Simultaneous measurement of Ca2+ and pH by laser cytometry using fluo-3 and SNARF-1.
We present a new convenient method for simultaneous measurement of intracellular calcium concentration ([Ca2+]i) and intracellular pH (pHi) using laser cytometry with a mixture of fluo-3 (for [Ca2+]i) and SNARF-1 (for pHi), with iso excitation (488 nm)-dual emission (530 nm for fluo-3 and > 630 nm for SNARF-1). By using this technique, we measured the changes in [Ca2+]i and pHi in A-431 human epidermoid carcinoma cells and HSG human salivary gland cells stimulated by ATP. We found that alkalization in A-431 cell occurred with the elevation of [Ca2+]i; in contrast, alkalization in HSG cells did not occur at all, even though the elevation of [Ca2+]i was observed. Using BAPTA (a chelating agent of Ca2+) and amiloride (an inhibitor of the Na+/H+ exchanger), we found that the elevation of pHi requires the elevation of [Ca2+]i but that the elevation of [Ca2+]i does not always require a rise in pHi. From our results we conclude that elevation of [Ca2+]i takes precedence over the elevation of pHi in ATP-stimulated signal transduction.
Atsumi
T
T
Department of Oral Physiology, Meikai University School of Dentistry, Saitama, Japan.
Sugita
K
K
Kohno
M
M
Takahashi
T
T
Ueha
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000584:Amiloride / Q000494:pharmacology; D000814:Aniline Compounds / Q000737:chemistry*; D001578:Benzopyrans; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D009284:Naphthols / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D014966:Xanthenes / Q000737:chemistry*
D000814:Aniline Compounds; D001578:Benzopyrans; D002614:Chelating Agents; D005456:Fluorescent Dyes; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; D014966:Xanthenes; C091666:seminaphthorhodaminefluoride; C059715:Fluo-3; D004533:Egtazic Acid; D000584:Amiloride; D000255:Adenosine Triphosphate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium