Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy.
Described is a microscopic spectral imaging approach to monitor pH and Ca2+ simultaneously from combined spectra of multiple ion indicators. Emitted light from a cell is focused onto a grating spectrograph and spectra are imaged with a cooled charge-coupled device camera. The combined spectral output of fura 2 and SNARF-1 was analyzed to follow changes in intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) simultaneously and to correct the Ca2+ signal for concurrent changes in pHi. Responses of individual hamster insulinoma (HIT-T15) cells to effectors of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pHi and transiently increased [Ca2+]i. Removal of NH4Cl induced cytosolic acidification concomitant with either no change or sustained increases in [Ca2-]i. Glucose treatment generally resulted in rapid and sustained increases in both [Ca2+]i and pHi but also heterogeneous pHi and [Ca2+]i responses. Corrections of the fura 2 signal for pH were important for following Ca2+ transitions elicited by NH4Cl but were less important for glucose-induced responses. The spectral imaging microscope provides a sensitive method for simultaneous measurements of pHi and [Ca2+]i in single cells.
Martínez-Zaguilán
R
R
Department of Physiology, University of Arizona, Tucson 85724, USA.
Gurulé
M W
MW
Lynch
R M
RM
D000818:Animals; D001578:Benzopyrans; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D005456:Fluorescent Dyes; D005470:Fluorometry; D016257:Fura-2; D005947:Glucose / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007425:Intracellular Membranes / Q000378:metabolism*; D009284:Naphthols; D012235:Rhodamines; D013997:Time Factors; D014407:Tumor Cells, Cultured
D001578:Benzopyrans; D005456:Fluorescent Dyes; D007328:Insulin; D009284:Naphthols; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride; D006859:Hydrogen; D005947:Glucose; D002118:Calcium; D016257:Fura-2