CARBONIC-ANHYDRASE-II MESSENGER-RNA EXPRESSION IN INDIVIDUAL OSTEOCLASTS UNDER RESORBING AND NONRESORBING CONDITIONS

Rabbit osteoclasts can be transformed from a nonresorbing state to a r esorbing state by transferring them from culture medium at pH 7.5 to o ne at pH 6.5. We evaluated whether expression of mRNA for carbonic anh ydrase (CA-II) could be used as an indicator of the state of activity of individual osteoclasts. A cDNA probe to rabbit carbonic anhydrase I I (CB-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 exp ressed higher levels of CA-II mRNA than ''spread'' osteoclasts with si milar numbers of nuclei. When maintained at pH 6.5 for 6 h, the level of CA-LI mRNA was increased significantly in osteoclasts of both morph ologies compared with those in parallel cultures maintained at pH 7.5. These results were confirmed by quantitating CA-II mRNA using the pol ymerase chain reaction (PCR). Oligonucleotide primers specific for rab bit CA-II were synthesized and used to amplify CA-II cDNA transcribed from mRNA prepared from single or small numbers (one to eight cells) o f osteoclasts that were collected with a micromanipulator. This genera ted a similar to 510 bp PCR product, corresponding to the predicted si ze of the CA-II fragment encompassed by the primers. For quantitation, CA-II mRNA levels were compared with the levels of a similar to 900 b p actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture. Th e ratio of CA-II mRNA expression to actin mRNA expression was signific antly increased in osteoclasts cultured at pH 6.5 for 6 h compared wit h osteoclasts maintained at pH 7.5 (1.89 +/- 0.12 versus 0.98 +/- 0.06 , n = 39, mean +/- SEM, of all assays combined; P < 0.001). Our result s demonstrate that CA-II mRNA expression is upregulated in osteoclasts in the resorptive state. The methods used provide a novel molecular a pproach for analyzing osteoclast activity with assays that are applica ble to single cells and obviate the problem of osteoclast impurity, al lowing investigation of osteoclast heterogeneity.