Dissociation of rat parotid gland

Abstract

Rat parotid gland was dissociated by sequential collagenase and hyaluronidase digestions, chelation with ethylenediaminetetraacetic acid, and mild shearing force to yield predominantly single cells. The isolated acinar cells retained their morphologic characteristics and their amylase activity. The functional integrity of the isolated cells was assessed by measuring their secretory response to isoproterenol, epinephrine, and carbamylcholine and by their ability to incorporate radioactively labeled leucine and thymidine. The discharge of amylase from the dissociated cells was not effected by isoproterenol or norepinephrine and the response to carbamylcholine was minimal. The data indicate a destruction or perturbation of hormone receptors during the dissociation procedure. The maintenance of the cells in culture for up to 18 hours failed to restore the responsiveness of the isolated parotid gland acinar cells to isoproterenol. The isolated cells incorporated 14C-leucine into proteins at a linear rate between 30 and 180 minutes. Chromatographic and electrophoretic profiles of newly synthesized proteins indicated that all major proteins synthesized in vivo were also synthesized by the isolated cells. The isolated cells incorporated tritiated thymidine into DNA. Furthermore, stimulation of DNA synthesis by isoproterenol in vivo was reflected by a higher rate of thymidine incorporation by the isolated cells as compared with controls. The dissociated parotid gland cells offer a convenient system for studying various cellular processes, particularly the synthesis of macromolecules with high specific activity. However, some functions, notably the response to beta- adrenergic agonists, are lost during the dissociation procedure.