Smooth muscle cell (SMC) contraction is regulated mainly by receptor and mechanical activation of the contractile proteins actin and myosin. Contraction can also be activated by a change in membrane potential or activation of stretch-dependent ion channels in the plasma membrane. Phosphorylation of the 20-kDa light chain of myosin by myosin light chain kinase (MLC kinase) enables the interaction between myosin and actin. Cross-bridging of myosin with actin uses the energy released from ATP by the activity of myosin ATPase.1
Removal of the high-energy phosphate from the myosin light chain by MLC phosphatase results in relaxation of smooth muscle. MLC phosphatase is regulated by the G protein RhoA and its target Rho kinase. When the myosin-binding subunit of MLC phosphatase is phosphorylated by Rho kinase, its activity is inhibited and the myosin light chain remains in the phosphorylated state, thus promoting contraction. Inhibition of Rho kinase therefore has the opposite effect, whereby relaxation of smooth muscle is promoted.1
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Initiation of smooth muscle contraction is by a Ca2+ mediated change in myosin. Intracellular concentration of Ca2+ increases in response to a specific stimulus, and this Ca2+ forms a complex with the acidic protein calmodulin. This complex activates MLC kinase, which then, as described above, phosphorylates the light chain of myosin. The release of Ca2+ from intracellular stores and influx of Ca2+ from extracellular space increases the cytosolic Ca2+. Binding of agonists to receptors coupled to a G protein stimulates the enzyme phospholipase C (PLC), which is specific for phosphatidylinositol 4,5-bisphosphate(PIP2). PLC catalyzes the formation of two second messengers from PIP2; inositol triphosphate (IP3) and diacylglycerol (DG). Ca2+ is released into the cytosol when IP3 binds to receptors on the sarcoplasmic reticulum. DG activates protein kinase C (PKC), which in turn phosphorylates target proteins.1
The activation of the SMC Rho kinase signalling pathways is reasonably constant, however the mechanisms for generating Ca2+ signals vary. The smooth muscle cells that make up the muscular part of the gastrointestinal tract (GI) are activated by pacemaker cells, the interstitial cells of Cajal (ICCs). These cells possess a cytosolic oscillator that produces repetitive Ca2+ transients, which in turn activate inward currents that distribute through the gap junctions connecting the ICCs. This supplies the depolarizing signal that promotes contraction.2
The GI smooth muscles displays autonomous behaviour, however the desired contractile response from higher regulatory systems e.g. enteric nervous systems (ENS) or hormones cannot be elicited if excitation-contraction (E-C) coupling mechanisms regulated by intrinsic regulatory pathways, which include the ICCs, are inactivated.3
The ICCs are the main regulators of the GI SMC activation. They produce pulses of Ca2+ responsible for slow waves, which are periodic depolarisations that activate the influxes that distribute through the gap junctions. The ICCs do not generate action potentials, and propagation of excitation through the network is via a potential-dependent mechanism to contemporize the individual oscillators. The oscillator of ICC differs from that of various SMCs in that it produces the depolarizing signal that triggers the muscle cells, whereas the endogenous oscillator of the airway and vascular SMCs supplies the internal Ca2+ signal that promotes contraction.2
The contractility of the GI tract can be affected by various substances, and this present study attempts to investigate the effects of oxygen free radicals on the contractile properties of the guinea pig ileum.
A free radical is a species with one or more unpaired electron, and this includes the hydrogen atom, transition metals, and the oxygen molecule. When oxygen accepts an electron, it will form the superoxide radical anion O2-. This superoxide radical anion is known to be produced by phagocytic cells monocytes, macrophages, neutrophils, and eosinophils. If a second electron is added to O2-, the peroxide ion O22- is formed. Formation of any O22- at physiological pH will result in production of hydrogen peroxide (H2O2) via protonation, since the pKa of H2O2 is very high.4
Previous studies have proven the importance of oxygen radicals (oxygen ions and peroxides) in normal functioning of tissues, as well as in the pathogenesis of various clinical disorders. Reactive oxygen radicals are implicated in pathophysiology of gastrointestinal, respiratory, and cardiovascular diseases and numerous studies have investigated their role in different types of smooth muscle injuries.
