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The refractory period of the presynaptic Na+ current (INa) of the frog neuromuscular junction before and after the block of the presynaptic delayed rectifier K+ conductance by 3,4-diaminopyridine (3,4-DAP) was studied by the perineurial recording technique. Application of 3,4-DAP 0.45 mM greatly prolonged the refractory period of the last nodes of Ranvier of frog motor axons. Suppression of the repetitive activity caused by 3,4-DAP by 3-aminobenzoic acid ethyl ester (tricaine) 0.46 mM (a local anesthetic) decreased the refractory period back towards normal values. These results indicate that 3,4-DAP impairs conduction of high frequency nerve impulses along the last nodes of Ranvier due to its block of presynaptic K+ conductance. The spontaneous activation of the most excitable, last nerve segments seemed to be the main factor causing such impairment. This phenomenon could explain in part the adverse motor effects shown by some patients treated with high doses of 3,4-DAP., (Copyright 1998 Elsevier Science B.V.)

The 3H-overflow from slices of the rabbit caudate nucleus preincubated with tritiated dopamine (DA), or choline, and then superfused and stimulated twice with 3,4-diaminopyridine (3,4-DAP; 25 microM, 1 min), was explored as an in vitro model for evoked release of DA, or acetylcholine (ACh), respectively. In both cases the 3,4-DAP-evoked 3H-overflow was tetrodotoxin-sensitive and Ca(2+)-dependent and hence most probably represents action potential-induced exocytotic release of DA or ACh, respectively. Using pairs of preferential agonists/antagonists it was shown, that evoked DA release was inhibited via presynaptic D2 autoreceptors (quinpirole/domperidone) and kappa-opioid receptors (U-50488H/norbinaltorphimine). No evidence was found for the presence of presynaptic adenosine A1 or A2 receptors on dopaminergic terminals. Moreover, 3,4-DAP-evoked DA release was unaffected by increased intracellular cyclic AMP levels or by drugs affecting the NO/guanylate cyclase pathway. In a similar manner it was shown that 3,4-DAP-evoked ACh release was inhibited via presynaptic muscarine autoreceptors (oxotremorine/atropine) and dopamine D2 heteroreceptors (quinpirole/domperidone). Again, no evidence for the involvement of the NO/guanylate cyclase system in the modulation of ACh release was found, whereas the presence of inhibitory adenosine A1 receptors, but not of facilitatory A2 receptors, could be clearly established. It is concluded, that 3,4-DAP-evoked 3H-overflow from rabbit caudate nucleus slices preincubated with [3H]DA or [3H]choline, represents a simple and useful in vitro model for action potential-induced DA or ACh release, respectively. Moreover, at least in this model or rabbit brain region, facilitatory adenosine A2 receptors and the NO/guanylate cyclase system seem not to be involved in the release of these transmitters.

We have investigated the participation of the N-type (omega-conotoxin GVIA-sensitive) and L-type (nifedipine-sensitive) calcium channels in the alpha 2-adrenoceptor mediated autoinhibition of the release of [3H]noradrenaline from chick sympathetic neurons in culture. Blockade of 3,4-diaminopyridine-sensitive potassium channels resulted in tetrodotoxin-sensitive and calcium-dependent increase of the release of [3H]noradrenaline evoked by electrical stimulation. Nifedipine attenuated the evoked release under control conditions by 20%, but in the presence of 3,4-diaminopyridine by 51%, while omega-conotoxin decreased the release under control conditions by 87% and in the presence of 3,4-diaminopyridine by only 43%. The L-type calcium channel activator Bay k 8644 increased the evoked release of the transmitter both in the absence and in the presence of 3,4-diaminopyridine. Under control conditions, the alpha 2-adrenoceptor agonist UK 14304 decreased the evoked release by 57% and the alpha 2-adrenoceptor antagonist rauwolscine increased it by 14%. Nifedipine did not prevent this modulation. In the presence of 3,4-diaminopyridine, UK 14304 lost its effect on the release of noradrenaline, but its inhibitory action was restored when nifedipine, but not omega-conotoxin, was added. Changes in the increase of intracellular calcium concentration ([Ca2+]i) evoked by electrical stimulation, measured in the cell processes by microfluorimetry, paralleled the changes in the release of [3H]noradrenaline. Under control conditions, nifedipine attenuated the rise of intracellular calcium by only 16%, while omega-conotoxin did so by 66%. 3,4-Diaminopyridine enhanced the evoked rise of [Ca2+]i; in its presence the rise of intracellular calcium was about equally reduced by nifedipine and omega-conotoxin (by 46 and 36%, respectively). These effects were additive. UK 14304 diminished the peak concentration of [Ca2+]i elicited by the standard electrical stimulation by 31% and rauwolscine antagonised this effect. UK 14304 did not measurably inhibit the stimulation-evoked rise of intraterminal [Ca2+]i in the presence of 3,4-diaminopyridine but it produced an inhibition by 26% if nifedipine had been applied together with 3,4-diaminopyridine. Our observations show that, under control conditions, the stimulated release of [3H]noradrenaline is mainly associated with the opening of N-type channels, while in the presence of 3,4-diaminopyridine the contribution of L-type channels becomes more important. The alpha 2-adrenoceptor stimulation by UK 14304 inhibits the release of [3H]noradrenaline but, in the presence of 3,4-diaminopyridine, the inhibition of release can only be observed if the massive influx through L-type calcium channels is prevented. These data suggest that presynaptic alpha 2-adrenoceptors of chick sympathetic neurons preferentially influence the N-type calcium channels.

