BIPHASIC INCREASE IN CYTOSOLIC-FREE CALCIUM-INDUCED BY BRADYKININ ANDHISTAMINE IN CULTURED TRACHEAL SMOOTH-MUSCLE CELLS - IS THE SUSTAINEDPHASE ARTIFACTUAL

The effects of bradykinin (BK) and histamine on intracellular Ca2+ ([C a2+](i)) were studied in fura-2-loaded guinea-pig tracheal smooth musc le cells in culture. BK, at 10 nM, and histamine, at 100 mu M, induced a rise in [Ca2+](i) which was inhibited by the B2 antagonist Hoe 140 and by the H1 antagonist triprolidine, respectively. This rise in [Ca2 +](i) is biphasic, consisting of a rapid transient phase followed by a sustained phase. The transient phase, induced by either BK or histami ne, was strongly inhibited by thapsigargin, a microsomal Ca2+-ATPase i nhibitor, usually used to deplete certain intracellular Ca2+-stores. N i2+ (4 mM) did not affect the transient phase but abolished the sustai ned phase when cells were stimulated by BK, further supporting the fac t that the transient phase was only dependent on intracellular Ca2+ po ols. The sustained phase was partially (for BK) and completely (for hi stamine) inhibited by 30 mu M This effect could be completely reversed by the addition of DTPA, a cell-impermeant chelator of Mn2+ indicatin g that the Mn2+ exerted its effect extracellularly. The presence of 1 mM probenecid (an inhibitor of a membrane organic anion transporter th at extrudes fura-2) drastically inhibited the sustained phase by more than 77% for BK and 88% for histamine. Our results suggest that the ef fects of BK and histamine on airway smooth muscle cells are mediated v ia bradykinin B2 receptors and histamine H1 receptors, respectively wh ose activation allows the rapid transient rise in [Ca2+](i) from thaps igargin-sensitive intracellular Ca2+ pools. The sustained phase is pro posed to be drastically influenced by an acceleration of fura-2 extrus ion during the increase of[Ca2+](i) via a probenecid-sensitive mechani sm.