Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles.
Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.
Seiler
S M
SM
Cragoe
E J
EJ
Jones
L R
LR
D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D009206:Myocardium / Q000378:metabolism*; D011796:Quinacrine / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013347:Subcellular Fractions / Q000378:metabolism
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000408:Alamethicin; D011813:Quinuclidinyl Benzilate; D012964:Sodium; D011796:Quinacrine; D011802:Quinidine