1. The effects were studied of the central neurone P-type Ca2+ channel blockers, w-agatoxin IVA, w-conotoxin MVIIC (polypeptide toxins) and synthetic funnel-web spider polyamine toxin on acetylcholine release from mouse motor nerve. 2. o-Agatoxin IVA decreased the quantal content of endplate potentials and blocked synaptic transmission in the nanomolar range in a reversible manner, whereas the other toxins depressed transmission in the hundred micromolar range. 3. The polyamine toxin, but not the polypeptide toxins, decreased the amplitude of the miniature endplate potential. The increase in the frequency of miniature endplate potentials evoked by high [K+], but not that evoked by a-latrotoxin, was effectively antagonized by wi-agatoxin IVA. 4. In the presence of w-agatoxin IVA, high frequency nerve stimulation produced facilitation of endplate currents and tetanic contractions.5. The results suggest that, under physiological conditions, the Ca2+ necessary for nerve action potential-evoked acetylcholine release is translocated via a subtype of the P-type Ca2+ channel sensitive to w-agatoxin IVA.The biochemical event of neurotransmitter release is initiated by an elevation of intraterminal Ca2c oncentration resulting from rapid Ca2+ influx through voltage-gated Ca2+ channels (Llina's, Steinberg & Walton, 1981). However, differences in channel proteins and dynamic interactions with lipid matrices endow Ca2+ channels with heterologous electrophysiological and pharmacological characteristics. In mammalian neuromuscular junctions the evoked release of acetylcholine (ACh) is resistant to w)-conotoxin GVIA and 1,4-dihydropyridine Ca2+ modulators, which affect N-and L-type Ca2+ channels and neurotransmission in a variety of other neurones (Kerr & Yoshikami, 1984;Sano, Enomoto & Maeno, 1987;Atchison, 1989;Hess, 1990 (Llinas, Sugimori, Hillman & Cherksey, 1992). We compared the effects of three P-channel blockers and a glutamate receptor-channel antagonist on synaptic transmission in a peripheral motor nerve, including the polypeptide toxins w-agatoxin IVA (Mintz, Adams & Bean, 1992) and w-conotoxin MVIIC (Hillyard et al. 1992) and a synthetic funnel-web spider polyamine toxin (sFTX) and argiotoxin. (Argiotoxin is a polyamine from the spider Argiope lobata that retains the sFTX moiety and blocks a glutamate-gated channel; Priestley, Woodruff & Kemp, 1989.) The results indicate that w-agatoxin IVA is a more potent and specific blocker than other toxins and suggest that a subtype of the P-type channel is involved in physiological release of ACh. METHODSNerve-muscle preparation Phrenic nerve hemidiaphragms were isolated from mice (ICR strain,(20)(21)(22)(23)(24)(25)). Mice were killed by a blow to the head followed by exsanguination. Preparations were bathed in Tyrode solution (composition (mM): NaCl, 137; KCl, 2-8; CaCl2, 1.8; MgCl2, 1P1; NaHCO3, 11; NaH2PO4, 0 33; and glucose,
1 Endplate potentials (e.p.ps) were investigated in the presence of geographutoxin II (GTXII) in the mouse phrenic nerve diaphragm preparation. This toxin preferentially blocks muscle Na+ channels which allows the study of e.p.ps in the absence of nicotinic receptor antagonists or substances to depress acetylcholine release. 2 GTXII abolished muscle action potentials and antagonized the depolarization of the muscle membrane produced by the crotamine-induced opening of Na+ channels. 3 E.p.ps as large as 19-25mV were observed after 2-4 ugmlP' GTXII. These concentrations of GTXII did not cause discernible changes of resting membrane potential and frequency and amplitude of miniature e.p.ps. 4 Lower concentrations (1-2pgml-1) of GTXII caused incomplete blockade of the muscle Na+ channel resulting in exaggerated 'e.p.ps', while higher concentrations of GTXII (8pgmlP1) abolished e.p.ps by a prejunctional effect. 5 Trains of e.p.ps on repetitive stimulation after GTXII neither ran down, as in tubocurarinetreated preparations, nor facilitated, as in low Ca2+ and/or high Mg2+-treated preparations, and were indistinguishable from those of untreated cut muscle preparation. 6 In cut muscle preparations, GTXII did not affect the rise and decay times, amplitude or rundown of e.p.ps. 7 It is concluded that GTXII is a useful agent for studying neuromuscular transmission. This method provides e.p.ps which are neither attenuated nor modified because manipulations that alter transmitter release and postjunctional receptor responses are avoided.
