The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings

Abstract

1 The importance of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2 Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.pf) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3 The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4 All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5 The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (3O-100,uM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel. 6 H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250pM). Thus H7 is likely to inhibit PK A in frog motor nerve endings. 7 H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release. 8 The results suggest: (i) that the adenylyl cyclase-cyclic AMP-PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP-PK A system modulates rather than mediates the inhibitory effects of adenosine.

Inhibition of spontaneous acetylcholine secretion by 2‐chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction

“…In all 12 experiments, neither 1 mM CADO alone nor 50 mM H7 alone had any statistically-signi®cant e ect on mepp frequency (n=12 experiments, see also Figure 1). Previously, a similar lack of e ect of H7 on ACh release was observed in the frog (Hirsh et al, 1990). However, in the continued presence of H7, 1 mM CADO decreased mepp frequency to 68% of the control value in the experiment of Figure 3, a highly signi®cant decrease in spontaneous ACh release (P50.05).…”

“…H7 did not signi®cantly alter the mean mepp amplitudes (n=12 preparations) in these studies. Thus H7 is unlikely to exert a the type of postjunctional nicotinic receptor/ion channel blocking action in the mouse as was observed in the frog (Hirsh et al, 1990;SebastiaÄ o & Ribeiro, 1990). inhibition of spontaneous ACh release by CADO.…”

“…Both H7 and the selective PKA inhibitor KT 5720 blocked the effect of a membrane permeable cyclic AMP analogue, but only H7 unmasked the inhibitory effect of CADO In the absence of protein kinase inhibitors, the membrane permeable cyclic AMP derivative, (8-4-(cyclopentylthio) cyclic AMP (CPT cyclic AMP; see Hirsh et al, 1990) increased spontaneous ACh release 1.8 fold (n=7 experiments ± compare the ®rst and last columns of Figures 5 and 6). H7 (50 mM, Figure 5, n=6 experiments) blocked the stimulatory e ects of CPT cyclic AMP, suggesting that H7 is inhibiting PKA in our system.…”

“…In order to determine if protein kinases play a role in this system, we investigated the e ects of H7, a widely-used nonselective protein kinase inhibitor (Hidaka et al, 1984) that has also been used in previous studies at the neuromuscular junction (see e.g. Chen et al, 1989;Hirsh et al, 1990;SebastiaÄ o & Ribeiro, 1990). …”

“…In amphibia, the inhibitory e ect of A 1 adenosine receptor activity is not mediated via cyclic AMP or its associated protein kinase A (PKA, Hirsh et al, 1990). In contrast to these results is the report that, in the mouse, pretreatment with the non-selective protein kinase inhibitor H7 prevented the inhibitory e ects of 2-chloroadenosine (CADO) on spontaneous ACh release re¯ected as miniature end-plate potential (mepp) frequency (Chen et al, 1989).…”