Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Ionic associations within 460 non-infection urinary stones. A quantitative chemical analytical study applying a new classification.

In addition to standard quantitative wet chemical and inductively coupled plasma atomic-emission spectrometric stone analytic techniques, elemental analysis for the determination of nitrogen, carbon and hydrogen was utilized in this study of 460 category I (non-infection) urinary stone samples from western Saudi Arabia. They were classified according to the percentage composition of detected ions, including trace or minimum amounts. The incidence of uric acid stones (24%) is higher than that reported from western countries but similar to those reported from eastern Europe and other parts of the middle east and most are in the group with the highest uric acid content (UrI4). Oxalate stones are the most common type (61%) and phosphate stones (15%) the least common. The results confirm the reliability of elemental microanalysis and support its use for the quick identification of stones especially those that weigh < 1 mg and are too small for wet chemical analysis. Within the various stone types, however, the ionic associations shown by wet chemical analysis denoted the presence of mutual indirect associations between the characterising ion oxalate and both uric acid and phosphate ions, but no association between the characterizing ion uric acid and phosphate ions. Factors that affect these ionic correlations may influence the processes of stone initiation and type of stone formed.