Characteristics of a 5-hydroxytryptamine-sensitive adenylate cyclase in intact and intracellularly perfused squid axons.

1. Cyclic AMP metabolism was studied in intact and intracellularly perfused axons. 2. Cyclic AMP content of intact axons lay within the range 10-100 nmol kg-1 axoplasm. This was increased by exposure to caffeine (2-fold) and to 5-HT (15-fold). The caffeine-sensitive cyclic AMP increase was 30-fold larger in the presence of 5-HT. 3. A reduction in sodium concentration from the sea water bathing intact axons attenuated the 5-HT-evoked increase in cyclic AMP content, but had little effect on resting cyclic AMP. This effect was partially reversed by exclusion of external calcium, and suggests that free calcium plays a role in cyclic AMP homeostasis. 4. Prolonged exposure of intact axons to 5-HT (up to 3 h) led to apparent desensitization of the cyclic AMP response. 5. Intracellular perfusion can be used as a method to study adenylate cyclase in a single axon, simply by measuring the cyclic AMP content of the emerging perfusate. 6. Intracellular perfusion revealed micromolar requirements for internal GTP (K0.5 approximately 1-10 microM) and external 5-HT (K0.5 approximately 1-10 microM); a detailed investigation of this observation was limited due to the progressive loss of 5-HT-evoked adenylate cyclase activity with time. This slow loss was not seen during Gpp(NH)p (guanylylimidodiphosphate), NaF or forskolin activation of cyclase activity. 7. In perfused axons, an increase in intracellular calcium stimulated cyclase activated by 100 microM-forskolin but inhibited cyclase activated by 500 microM-Gpp(NH)p or 10 mM-NaF. A reduction in intracellular magnesium from 10 to 4-5 mM attenuated the effects of 5-HT-evoked cyclase activity. 8. Study of the perfused axon allows characterization of the intracellular requirements of a plasmalemmal transduction system which activates adenylate cyclase, whilst maintaining ionic asymmetry across the cell membrane.