Effects of two putative endogenous digitalis-like factors, marinobufagenin and ouabain, on the Na+, K+-pump in human mesenteric arteries.
OBJECTIVE
Recently, in rat aortae, two putative digitalis-like factors, marinobufagenin and ouabain, were shown to interact with alpha-1 (sarcolemma) and alpha-3 (nerve endings) subunits of the sodium pump, respectively, and elicit vasoconstriction via inhibition of the Na+,K+-pump in vascular smooth muscle or norepinephrine release. The purpose of the present study was to investigate the effects of ouabain and marinobufagenin on vascular tone, activity of Na+,K+-pump, and the expression of isoforms of the Na+,K+-ATPase alpha-subunit in human mesenteric arteries.


METHODS
Arteries were obtained from male patients undergoing surgery due to intestinal adenocarcinoma. Vasoconstrictor effects of both inhibitors were studied in isolated vascular rings. Na+,K+-pump activity was measured using the 86Rb technique. Membrane fractions of sarcolemma and nerve endings plasmalemma were prepared via differential centrifugation of membranes in a sucrose density gradient Specific antibodies to the alpha-1 and alpha-3 subunits of Na+,K+-ATPase were used to detect alpha-1 and alpha-3 isoforms in the membrane fractions by Western blotting.


RESULTS
Marinobufagenin (EC50 = 88+/-15 nmoles/l) and ouabain (EC50 = 320+/-50 nmoles/l) elicited vasoconstriction in mesenteric artery rings. At a concentration of 1 nmol/l, both compounds stimulated the Na+,K+-pump, but inhibited its activity at 10-1000 nmoles/l. No stimulation of the Na+,K+-pump was observed in the presence of 5 micromol/l phentolamine; rather 1 nmol/l of marinobufagenin and ouabain inhibited the Na+,K+-pump by 30% and 13%, respectively. The alpha-1 polyclonal antibody detected alpha-1 isoform in membrane fractions from both sarcolemma and nerve endings. A monoclonal alpha-1 antibody detected the material in sarcolemmal membranes only. The alpha-3 isoform was detected in both membrane fractions by both antibodies, but staining for alpha-3 was more pronounced in the nerve endings.


CONCLUSIONS
These results demonstrate that, in physiologically 'realistic' concentrations, marinobufagenin and ouabain can significantly affect the Na+,K+-pump in human mesenteric artery, and illustrate the importance of interaction of digitalis-like Na+,K+-pump inhibitors with the Na+,K+-ATPase localized to the intravascular adrenergic terminals. Present observations are in accord with the previous data suggesting that marinobufagenin and ouabain display greater affinity to alpha-1 and alpha-3 isoforms of the Na+,K+-pump, respectively, and support the view that the differential responsiveness to endogenous digitalis-like inhibitors is one of the features of alpha-isoforms of Na+,K+-ATPase.
Bagrov
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AY
Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore, Maryland 21224, USA.
Fedorova
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OV
D000818:Animals; D002018:Bufanolides / Q000378:metabolism / Q000494:pharmacology*; D004071:Digitalis Glycosides / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects* / Q000201:enzymology* / Q000502:physiology; D008875:Middle Aged; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D014661:Vasoconstriction / Q000187:drug effects
D002018:Bufanolides; D004071:Digitalis Glycosides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C093896:marinobufagenin; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase