J. Physiol. Biochem., 62 (4), 227-236, 2006
K+ channels involved in contractility of rabbit small intestine V. Lamarca, L. Grasa, D.S. Fagundes, M.P. Arruebo, M.A. Plaza and M.D. Murillo* Departamento de Farmacología y Fisiología (Fisiología), Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain (Received on February 3, 2006)
V. LAMARCA, L. GRASA, D. S. FAGUNDES, M. P. ARRUEBO, M. A. PLAZA and M. D. MURILLO. K+ channels involved in contractility of rabbit small intestine. J. Physiol. Biochem., 62 (4), 227-236, 2006. Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K+ channels involved in the contractility of longitudinal smooth muscle of rabbit small intestine in vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions. Key words: K+ channels, Contractility, Small intestine, Longitudinal muscle, Rabbit.
Correspondence to M. D. Murillo (Tel.: +34 976 761 652; Fax. +34 976 761 612; e-mail:
[email protected]).
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The function of gastrointestinal smooth muscle is twofold: to mix intestinal content with digestive enzymes and thereby aid digestion, and to propel intestinal content. The organization of gastrointestinal motility in motor patterns is dependent on numerous neuronal functions in the gut that are stimulated by nutrients. Smooth muscle cells in the intestine are arranged into bundles that form two layers: the inner circular muscle layer and the outer longitudinal muscle layer. The smooth muscle contraction of the intestine exhibits two distinct types of contractions: tonic and rhythmic phase, which undertake the functions of mixing and propulsion (9, 20). Electromechanical coupling in smooth muscle serves to coordinate the contractile activity of syncytium. The electrical activity of smooth muscle in the gut is generated by ionic conductances that regulate – and in turn are regulated by – the membrane potential of smooth muscle cells (29). The functional unit underlying the setting status of the membrane potential is the ion channel. Ionic channels are pores in the cell membrane that allow the rapid transfer of ions across the cell membrane. Ion channels are the basic excitable unit in all cells, including smooth muscle. Individual ionic channels can respond to a specific stimulus that may be electrical, such as a membrane potential change; or chemical, such as a neurotransmitter; or mechanical. The main classes of ion channels are Na+, K+, Ca2+, nonselective cation, and anion channels (6). The contractile activity of gastrointestinal smooth muscle is regulated by Ca2+ and K+ currents and by excitatory and inhibitory mediators (10). Ca2+ participates in the activation of the contractile apparatus: it either enters the cytoplasmic compartment during periods of membrane depoJ. Physiol. Biochem., 62 (4), 2006
larisation, mechanical distortion, or stimulation by agonists, through voltagedependent Ca2+ channels, or it is released from intracellular stores increasing cytosolic [Ca2+] (8, 19, 26). A variety of inwardly rectifying voltage- (Kv), Ca2+- (KCa2+), and ATPdependent (KATP) K+ channels play a key roles in controlling the excitability of smooth muscles (1-3, 6, 16, 21, 27, 29). A number of different K+ channels have been identified in the gastrointestinal tract (29). In visceral smooth muscles, studies support the key role of the channels of the delayed rectifier family in the regulation of resting membrane potential. K+ channels are encoded by a superfamily of K+ channels genes (14). Ca2+-activated K+ channels are a diverse group of K+ channels that share a common property: an increase in opening probability as internal Ca2+ rises (6). The aim of this study was to examine the types of K+ channels that are involved in the contractility of longitudinal smooth muscle of rabbit small intestine in vitro. Material and Methods The equipments used and the handling and sacrifice of the animals complied with the European Council legislation 86/609/ EEC concerning experimental animal protection. Male New Zealand rabbits weighing 2-2.5 kg were maintained at a constant room temperature (22 ºC) with free access to water and standard rabbit fodder. The experimental protocols were approved by the Ethics Committee of the University of Zaragoza (Spain). Preparation of smooth muscle segments.– After 24 h of fasting, animals were humanely killed by a blow to the head. Pieces of rabbit duodenum (1-6 cm distal
