Inhibition of “spontaneous,” notochord-induced, and collagen-induced in vitro somite chondrogenesis by cyclic AMP derivatives and theophylline

Abstract

The present study represents a first step in investigating the possible involvement of cyclic AMP in the stimulation of somite chondrogenesis elicited by extracellular matrix components produced by the embryonic notochord. Dibutyryl cyclic AMP (db-cAMP) at 1.0 m M severely impairs “spontaneous” somite chondrogenesis, i.e., inhibits the formation of the small amount of cartilaginous matrix normally formed by embryonic somites in vitro in the absence of inducing tissues. This inhibition of cartilaginous matrix formation is reflected in a 30–40% reduction in sulfated glycosaminoglycan (GAG) accumulation. 8-Bromo-cyclic AMP also severely inhibits cartilage formation and sulfated GAG accumulation by somite explants. This impairment is limited to cyclic AMP derivatives; dibutyryl cyclic GMP, 5′-AMP, and 2′,3′-AMP have no effect. The inhibitory effect of cyclic AMP derivatives is mimicked by the cyclic AMP-phosphodiesterase inhibitor, theophylline, and potentiated by the addition of both db-cAMP and theophylline. Dibutyryl cyclic AMP and/or theophylline also inhibit the stimulation of cartilaginous matrix formation and sulfated GAG accumulation normally elicited by the embryonic notochord, reducing accumulation to a level similar to that found in somite explants without notochord. The inhibition of chondrogenesis by cyclic AMP in notochord-somite explants appears to result from an inability of somites to respond and not from an effect on the inductive capacity of the notochord, since db-cAMP has no detectable effect on the synthesis of molecules (sulfated GAG and collagen) by the notochord that have been implicated in its inductive activity. Finally, db-cAMP and/or theophylline inhibit the stimulation of somite chondrogenesis normally elicited by purified Type I collagen substrates. Dibutyryl cyclic AMP and theophylline reduce sulfated GAG accumulation by somites cultured on collagen to a level even below that accumulated by somites cultured in the absence of collagen, i.e., on Millipore filters.