Effects of the antidiuretic hormone,arginine vasotocin,theophylline, filipin and A23187 on cyclic AMP in isolated frog skin epithelium (Rana temporaria)

A method for measuring cAMP in frog skin epithelium was developed. The epithelia were isolated after collagenase-treatment. cAMP was extracted by boiling water and the extract was purified on dry AI₂O₃. The change with time of the cAMP level after addition of arginine vasotocin (AVT) was studied. The hormone caused a rapid increase in cAMP level with a maximum after 3–5 min, whereafter the cAMP level declined. Incubation with AVT made the epithelia refractory to a second dose of AVT, which indicates that the decline in cAMP level was caused by a feedback mechanism and not by inactivation of the hormone. cAMP appeared evenly distributed in all cell-layers of the epithelia both before and after stimulation with AVT. Theophylline caused a rapid increase in the cAMP level, which remained elevated for at least 45 min. Addition of the ionophore A23187 or of filipin had no effect on the cAMP level. However, in the presence of theophylline, A23187 enhanced the cAMP level, whereas filipin had no effect. Therefore the involvement of cAMP in the action of A23187 has to be considered.