Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects.

Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility.

Effects of detyrosinated tubulin on Na+,K+-ATPase activity and erythrocyte function in hypertensive subjects.

Formation of tubulin/Na(+),K(+)-ATPase (NKA) complex in erythrocytes of hypertensive subjects results in a 50% reduction in NKA activity. We demonstrate here that detyrosinated tubulin, which is increased in hypertensive erythrocytes membranes, enhances the inhibitory effect of acetylated tubulin on NKA activity. Moreover, we report a reduced content and activity of the enzyme tubulin tyrosine ligase in erythrocytes of hypertensive subjects. Such alterations are related to changes in erythrocyte deformability. Our findings indicate that the detyrosination/tyrosination cycle of tubulin is important in regulation of NKA activity, and that abnormalities in this cycle are involved in hypertension development.

High glucose levels induce inhibition of Na,K-ATPase via stimulation of aldose reductase, formation of microtubules and formation of an acetylated tubulin/Na,K-ATPase complex.

Our previous studies demonstrated that acetylated tubulin forms a complex with Na(+),K(+)-ATPase and thereby inhibits its enzyme activity in cultured COS and CAD cells. The enzyme activity was restored by treatment of cells with l-glutamate, which caused dissociation of the acetylated tubulin/Na(+),K(+)-ATPase complex. Addition of glucose, but not elimination of glutamate, led to re-formation of the complex and inhibition of the Na(+),K(+)-ATPase activity. The purpose of the present study was to elucidate the mechanism underlying this effect of glucose. We found that exposure of cells to high glucose concentrations induced: (a) microtubule formation; (b) activation of aldose reductase by the microtubules; (c) association of tubulin with membrane; (d) formation of the acetylated tubulin/Na(+),K(+)-ATPase complex and consequent inhibition of enzyme activity. Exposure of cells to sorbitol caused similar effects. Studies on erythrocytes from diabetic patients and on tissues containing insulin-insensitive glucose transporters gave similar results. Na(+),K(+)-ATPase activity was >50% lower and membrane-associated tubulin content was >200% higher in erythrocyte membranes from diabetic patients as compared with normal subjects. Immunoprecipitation analysis showed that acetylated tubulin was a constituent of a complex with Na(+),K(+)-ATPase in erythrocyte membranes from diabetic patients. Based on these findings, we propose a mechanism whereby glucose triggers a synergistic effect of tubulin and sorbitol, leading to activation of aldose reductase, microtubule formation, and consequent Na(+),K(+)-ATPase inhibition.

Involvement of membrane tubulin in erythrocyte deformability and blood pressure.

OBJECTIVE: To test the hypothesis that erythrocyte deformability is influenced by changes in the content of membrane tubulin (Mem-tub). METHODS AND RESULTS: Human erythrocytes contain tubulin distributed in three pools (membrane, sedimentable, soluble). Erythrocytes from hypertensive humans have a higher proportion of Mem-tub. Increased Mem-tub content in hypertensive patients was correlated with decreased erythrocyte deformability. Treatment of erythrocytes from normotensive individuals with taxol increased Mem-tub content and reduced deformability, whereas treatment of hypertensive patients erythrocytes with nocodazole had the opposite effect. In-vivo experiments with rats were performed to examine the possible relationship between Mem-tub content, erythrocyte deformability, and blood pressure. Spontaneously hypertensive rats (SHRs) showed lower erythrocyte deformability than normotensive Wistar rats. During the development of hypertension in SHR, tubulin in erythrocytes is translocated to the membrane, and this process is correlated with decreased deformability. In-vivo treatment (intraperitoneal injection) of SHR with nocodazole decreased Mem-tub content, increased erythrocyte deformability, and decreased blood pressure, whereas treatment of Wistar rats with taxol had the opposite effects. CONCLUSION: These findings indicate that increased Mem-tub content contributes to reduced erythrocyte deformability in hypertensive animals.

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na+, K+-ATPase activity.

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na(+), K(+)-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.