Fluorescence response in chlortetracycline-loaded neutrophils measures release of Ca2+ from intracellular membrane enclosed storage sites.
Chlortetracycline complexes with di- and trivalent cations resulting in an enhancement of its fluorescence emission intensity. Rabbit peritoneal neutrophils loaded with chlortetracycline gave a fluorescence response, even in the absence of extracellular Ca2+ and Mg2+, by a decrease in fluorescence intensity. The shift in the fluorescence emission maximum to lower wavelengths after the response suggested the response to be due to Ca2+ and not Mg2+ flux. The response was elicited by three mechanisms--a receptor-mediated mechanism by the chemotactic peptide, an ionophore-mediated one by lasalocid, and a detergent-mediated response by digitonin. These observations indicated that the response was due to transport of calcium across membranes in the intracellular compartments and may be physiologically significant. Whereas extracellular Ca2+ did not significantly affect the chemotactic peptide and lasalocid-mediated responses, Ca2+ inhibited the digitonin-mediated responses in a dose-dependent manner possibly due to extracellular Ca2+ flooding the cytosol through the digitonin-permeabilized plasma membrane and equilibrating the Ca2+ gradient across the intracellular membranes. The data collectively indicate that the fluorescence response is due to release of Ca2+ across intracellular membranes from a Ca2+ storage site into the cytosol.
Jacob
J
J
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
D000818:Animals; D002118:Calcium / Q000097:blood* / Q000494:pharmacology; D002751:Chlortetracycline / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007832:Lasalocid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008274:Magnesium; D002118:Calcium; D007832:Lasalocid; D002751:Chlortetracycline