Mechanisms of hydrogen sulfide (H 2 S) action on synaptic transmission at the mouse neuromuscular junction

Hydrogen sulfide (H2S) is a widespread gasotransmitter also known as a powerful neuroprotective agent in the central nervous system. However, the action of H2S in peripheral synapses is much less studied. In the current project we studied the modulatory effects of the H2S donor sodium hydrosulfide (NaHS) on synaptic transmission in the mouse neuromuscular junction using microelectrode technique. Using focal recordings of presynaptic response and evoked transmitter release we have shown that NaHS (300 lM) increased evoked end-plate currents (EPCs) without changes of presynaptic waveforms which indicated the absence of NaHS effects on sodium and potassium currents of motor nerve endings. Using intracellular recordings it was shown that NaHS increased the frequency of miniature endplate potentials (MEPPs) without changing their amplitudes indicating a pure presynaptic effect. Furthermore, NaHS increased the amplitude of end-plate potentials (EPPs) without influencing the resting membrane potential of muscle fibers. L-cysteine, a substrate of H2S synthesis induced, http://dx.doi.org/10.1016/j.neuroscience.2015.07.036 0306-4522/ 2015 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Address: Department of Human and Animals Physiology, Institute of Fundamental Biology and Medicine, Kazan Federal University, Kremlevskii Street 18, Kazan 420008, Russia. Tel: +7-8432337844. E-mail addresses: gerasimova.el.2011@yandex.ru (E. Gerasimova), julia.lebedevafg@yandex.ru (J. Lebedeva), alv.yakovlev@gmail.com (A. Yakovlev), zefiroval@rambler.ru (A. Zefirov), rashid.giniatullin@ uef.fi (R. Giniatullin), sitdikovaguzel@gmail.com, guzel.sitdikova@ kpfu.ru (G. Sitdikova). Abbreviations: AOAA, aminooxyacetic acid; BAPTA-AM, 1,2-bis(2-am inophenoxy)ethane-N,N,N0,N0-tetraacetic acid tetrakis acetoxymethyl ester; CBS, cystathionine b-synthase; CO, carbon monoxide; CSE, cystathionine c-lyase; EGTA-AM, ethylene glycol-bis(2-aminoethyle ther)-N,N,N0,N0-tetraacetic acid acetoxymethyl ester; EPCs, end-plate currents; EPPs, end-plate potentials; H2S, hydrogen sulfide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MDL 12330A, MDL 12,330A hydrochloride; MEPCs, miniature end-plate currents; MEPPs, miniature end-plate potentials; NaHS, sodium hydrosulfide; NO, nitric oxide; pCPT-cAMP, 8-(4-chlorophenylthio)-adenosine 30,50cyclic monophosphate; RyR, ryanodine receptors; b-CA, b-cyano-lalanine. 577 similar to NaHS, an increase of EPC amplitudes whereas inhibitors of H2S synthesis (b-cyano-L-alanine and aminooxyacetic acid) had the opposite effect. Inhibition of adenylate cyclase using MDL 12,330A hydrochloride (MDL 12,330A) or elevation of cAMP level with 8-(4chlorophenylthio)-adenosine 30,50-cyclic monophosphate (pCPT-cAMP) completely prevented the facilitatory action of NaHS indicating involvement of the cAMP signaling cascade. The facilitatory effect of NaHS was significantly diminished when intracellular calcium (Ca) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) and ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid acetoxymethyl ester (EGTA-AM). Activation of ryanodine receptors by caffeine or ryanodine increased acetylcholine release and prevented further action of NaHS on transmitter release, likely due to an occlusion effect. Inhibition of ryanodine receptors by ryanodine or dantrolene also reduced the action of NaHS on EPC amplitudes. Our results indicate that in mammalian neuromuscular synapses endogenously produced H2S increases spontaneously and evoked quantal transmitter release frommotor nerve endingswithout changing the response of nerve endings. The presynaptic effect of H2S appears mediated by intracellular Ca 2+ and cAMP signaling and involves presynaptic ryanodine receptors. 2015 IBRO. Published by Elsevier Ltd. All rights reserved.