ryanodine and 4-chloro-3-ethylphenol

We examined cytosolic concentration of Ca2+ ([Ca2+]i) in canine airway smooth muscle using fura 2 fluorimetry (global changes in [Ca2+]i), membrane currents (subsarcolemmal [Ca2+]i), and contractions (deep cytosolic [Ca2+]i). Acetylcholine (10(-4) M) elicited fluorimetric, electrophysiological, and mechanical responses. Caffeine (5 mM), ryanodine (0.1-30 microM), and 4-chloro-3-ethylphenol (0.1-0.3 mM), all of which trigger Ca2+-induced Ca2+ release, evoked Ca2+ transients and membrane currents but not contractions. The sarcoplasmic reticulum (SR) Ca2+-pump inhibitor cyclopiazonic acid (CPA; 10 microM) evoked Ca2+ transients and contractions but not membrane currents. Caffeine occluded the response to CPA, whereas CPA occluded the response to acetylcholine. Finally, KCl contractions were augmented by CPA, ryanodine, or saturation of the SR and reduced when SR filling state was decreased before exposure to KCl. We conclude that 1) the SR forms a superficial buffer barrier dividing the cytosol into functionally distinct compartments in which [Ca2+]i is regulated independently; 2) Ca2+-induced Ca2+ release is preferentially directed toward the sarcolemma; and 3) there is no evidence for multiple, pharmacologically distinct Ca2+ pools.

Topics: Acetylcholine; Animals; Caffeine; Calcium; Calcium-Transporting ATPases; Cell Membrane; Chlorophenols; Cytosol; Dogs; Electric Conductivity; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Indoles; Muscle Contraction; Muscle, Smooth; Ryanodine; Sarcoplasmic Reticulum; Trachea

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca2+ apparently through ryanodine-sensitive Ca2+ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca2+, by use of Ca2+ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells. 2. After phenylephrine-(PE, 10 microM) sensitive Ca2+ stores were depleted by maximal PE stimulation in Ca2+-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca2+ was restored for a 30 min refilling period. At the end of this period, Ca2+ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca2+ store. 3. In a concentration-dependent manner (30, 100 and 300 microM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca2+-free media. This suggests that Ca2+ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca2+-containing or Ca2+-free Krebs solution. 4. Restoring Ca2+ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca2+. The contraction of tissues pretreated with 300 microM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 microM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca2+-free and Ca2+-containing medium suggesting a rapid reversal of CEP effects. 5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K+ in the absence of and after 30 min pre-incubation with 30, 100 and 300 microM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K+ (77.6, 41.1 and 10.8% of control). 6. Some arterial rings were pre-incubated with ryanodine (30 microM) for 30 min before the construction of PE concentration-response curves. In Ca2+-free Krebs solution, ryanodine a

Topics: Animals; Calcium; Calcium Channel Agonists; Cells, Cultured; Chlorophenols; Dogs; Endothelium, Vascular; Fluorometry; In Vitro Techniques; Mesenteric Arteries; Muscle Contraction; Muscle, Smooth, Vascular; Phenylephrine; Potassium; Pulmonary Artery; Ryanodine; Serotonin