Differential modulation of voltage-activated conductances by intracellular and extracellular cyclic nucleotides in leech salivary glands

Abstract

1. Two-electrode voltage clamp was used to study the effects of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) on voltage-dependent ion channels in salivary gland cells of the leech, Haementeria ghilianii. 2. Intracellular cyclic 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 intracellular injection of cyclic AMP and its dibutyryl and 8-bromo analogues. Cyclic AMP appeared to be the second messenger for the putative neuroglandular transmitter, 5-hydroxytryptamine. 3. Intracellular injection of cyclic GMP specifically potentiated high-voltage-activated (HVA) Ca2+ current and the effect was mimicked by zaprinast, an inhibitor of cyclic GMP-dependent phosphodiesterase. 4. Extracellularly, cyclic GMP and cyclic AMP specifically decreased the amplitude and increased the rate of inactivation of HVA Ca2+ current. These effects of the cyclic nucleotides are identical to those known for extracellular ATP, which activates a presumed purinoceptor. The pyrimidine nucleotide, UTP, was almost equipotent to ATP (threshold dose < 10(-6) M), indicative of a vertebrate-type nucleotide receptor. However, suramin (5 x 10(-5) M), a non-specific P2-receptor antagonist, failed to block the effects of 5 x 10(-6) M ATP (higher suramin doses could not be reliably tested because of the depolarization and increase in membrane conductance produced by the drug). 5. Activation of the putative purinoceptor by ATP did not affect inward rectifier Na+/K+ current which is known to be potentiated by intracellular cyclic AMP and reduced by intracellular cyclic GMP. 6. The preparation may provide a useful model for study of nucleotide actions, and interactions, in channel modulation. It has technical advantages such as large cells (1200 microns in diameter) which lack intercellular coupling and may be individually dissected for biochemical studies.