DIFFERENTIAL MODULATION OF VOLTAGE-ACTIVATED CONDUCTANCES BY INTRACELLULAR AND EXTRACELLULAR CYCLIC-NUCLEOTIDES IN LEECH SALIVARY-GLANDS

1 Two-electrode voltage clamp was used to study the effects of adenosi ne 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) on voltage-dependent ion channels in saliva ry gland cells of the leech, Haementeria ghilianii. 2 Intracellular cy clic AMP specifically blocked delayed rectifier K+ channels. This was shown by use of 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor), forskolin (an activator of adenylyl cyclase) and intracel lular injection of cyclic AMP and its dibutyryl and 8-bromo analogues. Cyclic AMP appeared to be the second messenger for the putative neuro glandular transmitter, 5-hydroxytryptamine. 3 Intracellular injection of cyclic GMP specifically potentiated high-voltage-activated (HVA) Ca 2+ current and the effect was mimicked by zaprinast, an inhibitor of c yclic GMP-dependent phosphodiesterase. 4 Extracellularly, cyclic GMP a nd cyclic AMP specifically decreased the amplitude and increased the r ate of inactivation of HVA Ca2+ current. These effects of the cyclic n ucleotides are identical to those known for extracellular ATP, which a ctivates a presumed purinoceptor. The pyrimidine nucleotide, UTP, was almost equipotent to ATP (threshold dose <10(-6) M), indicative of a v ertebrate-type nucleotide receptor. However, suramin (5 x 10(-5) M), a non-specific P-2-receptor antagonist, failed to block the effects of 5 x 10(-6) M ATP (higher suramin doses could not be reliably tested be cause of the depolarization and increase in membrane conductance produ ced by the drug). 5 Activation of the putative purinoceptor by ATP did not affect inward rectifier Na+/K+ current which is known to be poten tiated by intracellular cyclic AMP and reduced by intracellular cyclic GMP. 6 The preparation may provide a useful model for study of nucleo tide actions, and interactions, in channel modulation. It has technica l advantages such as large cells (1200 mu m in diameter) which lack in tercellular coupling and may be individually dissected for biochemical studies.