Phrenic Nerve-hemidiaphragm Preparation Research Articles

Low quantal content of the endplate potential reduces safety factor for neuromuscular transmission in the diaphragm of the newborn rat

We have used phrenic nerve-hemidiaphragm preparations from rats aged 11-28 days to determine the electrophysiological basis of the three-fold increase in sensitivity of the neonatal neuromuscular junction to non-depolarizing neuromuscular blocking drugs. The mean resting membrane potential (RMP) of intact muscle fibres at 19-21 degrees C increased from -67.1 (SEM 0.6) mV at 11 days to -75.8 (0.06) mV at 28 days. Over the same age range the mean size of miniature endplate potentials (MEPP) decreased from 1.7 (0.1) mV to 1.4 (0.03) mV, and the threshold depolarization to produce a muscle action potential increased from 6.8 (0.4) mV to 9.5 (0.6) mV. The quantal content of the evoked endplate potential (EPP) calculated from EPP and MEPP measured in cut muscle fibres was only 6.6 (0.9) at 11 days, but this increased to 21.1 (2.3) at 21 days. As a result of the low quantal content of the EPP, the safety factor for neuromuscular transmission was only 1.7 at 11 days, compared with 2.8 at 20 days. A safety factor of 1.7 in the youngest rats indicates that block of only about 40% of postjunctional acetylcholine receptors would result in failure of neuromuscular transmission. Hence, a low quantal content of EPP, leading to a low safety factor for neuromuscular transmission, probably underlies the increased sensitivity to non-depolarizing neuromuscular blocking drugs of young rats and, by inference, human neonates.

Effect of hypoxia on neuromuscular transmission in the developing diaphragm

To study the effects of hypoxia on neuromuscular transmission in the developing diaphragm, phrenic nerve-hemidiaphragm preparations were obtained from newborn (4-9 days) and older (22-30 days) rats. Diaphragms were stimulated directly or indirectly (via the nerve) for 1 s at frequencies of 10-80 Hz. Force generated in response to stimulation was measured during perfusion of oxygenated Ringer solution (control) and Ringer solution bubbled with 95% N2-5% CO2 (hypoxia). After 45 min of hypoxia, the force response of the older diaphragms to direct stimulation had decreased to approximately 50% of control at > or = 40 Hz; however, when stimulation occurred via the nerve at these frequencies only 15-20% of control force was generated. In the newborn diaphragms, the force decrement after similar or longer periods of hypoxia (< or = 90 min) was 30- 40% irrespective of the route or frequency of stimulation. After 15 min of reoxygenation, the force response to both muscle and nerve stimulation recovered completely in the older diaphragms but only partially in the newborn diaphragms (range 77% of control at 50 Hz to 95% of control at 10 Hz). These data suggest that in the newborn diaphragm 1) neuromuscular transmission is more resistant to the effects of hypoxia than the older diaphragm and 2) the predominant effect of hypoxia is peripheral in the diaphragm muscle fibers, whereas in the older diaphragm the effect is before or at the neuromuscular junction.

The Effect of CO[2]-Induced Acid-Base Changes on the Potencies of Muscle Relaxants and Antagonism of Neuromuscular Block by Neostigmine in Rat In Vitro

We investigated the influence of CO2-induced acid-base changes on the potencies of the monoquaternary relaxants (rocuronium, vecuronium, and d-tubocurarine) and bisquaternary relaxants (pancuronium, pipecuronium, and metocurine), and on the antagonism of their neuromuscular block by neostigmine. Phrenic nerve-hemidiaphragm preparations of rats were used. The pH changes were induced by changing the CO2 concentration aerating the modified Krebs solution. The potencies of the monoquaternary relaxants increased at 9% CO2 (pH 7.2) and decreased at 2.5% CO2 (pH 7.6) from those of 5% CO2 (pH 7.4) both with and without neostigmine (P < 0.05), whereas the potencies of the bisquaternary drugs did not change significantly at different pH levels from control (pH 7.4) both with and without neostigmine. The slopes of the log concentration-percent response curves of each drug were not significantly different at each pH level. The ratios of inhibitory concentrations, 50% (IC50) values with and without neostigmine at each pH value for each drug were not significantly different indicating that the neostigmine-induced antagonism for each drug was not affected by the CO2-induced acid-base changes. But the ratios of the IC50 values of the steroidal relaxants (rocuronium, vecuronium, pancuronium, and pipecuronium) were significantly lower (P < 0.05) than those of the isoquinolinium drugs (d-tubocurarine and metocurine) at each pH level, suggesting that the antagonism is enhanced at each pH level for the isoquinolinium relaxants. The difference was independent of their monoquaternary or bisquaternary nature. These results suggest that CO2 increases the potency of the monoquaternary relaxants but does not affect the bisquaternary relaxants.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation by tonic A2a‐adenosine receptor activation of CGRP‐facilitated [3H]‐ACh release from rat motor nerve endings

