Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils.
Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.
Merritt
J E
JE
Department of Cellular Pharmacology, Smith, Kline and French Research Limited, Welwyn, Herts, United Kingdom.
Jacob
R
R
Hallam
T J
TJ
D001572:Benzofurans; D002118:Calcium / Q000097:blood*; D016257:Fura-2; D006801:Humans; D007975:Leukotriene B4 / Q000494:pharmacology; D008345:Manganese / Q000097:blood*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology
D001572:Benzofurans; D010972:Platelet Activating Factor; D007975:Leukotriene B4; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009532:Nickel; D002118:Calcium; D016257:Fura-2