Na,K-ATPase Inhibitors from Bovine Hypothalamus and Human Plasma Are Different from Ouabain: Nanogram Scale CD Structural Analysis

Abstract

The specific, high affinity binding of plant-derived digitalis glycosides by the mammalian sodium and potassium transporting adenosine triphosphatase (Na,K-ATPase, or sodium pump), a plasma membrane enzyme with critical physiological importance in mammalian tissues, has raised the possibility that a mammalian analog of digitalis might exist. We previously isolated and structurally characterized from bovine hypothalamus a novel isomer of the plant glycoside, ouabain, which differs structurally only in the attachment site and/or the stereochemistry of the steroid moiety [Tymiak et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8189-8193]. Hamlyn and co-workers reported a molecule purified from human plasma which by mass spectrometry could not be distinguished from plant ouabain [Hamlyn et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6259-6263]. Since rhamnoside cardiotonic steroids are not known as natural products from mammalian sources, it became important to compare these two pure isolates to determine if the same or structurally distinct compounds has been found. Our results indicate that the human and bovine Na,K-ATPase-inhibitors are identical, but different from plant ouabain. This supports the notion that the human sodium pump may be under specific physiological regulation by a mammalian analog of the digitalis glycosides.

Structure of 'endogenous ouabain'

“…With no conceivable candidate among sugar position isomers of ouabain, our study came to the point of reexamining the results obtained from the previous nanogram-scale naphthoylation of HIF and OLC (13,14).…”

“…Although the molecular ion peak of this HIF derivative was not clear, the product was described as HIF pentanaphthoate (13). Subsequent microscale derivatization of OLC (400 ng, gift from Upjohn Laboratories) also gave the same RP-HPLC and CD (''zero-CD'') profiles as those of HIF pentanaphthoate (14). This led to the conclusion that the ouabain-like sodium pump inhibitor is an ouabain isomer, often referred to as endogenous ouabain (2,4,5), even though the physiological discrepancies between HIF and other compounds still remained to be clarified (2): the term endogenous ouabain has been used to distinguish this compound from plant ouabain (2,4,5), although its true origin has not yet been clarified.…”

Na + pump low and high ouabain affinity α subunit isoforms are differently distributed in cells

“…This could be the rationale for colocalization of high ouabain affinity Na ϩ pumps and Na ϩ ͞Ca 2ϩ exchangers in these PM areas. This might reveal how an endogenous ouabain isomer (12,(37)(38)(39), which circulates at nanomolar concentrations (40) and is present in the brain (38,39), could, by modulating the activity of these high ouabain affinity Na ϩ pumps (28,37), play a role in many physiological, and even pathophysiological, processes (12). Finally, this may also explain why the isoformspecific features of the ouabain binding regions of ␣2 and ␣3 have been so well conserved during the evolution of higher animals (4,10).…”

Bovine Adrenals Contain, in Addition to Ouabain, a Second Inhibitor of the Sodium Pump

“…There has been an intensive search for such substances that has led to the isolation of an ouabain-like inhibitor of the sodium pump from human plasma (5) and an attempt to isolate an isomer of ouabain from bovine hypothalamus (6). Nanogram scale CD structural analysis indicates that both substances show similar properties but are not identical with ouabain (7). It has been shown in a number of investigations that both compounds show inotropic effects and increase the contractility of arterial blood vessels (8,9).…”