Abstract
The cytoplasmic free calcium ion concentration ([Ca2+]i) of cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent [Ca2+]i indicator, fura-2, was measured by digital ratio imaging microscopy. Spontaneous [Ca2+]i oscillations were observed in 25% to 80% of the cells, which differed with the batches of the cultured cells after 5 to 8 days in culture. The frequency and amplitude of the [Ca2+]i oscillations in each individual cell were usually regular, but heterogeneity between neighboring cells was observed. The spontaneous [Ca2+]i oscillations were also observed even after incubation of the cells under a serum-free condition for 72 hr. Exchange of extracellular solution to Ca2+-free solution containing EGTA or BAPTA immediately stopped the [Ca2+]i oscillations. The ratio of the oscillating cells was dependent on the extracellular calcium ion concentration ([Ca2+]o); and heterogeneity in the range of the [Ca2+]o to generate the [Ca2+]i oscillations was observed. An inorganic Ca2+-antagonist, LaCl3, immediately suppressed the [Ca2+]i oscillations, but the treatment with verapamil or nicardipine, Ca2+-channel blockers, did not have any effect on the [[Ca2+]i oscillations. An inhibitor of the intracellular Ca2+ pump, thapsigargin, induced a transient increase in [Ca2+]i and then inhibited the spontaneous [Ca2+]i oscillations. Neomycin, a compound known to inhibit phosphoinositide turnover, inhibited the [Ca 21]i oscillations. These results suggest the following: (1) The generation of the spontaneous [Ca2+]i oscillations is highly dependent on [Ca2+]o but not due to Ca2+ influx through voltage-dependent Ca2+ channels; (2) Thapsigargin-sensitive Ca2+ pumping pools, which may be inositol 1, 4, 5-trisphosphate (IP3)-releasable Ca2+ pools, play an important role in generating the spontaneous [Ca2+]i oscillations, and the uptake of Ca2+ into the pools is highly dependent on Ca2+ influx across the plasma membrane.
