Cholinergic and anticholinergic drug effects on survival during hypoxia: significant gender differences.

We examined central and peripheral components of cholinergic drug protection against hypoxia in male and female mice. Survival times were measured in groups of control and treated (i.p. injection) animals exposed to hypoxia (5% O2/95% N2). Body temperatures were also measured in separate groups of normoxic control and treated animals. Control (NaCl) animals of both sexes survived only 3.5 and 3.7 min of hypoxia. After physostigmine (0.2 mg/kg), however, significantly more females (82.4%) than males (40.5%) survived 35 to 60 min of hypoxia, although physostigmine hypothermia was equal in both sexes. Pilocarpine (5 mg/kg) also produced a gender difference (female > male) in survival, despite equal hypothermia. Hypothermia after neostigmine (0.2 mg/kg) was equal in males and females, yet neither sex survived longer than controls. The protective and hypothermic effects of physostigmine were blocked by atropine sulphate (5 mg/kg). In contrast, atropine methylnitrate (2 mg/kg) did not block physostigmine hypothermia in either sex, but markedly decreased physostigmine's protective effect in females. Beside the significant gender differences in physostigmine and pilocarpine protection, the results show that hypothermia alone is not responsible for protection or for the gender difference. Survival prolongation in males appears to depend solely on physostigmine's central actions. In females, peripheral actions (e.g., hormone release from pituitary and ovary) may contribute to protection and to the gender difference.

Some new observations on caffeine-induced rhythmic hyperpolarization in frog sympathetic ganglion cells.

Unstimulated bullfrog sympathetic ganglia were studied in vitro by intracellular and extracellular recording methods. In 80% of the cells impaled with K citrate microelectrodes, caffeine caused initial hyperpolarization (ICH) followed by rhythmic membrane hyperpolarization (RMH). Four different patterns of rhythmicity were observed, the most common being a regular beating pattern. RMH frequency depended on both caffeine and Ca2+. Tetraethylammonium reduced RMH amplitude, but did not affect frequency. Caffeine effects on cyclic AMP are not responsible for RMH since neither dibutyryl cyclic AMP nor phosphodiesterase inhibitors elicited RMH. However, the anion in the microelectrode filling solution is critical to both the incidence and amplitude of RMH, the order of effectiveness being: citrate much much greater than glutamate, acetate and chloride. In cells impaled by electrodes filled with K thiocyanate or K iodide, caffeine also caused large amplitude hyperpolarizing oscillations of membrane potential, suggesting that the effectiveness of citrate is not due to Ca2+ chelation. High gain extracellular DC recording revealed no sign of caffeine ICH, RMH or any hyperpolarizing effects. The absence of signs of caffeine hyperpolarization with extracellular recording has several interpretations, and these are discussed.

Adsorption of aminopyridines to phosphatidylserine membranes.

Aminopyridines belong to the class of compounds which facilitate synaptic transmission at low calcium concentration, an effect associated with the block of K+ channels, enhanced entry of calcium into presynaptic terminals and greater release of transmitter. We have measured the zeta-potential of phosphatidylserine vesicles in the presence of aminopyridines and some related compounds in order to relate the strength of association of the aminopyridines with their biological effectiveness. The dependence of zeta-potential on the concentration of aminopyridines was analyzed in terms of the Langmuir-Stern-Grahame adsorption model. The rank order of the association constants (in M-1) obtained in the study was as follows: 3,4-diaminopyridine (6.5), 4,5-diaminopyrimidine (3.8), 4-aminopyridine (2.6), 3-aminopyridine (1.8), 2-aminopyridine (1.6), 4-dimethylaminopyridine (0.5), 4-aminopyridine methiodide (0.2), and, as control, calcium (12.1). The comparison of association constants with published results of the electric potential maps obtained by the CNDO/2 method suggests that binding to phosphatidylserine membrane increases with the density of excess charge on the protonated aminopyridine ring. We find that the sequence of potencies of aminopyridines in blocking K+ channels, in releasing transmitter, and in the shifts of calcium concentration dependence of synaptic transmission are about the same as the sequence of association constants with the phosphatidylserine membrane. Assuming that the binding domain for aminopyridines in the presynaptic terminal has similar adsorption properties as the phosphatidylserine membrane, we estimate the electric potential difference between the domain and the external solution to be between -300 and -340 mV.

