A quantitative assay for lysosomal acidification rates in human osteoclasts
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A quantitative assay for lysosomal acidification rates in human osteoclasts. / Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld; Boutin, Jean A; Sørensen, Mette Grøndahl; Karsdal, Morten Asser; Henriksen, Kim Steen.
In: ASSAY and Drug Development Technologies, Vol. 9, No. 2, 04.2011, p. 157-64.Research output: Contribution to journal › Journal article › Research › peer-review
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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
Y1 - 2011/4
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
VL - 9
SP - 157
EP - 164
JO - Assay and Drug Development Technologies
JF - Assay and Drug Development Technologies
SN - 1540-658X
IS - 2
ER -
ID: 47555645