Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.

Protons resolve dual effects of calcium on miniature end-plate potential frequency at frog neuromuscular junctions.

Inhibition of transmitter release by protons (H+) was studied at the frog neuromuscular junction at various extracellular concentrations of calcium ([Ca++]o) and potassium ([K+]o) by recording miniature end-plate potential (MEPP) frequency with the intracellular microelectrode. H+ decreased K+ -stimulated MEPP frequency. A double logarithmic graph of MEPP frequency at 7.5 mM K+ vs. [H+]o yielded a straight line with negative slope. At 10 mM K+, there was a parallel shift to the right of the graph. According to the surface charge model, K+ acts solely to depolarize the prejunctional membrane in accordance with the Nernst equation. By decreasing the prejunctional negative surface charge, H+ decreases K+ -stimulated MEPP frequency by decreasing [Ca++]o at the Ca++ channel. An estimated pKa of 4.20 may represent an acidic site at the Ca++ channel associated with Ca++ influx. As [Ca++]o increased above 1 mM for pH 7.40 and 10 mM K+, MEPP frequency decreased, i.e., the inhibitory component of dual effects of Ca++ occurred. At pH 6.40, the inhibitory component was abolished, unmasking the stimulatory effect of Ca++ on MEPP frequency. Reversal of Ca++ action by H+ could not be explained by surface charge theory alone. A double logarithmic graph of MEPP frequency vs. [K+]o at 8.5-10.5 mM was linear with a slope of 4. There were parallel shifts to the right of this graph for changes in pH from 7.40 to 6.90 and in [Ca++]o from 1 to 2.5 mM. These results are explained on the hypothesis that K+ also acts at an acidic prejunctional site to increase Ca++ -dependent quantal transmitter release. This action of K+ was inhibited by H+ and raised Ca++. Based on kinetic theory, the estimated pKa of the acidic prejunctional K+ site was 6.31. Based on free energy calculations, its cation preference was H+ greater than K+ greater than Ca++.

Potentiation of aminoglycoside-induced neuromuscular blockade by protons in vitro and in vivo.

pH-dependent effects of 100 microM streptomycin and various aminoglycosides were examined at frog (Rana pipiens pipiens) sciatic sartorii in vitro by using the intracellular microelectrode recording technique. pH-dependent effects of streptomycin were also examined on indirectly elicited (nerve-stimulated) and directly elicited sartorius muscle twitches in vitro. Furthermore, in vivo effects of systemic pH alterations on neomycin-induced mortality were examined in Sprague-Dawley rats. There was a direct correlation between aminoglycoside potency and the number of basic groups per drug molecule (r = 0.95). At pH 7.2 and 9.0, the effect of pH on aminoglycoside potency correlated inversely with the pKa of the aminoglycoside (r = -0.98). At 0.7 and 2.2 pH units below 7.2, however, aminoglycoside-induced inhibitions of quantal content, end-plate potential amplitude and the indirectly elicited muscle twitch were potentiated by a pH-dependent mechanism that was independent of the pKa of the aminoglycoside. At these pH values, qualitatively similar drug effects were not observed on miniature end-plate potential amplitude and frequency or the directly elicited muscle twitch. Potentiation of aminoglycoside action was observed on mortality of rats, however, when the pH was 0.1 pH unit below 7.4. Thus, potentiation of aminoglycoside-induced neuromuscular blockade by protons in vitro and in vivo was demonstrated and appears to involve the pH-dependent activity of a prejunctional membrane component that regulates voltage-dependent, Ca++-mediated transmitter release.

Effects of primidone, phenobarbital and phenylethylmalonamide in the stimulated frog neuromuscular junction.

The effects of primidone (1.0 mM), phenobarbital (0.2 mM) and phenylethylmalonamide (PEMA) (1.0 mM) on nerve-stimulated transmitter release (quantal content) were determined for extracellular Ca++ concentrations ([Ca++]0) from 0.4 to 0.8 mM at 1.0 Hz nerve stimulation frequency. At these [Ca++]0, the relationship between the log of quantal content vs. the log of [Ca++]0 is linear. Both primidone and phenobarbital increased quantal content to 171% of controls. These drugs, however, caused parallel shifts of the log-log plot of quantal content vs. [Ca++]0 to the left. Thus, drug effects were not modified by varying [Ca++]0. These drugs were also examined on frequency facilitation. During frequency facilitation, the relationship between the log of quantal content vs. nerve-stimulation frequency (0.5-8.0 Hz) is linear. Both primidone and phenobarbital caused parallel shifts of this plot to the left. These drug effects, therefore, were not modified by nerve stimulation frequency. PEMA did not affect quantal content in either series of experiments. Finally, the sciatic nerve was not stimulated and spontaneous transmitter release was measured. Under these conditions, phenobarbital increased transmitter release in high external K+ (7.5 mM) (1.8 mM Ca++, no Mg++) and in normal K+ (2.5 mM) (1.8 mM Ca++, no Mg++) to the same magnitude (130% of control) in contrast to the reported effects of primidone and PEMA. In conclusion, the effects of primidone, phenobarbital and PEMA were different in the stimulated frog neuromuscular junction.

Primidone but not phenylethylmalonamide, a major metabolite, increases nerve-evoked transmitter release at the frog neuromuscular junction.

The fundamental responses of primidone and phenylethylmalonamide (PEMA), a major metabolite, were investigated electrophysiologically at the frog (Rana pipiens) neuromuscular junction. Concentrations of 0.2 to 1.,0 mM of each drug were used. Primidone significantly increased nerve-evoked transmitter release in a dose-dependent manner up to 186% of control at 1.0 mM concentration, whereas PEMA had no significant effect. In a separate set of experiments in which the sciatic nerve was not stimulated, primidone significantly increased transmitter release in high external K+ (7.5 mM) (no Mg++), but had no significant effect in normal K+ (2.5 mM (no Mg++). The effect of primidone in high K+ diminished in the presence of Mg++ or of decreased Ca++; PEMA also increased the frequency of MEPPs in high K+, but this effect was not sustained and diminished slowly to control values over a period of 50 min. In addition to its predominant presynaptic action, primidone also decreased MEPP amplitude to 79% of control compatible with the relatively small postjunctional depressant action, whereas PEMA had no effect. Propylene glycol, the solvent used for primidone, did not alter the effects of the drug. In conclusion, primidone but not PEMA has a predominant presynaptic action resulting in a dose-dependent increase in nerve-stimulated transmitter release and EPP amplitude.