Selective drug action on two Ca2+ uptake processes in normal and hyperexcitable presynaptic membrane.

Synaptic transmission in the bullfrog sympathetic ganglion in vitro reflects Ca2+-dependent presynaptic and Na+-dependent postsynaptic membrane excitabilities. Aminopyridines (e.g., 3-AP, 4-AP,3,4-DAP) produce Ca2+-dependent presynaptic membrane hyperexcitability (increased transmitter release), evident as synchronized, stimulus-bound repetitive postsynaptic spike responses (SBR) to each single preganglionic stimulus (0.1 Hz). The SBR induced by 3,4-DAP is selectively eliminated as the normal [Ca2+]0 of 1.8 mM is reduced to 0.6 mM, whereas failure of the primary spike response begins only at 0.33 mM [Ca2+]0 and is complete at 0.07 mM. These differences in Ca2+ dependence suggest that two separate presynaptic Ca2+ uptake processes are involved in transmission (the primary spike) and in SBR. The authors' present work with Ca2+-channel blockers (CCBs) reinforces the preceding evidence for quantitatively separate presynaptic Ca2+ uptake processes. Thus, aminopyridine-induced SBR is also selectively abolished by verapamil (V) or diltiazem (D). From threshold to complete abolition of SBR the effective CCB ranges are: V,0.04-0.15 mM; D,0.01-0.08 mM. Higher concentrations are required to block synaptic transmission (the primary spike), the effective CCB ranges being: V,0.25-0.75 mM; D,0.1-0.4 mM. The CCBs thus display considerable concentration selectivity in stabilizing the hyperexcitable presynaptic membrane and in depressing its normal excitability. This is fully analogous to the authors' earlier work, in which SBR induced by physostigmine or 3,4-DAP was selectively abolished by d-tubocurarine or lidocaine concentrations below transmission-blocking levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic facilitation by 3-aminopyridine and its antagonism by verapamil and diltiazem.

The effects of 3-aminopyridine (3-AP) on synaptic transmission in bullfrog sympathetic ganglion were studied in normal Ringer's solution and during graded reductions in extracellular Ca++ by means of intra- and extracellular recording techniques. 3-AP caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges (SBR). In the absence of 3-AP, synaptic transmission, measured as the amplitude of the postganglionic compound action potential, failed progressively as Ca++ was reduced from 1.8 to 0.47 mM. This Ca++ dependence curve of synaptic transmission was shifted to the left (lower Ca++) by 3-AP in dose-related fashion, with maximum shift (4- to 5-fold) at 1 mM 3-AP. The magnitude of the maximum shift produced by 3-AP was precisely the same as that produced by 3,4-diaminopyridine and 4-aminopyridine. Although 3-AP could prevent transmission failure at otherwise suboptimal Ca++ levels, its ability to generate SBR failed progressively as Ca++ was reduced from normal (1.8 mM) to 0.5 mM. Thus, there was a wide difference between the Ca++ dependence domains of synaptic transmission and of SBR in the presence of 3-AP. To confirm this difference in Ca++ dependence domains by a method other than reduction of [Ca++]0, we investigated the interactions between 3-AP and two Ca++ entry blockers, verapamil and diltiazem. 3-AP SBR was abolished by verapamil and by diltiazem at concentrations significantly below those required to block synaptic transmission in the presence of 3-AP. The results thus demonstrate a competitive interaction between aminopyridines and Ca++ entry blockers and further confirm the Ca++ dependent nature of the synaptic actions of aminopyridines.

DMSO effects on synaptic facilitation and calcium dependence in bullfrog sympathetic ganglion.

