Large scale preparation of rabbit aortic smooth muscle cells for use in calcium uptake studies

Rabbit aortic smooth muscle cells were prepared by enzymatic digestion of the aortic smooth muscle layer. The cells were subcultured up to Passage 22 starting from a cryogenically preserved stock (approximately 10(10) cells, Passage 8) and characterized morphologically and for 45Ca++ uptake. Microscopically the cells demonstrated the characteristics of vascular smooth muscle cells. 45Ca++ uptake by the cells plated on tissue culture flasks (25 cm2) was determined at 25 degrees C in physiological salt solution (PSS) containing 45Ca++ in low (5 mM) or high (50 mM) KCl concentrations. At the end of the incubation period (0 to 30 min), PSS was aspirated and the cells quickly washed, digested with 0.5 N NaOH, and counted for 45Ca++. High K+ increased the 45Ca++ uptake by 100% or more compared to the low K+ uptake of 45Ca++. This K+-induced 45Ca++ uptake was eliminated in osmotically shocked cells, and inhibited by nifedipine, verapamil, and diltiazem, in a dose-dependent manner. The extent of 45Ca++ uptake and the inhibitory activity of nifedipine were retained up to Passage 22. It is concluded that the developed methodology for scaled-up cultures of rabbit aortic smooth muscle cells provides morphologically intact and biochemically functioning cells suitable for calcium channel studies.