Receptor‐activated calcium influx in human airway smooth muscle cells.

1. Fluorescence measurements of intracellular calcium concentrations ([Ca2+]i) were made on cultured human airway smooth muscle cells using the dye Fura‐2. The response to either histamine (100 microM) or bradykinin (1 microM) was biphasic, with a transient increase in [Ca2+]i followed by a sustained [Ca2+]i increase lasting many minutes. The average steady‐state (plateau) [Ca2+]i following agonist activation was 267 +/‐ 5 nM, whereas the average basal [Ca2+]i was 148 +/‐ 4 nM. 2. The sustained rise in [Ca2+]i required the continued presence of either histamine or bradykinin and was dependent on extracellular Ca2+. The magnitude of the transient rise in [Ca2+]i was not dependent on extracellular Ca2+. Sustained, receptor‐activated rises in [Ca2+]i were rapidly abolished by chelation of extracellular Ca2+, or addition of non‐permeant polyvalent cations, whereas these agents had minor effects in the absence of agonist. These data indicate that the sustained increase in [Ca2+]i was dependent on receptor‐activated Ca2+ influx. 3. Receptor‐activated Ca2+ influx was not affected by treatment with organic Ca2+ channel antagonists (nifedipine (10 microM), nisoldipine (10 microM) or diltiazem (10 microM] or agonists (Bay K 8644 (500 nM to 10 microM) or Bay R 5417 (500 nM]. The magnitude of the sustained rise was also not affected by pre‐treatment with ouabain (100 microM) indicating little involvement of Na(+)‐Ca2+ exchange in the influx mechanism. 4. Receptor‐activated Ca2+ influx could be completely inhibited by several polyvalent cations (Co2+, Mn2+, Ni2+, ‐Cd2+ or La3+). Quantitative estimates of the potency of block were obtained for Ni2+ and La3+. These measurements indicate that the pKi for Ni2+ was 3.6 and for La3+ was 3.5. 5. Both Mn2+ and Co2+ ions caused a time‐dependent quench of intracellular Fura‐2; however, permeation of neither ion was increased following receptor activation, indicating that the influx pathway is not permeable to these cations. 6. Fura‐2 was used to monitor the rate of Ba2+ entry into airway smooth muscle cells by monitoring the Ca(2+)‐Fura‐2 and Ba(2+)‐Fura‐2 isosbestic points as well as the 340 and 380 nm signals. Cell activation did not increase the rate of Ba2+ entry indicating that the Ca2+ influx pathway was poorly permeant to Ba2+ ions. Ba2+ (2 mM) was able to inhibit Ca2+ entry as shown by its effects on the Ba(2+)‐independent, Ca(2+)‐dependent wavelength (371 nm). 7. The voltage dependence of Ca2+ influx was examined before and after agonist‐induced activation. The effect of KCl‐induced depolarization prior to cell activation was to cause a slight increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)