K + CHANNELS IN SMALL INTESTINE
the pylorus), jejunum (1-6 cm distal the ligament of Treitz) and ileum (1-6 cm proximal the sacculus rotundus) were removed, washed, freed from mesenteric attachment and cut into smaller segments. Whole thickness segments (10 mm long and 5 mm wide) were suspended in the direction of longitudinal smooth muscle fibers in a thermostatically controlled (at 37 ºC) organ bath (10 mL capacity) containing Krebs solution and, to maintain continuously gassed with 95% O2 and 5% CO2. Each segment of duodenum, jejunum, and ileum was connected to an isometric force transducer (Pioden UF1, Graham Bell House, Canterbury, U.K.) and stretched passively to an initial tension of 20 mN. Signal output of the mechanical activity was amplified, recorded on a computer for later analysis using The Mac Lab System/8e computer program (AD Instruments Inc., Milford MA, U.S.A) and digitized at two samples per second per channel. Before testing, segments were allowed to equilibrate in Krebs solution for 60 min. During that time, the nutrient solution was changed every 20 min. Each experimental protocol was systematically performed on two or three segments of duodenum, jejunum and ileum taken from each of three or four rabbits. Segments that did not show spontaneous activity were discarded. Thus, each preparation served as its own control. After the equilibration period, spontaneous contractions in longitudinal smooth muscle of small intestine were recorded for 5 min in the absence (control period) or presence of different drugs. In order to study the effects of cholinergic, adrenergic, purinergic, neural transmission and prostaglandins release on spontaneous contractions, the segments were incubated for 5 min with 10-6 M J. Physiol. Biochem., 62 (4), 2006
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atropine, 10-6 M phentolamine, 10-6 M propranolol, 10-5 M suramin, 10-6 M tetrodotoxin and 10-5 M indomethacin. To determine the participation of the different types of K+ channels on both spontaneous and high K+-induced contractions (80 mM, 3 min), these were measured in the absence and in the presence of different blockers or openers of several K+ channels (2). Apamin, (a blocker of smallconductance Ca2+-activated K+ channels); charybdotoxin (a selective blocker of intermediate- and large-conductance Ca2+-activated K+ channels); iberiotoxin (a blocker of large-conductance Ca2+-activated K+ channels); tetraetylammonium (a non-specific K+ channel blocker); NS1619 (an opener of large-conductance Ca2+-activated K+ channels); 4-aminopyridine (blocker of voltage-sensitive K+ channels), E4031 (a selective blocker of HERG K+ channels); glibenclamide (a blocker of ATP-sensitive K+ channels); levcromakalim and diazoxide (openers of ATP-sensitive K+ channels); and BaCl2 (a blocker of inward rectifier K+ channels) were all incubated for 5 min. Data analysis.– Most segments showed spontaneous contractions. For each segment of smooth muscle, the mean amplitude (in mN) of contractions was calculated as the average of peak-to peak differences over 5 min. The frequency of contractions was expressed as the number of contractions per minute (cpm) in a 5-min period, and the tone of spontaneous contractions as the mean of the tension values recorded in a 5-min period. Amplitude, frequency and tone of spontaneous contractions in the presence of drugs are expressed as a percentage of the values recorded in the absence of drugs (control period) (8, 22, 23). The responses of longitudinal smooth muscle to KCl were mea-
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sured as the integrated mechanical activity per second (milinewtons per second, mN s-1), and normalized per square millimeter of cross-sectional area (CSA, mm2). CSA was determined for each muscle strip using the equation: CSA (mm2) = mass (mg) [length (mm) density (mg mm-3]-1, where rabbit intestinal muscle density was assumed to be 1.05 mg mm-3 and length and mass (wet weight) of each segment were noted on completion of experiments. Values are expressed as mean ± SEM. Comparisons between means were made using one-way variance analysis (ANOVA) tests and P-values were determined using the Scheffé F test. Differences with P-values
