinergic antagonists, and was therefore not caused by the release of endogenous acetylcholine within the tissue. In contrast, exposure of guinea-pig pancreas fragments to high extracellular [K+], which does not increase cell-surface Ca2+ permeability or evoke secretion, did not cause an increase in phosphatidylinositol turnover, even though such an increase was triggered by carbamoylcholine, which is a secretagogue. These observations are consistent with a suggested function for phosphatidylinositol breakdown in the mechanisms of cell-surface Ca2+ gates

Muscarinic cholinergic stimulation of phosphatidylinositol turnover in the longitudinal smooth muscle of guinea pig ileum

1. The metabolism of phosphatidylinositol and phosphatidate was investigated in fragments of longitudinal smooth muscle from guinea-pig ileum incubated with cholinergic and anticholinergic drugs. 2. Incorporation of Pi into these lipids was enhanced by acetylcholine and carbamoylcholine. 3. The receptor responsible for triggering this response was of the muscarinic type, since (a) the response was also produced by the muscarinic agonists acetyl-beta-methylcholine, carbamoyl-beta-methylcholine and pilocarpine, and (b) the response was prevented by atropine and prophylbenzilylcholine mustard, but not by tubocurarine. 4. Increased phosphatidylinositol labellin was clearly observed within 5 min in tissue treated with a high concentration of carbamoylcholine. 5. Halfmaximal stimulation of phosphatidylinositol labelling occurred at approx. 10 muM-muM-carbamoylcholine. 6. Incubation of muscle fragments with carbamoylcholine provoked a decrease in phosphatidylinositol concentration, as would be expected if phosphatidyl-inositol breakdown is the reaction controlled by agonists. 7. This information all appears consistent with the proposal that phosphatidylinositol breakdown may be a reaction intrinsic to the mechanisms of muscarinic cholinergic receptor systems

Effects of calcium-antagonistic drugs on the stimulation by carbamoylcholine and histamine of phosphatidylinositol turnover in longitudinal smooth muscle of guinea-pig ileum.

A number of drugs classed as calcium antagonists, spasmolytics, non-specific receptor antagonists or receptor antagonists with multiple sites of action were tested to determine whether they prevent the stimulation of phosphatidylinositol turnover caused in various tissues by the activation of receptors which increase cell-surface Ca2+ permeability. The experiments were done with fragments of longitudinal smooth muscle from guinea-pig ileum; these were incubated in vitro with 32Pi and either 100 muM-carbamoylcholine or 100 muM-histamine, in the presence of antagonistic drugs at concentrations at least sufficient to cause complete blockade of smooth-muscle contraction. The phosphatidylinositol response to carbamoylcholine was not changed by cinchocaine, papaverine, nifedipine, dibenamine, amethocaine, cinnarizine, lidoflazine, methoxyverapamil, prenylamine or two antimuscarinic alkane-bis-ammonium compounds, and the response to histamine was unaffected by the first four drugs. In contrast, phenoxybenzamine prevented the increase in phosphatidylinositol labelling caused by either carbamoylcholine or histamine. The insensitivity of the phosphatidylinositol response to most of the drugs provides further experimental support for the conclusion that the receptor-stimulated phosphatidylinositol breakdown which initiates the increase in phosphatidylinositol turnover is not caused by an increase in intracellular Ca2+. The simplest interpretation of the available information appears to be that phosphatidylinositol breakdown plays a role in the coupling between the receptor-agonist interaction and the opening of cell-surface Ca2+ gates [Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147]. If this is correct, then phenoxybenzamine must exert its inhibitory effects on phosphatidylinositol breakdown early in this sequence of events, but the drugs must act at a stage later than phosphatidylinositol breakdown. The unexpected difference in the effects of dibenamine and phenoxybenzamine, which are chemically very similar, may provide a useful experimental tool with which to explore the way in which activated receptors provoke the opening of cell-surface Ca2+ gates