THE CELL-SURFACE OF ISOLATED CARDIAC MYOCYTES - A LIGHT-MICROSCOPE STUDY WITH USE OF FLUOROCHROME-COUPLED LECTINS

In cells isolated from guinea-pig or rat ventricular muscle occurrence and distribution of carbohydrate components of the surface coat were monitored using fluorochrome-coupled lectins. Fluorescence of membrane-bound lectins was assayed by an image analysis system. The lectins ConA† † Abbreviations: BSA, bovine serum albumin; ConA, Concanavalin A; DBA, Dolichos biflorus agglutinin; DMSO, dimethyl sulfoxide; EGTA, ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; FITC, fluorescein isothiocyanate; Gal, d-galactose; GalNAc, N-acetyl-d-galactosamine; Glc, d-glucose; GlcNAc, N-acetyl-d-glucosamine; HEPES, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); LFA, Limax flavus agglutinin; Man, d-mannose; met-Glc, methyl-d-glucoside; met-Man, methyl-d-mannoside; NeuNAc, N-acetylneuraminic acid; NeuNGc, N-glycoloylneuraminic acid; PNA, peanut agglutinin; RCA-I, Ricinus communis agglutinin-I; SBA, soy bean agglutinin; TMA-DPH, 1-[4(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene; TRITC, tetramethylrhodamine isothiocyanate; UEA-I, Ulex europaeus agglutinin-I; WGA, wheat germ agglutinin; sWGA, succinylated WGA., WGA, sWGA, LFA and RCA-I showed specific binding to the whole myocyte surface, indicating a homogeneous distribution of α-mannosyl, α-glycosyl, N-acetylglucosaminyl, N-acetylneuraminate and β-galactosyl residues. Binding of DBA and SBA, with specific affinity for N-acetylgalactosaminyl residues, to guinea-pig cardiac myocytes was mainly at the cell poles corresponding to intercalated discs in intact tissue. Both lectins failed to interact with rat myocytes. UEA-I, specific for α-l-fucose, bound slightly to rat and not to guinea-pig myocytes. Binding of PNA to guinea-pig myocytes was observed only after cleaving off sialic acids from cell surface, suggesting that sialic acids mask galactosyl-β(1,3)-N-acetylgalactosamine residues. Specifity of lectin-cell interaction was tested by an inhibition assay where free sugars were tested for their capacity to inhibit lectin binding to the myocytes. When comparing different isolation procedures based on different proteolytic enzymes, the myocytes' affinity to any lectin was found to be qualitatively unchanged. Investigation of lectin-decorated myocytes by means of confocal laser scan microscopy showed that lectin binding sites are not confined to the cell surface but are also present in sarcolemmal invaginations, i.e. transverse tubules. This suggests that the tubular system is lined with a carbohydrate layer similar to, and continuous with, that of the peripheral cell surface. © 1990.