Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin 2

Abstract

The Ca2+-sensitive dye quin 2 was used to monitor Ca2+ levels and to follow Ca2+ transients in suspensions of isolated toad (Bufo marinus) smooth muscle cells, whose contractile activity was monitored with a Coulter counter. Single cells were not utilized to follow Ca2+ changes involved with contraction because of problems of rapid dye bleaching and due to photosensitization of smooth muscle cells loaded with quin 2. High levels of quin 2 loading (2-5 mM) severely prolonged (time course increase greater than 3 times), or completely inhibited, contractile responses to carbachol or potassium depolarization. Lower levels (less than 1 mM) produced adequate fluorescent signals, even at the single cell level, and allowed contractile responses of normal magnitude, although with somewhat prolonged (2-3 times) time course. Resting Ca2+ concentrations determined using quin 2 at these lower levels were 129 +/- 3 nM, a value that closely coincided with that measured in the same cell type using Ca-sensitive microelectrodes, or an alternate, more highly fluorescent dye, Fura-2. Resting Ca2+ was highly dependent on the extracellular Ca2+ concentration that appeared to effect intracellular Ca2+ (Cai2+) both by altering the driving force on Ca2+ cross the membrane, as well as the Ca2+ permeability of the cell itself. A small but significant relaxation was observed in response to the lowering of cytoplasmic Ca2+ below resting levels. After carbachol or K+ addition, fluorescent transients peaked significantly before the onset of contraction, which was also transient. Isoproterenol, a known relaxant of these cells, caused a small decrease in Cai2+ (approximately 40 nM) below rest, when applied in maximal dosage (10(-4) M). Isoproterenol also consistently diminished the Ca2+ transient induced by excitatory stimuli such as carbachol or K+. These results indicate that changes in contractility may be directly linked to changes in free cytoplasmic Ca2+ in smooth muscle cells.