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) Ca21 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-M), a non-specific P2-receptor antagonist, failed to block the effects of 5 x 1O6 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 pm in diameter) which lack intercellular coupling and may be individually dissected for biochemical studies.

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system

“…In the presence of 50 µM PPADS the 2 mM ATP-induced depolarization decreased by 24.8 Ϯ 10.3% (n ϭ 5) of its control amplitude and it was completely suppressed by 200 µM PPADS (n ϭ 7). The putative P2X receptor antagonist suramin (500 µM; Dunn and Blakeley, 1988) itself acted agonistically and depolarized neuropile glial cells by 4.5 Ϯ 3.1 mV (n ϭ 6) as has already been reported for leech neurons (Backus et al, 1994) and salivary gland cells (Everill and Berry, 1995).…”

“…For example, purinoceptors have been identified in Taenia coli (Hourani et al, 1985) and in salivary gland cells of the leech Haementeria ghilianii (Everill and Berry, 1995). In the central nervous system of the medicinal leech extracellular ATP depolarizes annulus erector (AE), anterior pagoda (AP), Retzius, noxious (N), pressure (P) and touch (T) neurons (Backus et al, 1994).…”