Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts

Alterations in receptor‐independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn 2+ , forskolin, and Gpp(NH)p. Analyses were performed 3,6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn 2+ decreases with the age of the culture; the apparent activation constant for Mg 2+ does not. Bimodal activation by Mn 2+ , i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg 2+ , forskolin activation occurs with low‐ and high‐affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low‐ and high‐affinity activation only in the presence of high Mg 2+ . In contrast, the Vmax for forskolin increases with increasing Mg 2+ in all culture ages. Although Gpp(NH)p‐dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg 2+ , low Mg 2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 μM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera‐ and pertussis toxin‐dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.