The action of cysteinyl-leukotrienes on intracellular calcium mobilizationin human detrusor myocytes

Objective To investigate the presence of leukotriene D-4 receptors in fura- 2-loaded human detrusor smooth muscle cells (DSMCs) by examining the abilit y of leukotriene D-4 to raise intracellular-free Ca2+ concentration ([Ca2+] (i)), to determine the origin of the leukotriene D-4-mediated rise in [Ca2](i) and to investigate whether the specific leukotriene D-4 receptor antag onist montelukast inhibits the Ca2+ response induced by leukotriene D-4. Materials and methods Detrusor muscle biopsies were obtained from patients with benign noninvasive bladder diseases undergoing cystoscopy. DSMCs were isolated using an explant technique and maintained in culture, Only primary cultures or cells passaged up to three times were used for experiments. DS MCs were characterized with immunohistochemical staining and their identity confirmed by transmission electron microscopy. [Ca2+](i) was measured in s ingle DSMCs using the Ca2+ probe fura-2 and fluorescence-ratio microscopy. Results Immunohistochemical staining showed that 80-99% of the cells were p ositive for smooth muscle alpha- actin. The ultrastructural features of the cultured cells were those of smooth muscle cells and showed no differentia tion in a fibroblastic or myofibroblastic direction. Leukotriene D-4 increa sed the level of [Ca2+](i) in a dose-dependent [Ca2+](i) in a dose-dependen t manner. Calcium was mobilized almost exclusively from intracellular Ca2stores. There was a dose-dependent inhibition of the increase in [Ca2+](i) by montelukast. Conclusion The present study is the first to show the presence of specific leukotriene D-4 receptors in human detrusor myocytes. This may have implica tions for a potential pathophysiological role of leukotriene D-4 in patient s with interstitial cystitis and other functional or inflammatory bladder d isorders.