Effects of Bay K 8644 on spontaneous and evoked transmitter release at the mouse neuromuscular junction (original) (raw)
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The Journal of Physiology, 1995
The effects of the calcium channel blockers, funnel-web spider toxin (FTX), w-agatoxin IVA (w-Aga IVA) and w-conotoxin GVIA (w-CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50= 0o02 #l ml-') and w-CgTX (1 #M) but was not affected by w-Aga IVA (0'5/M). When FTX (0 1 #l ml-') was assayed on spontaneous release either in normal Ringer solution or in low Ca2+-high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by-2-fold in both conditions. 3. Presynaptic calcium currents (Ica), measured by the perineurial technique in the presence of 10 mm tetraethylammonium chloride (TEA) and 200 juM BaCl2 to block K+ currents, were blocked by w-CgTX (5 ,UM), partially blocked by FTX (1 1d ml-') and not affected by w-Aga IVA (0 5,UM). 4. The presynaptic calcium-activated potassium current (IK(ca)) measured by the perineurial technique in the presence of 0 F5jM 3,4-aminopyridine (DAP) to block voltage-dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 juM). This current was also blocked by w-CgTX (5 FM) and by CdC12 (200 FM) but was not affected by FTX (1 Fl ml-'). The blockade by w-CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two w-CgTX-sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a w-CgTXand FTX-sensitive population.
British Journal of Pharmacology, 1997
1. The effects of the voltage-dependent calcium channel (VDCC) blockers omega-agatoxin IVA (omega-AgaIVA), omega-conotoxin GVIA (omega-CgTx), omega-conotoxin MVIIC (omega-MVIIC) and omega-conotoxin MVIID (omega-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of omega-AgaIVA and omega-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker omega-AgaIVA (100 nM) and P- Q- and N-type channel blockers omega-MVIIC (1 microM) and omega-MVIID (3 microM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker omega-CgTx (5 microM) was ineffective. 3. The process of release was much more sensitive to omega-MVIIC (IC50 = 39 nM) than to omega-MVIID (IC50 = 1.4 microM). After almost completely blocking transmitter release (quantal content approximately 0.3% of its control value) with 3 microM omega-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of approximately 20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 +/- 0.04 to 4.8 +/- 1.4). 4. Nerve-evoked transmitter release in a low Ca(2+)-high Mg2+ medium (low release probability, quantal content = 2 +/- 0.1) had the same sensitivity to omega-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K(+)-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of omega-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33 degrees C. Conversely, the effect of omega-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10-30 nM) of omega-AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 microM omega-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with omega-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.
Spontaneous acetylcholine release in mammalian neuromuscular junctions
American Journal of Physiology-cell Physiology, 1997
Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca 2ϩ content of nerve terminals. The Ca 2ϩ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca 2ϩ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker,-conotoxin GVIA (-CgTx, 5 µM). On the other hand, nifedipine and-CgTx had no effect on K ϩ-induced transmitter secretion.-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K ϩ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca 2ϩ enters nerve terminals through at least three different channels, two of them (L-and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca 2ϩ-binding molecule-related spontaneous release apparently binds Ca 2ϩ very rapidly and would probably be located very close to Ca 2ϩ channels, since the fast Ca 2ϩ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca 2ϩ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca 2ϩ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by ''bulk'' intracellular Ca 2ϩ concentration increases.
Journal of Neuroscience, 2006
Ca 2ϩ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of Ϫ80 mV in the absence of extracellular Ca 2ϩ elicited Ca 2ϩ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca 2ϩ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca 2ϩ syntillas, which are brief, focal Ca 2ϩ transients, or an increase in global [Ca 2ϩ ]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of Ϫ80 to Ϫ60 mV, in which there was no detectable voltage-gated inward Ca 2ϩ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd 2ϩ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca 2ϩ channels, having no effect on gating charge movement. At 0 mV, the IC 50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca 2ϩ . Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca 2ϩ .
Brain Research, 1990
The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release process was studied in presence of different extracellular Ca 2÷ concentrations, in the mouse phrenic-diaphragm preparation. Hemidiaphragms were incubated for 2 h at room temperature in the presence or absence of TPA. TPA increased the basal frequency of miniature end plate potentials (mepp's) in a dose-dependent manner, resulting in a maximal increase of 280% at a concentration of 0.5/~M. An inverse relationship between extracellular Ca 2÷ concentration and TPA effect was observed: at high extracellular concentrations of Ca 2÷ the action of TPA decreased significatively, while at low Ca 2÷ concentrations the effect of TPA was remarkably augmented. The highest effect of TPA was obtained when tested in a calcium-free medium. TPA also increased mepp frequency stimulated by 10 mM K ÷. As at basal conditions, the effect of TPA was higher at lower concentrations of extracellular calcium. The results suggest that the effect of stimulation of PKC on neurotransmitter release at the mice neuromuscular junction is not exerted at the level of calcium influx to the nerve terminal. Moreover the action of calcium and TPA seems to be superimposed. The effect of K ÷ on neurotransmitter release could be explained not only by depolarization of the nerve terminal but by increasing the pool of activable PKC.