Changes in adenylate cyclase activity during chondrogenesis in cultured limb mesenchyme

Abstract

An in vitro assay to examine the effects of forskolin on adenylate cyclase (AC) activity and chondrogenesis in micromass cultures of mesenchymal cells derived from the distal tip of stage 25 chick limb buds is described. Forskolin activation of AC increased 6 fold in prechondrogenic cells cultured for 48 hours relative to uncultured cells at the time of cell dissociation (Day 0) and remained elevated and unchanged in differentiated cells during the remaining four days of culture. Equivalent increases occurred in cyclic AMP (cAMP) concentrations over the six day period examined. Forskolin also accelerated chondrogenesis in these cultures. Quantitative analysis of Alcian green binding to cartilage matrix showed approximately 2 fold increases in forskolin-treated cultures relative to controls on each of days 2–4 of culture. These results demonstrate that dramatic increases in the total pool of AC activity occur in prechondrogenic mesenchyme prior to overt chondrogenesis which, through increases in cAMP, may play an important role in cartilage differentiation.