Measurement of intracellular calcium ions and oxygen radicals in polymorphonuclear leucocyte-erythrocyte 'ghost' hybrids

Abstract

Sendai virus induced fusion between rat polymorphonuclear leucocytes and human erythrocyte 'ghosts' containing the Ca-activated photoprotein obelin. This resulted in the production of more than 90% of the cell population as viable hybrid cells, containing active photoprotein. Substances entrapped originally within the 'ghosts' could be transferred to the hybrids as shown morphologically by fluorescence microscopy, and immunologically by the use of antibodies specific for each of the cell types. Resting free Ca2+ in the hybrids was estimated to be 0.1-0.3 microM. The relationship between intracellular Ca2+ within the hybrids and the production by the hybrids of oxygen radicals, as measured by luminol chemiluminescence, was investigated. The Ca ionophore A23187 stimulated both a rise in intracellular Ca2+ and oxygen radical production, the maximum rate of oxygen radical production being dependent upon the intracellular Ca2+ concentration. Complement activation at the cell surface, chemotactic peptide, and concanavilin A stimulated a rise in intracellular Ca2+, each with different characteristics: complement activation increased intracellular Ca2+ to 8 microM for at least 60 sec; chemotactic peptide raised it to approximately 0.6 microM for a prolonged period (at least 10 min) and concanavilin A stimulated a transient (t1/2 = approximately 80 sec) rise to approximately 0.6 microM. Un-opsinized particles, latex beads (diam. approximately equal to 1 micron), which stimulate oxygen radical production, did not produce a detectable rise in intracellular Ca2+. Intracellular EGTA (approximately, 2.5 mM) inhibited both the oxygen radical production and the rises in intracellular Ca2+ induced by chemotactic peptide and concanavilin A, and delayed both the rise in intracellular Ca2+ and the onset of oxygen radical production induced by complement. Intracellular EGTA had no effect on oxygen radical production stimulated by latex particles. An increase in intracellular Ca2+ triggered oxygen radical production by the hybrids in response to the following stimuli: concanavilin A, chemotactic peptide, complement and the Ca ionophore A23187. However, the extent and duration of the increase in cytoplasmic Ca2+ was different for each stimulus, and the apparent relationship between cytoplasmic Ca2+ and oxygen radical production was different for each stimulus. Un-opsinized particles stimulated oxygen radical production without requiring a rise in intracellular Ca2+ concentration.