1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors. Since both CGRP and A2a-adenosine receptors are positively coupled to the adenylate cyclase/cyclic AMP system, cooperation between these receptors might occur at the second messenger transduction system level.

Contribution of direct actions of the oxime HI-6 in reversing soman-induced muscle weakness in the rat diaphragm

The actions of the bispyridinium oxime HI-6 ([[[(4-aminocarbonyl)pyridino]-methoxy]methyl]-2-[(hydroxyimino)methyl]-pyridinium dichloride) were investigated in vitro on rat phrenic nerve-hemidiaphragm preparations. Isometric twitch and tetanic tensions were elicited at 37°C with supramaximal nerve stimulation at frequencies of 20 and 50 z. To approximate normal respiration patterns, trials consisting of 30 successive 0.55 s trains were alternated with 1.25 s rest periods. Under control conditions, the above stimulation pattern generated tensions that were well maintained at both frequencies. In contrast, a marked depression of muscle tension was observed in diphragms removed from rats administered 339 μg/kg soman (3 LD 50) and tested in vitro. Addition of HI-6, 4 h after soman exposure, led to a nearly complete recovery of muscle tension at 20 Hz. At 50 Hz, muscle tensions still declined especially when trains were elicited at 1.25 and 3 s intervals. The recovery by HI-6 observed in this study appears to be mediated by mechanisms unrelated to acetylcholinesterase reactivation since no increase of enzymatic activity was detected and the effect was reversed by a brief washout in oxime-free physiological solution. The results suggest that the direct action of HI-6 may play a role in restoring soman-induced diaphragmatic failure but this effect woul be significant primarily under low use conditions.

Diabetic rats show more resistance to neuromuscular blockade induced by aminoglycoside antibiotics

1. 1. Neuromuscular blocking effect of gentamicin and neomycin evaluated in phrenic-nerve hemidiaphragm preparation in normal and diabetic rats and IC 50 of both drugs determined from LDR curves. 2. 2. IC 50 of gentamicin was 0.91 ± 0.05 and 1.67 ± 0.08 mmol·1 −1 for normal and diabetic rats respectively. 3. 3. IC 50 of neomycin was 0.42 ± 0.02 and 0.61 ± 0.06 mmol·1 −1 for normal and diabetic rats respectively. 4. 4. The results show diabetic rats are more resistant to neuromuscular blocking action of aminoglycoside antibiotics (AGs). 5. 5. Replacement of sodium by lithium (1.5 and 5 mmol·1 −1) in the media, reduced this effect of aminoglycosides (AGs) significantly. 6. 6. It appears that the prevention of AGs induced-neuromuscular blockade by lithium is not significantly different in both groups.

Ion channel blockade by oximes and recovery of diaphragm muscle from soman poisoning in vitro

1. The actions of oximes and related compounds on the nicotinic acetylcholine receptor ion channel at the adult mouse muscle endplate were investigated by use of single-channel recording techniques. The aim of the study was to determine whether the channel-blocking properties of the compounds could contribute to their therapeutic effectiveness against soman poisoning in vitro. 2. Therapeutic effectiveness was assessed in guinea-pig phrenic nerve-hemidiaphragm preparations by measuring the degree of recovery of neuromuscular function produced by the compounds following poisoning by soman. A number of the compounds, including some which lacked the oxime group, produced a significant recovery of neuromuscular function which was unrelated to acetylcholinesterase (AChE) reactivation; this was reversed by washing off the compound, and was therefore attributed to a direct pharmacological action on the muscle. 3. Single channel recordings showed that some of the compounds blocked open nicotinic receptor ion channels in preparations of mouse muscle fibres. The compounds which showed the greatest direct pharmacological actions in diaphragms produce a very fast, flickering blockade of the channels. Several quantitative measures of channel-blocking activity correlated very well with the direct pharmacological action. Furthermore, for two compounds studied in greater detail, the direct action and channel-blocking showed similar concentration-response relationships. 4. The results of this study indicate that the direct pharmacological action of oximes and their analogues against neuromuscular blockade by soman in vitro is due to their channel-blocking activity. The direct action does not correlate well with protection against soman poisoning in vivo, however, which suggests that additional non-reactivating properties of these compounds, at sites other than the neuromuscular junction, may also be important for their therapeutic effectiveness.

