Cigarette smokers develop altered erythrocyte membrane composition: an investigation unmasking the role of membrane bound integral protein GLUT 1

Abstract

Erythrocytes in cigarette smokers are prone to oxidative damage. Here, we sought to elucidate the facts behind modifications and possible defense system developed in erythrocyte of cigarette smokers. We observed significant increase in stomatocytes and spherocytes, and osmotic fragility of erythrocyte, along with reduced level of protein thiol and increased fluorescence anisotropy in isolated membrane. Denaturing gel electrophoresis indicated alterations in band 3, band 4.2 and band 4.5. Among those, Glut 1 (i.e. band 4.5), which transports glucose (insulin independent) and dehydroascorbate (DHA), was selectively chosen for its long history in reducing reactive oxygen species (ROS). The increased Glut 1 level in smokers was confirmed by immunoblotting and immunocytochemistry. Furthermore, smokers showed significantly higher glucose uptake in whole blood. The intracellular (Ic) ROS (as indicated by 2′,7′-dichlorofluorescin) was significantly higher in smokers as evidenced by flow cytometric assay. Glucose and DHA alone or together significantly reduced IcROS at higher rate in smokers. However, in presence of Glut 1 specific blocker, phloretin, neither glucose nor DHA could reduce IcROS in both non-smokers and smokers. This confirms that Glut 1 by transporting glucose or DHA attenuates IcROS. Therefore, we conclude that erythrocytes, although altered morphologically, also develop a defense system by upregulating Glut 1 to combat with enhanced Ic oxidative insult in cigarette smokers.

Keywords:

• Cigarette smoking
• erythrocyte membrane
• Glut 1
• dehydroascorbate
• flow cytometry

Acknowledgements

Authors indebted to CRNN, University of Calcutta for providing the access of BD FACS VERSE, to the Central Instrumental Facility of The Bose Institute, Kolkata for fluorescence anisotropy; and also to Ms. Boni Halder, technical officer, DBT-CU-IPLS, central facility of University of Calcutta for cooperating in accessing the confocal laser scanning microscopy. The authors are also grateful to Dr. Sreya Chattopadhyay, Assistant Professor of the department of Physiology, University of Calcutta, and her research fellow, Mr. Sayan Ghosh for helping in immunoblotting.

Disclosure statement

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

The authors are thankful to University Grants Commission India, New Delhi [F. No. 41-92/2012 (SR), dated 13.07.2012] for partially funding the work, and also for providing Rajiv Gandhi National fellowship [F1-17.1/2012-13/RGNF-2012-13-SC-WES-29259] to Jyotirmoy Sikdar. Authors are also thankful to Centre for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta [No. Conv/103/Nano Pr (2011), dated 01.07.2011] for partially funding the work.