Actions of 4‐chloro‐3‐ethyl phenol on internal Ca 2+ stores in vascular smooth muscle and endothelial cells

1 Recently, 4‐chloro‐3‐ethyl phenol (CEP) has been shown to cause the release of internally stored Ca 2+ , apparently through ryanodine‐sensitive Ca 2+ 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 Ca 2+ , by use of Ca 2+ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells. 2 After phenylephrine‐(PE, 10 μ M ) sensitive Ca 2+ stores were depleted by maximal PE stimulation in Ca 2+ ‐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 Ca 2+ was restored for a 30 min refilling period. At the end of this period, Ca 2+ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca 2+ store. 3 In a concentration‐dependent manner (30, 100 and 300 μ M ), CEP significantly reduced the size of the post‐refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca 2+ ‐free media. This suggests that Ca 2+ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca 2+ ‐containing or Ca 2+ ‐free Krebs solution. 4 Restoring Ca 2+ in the presence of PE caused a large contraction, which reflects PE‐induced influx of extracellular Ca 2+ . The contraction of tissues pretreated with 300 μ M CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μ M CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca 2+ ‐free and Ca 2+ ‐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 μ M 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 μ M ) for 30 min before the construction of PE concentration‐response curves. In Ca 2+ ‐free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 m M K + contraction, n =11) in the presence of external Ca 2+ . EC 50 values for PE in ryanodine‐treated tissues (1.7±0.25 μ M , n =16) were not significantly different from controls (2.5±0.41 μ M , n =22). Maximum contractions to PE (118.5±4.4% of 100 m M K + contraction, n =16) were also unaffected by ryanodine when compared to controls (129±4.2%, n =23). 7 When fura‐2 loaded smooth muscle cells ( n =13) and endothelial cells ( n =27) were imaged for Ca 2+ distribution, it was observed that 100 and 300 μ M CEP in Ca 2+ ‐free medium caused Ca 2+ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 m M ) reversed the effect of CEP on intracellular Ca 2+ to control values. 8 These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca 2+ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5‐HT and K + . The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca 2+ elevation.British Journal of Pharmacology (1997) 122 , 504–510; doi: 10.1038/sj.bjp.0701389