Presynaptic A1-purinoceptor-mediated inhibitory effects of adenosine and its stable analogues on the mouse hemidiaphragm preparation

1. The effect of adenosine or its stable analogues (2-chloroadenosine, CADO; 5'-N-ethylcarboxamidoadenosine, NECA; and N6-cyclopentyladenosine, CPA) on the release of [3H]-acetylcholine ([3H]-ACh), and on the development of force of contraction evoked by electrical stimulation of the nerve, were studied in the mouse phrenic nerve-hemidiaphragm preparation. Evidence was obtained that the release of ACh is subject to presynaptic modulation through presynaptic A1(P1)-purinoceptors. 2. Adenosine or its stable analogues (CADO, NECA, CPA) inhibited, in a concentration-dependent manner, both the release of ACh and the force of the indirectly elicited contraction of hemidiaphragm preparation, provided in the latter case that the margin of safety was reduced by (+)-tubocurarine or magnesium. The order of potency in reducing ACh release was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.08 +/- 0.01, 0.74 +/- 0.05, 9.05 +/- 0.20, and 410.2 +/- 42.5 mumol/l, respectively. The order of potency in reducing twitch tension was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.11 +/- 0.02, 0.48 +/- 0.03, 2.07 +/- 0.49, and 240.4 +/- 20.0 mumol/l, respectively. 3. 8-Phenyltheophylline (8-PT) antagonized the inhibitory effects of the adenosine receptor agonists on ACh release and twitch tension.(ABSTRACT TRUNCATED AT 250 WORDS)

Dendrotoxin-like effects of noxiustoxin

Noxiustoxin from the Mexican scorpion (Centruroides noxius Hoffmann) is known to block neuronal K+ channels. Noxiustoxin facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations, but not in mouse phrenic nerve-hemidiaphragm preparations. Noxiustoxin displaced binding of a radiolabelled dendrotoxin from synaptosomal membranes from rat brain, with a Ki of 10−10M. It is concluded that noxiustoxin shares some pharmacological properties with the K+ channel blocking dendrotoxins.

Impaired neuromuscular transmission during partial inhibition of acetylcholinesterase: the role of stimulus-induced antidromic backfiring in the generation of the decrement-increment phenomenon.

Neuromuscular transmission was studied in the rat phrenic nerve-hemidiaphragm preparation with acetylcholinesterase (AChE) partially inactivated. Enzyme inhibition resulted in (1) increased single-twitch tension of the diaphragm; (2) compound muscle action potential (CMAP) containing repetitive discharges; (3) stimulus-induced antidromic backfiring (SIAB) seen in the phrenic nerve; and (4) repetitive nerve stimulation (RNS) eliciting a decrement-increment (D-I) phenomenon (i.e., amplitude reduction maximal with the second CMAP). Using a high-calcium and low-magnesium solution, SIAB and the decrement of the second CMAP during RNS were intensified, whereas closely spaced trains and (+)-tubocurarine (TC) abolished SIAB and simultaneously prevented the decrement of the second CMAP. Importantly, low concentrations of (+)-TC prevented SIAB in the phrenic nerve, while the repetitive discharges of the CMAP and the increase in twitch tension remained unaffected. This observation suggests that preterminal nicotinic receptors stimulated by released acetylcholine induce SIAB, whereas postsynaptic events are less important in the generation of SIAB. SIAB, a presynaptic event, appears to be responsible for the transient impairment of the neuromuscular transmission, i.e., the D-I phenomenon.

Neuromuscular pharmacology in rat neonates: development of responsiveness to prototypic blocking and reversal drugs.

The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine. The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incomplete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc.

Presynaptic A1-purinoceptor-mediated inhibitory effects of adenosine and its stable analogues on the mouse hemidiaphragm preparation.

