Reactive oxygen species (H₂O₂): effects on the gallbladder muscle of guinea pigs

Abstract

The level of lipid peroxidation as well as the PGE2 content was significantly increased after H2O2 pretreatment. Unlike superoxide dismutase, the free radical scavenger catalase prevented the H2O2 induced contraction, and its inhibition of the CCK-8 induced contraction. It is concluded that ROS cause damage to the plasma membrane of the gallbladder muscle and contraction through the generation of PGE2 induced by cPLA2-cyclooxygenase and probably mediated by the PKC-MAPK pathway. lipid peroxidation; prostaglandin E2 content; smooth muscle REACTIVE OXYGEN SPECIES (ROS) such as superoxide radical anion, hypochlorous acid, and hydrogen peroxide (H 2 O 2 ) are by-products of oxidative metabolism, in which energy activation and electron reduction are involved. Inflammatory processes enhance their production, which could affect cell functions (20). The mechanisms whereby ROS act at the cellular level are not fully understood. Their pathogenic role on cell membranes and signal-transduction pathways may explain the early events that are associated with cell dysfunction caused by inflammation. Therefore, the understanding of these mechanisms is important for developing therapeutic strategies at cellular sites of dysfunction.Acute cholecystitis is characterized by an inflammatory process that involves the gallbladder wall with polymorphonuclear cells (23). ROS play a major role in mediating cell injury during inflammatory processes. Disorders of gallbladder motility caused or aggravated by inflammatory processes may be due to the actions of ROS. They may also contribute to the inhibition of agonist-induced contraction (25, 37). The actions of ROS are mediated in part by cyclooxygenase by-products that are important participants in the inflammatory process (16). The contraction of tracheal and ileal muscle strips induced by ROS in vitro appear to be mediated by prostaglandins (24,29).Prostaglandins have varied effects on gastrointestinal motility (5,12,17,32). Muscle strips from various segments of guinea pig stomach exhibited different responses to PGE 1 , PGE 2 , and PGF 2␣ (22). In rat stomach, PGE 2 caused a contraction in the longitudinal preparation but had no effect on the circular muscle (35). PGE 1 , PGE 2 , and PGF 2␣ potentiated the contractile responses of guinea pig gallbladder to transmural stimulation and ACh (28). Prostaglandins may also be involved in muscle cytoprotection since PGE 2 and other prostaglandins appear to participate in the pathway responsible for increased formation of scavengers of free radicals (2). These studies therefore were aimed at examining the effects of ROS on gallbladder muscle function utilizing H 2 O 2 on enzymatic digested single muscle cells from normal guinea pig gallbladders. MATERIALS AND METHODSAnimals. Adult male guinea pigs were purchased from Elm Hill Breeding Laboratory (Chelmsford, MA). The Animal Welfare Committee of Rhode Island Hospital approved their use. Animals were housed in thermoregulated rooms and had free access to food and water. After an overnight fast, th...

PAF-like lipids- and PAF-induced gallbladder muscle contraction is mediated by different pathways in guinea pigs

“…Gallbladder muscle cells were prepared as previously described (24,25,27,28). After the gallbladder was removed from the animal, it was immediately placed in ice-cold preoxygenated Krebs buffer solution, washed, and transferred to a dissecting pan containing the same buffer continuously aerated with 95% O 2-5% CO2.…”

“…Muscle cells were permeabilized with saponin (2,24,25,27,28). After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed and incubated in a collagenase-free medium resembling cytosol (cytasolic buffer), which had the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 5.0 MgSO4, 0.96 NaH2PO4, 0.48 CaCl2, and 1.0 EGTA.…”

“…After muscle cells were equilibrated for 20 min, 0.25-ml aliquots of cell suspension were added to test tubes containing 0.1 ml of buffer with appropriate concentrations of an agonist (24,25,27,28). Receptor protection studies were performed in muscle cells first incubated with PAF or H 2O2 for 2 min, followed by a 15-min incubation with 5 M of N-ethylmaleimide (NEM) (5).…”

“…The initial injury in the gallbladder muscle caused by ROS appears to be confined to the plasma membrane (27). These free radicals induce nonenzymatic oxidation of phospholipids (lipid peroxidation) and damage membrane protein constituents by oxidizing transmembrane receptors and calcium channels (3,9).…”

“…H 2 O 2 causes muscle cell contraction and increases PGE 2 formation (27). The pathways responsible for these cellular responses are not known.…”