Two-photon permeabilization and calcium measurements in cellular organelles.

Abstract

Inositol trisphosphate and cyclic ADP-ribose, main intracellular Ca(2+) messengers, induce release from the intracellular Ca(2+) stores via inositol trisphosphate and ryanodine receptors, respectively. Recently, studies using novel messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releasing Ca(2+) from calcium stores in organelles other than endoplasmic reticulum (ER) have been conducted. However, technical difficulties of Ca(2+) measurements in relatively small Ca(2+) stores prompted us to develop a new, more sensitive, and less damaging two-photon permeabilization technique. Applied to pancreatic acinar cells, this technique allowed us to show that all three messengers - IP(3), cADPR, and NAADP - release Ca(2+) from two intracellular stores: the endoplasmic reticulum and an acidic store in the granular region. This chapter describes a detailed procedure of using this technique with pancreatic acinar cells.