Int. J. Biochem. Vol. 20, No. 3, pp. 285-289, 1988 0020-711X/88 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1988 Pergamon Journals Ltd ALTERED Na÷-K+-ATPase, CELL Na ÷ AND LIPID PROFILES IN CANINE ARTERIAL WALL WITH CHRONIC CIGARETTE SMOKING* THOMAS N. TULENKO l, JOSEPH L. RABINOWITZ 2, ROBERT H. COX3 and WILLIAM P. SANTAMORE 3 ~Department of Physiology and Biochemistry, The Medical College of Pennsylvania, Philadelphia, PA 19129, U.S.A. [Tel. (215) 842-7045] 2Veterans Administration Hospital, and Department of Biochemistry, University of Pennsylvania, Philadelphia, PA, U.S.A. 3The Bockus Research Institue, The Graduate Hospital, and Department of Physiology, University of Pennsylvania, Philadelphia, PA, U.S.A. (Received 19 May 1987) Abstract--1. We evaluated the influence of cigarette smoking on arterial wall membranes, using Na+-K+-ATPase activity, free cholesterol (FC) and phospholipid (PL) contents as indices of membrane structural and functional integrity. 2. Segments of aorta, carotid and femoral arteries were obtained from normal dogs (controls) and dogs subjected to chronic cigarette smoking for 2 yr (12 cigarettes a day). 3. Na+-K+-ATPase activity was assessed in segments of carotid and femoral arteries using a ouabain-sensitive 86Rb uptake procedure for intact tissues. 4. Free cholesterol and phospholipids were separated, identified, and quantitated from extracts of aortic samples by means of two dimensional thin-layer chromatography. 5. Na+-K+-ATPase activity was reduced in the smoker group in both carotid and femoral arteries. This reduced enzyme activity was accompanied by a rise in cell Na ÷ levels at both arterial sites. 6. Aortic FC was elevated and the PL profile was altered in the smoker group; as a result, phosphatidylcholine was reduced, whereas lysophosphatidylcholine, phosphatidic acid, and cardiolipin were elevated. 7. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingolipid levels were unchanged. In addition, the FC/PL ratio was increased in the smokers. 8. Taken together, the changes in Na+-K÷-ATPase activity, FC/PL ratio and phospholipid profiles observed are consistent with the hypothesis that chronic cigarette smoking causes a reorganization of the phospholipid bilayer in the smooth-muscle cell membrane of the arterial wall. INTRODUCTION Cigarette smoking has been identified as a significant risk factor linked with a number of arterial wall dis- orders including atherosclerosis (Auerback et al., 1976; Straug and Richards, 1976), aortic aneurysm (Auerback and Garfinkel, 1980), and fibrotic initial thickening (Bylock et al., 1979). In addition to these vascular changes, cigarette smoking produces subtle changes in lipid metabolism and significantly in- creases blood cholesterol and very low-density lipo- protein (VLDL) levels (Brischetti et al., 1983), re- duces plasma high-density lipoprotein (HDL) levels (Bruni et al., 1975), and alters the phospholipid and apoprotein composition of HDL (Berg et al., 1979; Hagarty et al., 1982). Lipoproteins, in turn, are thought to participate in regulating the cholesterol and phospholipid content in peripheral cell mem- branes and, indirectly, the physical state of bio- membranes (Van Deenan, 1981). An important enzyme system associated with the plasma membrane is the Na÷-K+-activated ATPase enzyme. By main- taining Na ÷ and K ÷ gradients across cell membranes, this enzyme system preserves the excitable nature of nerve and muscle cells. In addition, Na+-K÷-ATPase *Supported in part by NIH Grants HL-30496, AG-04908, and HL-28476, and a grant-in-aid from the American Heart Association, Southeastern Pennsylvania Chapter. activity has been frequently used as a marker for plasma membranes and has been followed as a probe for monitoring membrane integrity following alter- ations in the physical state of various biological mem- branes (Zakin and Vassey, 1980). Since Na+-K ÷- ATPase is an integral membrane protein that plays an important role in regulating vascular smooth muscle function, our study was undertaken to evalu- ate Na+-K+-ATPase activity of the canine arterial wall as well as neutral lipid and phospholipid com- position in arteries that is associated with chronic exposure to cigarette smoking. Because intracellular Na ÷ is one of the primary factors determining Na+-K+-ATPase activity, we also determined the cellular Na ÷ concentration. METHODS AND MATERIALS We performed these experiments using arteries from beagle dogs 3.5 yr old that had been exposed for 2 yr to cigarette smoke. Airway tracbeostomies were performed, and they were fitted with tracheostomy collars attached by tubing to a piston-type smoking pump (A. D. Little). Cigarettes containing 1.5 mg of nicotine and 30-35 mg of tar (N.C.I. No. AI) were inserted into the smoking pump. The cigarettes of the test group were lit so that the smoke could be inhaled, but the cigarettes of the control dogs remained unlit. The test animals smoked 12 cigarettes per day for 2 yr (34 ml/puff volume; 2 see duration every 30 sec) and con- tinued to smoke until ! day before they were killed. At the 285