Arch Toxicol (1995) 69: 149-159 9Springer-Verlag 1995 Charles A. Santos-Buch 9 Harry R. Hall 9 Fausto Farfan Irene Orlow 9 Adolfo Firpo 9 Betsy F. yon Kreuter Carl G. Becker Characterization of the tobacco glycoprotein surface binding property of heart and skeletal muscle cells. I. Modulation of the heart cell membrane TGP interaction by anti-TGP IgG Received:3 January / Accepted: 5 July 1994 Abstract Monolayers of L6 rat skeletal myoblast cells formed surface binding isotherms with the purified to- bacco leaf glycoprotein TGPI and the enriched cigarette tar glycoprotein TGP2. Scatchard analysis showed that the binding in the range of the limited concentrations tested was to a single class molecule and the calculated affinity constant (Kd) for TGPt and TGP2 showed similar values (9.78 • 10 -t3 M and 3.09 x 10 -13 M, respectively). The bound TGPs were almost totally displaced by excess non- radiolabeled molecules. The calculated Bmax of the L6 myoblast monolayer was 2.93 fmol for TGPt and 0.217 fmol for TGP2 per 32.2 mm2. Guinea pig heart sarcolemma binding isotherms were also formed with radiolabeled TGPr and TGP2. The interaction of tobacco leaf TGP[ with the heart cell membranes was irreversible because only 15-20% of the bound TGPl was displaced by 100-fold, non-labeled molecules but the interaction of tar TGP2 with heart sarcolemma was reversible and probably saturable. The heart sarcolemma TGP2 affinity constant (Kd) was 5.88 • 10 -7 M and the Bronx, 2.45 x 10 -8 M per 12.5 ktg sarcolemma. Pretreatment of heart sarcolemma with increasing concentrations of leaf TGPl did not dis- place tar TGP2 binding but its absorption on the membrane resulted in increased TGP2 sarcolemma attachment by a complex and unexplained mechanism. Increasing concen- trations of the sera of 10 of 15 guinea pigs (67%) that received mainstream emissions of tobacco smoke from a University of Kentucky cigarette smoking machine for 152 days, displaced cigarette tar TGPz heart cell sarcolemma attachment and this inhibition was significantly different from that produced by the sera of sham smoked and of non- exposed animals (Mann-Whitney test, p = 0.0082). Staphy- lococcus protein A inhibited the displacement of TGP2 produced by the sera of cigarette smoke exposed guinea c. A. Santos-Buch (I:~) Department of Pathology, Cornell University Medical College, 1300 York Avenue, New York, New York pigs and this observation indicated that this action was mediated by IgG molecules. The specific immunoprecipita- tion of a radiolabeled surface epitope of the L6 myoblast monolayers pretreated with TGP1 or TGP2 by immune IgG against TGPz and by the IgG of an antiserum against standard TGP showed that the tobacco glycoproteins attached to a unit polypeptide of the plasma membrane of the muscle cells of approximately 76 kDa. These data support the notion that TGP molecules in cigarette smoke are absorbed systemically on smoking and may have a direct toxic effect when they attach to the surface TGP binding proteins of heart and skeletal muscle cells. Key words Tobacco toxicity - Tobacco glycoprotein Heart and skeletal muscle tobacco binding protein Introduction Cigarette smoke may be toxic to striated muscle. This supposition is supported by the anecdotal observation that athletes shave off seconds from prior timed events shortly after stopping cigarette smoking, often as soon as a few days or weeks of discontinuance. The interaction of cigarette smoke with striated muscle and the heart may also result in clinical dysfunction. For example, there are reports of reduced frequency of episodes of atrial premature depolarization among young persons after quitting cigarette smoking (Bigger 1988). These observations in healthy men and women with no apparent coronary artery disease or other cardiovascular lesion imply that biologically active molecules in cigarette smoke rapidly and reversibly act on the muscle cell. The interaction of cigarette smoke with the heart may result later in serious irreversible injury because significant associations have been found between passive smoking and heart disease (Glantz and Parmley 1991). Moreover, there are data that indicate that cigarette smok- ing may result in cardiomyopathy in absence of obstructive coronary artery disease and this observation may suggest that tobacco molecules could have a direct toxic effect on