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 1010cells, Passage 8) and characterized morphologically and for45Ca++ 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°C in physiological salt solution (PSS) containing45Ca++ in low (5 mM) or high (50mM) KCl concentrations. At the end of the incubation period (0 to 30 min), PSS was aspirated and the cells quickly washed, digested with 0.5N NaOH, and counted for45Ca++. High K+ increased the45Ca++ uptake by 100% or more compared to the low K+ uptake of45Ca++. This K+-induced45Ca++ uptake was eliminated in osmotically shocked cells, and inhibited by nifedipine, verapamil, and diltiazem, in a dose-dependent manner. The extent of45Ca++ 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.