Extracellular acidosis suppresses endothelial function by inhibiting store-operated Ca2+ entry via non-selective cation channels.
OBJECTIVE
Hypoxia, ischaemia, and exogenous chemicals can induce extracellular and intracellular acidosis, but it is not clear which of these types of acidosis affects endothelial cell function. The synthesis and release of endothelium-derived relaxing factors (EDRFs) are linked to an increase in cytosolic Ca(2+) concentration, and we therefore examined the effects of extracellular and intracellular acidosis on Ca(2+) responses and EDRF production in cultured porcine aortic endothelial cells.


RESULTS
Cytosolic pH (pH(i)) and Ca(2+) were measured using fluorescent dyes, BCECM/AM (pH-indicator) and fura-2/AM (Ca(2+)-indicator), respectively. EDRFs, nitric oxide (NO) and prostaglandin I(2) (PGI(2)) were assessed using DAF-FM/DA (NO-indicator dye) fluorometry and 6-keto PGF(1alpha) enzyme immunoassay, respectively. HEPES buffers titrated to pH 6.4, 6.9, and 7.4 were used to alter extracellular pH (pH(o)), and propionate (20 mmol/L) was applied to cause intracellular acidosis. Extracellular acidosis strongly suppressed bradykinin (BK, 10 nmol/L)- and thapsigargin (TG, 1 micromol/L)-induced Ca(2+) responses by 30 and 23% at pH(o) 6.9, and by 80 and 97% at pH(o) 6.4, respectively. During the examinations, there were no significant differences in pH(i) among the three groups at pH(o) 7.4, 6.9, and 6.4. Extracellular acidosis also inhibited BK-stimulated PGI(2) production by 55% at pH(o) 6.9 and by 77% at pH(o) 6.4, and NO production by 38% at pH(o) 6.9 and by 91% at pH(o) 6.4. The suppressive effects of extracellular acidosis on Ca(2+) responses and NO production were reversible. Propionate changed pH(i) from 7.3 to 6.9, without altering pH(o) (7.4). Intracellular acidosis had no effect on BK- and TG-induced Ca(2+) responses or NO production.


CONCLUSIONS
These results indicate that extracellular, but not intracellular, acidosis causes endothelial dysfunction by inhibiting store-operated Ca(2+) entry, so helping to clarify the vascular pathophysiology of conditions such as ischaemia, hypoxia, acidosis, and ischaemia-reperfusion.
Departments of Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Japan.
Masayoshi
M
Asai
Kazuhiko
K
Takeuchi
Masao
M
Saotome
Tsuyoshi
T
Urushida
Hideki
H
Katoh
Hiroshi
H
Satoh
Hideharu
H
Hayashi
Hiroshi
H
Watanabe
D000138:Acidosis / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D045462:Endothelium-Dependent Relaxing Factors / Q000378:metabolism; D011464:Epoprostenol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology
D045462:Endothelium-Dependent Relaxing Factors; D007473:Ion Channels; D009569:Nitric Oxide; D019284:Thapsigargin; D011464:Epoprostenol; D001920:Bradykinin; D002118:Calcium