1. The effect of adenosine or its stable analogues (2-chloroadenosine, CADO; 5'-N-ethylcarboxamidoadenosine, NECA; and N6-cyclopentyladenosine, CPA) on the release of [3H]-acetylcholine ([3H]-ACh), and on the development of force of contraction evoked by electrical stimulation of the nerve, were studied in the mouse phrenic nerve-hemidiaphragm preparation. Evidence was obtained that the release of ACh is subject to presynaptic modulation through presynaptic A1(P1)-purinoceptors. 2. Adenosine or its stable analogues (CADO, NECA, CPA) inhibited, in a concentration-dependent manner, both the release of ACh and the force of the indirectly elicited contraction of hemidiaphragm preparation, provided in the latter case that the margin of safety was reduced by (+)-tubocurarine or magnesium. The order of potency in reducing ACh release was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.08 +/- 0.01, 0.74 +/- 0.05, 9.05 +/- 0.20, and 410.2 +/- 42.5 mumol/l, respectively. The order of potency in reducing twitch tension was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.11 +/- 0.02, 0.48 +/- 0.03, 2.07 +/- 0.49, and 240.4 +/- 20.0 mumol/l, respectively. 3. 8-Phenyltheophylline (8-PT) antagonized the inhibitory effects of the adenosine receptor agonists on ACh release and twitch tension.(ABSTRACT TRUNCATED AT 250 WORDS)

PROPOFOL POTENTIATES BOTH PRE- AND POSTSYNAPTIC EFFECTS OF VECURONIUM IN THE RAT HEMIDIAPHRAGM

We have measured twitch tension in response to train-of-four stimulation in rat isolated phrenic nerve-hemidiaphragm preparations. Propofol inhibited nerve evoked twitch tension, with 50% inhibition occurring at 420 (SD 29) mumol litre-1. Although propofol 100 mumol litre-1 by itself had no effect on nerve evoked twitch tension, it potentiated the neuromuscular blocking effects of vecuronium. The decrease in train-of-four ratio with vecuronium was directly proportional to the degree of twitch inhibition, regardless of whether twitch was depressed by vecuronium alone or in combination with propofol. The finding that the train-of-four ratio was a function of the degree of block, rather than simply a function of vecuronium concentration, indicates that propofol also contributed to train-of-four fade and potentiated both pre- and postsynaptic effects of the neuromuscular blocker. The concentrations of propofol used in this study are much greater than human therapeutic blood concentrations, which are typically 25-35 mumol litre-1 (4-6 micrograms ml-1) immediately after a bolus dose of 2 mg kg-1, suggesting that neither muscle weakness nor potentiation of vecuronium-induced neuromuscular block should be of concern at propofol concentrations occurring clinically.

AGE-DEPENDENT VARIATION IN RESPONSE TO TUBOCURARINE IN THE ISOLATED RAT DIAPHRAGM

The EC50 of tubocurarine was determined in phrenic nerve-hemidiaphragm preparations obtained from 35 Sprague-Dawley rats aged 0-46 days. We measured also the ratio of the fourth to the first twitch in the train-of-four (T4:T1) when the first twitch of the train was depressed to 50% of control. The preparation was not unduly sensitive to tubocurarine at 0 days and there was little evidence of T4:T1 fade. However, by age 11 days the preparation exhibited fade and a three-fold sensitivity to tubocurarine similar to that in the human neonate. We conclude that the phrenic nerve-hemidiaphragm preparation from 11-day-old rats should be a suitable model in which to investigate the biochemical and electrophysiological basis of the sensitivity seen in humans.

INTERACTION BETWEEN BETAMETHASONE AND VECURONIUM

A possible interaction between betamethasone and vecuronium was examined in 20 rat phrenic nerve-hemidiaphragm preparations. Ten preparations were bathed in a physiological solution with betamethasone 1 mumol litre-1 added and, after a 30-min period were exposed to vecuronium at concentrations of 4, 6, 8 and 10 mumol litre-1 with vecuronium free washings between each exposure. Ten control experiments were performed also using a betamethasone-free bathing solution. In comparison with control, the betamethasone group had significantly (P = 0.0008) less depression of muscle contraction (twitch) force at all concentrations of vecuronium. The calculated ED50 (50% depression of muscle contraction force) was 5.65 mumol litre-1 for controls and 7.39 mumol litre-1 for betamethasone-pretreated preparations. This study confirms our previous clinical observations that an interaction occurs between vecuronium and betamethasone which is characterized by resistance to neuromuscular block.

