Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing" conditions.
Rabbit osteoclasts can be transformed from a nonresorbing state to a resorbing state by transferring them from culture medium at pH 7.5 to one at pH 6.5. We evaluated whether expression of mRNA for carbonic anhydrase (CA-II) could be used as an indicator of the state of activity of individual osteoclasts. A cDNA probe to rabbit carbonic anhydrase II (CA-II) was prepared and used for in situ hybridization analysis of osteoclasts isolated from neonatal rabbit long bones. Quantitation by grain counting revealed heterogeneity within the osteoclast population: osteoclasts with a "compact" (rounded, less spread) morphology expressed higher levels of CA-II mRNA than "spread" osteoclasts with similar numbers of nuclei. When maintained at pH 6.5 for 6 h, the level of CA-II mRNA was increased significantly in osteoclasts of both morphologies compared with those in parallel cultures maintained at pH 7.5. These results were confirmed by quantitating CA-II mRNA using the polymerase chain reaction (PCR). Oligonucleotide primers specific for rabbit CA-II were synthesized and used to amplify CA-II cDNA transcribed from mRNA prepared from single or small numbers (one to eight cells) of osteoclasts that were collected with a micromanipulator. This generated a approximately 510 bp PCR product, corresponding to the predicted size of the CA-II fragment encompassed by the primers. For quantitation, CA-II mRNA levels were compared with the levels of a approximately 900 bp actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)
Asotra
S
S
MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Gupta
A K
AK
Sodek
J
J
Aubin
J E
JE
Heersche
J N
JN
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001862:Bone Resorption / Q000201:enzymology*; D002256:Carbonic Anhydrases / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D007668:Kidney / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D018076:DNA, Complementary; D012333:RNA, Messenger; D002256:Carbonic Anhydrases