Differences in sarcolemmal preparations: cell surface material and membrane sidedness.
Two different procedures were employed for the isolation of sarcolemma from the rat heart and the membranes were studied with respect to the presence of cell surface material as well as their functional characteristics. Both hypotonic shock-LiBr treatment method (fraction HL) and sucrose density gradient method (fraction S) yielded membranes enriched 8 to 13 fold with respect to Na+-K+ ATPase and adenylate cyclase activities in comparison to heart homogenate. Cell surface material was demonstrated on the outer surface of the vesicles only in fraction HL with cationic dyes, lanthanum and ferritin, applied either to the isolated fractions or perfused in the heart through coronaries. Fraction HL also had high sialic acid content. ATP independent Ca2+ binding in fraction HL was about 6 times more than that in fraction S which had little sialic acid and showed no cell surface staining with cationic dyes. On the other hand, ATP-dependent Ca2+ binding and Ca2+-stimulated Mg2+ dependent ATPase activities in fraction S were 4 to 6 times higher than those in fraction HL. Epinephrine stimulated adenylate cyclase in fractions HL and S by 24 and 3% whereas ouabain was found to inhibit Na+-K+ ATPase in these fractions by 80 and 10% respectively. A mild treatment of the membranes with deoxycholate to eliminate the semipermeable characteristics or effects of sidedness of the vesicles resulted in an almost complete ouabain inhibition of Na+-K+ ATPase in both fractions. These data suggest that presence of cell surface material as well as membrane sidedness has an important role in in vitro expression of functional characteristics of sarcolemma. It is emphasized that sarcolemmal preparations containing cell surface material will provide information more realistic to the native conditions in situ.
D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000032:analysis; D002462:Cell Membrane / Q000032:analysis; D005293:Ferritins; D006651:Histocytochemistry; D007811:Lanthanum; D008297:Male; D008722:Methods; D009206:Myocardium / Q000648:ultrastructure*; D051381:Rats; D011919:Rats, Inbred Strains; D012508:Sarcolemma / Q000032:analysis* / Q000201:enzymology; D012794:Sialic Acids / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods
D012794:Sialic Acids; D007811:Lanthanum; D005293:Ferritins; D000262:Adenylyl Cyclases; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase