Mammalian Bufadienolide Is Synthesized From Cholesterol in the Adrenal Cortex by a Pathway That Is Independent of Cholesterol Side-Chain Cleavage

Abstract

Abstract-An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na ϩ ,K ϩ -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.

Marinobufagenin Stimulates Fibroblast Collagen Production and Causes Fibrosis in Experimental Uremic Cardiomyopathy

“…That said, both PNx-IM and PNx-ADx, which reduce circulating MBG, substantially attenuate the cardiac functional and morphological changes without significantly affecting blood pressure. We should point out that experiments in the PNx-ADx model were performed because we reasoned that as adrenal cells grown in culture seem to make MBG, 20,21 it was likely that this procedure would lower the circulating levels of this hormone. However, whereas our data in the PNx-ADx animals support the concept that the adrenal gland is the major (but not the only) site of MBG production in vivo, it is also possible that other hormones made in the adrenal gland modulate MBG production elsewhere.…”

Cloning and characterization of cDNAs encoding steroidogenic acute regulatory protein from freshwater stingrays (Potamotrygon spp.)

“…Although their biosynthetic pathways have not been fully elucidated (Hamlyn 2004), there is evidence that several enzymes (including SCC) may be common to the synthesis of some cardiac glycosides and corticosteriods (Hamlyn et al 2003). However, the synthesis of bufadienolide in mammals appears to be independent of both StAR and SCC, as indicated by studies using mouse Y-1 adrenocortical cells transfected with genes that inhibit the expression of StAR and SCC (Dmitrieva et al 2000). Recent work suggests that endogenous cardiac glycosides may have an important role in the regulation of ionoregulation in fishes.…”

The Role of Cardiotonic Steroids in the Pathogenesis of Cardiomyopathy in Chronic Kidney Disease

“…Evidence is accumulating that most CTS are synthesised from cholesterol in the adrenal glands [37,38] and possibly hypothalamus [39] but the biosynthetic pathways for cardenolides and bufadienolides seem to differ. The trigger for biosynthesis initiation is complex, with serum concentrations of ouabain and marinobufagenin increasing in response to volume expansion [40], salt accumulation in the brain, adrenocorticotropic hormone, angiotensin II, vasopressin and phenylephrine [37,41].…”