Optical Measurement of Changes in Intracellular Calcium

Main Text

Ionized calcium is widely recognized as commonly involved in signal transduction in many cell types, from bacteria to mammalian neurons. The intracellular concentration of calcium plays central roles in cellular processes as diverse as neuronal transmission, exocytosis and secretion, excitation-contraction coupling in muscle, cell-cell communication, enzymatic reactions, modulation of various ion channels, control of luminescence, control of photoreceptor excitation and light adaptation, and bone formation, to name a few. Time-resolved measurement of changes in intracellular calcium is therefore critical to our understanding of cellular and intercellular function. When the article entitled “Rapid Changes of Intracellular Free Calcium Concentration” (1) appeared, direct detection of [Ca²⁺]_i was difficult, the preferred method requiring intracellular injection of the calcium-sensitive photoprotein aequorin (2, 3). As the authors acknowledged, that technique is dogged by difficulties, including slowness of the luminescence reaction between calcium ions and aequorin (4), its uncertain stoichiometry (5), and aequorin’s high molecular weight (6) precluding rapid diffusion. The requisite instrumentation is expensive: a cooled high-voltage photomultiplier tube is usually needed. Advent of the metallochromic indicator dyes arsenazo III and chlorophosphonazo III removed all these limitations: the instrumentation only requires a photodiode.

In the spring of 1974, arsenazo III was suggested as a possible indicator for changes in calcium ion concentration by John Cooper, professor of chemistry at Purdue University, in answer to an inquiry by Larry Pinto, then professor of biology at that university. Arsenazo III is a more sensitive derivative (with a second 2-arsonophenylazo group) of arsenazo I (2-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate, also called uranone or neothorin), which was first synthesized in 1941 by V. I. Kuznetsov and widely used in the determination of uranium (7). Arsenazo III forms colored complexes with, and is used in assays for, many metals (including some of the rare-earth metals). Pinto relayed Cooper’s suggestion to J.E.B., who then ordered arsenazo III from Sigma Chemical Company (St. Louis, MO), dissolved it in 3 M KCl and, in the spring of 1974, pressure injected that solution through a micropipette into a Limulus ventral photoreceptor cell. The injection immediately collapsed the photoreceptor’s sensitivity to light. However, over succeeding minutes, the light sensitivity recovered as the cell successfully pumped down the calcium (8) present in the commercial dye (the dye’s dissociation constant for calcium is much higher than typical intracellular concentration levels of free calcium). The injection of dye freed of contaminating calcium by ion exchange chromatography had no effect on light sensitivity. Because the relatively small photoreceptor cell was a less-studied preparation, Brown et al. (9) decided to attempt using the dye in voltage-clamped squid giant axons where previous studies using aequorin had shown voltage-sensitive calcium entry. Arsenazo III was dissolved in an isotonic solution mimicking squid axoplasm and injected axially over a 1–2-cm stretch into giant axons of Loligo pealei, using a device designed by Caldwell et al. (10). The squid axons were then voltage clamped and studied optically using methods developed for voltage-sensitive dye assays (11). Initial experiments were carried out at the Marine Biological Laboratory in Woods Hole, MA, during the summer season of 1974, and definitive experiments were performed in 1975.

The article by Brown et al. (1) was almost not published in the Biophysical Journal; in 1975, we submitted the article to Nature but, in their wisdom, reviewers opined that optical measurement of intracellular calcium was not of general interest. However, interest did grow: within a year, starting even before the publication of Brown et al. (1), dozens of laboratories used arsenazo III to conduct research on squid axons, giant synapses, synaptosomes, photoreceptors, barnacle and skeletal muscle, red blood cells, leukocytes, platelets, mitochondria, microsomes, fertilized eggs, and the like. A recent Google search for “calcium indicator dye” yielded more than six million entries. Arsenazo III was the first of, and model for, a still evolving series of low-toxicity small-molecule intracellular calcium indicator dyes, which began 5 years later with Tsien’s introduction (12) of the fluorescent calcium chelators based on EGTA [ethylene glycol bis(β-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid] and Bapta [1,2-bis(o-aminophenoxy)ethane-N,N,-N’,N’-tetraacetic acid]. Furthermore, despite the advent of genetically encodable calcium indicators, small molecular probes of rapid changes in intracellular calcium have retained their utility, even finding applications in studies of such medically important conditions as Alzheimer’s disease (13) and malaria (14).

Editor: Vasanthi Jayaraman.