A quantitative assay for lysosomal acidification rates in human osteoclasts

TY - JOUR

T1 - A quantitative assay for lysosomal acidification rates in human osteoclasts

AU - Jensen, Vicki Kaiser

AU - Nosjean, Olivier

AU - Dziegiel, Morten Hanefeld

AU - Boutin, Jean A

AU - Sørensen, Mette Grøndahl

AU - Karsdal, Morten Asser

AU - Henriksen, Kim Steen

PY - 2011/4/1

Y1 - 2011/4/1

N2 - 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.

AB - 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.

KW - Acids

KW - Adenosine Triphosphatases

KW - Animals

KW - Biological Assay

KW - Cattle

KW - Chloride Channels

KW - Chromaffin Cells

KW - Enzyme Inhibitors

KW - Humans

KW - Leukocytes, Mononuclear

KW - Lysosomes

KW - Osteoclasts

U2 - 10.1089/adt.2010.0272

DO - 10.1089/adt.2010.0272

M3 - Journal article

C2 - 21050068

SN - 1540-658X

VL - 9

SP - 157

EP - 164

JO - ASSAY and Drug Development Technologies

JF - ASSAY and Drug Development Technologies

IS - 2

ER -