Synaptic transmission in the bullfrog sympathetic ganglion was studied by means of extra- and intracellular recordings. DMSO (3-10%) caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges. DMSO also partially reversed a preexisting transmission failure in low Ca2+ medium. Ganglia were exposed to gradual reductions in extracellular Ca2+, in the absence and in the presence of DMSO. The recorded amplitude of the postganglionic compound action potential (CAP) was plotted as a function of Ca2+ concentration. In the absence of DMSO transmission failed progressively as Ca2+ was reduced from 1.8 to 0.47 mM but DMSO (3% and 10%) shifted the curve of transmission failure to the left (lower Ca2+ concentration). DMSO inhibits ganglion cholinesterase activity, but this is not the mechanism of its facilitatory effect on Ca2+ entry, since physostigmine did not shift the curve of transmission failure in low Ca2+ to the left. The data suggest that DMSO maintained transmitter release in low Ca2+ by a direct, nonspecific enhancement of Ca2+ influx into the presynaptic nerve terminal.

Effects of several aminopyridines and analogs on the calcium dependence of synaptic transmission.

The effects of 4-aminopyridine (4-AP), 4-AP methiodide (4-APMI), 3,4-diaminopyridine (3,4-DAP), 4,5-diaminopyrimidine (4,5-DAPM) and 4-dimethylaminopyridine (4-DMAP) on synaptic transmission in isolated frog sympathetic ganglia were examined by means of extracellular recording. Ganglia were exposed to graded reductions in extracellular Ca++, from the normal 1.8 mM in the absence and in the presence of drugs while recording amplitudes of the transmitted postganglionic compound action potential. The amplitudes of compound action potential relative to those in normal Ca++ were then plotted as a function of log Ca++ concentration. In the absence of drugs, transmission failed progressively as Ca++ was reduced from 1.8 to 0.47 mM. However, the curve of the compound action potential amplitude vs. log Ca++ was shifted to the left (lower Ca++) by all drugs in a dose-dependent fashion. The drugs varied in effectiveness in shifting the Ca++ dependence curve to the left, the order being: 3,4-DAP greater than or equal to 4-AP greater than or equal to 4,5-DAPM greater than 4-APMI greater than 4-DMAP. 4-AP, 3,4-DAP and 4,5-DAPM were also able to generate repetitive postganglionic discharge in response to a single orthodromic stimulus. All drugs, except 4,5-DAPM, were able to block synaptic transmission at higher concentrations, and their blocking potencies were in the following order: 4-DMAP greater than or equal to 4-APMI greater than or equal to 3,4-DAP greater than or equal to 4-AP.(ABSTRACT TRUNCATED AT 250 WORDS)

NaI prevents and reverses transmission failure in low Ca2+ Ringer.

Synaptic transmission in the bullfrog sympathetic ganglion fails progressively as Ca2+ is reduced from the normal (1.8 mM) to 0.47 mM in an NaCl Ringer. This Ca2+ dependence of synaptic transmission is shifted to the left (lower Ca2+ levels) when NaCl is replaced by NaI. Replacement of NaCl by NaI also reverses pre-existing transmission failure in low Ca2+. As reported previously by us, NaI also causes repetitive postganglionic responses to each single preganglionic stimulus. It is of interest that iodide's ability to cause repetitive postganglionic responses and to shift the Ca2+ dependence of synaptic transmission duplicate the effects of aminopyridines, notably 3,4-diaminopyridine.

Synaptic facilitatory and depressant actions of 3,4-diaminopyridine: correlation with anticurare properties.

