Separation and culture of living adrenaline- and noradrenaline-containing cells from bovine adrenal medullae.
Separation of viable adrenaline-containing from noradrenaline-containing chromaffin cells in large amounts has been achieved. The procedure involves collagenase digestion of bovine adrenomedullary tissue, isolation of cells through gentle filtration, separation of chromaffin from nonchromaffin cells on discontinuous gradients of the radiopaque contrast Renografin, and separation of adrenaline-enriched from noradrenaline-enriched fractions after centrifugation on self-generated Percoll gradients. Collection of 1-ml Percoll fractions gave two clear-cut catecholamine peaks. The denser peak was enriched in adrenaline and phenylethanolamine-N-methyltransferase (PNMT), suggesting that over 90% of cells were adrenergic. The lighter peak was preferentially enriched in noradrenaline but not in PNMT. With this information, we could collect by gentle aspiration two main fraction layers of larger volumes; one at the bottom of the Percoll gradient, which contained essentially adrenaline-storing cells and the other at the top of the gradient, enriched in noradrenaline cells. Those cells could be maintained viable for at least 1 week in primary monolayer cultures, as shown by neutral red staining and trypan blue exclusion. This method will allow the identification of chemical components, receptors, or ionic channels present in one specific type of cell, to determine their relevance to the regulation of the differential secretion of specific materials present in one but not in the other cell type and to ascertain whether the released materials from one cell type affect the functions of the other.
Moro
M A
MA
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma, Madrid, Spain.
López
M G
MG
Gandía
L
L
Michelena
P
P
García
A G
AG
D000313:Adrenal Medulla / Q000166:cytology*; D000818:Animals; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D002838:Chromaffin System / Q000166:cytology; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D004837:Epinephrine / Q000032:analysis*; D009638:Norepinephrine / Q000032:analysis*; D010625:Phenylethanolamine N-Methyltransferase / Q000378:metabolism
D004299:Dopamine beta-Hydroxylase; D010625:Phenylethanolamine N-Methyltransferase; D009638:Norepinephrine; D004837:Epinephrine