Inhibitors of TRP channels reveal stimulus-dependent differential activation of Ca2+ influx pathways in human neutrophil granulocytes.
A pharmacological characterization of Ca(2+) influx pathways in neutrophil granulocytes is problematic because of the lack of specific inhibitors. The activation of transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channels by intracellular adenosine diphosphate ribose (ADPR), well characterized in neutrophils, is reportedly inhibited by 8-bromo-ADPR (8Br-ADPR). TRPM2 is blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA) interfering with the pore, but ACA is as well effective on other transient receptor potential channels, especially transient receptor potential canonical (TRPC) channels. We wished to analyze whether ACA and 8Br-ADPR were suitable probes to demonstrate that different Ca(2+) entry pathways are activated in human neutrophil granulocytes by the receptor-dependent stimuli N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and platelet-activating factor (PAF) and the receptor-independent thapsigargin. Ca(2+)-influx-related increases in [Ca(2+)](i) were calculated by comparing aliquots of fluo-3-loaded neutrophils in the presence and absence of extracellular Ca(2+). Moreover, Mn(2+) quenching was used in fura-2-loaded cells. We compared 8Br-ADPR with ACA. 8Br-ADPR was exclusively effective when Ca(2+) influx (or Mn(2+) quenching) was induced by fMLP; it did not affect influx when PAF or thapsigargin was the stimulus. ACA inhibited Ca(2+) influx significantly more strongly when this was induced by PAF than by fMLP. Moreover, it reduced thapsigargin-induced Ca(2+) influx. The contribution of TRPM2 to Ca(2+) influx in neutrophils strongly depends on the stimulus; it is sizeable in the case of fMLP and minimal in the case of PAF. PAF induces Ca(2+) entry pathways different from TRPM2; the inhibition by ACA suggests the contribution of channels of the TRPC family.
Pantaler
Elena
E
Institut für Physiologie, Medizinische Fakultät der Reinisch-Westfälischen Technischen Hochschule, Pauwelsstrasse 30, Aachen, Germany.
Lückhoff
Andreas
A
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000494:pharmacology; D000814:Aniline Compounds / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D002934:Cinnamates / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008345:Manganese / Q000737:chemistry; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D050053:TRPM Cation Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000494:pharmacology*
C526196:8-bromo-adenosine diphosphate ribose; D000814:Aniline Compounds; D002934:Cinnamates; D005456:Fluorescent Dyes; D010972:Platelet Activating Factor; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D014966:Xanthenes; D062367:ortho-Aminobenzoates; C085901:4-amylcinnamoylanthranilic acid; D000246:Adenosine Diphosphate Ribose; C059715:Fluo-3; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D002118:Calcium