An increasing body of evidence suggests that an endogenous mammalian
bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-
ATPase activity and the pathogenesis of arterial
hypertension. We developed a purification scheme for
marinobufagenin (MBG), an amphibian
cardiotonic BD, and applied it to purify and characterize material in human plasma,
culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous
cholesterol was reduced by treatment of cultures with
mevastatin, a 3-hydroxy-3-methylglutaryl
coenzyme A reductase inhibitor. Supplementation of the serum and
cholesterol-free cell culture medium with the
LDL fraction of human plasma increased the production of MBG material in the presence of
mevastatin, supporting its origin from
cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through
cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from
cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of
cholesterol to the side-chain cleavage
enzyme complex mediated by
steroidogenic acute regulatory protein, and that neither cAMP nor
protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.