Identification of digitalis‐like compounds in human cataractous lenses

Abstract

Human cataractous lens nuclei extract inhibited, in a dose-dependent fashion, [3H]ouabain binding to rat brain synaptosomes and microsomal Na+-and K+-dependent adenosine triphosphate (Na+ , K+-ATPase) activity and interacted with anti-digoxin antibodies. The compounds responsible for these activities, termed digitalis-like compounds (DLC), were also detected in bovine, rat, cat and rabbit, normal, transparent lenses, but the levels were only 0.7-5.4% of the average levels in the cataractous human lenses. DLC from the human cataractous lenses were purified by a procedure consisting of organic extractions and batch chromatography followed by filtration through a 3000 Da cut-off filter and subsequent separations using reverse-phase high-performance liquid chromatography. The presence of DLC in the different fractions obtained in the chromatograms was monitored by their ability to inhibit [3H]ouabain binding and Na+, K+-ATPase activity. Based on chemical ionization mass spectrometry together with ultraviolet spectrometry and biological characterization, it is suggested that new bufodienolides, 19-norbufalin and 19-norbufalin peptide derivatives are responsible for the endogenous DLC activity. It is proposed that these compounds may regulate Na+, K+-ATPase activity in the lens under some physiological and pathological conditions.The Na+-and K+-dependent adenosine triphosphate (Na+, K'-ATPase) is an integral plasma membrane protein responsible for the maintenance of Na+ and K' concentration gradients in all eukaryotic cells. Since Na+, K'-ATPase has a high-affinity receptor for digitalis steroids, it has been postulated that there are endogenous ligands for these receptors which regulate the Na+, K' pump activity. Indeed, based on their ability to inhibit ['Hlouabain binding and Na+, K+-ATPase activity, digitalis-like compounds (DLC) have been shown to be present in the brain [l-31, heart [4] [24]. However, none of these compounds appears to be the natural ligand of the digitalis receptor of the Na+, K+-ATPase because of their limited specificity and affinity. Bufodienolides, which resemble the structure of the plant cardiac glycosides, have been identified in the plasma, brain and other tissues of toads [14, 15, 25, 261, however, their

A Specific Binding Protein for Cardiac Glycosides Exists in Bovine Serum

“…Consistent with this hypothesis, a number of cardiotonic steroids have recently been isolated from various mammalian tissues and fluids (4,5). Among those are the cardenolides ouabain or its isomer (6 -9), digoxin (10), and the bufadienolides 19-norbufalin (11) and marinobufagenin (12,13). Additional, yet unknown, substances seem to exist (14).…”

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

“…Apparently, the type of action of inhibitor A differs from that of ouabain. It is unclear at the present time why several inhibitors of the sodium pump with cardiac glycoside-like action exist (17)(18)(19). Do they address different target isoforms of Na ϩ /K ϩ -ATPase in different target cells?…”

“…Therefore, it was concluded that at least two different types of endogenous inhibitors of the sodium pump in the mammalian body exist (16). In fact, other inhibitors of the sodium pump of the bufadienolide type of cardiotonic steroids have been identified; a substance was isolated from human cataractous lenses that was identified as 19-norbufalin and a peptide derivative thereof (17). A compound similar to the amphibian bufadienolide marinobufagenin was isolated and identified from human urine (18), and a substance from human placentas with a molecular mass of 370 Da was tentatively identified as a dihydropyrone-substituted steroid, possibly a 3␤,14␣,20:21-bufadienolide (19).…”

Endogenous digitalis: pathophysiologic roles and therapeutic applications

“…Initially, bufalinlike immunoreactive material was detected in human bile and plasma. 68-71 Lichtstein et al 13 detected bufalin derivatives in the lenses of several mammalian species by using mass spectroscopy. Other workers 14,15 demonstrated marinobufagenin in mammalian plasma and urine by the use of specific immunoassays, mass spectrometry and, subsequently, nuclear magnetic resonance spectrometry.…”