Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study

Abstract

Using high sensitivity fluorescence imaging with shutter speeds ∼600,000 times faster than those of video frames, we have characterized Ca2+ waves within cells in exquisite detail to reveal Ca2+ signaling routes. Polarized neutrophils exhibited a counterclockwise rotating ryanodine-sensitive juxtamembrane Ca2+ wave during temporal calcium spikes. During stimulation with fMLP, a chemotactic factor, two Ca2+ waves traveling in opposite directions around the perimeter of the cell emanated from sites of stimulation (the clockwise wave is verapamil sensitive). Phagocytosed targets exhibit counterclockwise Ca2+ waves traveling about their periphery originating from the plasma membrane. This study: 1) outlines the technology to observe Ca2+ signaling circuitry within small living cells; 2) shows that extracellular spatial information in the form of a chemotactic factor gradient is transduced into intracellular chemical patterns, which provides fresh insights in signaling; 3) suggests that a line of communication exits between the cell surface and phagosomes; and 4) suggests that spatiotemporal Ca2+ patterns contribute to drug actions.

Cutting Edge: Optical Microspectrophotometry Supports the Existence of Gel Phase Lipid Rafts at the Lamellipodium of Neutrophils: Apparent Role in Calcium Signaling

“…Cells were observed using an axiovert fluorescence microscope (Carl Zeiss, New York, NY) (10). A narrow bandpass filter set (Omega Optical, Brattleboro, VT) was used with excitation at 485/22 nm and emission at 530/30 nm for FITC, and an excitation of 540/20 nm and emission at 590/30 nm for tetramethylrhodamine isothiocyanate.…”

“…High-speed microscopy was performed as previously described (10). To detect fluorescence changes in the short wavelength emission region of indo-1, a 355HT15 exciter, a 390LP dichroic reflector, and a 405DF43 emission filter were used.…”

“…This lipid raft-like structure disappears after treatment with m␤CD. This is also the site of Ca 2ϩ wave ignition in polarized neutrophils (10), and propagation of these Ca 2ϩ waves is abrogated by m␤CD. Thus, the existence of large lipid raft-like domains participating in Ca 2ϩ signaling can be directly observed in living polarized neutrophils.…”

Stretch‐activated calcium channels relay fast calcium waves propagated by calcium‐induced calcium influx

“…Fourthly, in a truly remarkable paper, Kindzelskii and Petty (2003) observed 90 µm/s subsurface calcium waves moving around human blood neutrophil cells. Some moved clockwise, as seen from the basalto-apical surface, whereas some moved counterclockwise.…”

“…Cricket 100 Rikmenspoel (1978) Drosophila 150 Bressac et al (1991) Other fruit fly (swimming forward) 100 Baccetti et al (1989) Tenebrio (beetle) (long wave) 400 Baccetti et al (1973b) Aleochara ( Rodent heart myocyte 80 Kaneko et al (2000) In situ 90 Takamatsu et al (1991) Isolated 60 Lipp and Nigglii (1994) 80 Cheng et al (1996) 100 Wussling and Salz (1996) 80 Ishide et al (1990) 100 Trafford et al (1995) 60 Wussling and Mair (1999) Rodent heart endothelial cell 60 Isshiki et al (2004) Mammalian cancer cell line HT 1080 fibrosarcoma 100 Huang et al (2004) HeLa carcinoma 80 Rintoul and Bainbridge (2003) Human blood neutrophil 100 Kindzelskii and Petty (2003) Ferret retina 200 Feller et al (1997) Cultured mouse neuron 200 Charles et al (1996) PC12 neurite 80 Reber and Schindelholz (1996) Secondly, Reber and Schindelholz (1996) observed 80 µm/s calcium waves moving along PC12 neurites towards the growth cone. Although they did not investigate the role of calcium influxes, these neurites are only approx.…”