Activity and expression of the Na+/H+ exchanger in human endothelial cells cultured in high glucose.
Establishing whether high ambient glucose affects the plasma membrane Na+/H+ exchanger is relevant to understanding the adverse effects of high glucose on cell replication and the mechanisms of the increased exchanger activity encountered in diabetic patients with nephropathy. In 8 primary and 15 first-passage isolates of human endothelial cells cultured in 30 mmol/l glucose for 8.7 +/- 2.3 and 15.8 +/- 2.3 days, respectively, we determined Na+/H+ exchanger activity and mRNA levels. Activity was determined by measuring 22Na+ influx in the presence or absence of dimethylamiloride (DMA) after intracellular acidification. We also measured fibronectin mRNA because fibronectin provides signals for cell replication through the Na+/H+ antiporter. Control cells grown in 5 mmol/l glucose showed at morphologic confluency a total Na+ influx (in nmol.mg protein-1.min-1) of 10.1 +/- 3.2 in primary and 11.7 +/- 2.2 in first subculture, which was reduced to 5.3 +/- 0.3 in the presence of DMA. Paired cultures exposed to 30 mmol/l glucose and exhibiting pHi and cell densities identical to controls showed in both primary and first subculture a reduction in total Na+ influx (delta = -0.98 +/- 0.93 nmol.mg protein-1.min-1 p < 0.005) whereas DMA-resistant Na+ influx was identical to that of control. Neither chronic hypertonicity nor acute exposure to high glucose mimicked the effects of chronic high glucose. The level of the Na+/H+ exchanger isoform 1 (NHE-1) mRNA was unchanged by high glucose whereas fibronectin mRNA levels were increased 1.5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
Zerbini
G
G
Endocrine-Hypertension Division, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Roth
T
T
Podestá
F
F
Cagliero
E
E
Doria
A
A
Canessa
M
M
Lorenzi
M
M
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D003928:Diabetic Nephropathies / Q000503:physiopathology; D004232:Diuretics / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D006982:Hypertonic Solutions; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D014471:Umbilical Veins
D004232:Diuretics; D005353:Fibronectins; D006982:Hypertonic Solutions; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D000584:Amiloride; D012964:Sodium; D005947:Glucose