Simultaneous measurement of intracellular ph and ca2+ in insulin
secreting cells by spectral imaging microscopy.
Mart[acute]inez-Zaguil[acute]an, Raul, Mark W. Gurul[acute]e, and
Ronald M. Lynch.
Departments of Physiology, Pharmacology, and Toxicology, University
of Arizona, Health Sciences Center, Tucson, AZ 85724
APStracts 2:0355C, 1995.
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 CCD camera. The combined spectral
output of Fura-2 and SNARF-1 was analyzed to follow changes in
[Ca2+]in and pHin simultaneously, and to correct the Ca2+ signal for
concurrent changes in pHin . Responses of individual Hamster
insulinoma (HIT-T15) cells to effectors of ion homeostasis were
heterogeneous. Treatment with NH4Cl increased pHin and transiently
increased [Ca2+]in. Removal of NH4Cl induced cytosolic acidification
concomitant with either no change or sustained increases in [Ca2+]in.
Glucose treatment generally resulted in rapid and sustained increases
in both [Ca2+]in and pHin, but also heterogeneous pHin and [Ca2+]in
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 pHin and
[Ca2+]in in single cell.
Received 21 April 1995; accepted in final form 29 September 1995.
APS Manuscript Number C224-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 November 95