Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Abstract

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.

Independent pathways regulate the cytosolic [Ca2+] initial transient and subsequent oscillations in individual cultured arterial smooth muscle cells responding to extracellular ATP.

“…Vascular smooth muscle cells were isolated from swine aorta with minor changes in the procedure described previously (Goldman et al, 1983;Linderman et al, 1990 .0 ,ug/ml insulin, and 0.04 mg/ml ascorbic acid (defined medium) (Libby and O'Brien, 1983 (Linderman et al, 1990).…”

“…Solution exchange was complete in -2 s (Cheyette and Gross, 1991 Because we previously showed that prestimulus [Ca2+]i in these cells is near 100 nM (Linderman et al, 1990), we have set each pCa prestimulus level to a value of 7. The amplitudes of transients and oscillations so calibrated agree with previous findings (Linderman et al, 1990). …”

“…Isolated smooth muscle cells from rabbit ear artery were shown to express ATP-activated Ca2+-permeable plasma membrane channels that mediate a Ca2+ influx in response to ATP (Benham and Tsien, 1987;Benham, 1989). We have previously reported that 50 ,uM extracellular ATP induces a robust initial transient rise in [Ca2+]i followed by semiperiodic fluctuations from a baseline level in individual porcine aortic smooth muscle cells (Linderman et al, 1990). The initial rise occurs rapidly and nearly synchronously within the temporal resolution of the experiment (5 s).…”

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

“…Measurement of [Ca" ]I in individual cells during attachment and spreading was carried out by plating fura 2-loaded cells on gelatin-coated polystyrene plastics (0.8 mm) set on microscopic stage at 37°C and by a digital image analysis of fura 2 fluorescence. The instrumentation has been described previously by Linderman et al (1990). Briefly, cells on the stage of a Zeiss IM-35 inverted microscope (Carl Zeiss, Thomwood, NY) were illuminated alternatively at 334 and 365 nm using a Hg-Xe arc lamp (Hamamatsu, Bridgewater, NJ) and a pair of monochromators (model H20, Instruments SA, Metuchen, NJ).…”

“…Fluorescence images were detected by a charge coupled device instrumentation camera (Photometrics, Tucson, AZ) every 15 or 20 s up to 30 min as soon as cells attach to a gelatin substratum (<2 min). Image collection and processing was accomplished through a menu-driven software package (Paragon-IPS, Paragon Imaging, Lowell, MA), which has been previously described (Linderman et al, 1990).…”