Dispersed Aortic Smooth Muscle Cells

Studies on hormone induced contraction of vascular smooth muscle strips in vitro have yielded a large body of pharmacological and biochemical data (1). Unfortunately, vascular strips possess inherent limitations for answering certain questions concerning sites of hormone binding on their constituent smooth muscle cells. Diffusion barriers in the tissue may hinder the use of macromolecular receptor probes and extracellular matrix elements may cause nonspecific binding of radiolabeled receptor probes. In intact tissue, it is also impossible to determine differences in receptor density and contractile responses among individual smooth muscle cells. The use of isolated cell preparations may eliminate these problems. Previous studies have utilized two approaches for obtaining isolated cells from vascular smooth muscle. First, cultures of such cells have been obtained by outgrowth from explants of large vessels (2-5) or from dissociated cells (5-7). While cells cultured for long term generally retain some of the structural and biochemical features of vascular smooth muscle cells in situ, they appear to be phenotypically altered in that their content of myosin is markedly reduced (7) and they are not responsive to contractile stimuli (3, 4). It is not yet known whether receptors for vasoactive agents are present on these cells. Second, freshly dispersed vascular smooth muscle cells have been prepared by enzymatic digestion. Peters et al. (8) used crude collagenase, elastase, and hyaluronidase to dissociate the rabbit aorta into single smooth muscle cells many of which were highly damaged, while Gimbrone and Cotran (6) used crude collagenase alone to generate a mixed population of endothelial and smooth muscle cells from human umbilical veins or arteries. Neither of these groups described contractile responses of their cells and we have found the procedure of Peters et al. to result in small numbers of viable but hormonally unresponsive cells. Several groups (9-1t) have described dissociation procedures yielding contractile smooth muscle cells, but from nonvascular tissue. Given the limitations of these various procedures, we undertook to devise a tissue dissociation procedure which would produce good yields of hormonally responsive vascular smooth muscle cells from rabbit aorta (12). While our studies were underway, Chamley et al. (7) reported on the use of enzymatic digestion to obtain smooth muscle cells from a variety of mammalian blood vessels. An unstated percentage of these cells