1 The Ca2" channel subtypes of the autonomic nerves of guinea-pig atria were elucidated by monitoring the effects of specific Ca2+ channel blockers on the negative and positive inotropic responses associated respectively, with stimulation of the parasympathetic and sympathetic nerves. 2 In left atria paced at 2-4 Hz, the negative inotropic effect induced by field stimulation of parasympathetic nerves (in the presence of propranolol) was abolished by w-conotoxin MVIIC, a blocker of N-type and OPQ subfamily Ca2" channels. .o-Conotoxin GVIA (an N-type blocker), o-agatoxin IVA (a P-type blocker), nifedipine (an L-type blocker) and Ni2+ (a T-and R-type blocker) were much less effective. 3 The positive inotropic response resulting from field stimulation of the sympathetic nerves (in the presence of atropine) was abolished by both co-conotoxins, while co-agatoxin IVA, nifedipine and Ni2+ were ineffective. 4 In the spontaneously beating right atria, the early negative inotropic effect produced by 1,1-dimethyl-4-phenylpiperazinium was abolished by co-conotoxin MVIIC; whereas the late positive inotropic effect was partially reduced, but not abolished, by a high concentration of o-conotoxin GVIA. 5 None of the peptide toxins affected the chronotropic and the inotropic responses evoked by carbachol and isoprenaline. 6 These results suggested that, under physiological conditions, the release of acetylcholine from paraysmpathetic nerves is dominated by an OPQ subfamily Ca2+ channel while that of noradrenaline from sympathetic nerves is controlled by an N-type Ca2+ channel. Ligand-induced noradrenaline release appeared to recruit additional type(s) of Ca2+ channel.
1 Whether the function of the postsynaptic acetylcholine receptor is use-dependently affected by repetitive nerve stimulation in the presence of competitive antagonists was studied in the mouse phrenic nervehemidiaphragm preparation.2 For electrophysiological experiments, the preparation was immobilized by synthetic p-conotoxin, which preferentially blocks muscular Na-channels causing neither depolarization of the membrane potential, inhibition of quantal transmitter release, nor depression of nicotinic receptor function. 3 High concentrations of cobratoxin depressed indirect twitches and endplate potentials (e.p.ps) without inducing waning of contractilities or run-down of trains of e.p.ps evoked at 10-100 Hz. However, waning and run-down were accelerated after washout of the toxin despite diminished postsynaptic receptor blockade. Once the run-down of e.p.ps was produced by washout or low concentrations of cobratoxin, further depression of e.p.p. amplitude with high concentrations of cobratoxin did not attenuate the e.p.p. rundown. 4 The degrees of waning of tetanus and trains of e.p.ps produced by a very high concentration of tubocurarine (20pM) were also less than that caused at a 100 fold lower concentration, albeit the amplitudes of twitches and the first e.p.p. were depressed more rapidly and markedly. 5 Tubocurarine, like cobratoxin, depressed the amplitude of miniature endplate potentials (m.e.p.ps) more than e.p.ps. 6 In contrast to the steepened run-down of successive e.p.ps in the presence of low concentrations of either nicotinic antagonists, the amplitude of m.e.p.ps observed during repetitive stimulation was uniform and was not different from that before stimulation. 7 The results suggest that the e.p.p. run-down and tetanic fade induced by nicotinic antagonists are due to a slow kinetic blockade of presynaptic receptors and confirm that the e.p.p. run-down is not produced by a use-dependent failure of postsynaptic nicotinic receptors. The roles of the presynaptic nicotinic receptor in positive or negative feedback modulations of transmitter release are discussed.
1 Neostigmine (0.5-2pLM) caused fade of tetanic contractions (Wedensky inhibition) evoked by repetitive nerve stimulation. The mechanism underlying this action was studied in intact and cut isolated phrenic nerve-diaphragm preparations of mice. 2 The fade was brought about by failure to elicit muscle action potentials. During fade, the muscle was unable to conduct directly evoked action potentials across the central endplate zone. Recovery of excitability occurred in 5 s with continued stimulation. 3 In the presence of neostigmine, the resting membrane potential at endplate areas during repetitive stimulation decreased from -80 mV to less than -50 mV within the first 10 pulses at 75-200 Hz and thereafter recovered gradually to about -60 mV in the following 5 s during continuous stimulation. 4 The quantal content of endplate potentials evoked by single stimulation was not reduced by neostigmine whereas that evoked by high frequency stimuli (75 Hz) was reduced to about 1/3 in 10 pulses. 5 It is concluded that the fade of tetanic contraction caused by inhibition of acetylcholinesterase is induced by the inactivation of sodium channels in the area surrounding the endplates and that the sustained fade is due to a decrease of transmitter release. Both effects are the result of acetylcholine accumulation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.