The involvement of nitric oxide (NO) in the evoked release of noradrenaline (NA) was studied in rat hippocampal slices preincubated with [3H]NA and stimulated with 3,4-diaminopyridine (3,4-DAP; 200 microM) for 2 min. The 3,4-DAP-evoked [3H]overflow was enhanced by the NO synthase substrate L-arginine, but not by D-arginine; it was reduced by the NO synthase inhibitor NG-nitro-L-arginine, which also antagonized the effects of L-arginine. The corresponding nitro derivative of D-arginine was inactive and unable to block the effects of L-arginine. Also drugs known to produce NO in-vitro, like sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) enhanced the 3,4-DAP-evoked NA release. The NO scavenger hemoglobin showed no significant effects when given alone, but reduced or abolished, respectively, the facilitatory effects of SNP, or SNAP and L-arginine. The cyclic GMP derivatives 8-Br-cGMP and Sp-8-p-chlorophenylthioguanosine-3',5'-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) also acted facilitatory, whereas the corresponding Rp-enantiomer of the latter compound was inactive, but antagonized the effect of Sp-8-pCPT-cGMPS. NA release evoked by 3,4-DAP (10 microM) from rat hippocampus synaptosomes was not affected by L-arginine or NG-nitro-L-arginine but slightly increased by SNAP and Sp-8-pCPT-cGMPS. Antagonists at NMDA, non-NMDA and metabotropic glutamate receptors neither affected the 3,4-DAP-evoked NA release nor the facilitatory effect of L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of Zn2+ on presynaptic currents was investigated on frog cutaneous pectoris nerve-muscle preparations. Nerve terminal spikes were recorded with extracellular electrodes placed in the perineurial sheaths of motor nerves. Zinc ions reversibly suppressed the component of the waveform associated with K+ currents--unmasking an inward current at the terminal--and induced repetitive firing when were applied to preparations perfused with calcium containing solutions. In experiments in which delayed rectifier channels were blocked by 3,4-diaminopyridine, Zn2+ caused a prolonged and reversible inward current associated with a slight decrease in the peak calcium current generated by 3,4-diaminopyridine. Zinc ions abolished the plateau calcium current produced by the simultaneous action of 3,4-diaminopyridine and tetraethylammonium. Finally, in all the preparations in which the exposure to Zn2+ was prolonged it was observed a dramatic and irreversible reduction of the presynaptic currents. These results suggest that Zn2+ has, at least, four different effects on presynaptic currents: (1) blockade of delayed rectifier potassium currents, (2) blockade of calcium-activated potassium currents, (3) blockade of calcium currents and (4) a delayed and irreversible disruption of all ionic conductances of the terminal.

