Action potential generation in reaggregates of rat aortic smooth muscle cells in primary culture.

Cultured vascular smooth muscle cells were prepared from adult rat aortas. The cells were dispersed by collagenase/elastase, and allowed to reaggregate into small spheres (50-200 micron in diameter) by plating on to cellophane. These primary cultures were incubated for 5-14 days, and then impaled with microelectrodes. The mean resting potential was -55 mV, and the mean input resistance was 9.0 M omega. The cells were quiescent, electrically and mechanically, and electrical stimulation usually did not elicit responses. However, addition of Ba++ (1 mM) or tetraehtylammonium ion (TEA; 5-15 mM) induced excitability (with accompanying contractions), either as spontaneous action potentials or by allowing responses to electrical stimulation. The cells became partially depolarized (e.g., to -36 mV) by these agents, and the input resistance increased. The frequency of spontaneous firing of the Ba++-induced spikes was affected by polarizing current pulses, as expected for pacemaker behavior. Elevation of Ca++ in the bathing solution increased the amplitude (overshoot) of the action potentials, and the spikes were blocked by verapamil (10(-5) M). Electron microscopy showed that the reaggregates consisted of a tight packing of elongated small-diameter cells, some of which exhibited thick and thin myofilaments and 'dense bodies'; many cells possessed surface caveolae. The results of this study demonstrate that reaggregates of arterial smooth muscle in primary culture can maintain functional and morphological characteristics of intact arterial smooth muscle, and therefore provide a useful preparation for the study of vascular smooth muscle function and control.