This study aimed to define the complete concentration-effect relationship for anticurare effects of 3,4-diaminopyridine (3,4-DAP) in the isolated sympathetic ganglion of the bullfrog. Synaptic transmission was monitored by extracellular and intracellular recordings of the postganglionic response to preganglionic stimulation. A previous study showed that in the bullfrog sympathetic ganglion 3,4-DAP caused stimulus-bound repetitive postganglionic responses (SBR) to each single preganglionic stimulus. The concentration-effect relationship for 3,4-DAP-induced SBR was bell-shaped, and the descending limb of the curve reflected progressive suppression of SBR while normal synaptic transmission was maintained. In the present study a detailed concentration-effect analysis of 3,4-DAP's anticurare action also resulted in a bell-shaped curve nearly congruent with that for SBR. SBR and anticurare effects of 3,4-DAP therefore occupy a common concentration-effect domain, and this suggests that a common mechanism (increased transmitter release) may account for both effects.

Synaptic transmission in low extracellular calcium is preserved by 3,4-diaminopyridine.

Synaptic transmission in the isolated bullfrog sympathetic ganglion was studied during graded reductions in extracellular Ca++, from the normal of 1.8 mM, in the absence and in the presence of different concentrations of 3,4-diaminopyridine (3,4-DAP). In drug-free Ringer's synaptic transmission, measured as the amplitude of the postganglionic compound action potential, failed progressively as Ca++ was reduced from 1.8 to 0.47 mM. This Ca++-dependence curve of synaptic transmission was shifted to the left (lower Ca++) by 3,4-DAP in dose-related fashion with threshold at 0.1 microM and maximum shift at 10 microM 3,4-DAP. At maximum shift (4- to 5-fold) in the Ca++-dependence curve, compound action potential amplitude was normal at 0.33 mM Ca++ then failed progressively as Ca++ was reduced to 0.12 mM. Also 3,4-DAP causes stimulus-bound repetitive postganglionic responses (SBR) to single preganglionic stimuli (Apatoff and Riker, 1982). SBR were selectively abolished as Ca++ was reduced form 1.8 to 0.47 mM. The data reveal that 3,4-DAP facilitates presynaptic influx or binding of Ca++. Furthermore, the high Ca++ requirement for 3,4-DAP-induced SBR, as well as the difference between threshold drug concentrations for preserving transmission (0.1 microM) and for generating SBR (2-5 microM), lead to the speculation that there may be two presynaptic receptors for 3,4-DAP.

N-ethylmaleimide effects on synaptic transmission in frog sympathetic ganglion.

In the isolated bullfrog sympathetic ganglion N-ethylmaleimide (NEM) produced time- and concentration-dependent effects that were irreversible by bathing in drug-free Ringer's solution. Initially, synaptic transmission was facilitated; this was evident as augmented action potential amplitude, repetitive synaptic firing and partial reversal of low [Ca++] transmission failure. Subsequently, transmission was depressed progressively to the point of complete block. Conduction in B and C fiber groups of the preganglionic nerve trunk was also blocked by NEM, without initial facilitation, but at a rate slower than the block of synaptic transmission. NEM had no effect on the amplitude of ganglion cell membrane depolarization produced by acetylcholine, suggesting that its effects were not exerted on postsynaptic nicotinic receptors. None of the effects of NEM were produced by the saturated analog N-ethylsuccinimide. Facilitatory effects of NEM may be attributable to inhibition of Na+ inactivation, or to increased entry of Ca++, at presynaptic nerve terminals. Because the late depressant stages of NEM action involve multiple sites (presynaptic, postsynaptic and axonal), the clarification of possible mechanisms will be a difficult experimental problem.

Effects of dithiothreitol, 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide on synaptic transmission at sympathetic ganglion cells of frog.

The effects of disulfide bond and sulfhydryl group reagents on synaptic transmission were studied in the bullfrog sympathetic ganglion. Ten millimoles of dithiothreitol (DTT), a disulfide bond reducing agent, decreased the amplitude of the transmitted postganglionic compound action potential (CAP) to 41% of control. More than one hour wash-out of DTT with normal Ringer's solution did not reverse the block, but 1 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), an oxidizing agent, rapidly restored the compound action potential amplitude. N-Ethylmalemide (NEM), an alkylating agent, added before DTNB, prevented the restoration of transmission by DTNB. Block of transmission induced by dithiothreitol was also reversed rapidly by 50 microns 3,4-diaminopyridine (3,4-DAP), to 87% of control compound action potential amplitude, and subsequent addition of DTNB restored transmission completely and this was accompanied by the stimulus-bound repetitive firing (SBR) which diaminopyridine produced in otherwise untreated ganglia. One to 3 mM dithiothreitol decreased the amplitude of excitatory post-synaptic potentials (EPSP), and greater concentrations slightly depolarized the cell membrane and decreased membrane resistance. Washout with Ringer's solution reversed the depolarization, but not the block of excitatory postsynaptic potentials. The amplitude of excitatory post-synaptic potentials was restored to threshold by 1 mM DTNB, which also slightly increased membrane resistance and the resting potential. It is concluded that the actions of dithiothreitol and DTNB in the bullfrog sympathetic ganglion are generally similar to those previously observed in other junctional tissues. Interactions between these drugs and 3,4-diaminopyridine in the present study also raise the question whether dithiothreitol and DTNB have any presynaptic effects.

The actions of 3,4-diaminopyridine in bullfrog sympathetic ganglia.

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.

A study of the actions of bromide on frog sympathetic ganglion.

The effects of sodium bromide on the bullfrog sympathetic ganglion were studied by extracellular and intracellular recording techniques. Equimolar replacement of sodium chloride (112 mM) by sodium bromide in Ringer's solution caused hyperpolarization (means = 7.4 mV) of ganglion cells, and antidromically evoked spikes showed increased rates of rise as well as prolonged post-spike positivity. These effects, rapid in onset, returned to control values after approximately 30 min of continued exposure to bromide Ringer. Orthodromic synaptic transmission was not impaired by bromide (84-112 mM); instead it produced increased post-spike negativity and occasionally, stimulus-bound repetitive postganglionic responses (SBR) to each single preganglionic stimulus. This synaptic enhancement persisted as long as exposure to bromide continued. Since ethanol also causes responses, its interaction with bromide was tested and pronounced synergism was found in which 95% of ganglion cells displayed repetitive orthodromic responses to each single preganglionic stimulus. The present and previous studies suggest that bromide and ethanol act at the unmyelinated presynaptic nerve terminal to generate stimulus-bound repetitive responses. The transient nature of the hyperpolarization produced by bromide argues against a mechanistic role in its anticonvulsant action. The persistent synaptic excitatory effects of bromide, however, may have implications for the role of chloride channels in anticonvulsant mechanisms or in epileptogenesis.

A comparative study of the effects of phenytoin and phenobarbital on electrically induced maximal seizures in frogs and mice.

This study was undertaken to compare seizure patterns and anticonvulsant effects of phenytoin in frogs (Rana pipiens) and the CF-1 strain of mice. Maximal seizures were induced with electroshock via corneal electrodes, and phenytoin anticonvulsant effects were determined by measuring the duration of tonic hindlimb extension (THE) and prevention of the THE. To control for any possible species difference that might be unique to phenytoin, the anticonvulsant effects of phenobarbital also were compared in frogs and mice. It was found that the dose-effect relationships for shortening THE duration and for prevention of THE by phenobarbital in frogs and mice were not significantly different in terms of slopes and potencies. With phenytoin, however, the dose-effect relationship for prevention of THE in mice was significantly steeper than that in frogs, whereas the slopes of the dose-effect relationships for shortening THE duration were not significantly different in these two species. Frogs decapitated 1.5 sec after electroshock exhibited THE durations equal to those in intact frogs, and phenytoin and phenobarbital efficacies in shortening THE duration were unchanged by decapitation. These results show that shortening of THE duration by both phenytoin and phenobarbital may reflect drug action in spinal or peripheral neural pathways. Also, it is suggested that phenytoin, but not phenobarbital, prevents THE by a selective action on cortical structures which are well developed in mice but are poorly developed or absent in frogs.

Dose-response analysis of phenytoin on electrically induced seizures and spontaneous activity of cerebellar purkinje cells in the frog.

The hypothesis that phenytoin exerts its anticonvulsant effect by increasing the spontaneous firing rate of cerebellar Purkinje cells was tested in frogs (Rana pipiens). Time-dose-effect relationships were first established for the anticonvulsant effect of phenytoin in intact frogs. Maximal seizures were induced by corneal electroshock (MES), and phenytoin was injected into the ventral lymph sac. At the time of peak effect (3 h), 20-40 mg/kg phenytoin protected 60-80% of frogs against tonic hindlimb extension (THE). With this functional data base, experiments were then undertaken to test the effect of phenytoin on Purkinje cell firing rates. Phenytoin was injected into the ventral lymph sac 30-45 min prior to anesthesia with tricaine. The cranium was opened, the dura mater overlying the cerebellum was removed, and the frog was then curarized. Single-unit extracellular recordings from Purkinje cells were made with NaCl-filled glass micropipettes 2-6 h after phenytoin injection, the expected time of maximum anticonvulsant effect. Effective anticonvulsant doses (20-40 mg/kg) of phenytoin produced no alteration in the spontaneous firing rates of cerebellar Purkinje cells compared to the rates in solvent-injected controls. Consequently, the hypothesis that the anticonvulsant effect of phenytoin is mediated by an action on Purkinje cell firing rates is not supported by the results of this study.

Phenytoin antagonism of electrically induced maximal seizures in frogs and mice and effects on central nervous system levels of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate.

Phenytoin antagonized the electroshock-induced increase in levels of cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) in cerebrum and cerebellum, respectively, from CF-1 mice. However, the effective dose range of phenytoin for significant reduction of the elevated levels of cAMP and cGMP was 2 to 5 times higher than that for prevention of tonic hindlimb extension in 95% of mice. The effective dose range of phenytoin for alteration of cyclic nucleotide levels was nearer to that for preventing tonic flexion and clonus; endpoints which are less relevant to anticonvulsant efficacy than is prevention of tonic hindlimb extension. Also , the greatest reduction in cyclic nucleotide levels occurred at a dose (100 mg/kg) which produced toxic signs in mice. Quaking mice (qk/qk), a mutant strain which exhibits spontaneous seizures, did not have abnormal levels of cAMP or cGMP in cerebrum or cerebellum, and a dose of phenytoin (15 mg/kg) which abolished all seizure activity did not alter levels of these cyclic nucleotides. In frogs, the electroshock-associated increase in levels of cAMP in the central nervous system was not altered by phenytoin even when the doses administered were up to twice the ED95 for prevention of tonic hindlimb extension. Because these data from mice and frogs show that the anticonvulsant effect of phenytoin is dissociated, by dose, from effects on central nervous system cyclic nucleotide levels, it is doubtful that the alteration of cyclic nucleotide levels is a mechanism by which phenytoin exerts its anticonvulsant effect

The solubility of phenytoin in a physiological salt solution.

The solubility of phenytoin (PHT) was examined at room temperature (23 degrees C) one hour and 16 hours after solutions were made. PHT was considered to be dissolved if it could be centrifuged at 1500 c g for 30 minutes without loss of supernatant radioactivity produced by 14C-PTH. Radio-labelled PHT dissolved in 50 mM NaOH (pH 12.6) was used as control. One hour after mixing, PHT concentrations greater than 200 microM were incompletely dissolved. Sixteen hours after mixing, concentrations greater than 150 microM were incompletely dissolved. Solutions made with an excess of Na-PHT yielded a supernatant concentration of 105 microM measured 24 hours after mixing. It is concluded that PHT concentrations higher than 105 microM may form supersaturated solutions that remain homogeneous for 16 hours if the total concentration does not exceed 150 microM. The 150 microM solution is considered to be a relatively stable supersaturated solution since the addition of a small amount of Na-PHT caused only a 7% reduction in supernatant radioactivity after 13 hours.