Characterization of signaling pathways to Na+/H+ exchanger activation with epidermal growth factor in hepatocytes.
To investigate the signaling pathways to Na+/H+ exchanger activation with epidermal growth factor in hepatocytes, we measured changes in cytosolic free calcium and intracellular pH levels at the single-cell level using digital imaging fluorescence microscopy of fura-2- or BCECF-loaded hepatocytes in primary culture. Epidermal growth factor induced cytosolic free calcium oscillations consisting of periodic trains of spikes with a latency period of up to several minutes. These calcium responses were inhibited by tyrosine kinase inhibitor genistein (100 mumol/L) and abolished by emptying of intracellular Ca2+ pools with 3 mumol/L thapsigargin, an inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum. Epidermal growth factor (1 nmol/L) induced an intracellular pH increase of 0.12 +/- 0.07 units from the basal level of 7.25 +/- 0.09 units after several minutes of latency. This effect was completely abolished by 1 mmol/L amiloride, an inhibitor of the Na+/H+ exchanger. The epidermal growth factor-induced intracellular pH increase was inhibited by pretreatment of hepatocytes with genistein (100 mumol/L), thapsigargin (3 mumol/L) or calmodulin inhibitor W-7 (25 mumol/L), but not with protein kinase C inhibitor H-7 (50 mumol/L) or with cyclic AMP-dependent kinase inhibitor H-8 (60 mumol/L). Phorbol ester PMA (phorbol 12-myristate 13-acetate), a potent activator of protein kinase C, induced a slight intracellular pH increase significantly smaller than that with epidermal growth factor, whereas this effect was completely blocked by pretreatment with H-7, indicating that PMA-induced intracellular pH increase is mediated by protein kinase C pathways, unlike epidermal growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)
Tanaka
Y
Y
First Department of Medicine, Osaka University School of Medicine, Japan.
Hayashi
N
N
Kaneko
A
A
Ito
T
T
Horimoto
M
M
Sasaki
Y
Y
Kasahara
A
A
Fusamoto
H
H
Kamada
T
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D019833:Genistein; D006863:Hydrogen-Ion Concentration; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010656:Phenylephrine / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin
D002147:Calmodulin; D007529:Isoflavones; D007546:Isoquinolines; D010879:Piperazines; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; D013729:Terpenes; D010656:Phenylephrine; D004815:Epidermal Growth Factor; C017967:W 7; D019284:Thapsigargin; D000584:Amiloride; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium