Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that is independent of cholesterol side-chain cleavage

An increasing body of evidence suggests that an endogenous mammalian bufadi enolide (BD) may be involved in the regulation of Na+,K+-ATPase activity an d the pathogenesis of arterial hypertension. We developed a purification sc heme for marinobufagenin (MBG), an amphibian cardiotonic ED, and applied it to purify and characterize material in human plasma, culture medium condit ioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreacti vity purified from plasma and measured by ELISA showed important similariti es (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulat ing mammalian ED may have an adrenocortical origin. Release of mammalian ED from adrenocortical cells grown in the absence of exogenous cholesterol wa s reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglu taryl coenzyme A reductase inhibitor. Supplementation of the serum and chol esterol-free cell culture medium with the LDL fraction of human plasma incr eased the production of MBG material in the presence of mevastatin, support ing its origin from cholesterol. We used Y-1 cell lines transfected with ge nes shown to inhibit steroidogenesis through cholesterol side-chain cleavag e (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian ED is synth esized in the adrenal cortex from cholesterol and shares important similari ties with the amphibian ED MEG, that its biosynthesis is independent of tra nsfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein k inase A appears to be a critical component of the pathway controlling its b iosynthesis.