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

Abstract

1 Previous studies have reported discrepancies in the potencies of A 1 adenosine receptor agonists at mouse motor nerve terminals. In addition, con¯icting results on the role of protein kinase A (PKA) in mediating the inhibitory e ects of A 1 receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2 CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 mM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 mM) produced highly statistically-signi®cant reductions in spontaneous ACh release. 3 In the presence of the non-selective protein kinase inhibitor, H7 (50 mM), the potency of CADO was increased such that 1 mM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4 Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 mM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory e ect of 1 mM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5 The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A 1 adenosine receptor and that the inhibitory e ects of CADO are independent of cyclic AMP accumulation or PKA inhibition.

The Mechanism for Prejunctional Enhancement of Neuromuscular Transmission by Ethanol in the Mouse

“…Presynaptic modulators of secretion that stimulate the cAMP or Munc13/syntaxin signaling cascades have been shown to produce increases in both evoked and spontaneous ACh release (Shapira et al,1987;Hirsh and Silinsky, 2002;Searl and Silinsky, 2008), effects similar to those described above for ethanol. Moreover, these signal transduction pathways have been implicated in the actions of ethanol (Moore et al, 1998;Lai et al, 2007).…”

“…Activation of the cAMP signaling cascade can enhance evoked and spontaneous neurotransmitter release at skeletal neuromuscular junctions (Hirsh and Silinsky, 2002;Searl and Silinsky, 2008) and does so through phosphorylation via PKA and activation of specific presynaptic proteins that are not phosphorylated by PKA (Searl and Silinsky, 2008). In these experiments, forskolin was used to stimulate adenylate cyclase and hence produce both PKA-dependent and PKAindependent actions of cAMP (Botelho et al, 1988;Ozaki et al, 2000;Seino and Shibasaki, 2005).…”

Adenosine decreases both presynaptic calcium currents and neurotransmitter release at the mouse neuromuscular junction

“…7), or, to determine non‐selective effects of adenosine on ionic currents other than those through P/Q‐type Ca 2+ channels (Figs 4 and 5). It should be stressed that changes in EPP amplitudes after adenosine treatment are due to changes in ACh release and not to changes in postjunctional sensitivity to ACh as MEPP amplitudes are not affected by adenosine in any species studied (Ginsborg & Hirst, 1972; Silinsky, 1980, 1984; Nagano et al 1992; Hirsh & Silinsky, 2002; Hirsh et al 2003).…”

Neurobeachin Is Essential for Neuromuscular Synaptic Transmission

“…Calcium-dependent facilitation of presynaptic acetylcholine release is modulated by PKA activity, and AKAPs have been suggested to modulate presynaptic differentiation and synaptic localization of PKA (Ghirardi et al, 1992;Losavio and Muchnik, 2000;Hirsh and Silinsky, 2002). Intriguingly, RI␣ is colocalized with synaptophysin to presynaptic vesicles (Perkins et al, 2001).…”