The ability of acute application of the neurotoxicant methylmercury (MeHg) to disrupt the function of presynaptic Ca2+ and Na+ channels at intact neuromuscular junctions was examined using mouse triangularis sterni motor nerves. In Ba(2+)-containing solutions, potential changes arising from Na+ and Ca2+ channel function could be recorded from the perineurial sheath surrounding motor neurons when K+ channels were blocked by tetraethylammonium chloride and 3,4-diaminopyridine. MeHg (100 microM) reduced both Na(+)- and Ba(2+)-dependent components to block within 3-5 min at apparently equivalent rates. Time to block was approximately 7 min after exposure to 50 microM MeHg. In 2 of 5 preparations exposed to 50 microM MeHg, the Ca2+ channel-mediated component was blocked prior to the Na+ channel-mediated component. In the remaining three preparations, Na(+)- and Ba(2+)-dependent potentials were blocked at similar times. Following block by MeHg, neither perfusing the preparation in MeHg-free solutions nor increasing the intensity and/or duration of stimulus to the intercostal nerves resulted in recovery of Na+ or Ca2+ potentials. In the presence of K+ channel blockers, repetitive firing of nerves in response to a single stimulus was observed in 20-30% of the triangularis preparations; in the two preparations treated with MeHg in which repetitive firing was observed, it decreased prior to block of the stimulus-induced Na+/Ba2+ potentials. These results corroborate the results obtained in isolated synaptosomes and pheochromocytoma cells, and suggest that MeHg decreases motor nerve excitability by disrupting Na+ channel function and may block neurotransmitter release by disrupting Na+ and Ca2+ channel function.

We investigated the effects of 3,4-DAP on ACh release from rat striatal slices superfused with or without choline, at rest and during electrical stimulation. In a choline-free medium, 3,4-DAP increased basal and stimulated ACh release while lowering the net efflux of choline; thus while the sum of ACh plus choline released remained constant, the ratio of released ACh to that of choline was increased. The drug failed to affect tissue ACh, choline or membrane phospholipid levels (including those of phosphatidylcholine). In a choline-containing medium, 3,4-DAP potentiated the enhancement by choline of both basal and electrically stimulated ACh release. Electrical stimulation alone increased ACh release from the slices without altering choline efflux or depleting tissue choline or ACh stores; however, this treatment did deplete membranes of phosphatidylcholine and of other major phospholipids. Superfusion of the slices with 3,4-DAP protected the slices from stimulation-induced phospholipid depletion. Calcium-dependent activation of high-affinity choline uptake may underlie the observed effects of 3,4-DAP.

The inhibition by the bis-benzyl-isoquinoline alkaloid tetrandrine of motor terminal calcium currents has been studied using extracellular, perineuronal electrodes in the M. triangularis preparation of the mouse. The calcium plateau current was irreversibly blocked, whereas the fast calcium current remained unaffected. From these results a calcium antagonism on neuronal calcium channels involved in transmitter release at motor nerve terminals is suggested.

4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) when injected intracisternally to anesthetized rats induced qualitatively similar central nervous system stimulant and convulsant effects at equimolar concentrations. Overall penetrability into cerebrospinal fluid of 4-AP is significantly higher than that of 3,4-DAP after single i.v. administration as evaluated by a high-performance liquid chromatographic determination. The present results can account for the lower central nervous system toxicity of 3,4-DAP when compared to 4-AP previously described after systemic administration.

The effects of 3,4-diaminopyridine (3,4-DAP) on isolated sympathetic ganglia were studied by means of intracellular and extracellular recording techniques. 3,4-DAP in micromolar concentrations caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges. Such stimulus-bound repetition (SBR) appeared to represent a presynaptic action of 3,4-DAP, as no repetitive firing could be evoked by antidromic or direct stimulation of ganglion cells. 3,4-DAP also increased the latency to the onset of the synaptic potential at concentrations paralleling those responsible for SBR. The actions of 3,4-DAP on synaptic transmission extended over a 10,000-fold concentration range, beginning with synaptic facilitation at micromolar concentrations (1-500 microM), and proceeding to depressant effects at millimolar concentrations (0.5-10 mM). The postganglionic repetitive discharges (SBR) induced by 3,4-DAP could be selectively suppressed by D-tubocurarine (D-Tc) or tetraethylammonium (TEA), at concentrations of these antagonists below those required to block transmission. 3,4-DAP had significant anti-curare effects, producing a four-fold shift of the D-Tc concentration-effect curve for transmission block. In contrast, 3,4-DAP neither antagonized nor enhanced the transmission block produced by TEA. These results are incompatible with traditional concepts of ganglionic blockade by D-Tc and TEA, and suggest the possibility of presynaptic sites of action in ganglion block.

Injections of quinolinic acid (QUIN) alone or combinations of QUIN with dipicolinic acid, Mg2+ or 3,4-diaminopyridine were made into the nucleus basalis magnocellularis of the rat. Choline acetyltransferase activity in the cortex decreased following injections of QUIN and this was antagonized by co-injections of QUIN with dipicolinic acid or Mg2+. Co-injections of QUIN with 3,4-diaminopyridine increased the neurotoxicity of quinolinic acid, while diaminopyridine alone was not toxic.