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There are numerous sources from which oxygen radicals such as superoxide anions and hydrogen peroxide may be formed, and in biological systems these may arise from enzymatic and nonenzymatic sources. The major sources of superoxide radicals include xanthine oxidase activity, membrane-bound neutrophilic NADPH oxidase, ââ‚¬Å“leakageââ‚¬Â from mitochondrial electron transport chain, and autoxidation of several compounds and xenobiotics such as catecholamines, ascorbate, glutathione, and reduced metals and metal-complexes. Other sources include activities of monoamine oxidase and nitric oxide, activated macrophages, and lipid peroxidation, Fenton-type reactions5, inflammation, and ischemia and reperfusion. There are also externally generated sources of oxygen radicals which include cigarette smoke, environmental pollutants, radiation, ultraviolet light, certain drugs, pesticides, anaesthetics and industrial solvents, and ozone.6
Besides playing a role as regulatory mediators in signalling processes, oxygen radicals are also involved in important physiological functions including the regulation of vascular tone, monitoring and sensing of oxygen concentration, regulation of oxygen concentration-controlled functions, transduction of signals from various membrane receptors, and maintenance of redox homeostasis.7 Numerous experimental data indicate that H2O2 and superoxide anions also play an important role in oxygen-dependent microbicidal activity by granulocytes.8, 9
Not only superoxide and H2O2 are precursors of more damaging substances, these polymorphonuclear-derived oxidants are mediators of enhanced mucosal permeability, electrolyte transport, and epithelial cell injury implicated in acute inflammation of the bowel.10 It has been shown that in biopsies of the colonic mucosal of inflammatory bowel disease patients, there is increased reactive oxygen intermediates, and attenuated antioxidant defenses.11
In the gut, the two main sources of reactive oxygen metabolites associated with gastrointestinal diseases are phagocytes and xanthine oxidase.12 Phagocytosis by mammalian granulocytes causes release of H2O2.9 During phagocytosis, there is a marked burst in oxygen consumption, and this results in the formation of superoxide anions and H2O2. Root et al (1975) investigated the release of H2O2 from human granulocytes during phagocytosis, and found that under basal conditions, granulocytes released less than 0.01 nmol/ml of H2O2 (2.5 x 106 polymorphonuclear leukocytes/ml), and this was measured by the scopoletin assay. They used latex, opsonised yeast, and staphylococci as phagocyte particles to investigate the effects on the release of H2O2, and it was observed that upon the addition of these phagocyte particles, there was an abrupt increase in extracellular peroxide concentration, which was more than 50-fold above basal levels. Their findings paralleled the respiratory burst, and there was no release of peroxide when phagocytosis was prevented, or when cells from patients with chronic granulomatous disease were used, which are incapable of formation of H2O2. This suggests that this phenomenon depends on the increased amounts of H2O2 being synthesised during phagocytosis. It was also observed that the rates at which H2O2 was produced and released were close to those of phagocytosis. Xanthine oxidase, which is implicated in the pathogenesis of ischemia reperfusion, produces superoxide, and is generated during ischaemia from xanthine dehydrogenase.12
At moderate concentrations, oxygen radicals actually serve a protective role against oxidative stress; however at high concentrations they may damage major constituents of cells. Excessive stimulation of NADPH oxidases, or poorly regulated sources such as the mitochondrial electron transport chain may result in excessive amounts of reactive oxygen species, which in turn are implicated in various diseases and tissue injuries.7 The toxicity of H2O2 and O2- is due to the ability of these radicals to produce other reactive oxygen species e.g. the hydroxyl radical and singlet oxygen, which in turn initiate a radical chain reaction, and subsequently results in formation of lipid and organic peroxide.13
Numerous studies have investigated the effects of oxygen radicals on the contractile response of the intestine. Experimental data suggest that the inflamed intestine associated with free radicals have altered contractility.14 Recently Peluso et al (2002) studied the effect of simulated oxidative stress on the motility of isolated preparations of rabbit jejunum and guinea pig ileum. The intestinal segments were pre-treated with 2,2ââ‚¬â„¢-Azobis (2-amidinopropane) dihydrochloride (ABAP), which generates peroxyl radicals by thermal decomposition. Subsequent contractile response to acetylcholine decreased in a dose-dependent manner, but the effects were reversible after washout, suggesting an atropine-like activity. Peluso also investigated the effects of ABAP on BaCl2 ââ‚¬"induced contractions of guinea pig ileum preparations, and ABAP was found to inhibit the contractions in a non-dose-dependent manner, suggesting that the inhibition of contractions were via the inhibition of influx of Ca2+ into cells through the L-type Ca2+ channels.
