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. 1991 Apr;435:123–144. doi: 10.1113/jphysiol.1991.sp018501

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

R K Murray 1, M I Kotlikoff 1
PMCID: PMC1181453  PMID: 1663158

Abstract

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)

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Selected References

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