Ca2+ mobilization by caffeine in single smooth muscle cells of the rat tail artery

The fluorescent dye fura-2 was used to study the effects of caffeine on cytosolic free Ca2+ level ([Ca2+]i) in freshly isolated single cells from the rat tail artery. Caffeine caused a concentration-dependent transient increase in [Ca2+]i and shortening of the cell. At higher concentrations (> 2 mM), a tonic increase in [Ca2+]i was also observed. The caffeine-induced changes in [Ca2+]i were reproducible with repeated challenges, even though the cells had contracted due to previous exposure to caffeine. Removal of extracellular Ca2+ reduced the resting [Ca2+]i to about half and abolished the tonic Ca2+ increase to caffeine. The transient component was not significantly affected to the first caffeine challenge after Ca2+ removal, but was abolished to the second challenge. Ryanodine (10 microM) significantly inhibited the responses to caffeine while nifedipine and TMB-8-(8-(diethylamino)octyl ester of 3,4,5-trimethoxybenzoic acid) were not effective. Thapsigargin (10-100 microM) induced a sustained increase in [Ca2+]i to 67 nM. The response of caffeine was not affected by thapsigargin. Pretreatment of the cells with noradrenaline (10 microM) abolished subsequent response to caffeine. These results show that Ca2+ responses to caffeine in single cells from the rat tail artery are reproducible with repeated caffeine challenge. Therefore, single cells can be used for comparison studies of the effects of pharmacological agents.