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A study by Van der Vliet et al (1989)15 shows similar effect of oxygen radicals on intestinal motility, in that a reduction in contraction was also observed. In one of the functional experiments, they studied the effects of H2O2 on contractile response induced by methacholine. Methacholine induced a contraction of longitudinal muscle from rat intestine. Addition of H2O2 resulted in a short-lived contraction of about one minute, which was followed by a slowly developing relaxation. Similar results were also observed for cumene hydroperoxide. The peroxides also diminished the spontaneous motility previously displayed by segments of the colon and small intestine. Pretreatment with H2O2 and cumene hydroperoxide decreased methacholine response in all parts of the gastrointestinal tract. In another functional experiment, the effects of H2O2 and cumene hydroperoxide on the contractions induced by depolarization with K+ were investigated. A decrease in contractions after depolarization with K+ was observed when the intestinal segments were pretreated with the peroxides. The authors conclude that the effects could be due to damage to membrane function or contractile apparatus due to the pretreatments.
Similar observations were made by Moummi et al (1991) with neurally stimulated guinea pig ileum, using concentrations of reactive oxygen metabolites (ROM) that were known to be produced by activated neutrophils.16 Upon addition of NH2Cl, a transient increase in electrically evoked twitch contraction followed by a marked inhibition of response was observed. HOCl and H2O2 produced the same pattern of response. However, a different observation was made with nonstimulated ileum. All three oxidants induced concentration dependent increases in the resting tension of ileum, with NH2Cl producing the most contractions and HOCl the least. There was however a depression of resting tones of the ileum after application of a higher concentration of the three oxidants. The oxidant-induced biphasic response of neurally stimulated ileum was tested with the cyclooxygenase inhibitor piroxicam, the muscarinic receptor antagonist atropine, the ganglionic blocker hexamethonium, and the histamine-1 receptor pyrilamine. No one of these inhibitors was able to modify the biphasic response evoked by the ROM. The study suggested that cyclooxygenase metabolites as well as the neurotransmitters histamine and acetylcholine are not involved in mediating the responses to oxidant-induced responses in electrically stimulated guinea pig ileum.
Different smooth muscle tissues respond differently to reactive oxygen species. While the ileum has been observed to produce a transient increase in contraction followed by relaxation, the trachea exhibits different response to different reactive oxygen species. Tissues with intact epithelium have been observed to being unresponsive to superoxide radical but produce a prolonged contraction to singlet oxygen. Removal of the tracheal epithelium results in a significant increase of the contraction induced by reactive oxygen species and in a reduction of reactive oxygen species-induced relaxation. Vascular smooth muscles may be affected by ROS directly, or indirectly via damage to the endothelium. Injury to the epithelium results in a prolonged contraction of smooth muscle induced by reactive oxygen species.17
The aim of this study is to investigate the effects of oxygen radicals on the guinea pig ileum contractile properties, and to elucidate possible mechanisms of action of oxygen radicals in inducing contraction/relaxation in ileal smooth muscle.