A quantitative assay for lysosomal acidification rates in human osteoclasts.
The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption lacunae. The electroneutrality of the lacunae is maintained by chloride transport through the chloride-proton antiporter chloride channel 7. Inhibition of either proton or chloride transport prevents bone resorption. The aims of this study were to validate the human osteoclastic microsome- based influx assay with respect to lysosomal acidification and assess whether it is a reliable test of a compound's ability to inhibit acidification. Investigated were the expression levels of the lysosomal acidification machinery, the activation of the assay by adenosine triphosphate, H(+) and Cl(-) dependency, the effect of valinomycin, inhibitor sensitivity, and the ion profile of the human osteoclast microsomes. The expression level of chloride channel 7 was increased in the human osteoclastic microsomes compared with whole osteoclasts. Acid influx was induced by 1.25 mM adenosine triphosphate. Further 1.1 μM valinomycin increased the acid influx by 129%. Total abrogation of acid influx was observed using both H(+) and Cl(-) ionophores. Finally, investigation of the anion profile demonstrated that Cl(-) and Br(-) are the preferred anions for the transporter. In conclusion, the acid influx assay based on microsomes from human osteoclasts is a useful tool for detection of inhibitors of the osteoclastic acidification machinery, and thus may aid the identification of effective drugs for osteoporosis that target the acid secretion by osteoclasts.
Nordic Bioscience A/S, Herlev, Denmark.
Vicki Kaiser
VK
Jensen
Olivier
O
Nosjean
Morten Hanefeld
MH
Dziegiel
Jean A
JA
Boutin
Mette Grøndahl
MG
Sørensen
Morten Asser
MA
Karsdal
Kim
K
Henriksen
D000143:Acids; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002417:Cattle; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D019439:Chromaffin Cells / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D010010:Osteoclasts / Q000737:chemistry* / Q000378:metabolism
D000143:Acids; C097540:CLCN7 protein, human; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D000251:Adenosine Triphosphatases