Developmental changes in neuromuscular transmission in the rat diaphragm.

Neuromuscular transmission was studied in diaphragms from rats of three ages, 4-7 days old, 11-12 days old, and adults with the use of an in vitro phrenic nerve-hemidiaphragm preparation. Each hemidiaphragm was stimulated via either muscle or nerve with 1-s stimulus trains at frequencies from 10 to 100 Hz. The patterns of force development obtained in response to the two routes of stimulation were compared for each group. Diaphragms from adults developed maximum force in response to stimulation of approximately 40 Hz with no significant decrease in force at higher frequencies. Within each stimulus train, once peak force was achieved, it was maintained for the remainder of the stimulus and responses to nerve and muscle stimulation were almost identical. In contrast, diaphragms from 4- to 7-day-old rats developed maximum force at approximately 20 Hz; stimulation at greater than or equal to 60 Hz induced significantly less peak force. This decrease in peak force at higher frequencies was significantly larger for nerve than for muscle stimulation. In addition, during each nerve stimulus train diaphragms from 4- to 7-day-old rats were unable to maintain peak force, which decreased at frequencies greater than 20 Hz. The decrease in force reached approximately 50% of peak at stimulation frequencies greater than or equal to 60 Hz. Diaphragms from 11- to 12-day-old rats showed intermediate responses. Based on the responses to phrenic nerve stimulation, we conclude that the neonatal rat diaphragm shows marked neuromuscular transmission failure that is not seen in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that catecholamines increase acetylcholine release from neuromuscular junction through stimulation of alpha-1 adrenoceptors.

1. The effects of alpha-1 adrenoceptor agonists on 3H-acetylcholine release from mouse phrenic nerve-hemidiaphragm preparation were studied. 2. In preparation which had been incubated with 3H-choline, electrical stimulation (50 Hz, 0.8 s trains every 10 s) released 3H-acetylcholine. Neurochemical evidence was obtained that noradrenaline, adrenaline and phenylephrine facilitated the stimulation evoked release of 3H-acetylcholine. This effect was much more marked when the prejunctional nicotinic receptors mediating positive feedback modulation were blocked by (+)-tubocurarine. In the presence of this drug, when the release of 3H-acetylcholine was reduced, alpha-1 adrenoceptor agonists enhanced the release in a prazosin-sensitive manner. 3. The rank order of potency of alpha-1 adrenoceptor agonists was adrenaline greater than noradrenaline greater than phenylephrine. By contrast, methoxamine had no effect on the release of 3H-acetylcholine. It is suggested, therefore, that circulating catecholamines may be able to affect neuromuscular transmission through stimulation of presynaptic alpha-1 adrenoceptors.

Run‐down of neuromuscular transmission during repetitive nerve activity by nicotinic antagonists is not due to desensitization of the postsynaptic receptor

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 nerve-hemidiaphragm preparation. 2. For electrophysiological experiments, the preparation was immobilized by synthetic mu-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. run-down. 4. The degrees of waning of tetanus and trains of e.p.ps produced by a very high concentration of tubocurarine (20 microM) 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.

Backfiring of the isolated rat phrenic nerve does not collide with impulse propagation following repetitive nerve stimulation at 1?50 Hz

Acetylcholinesterase inhibition with neostigmine in the isolated rat phrenic nerve-hemidiaphragm preparation induced axonal backfiring and repetitive compound muscle action potentials following single nerve stimulation. The duration of backfiring and the repetitive compound muscle action potentials did not exceed 55 ms. With repetitive nerve stimulation at frequencies ranging from 1 to 50 Hz, backfiring was present only with the first stimulus and the amplitude of the second compound muscle action potential was maximally reduced, while the subsequent responses recovered gradually. However, the amplitudes of the concomitant antidromic nerve action potentials remained unchanged during the entire train of stimulation. Lack of nerve action potential amplitude changes and the short duration of backfiring of the first nerve action potential exclude a collision phenomenon of backfiring with the nerve action potential induced by the second stimulus. Moreover, the duration of the repetitive compound muscle action potentials did not exceed the duration of backfiring. Therefore, the prolongation of the muscle membrane refractory period by reexcitation following backfiring cannot explain the decrement of the second compound muscle action potential.

The pre- and postjunctional components of the neuromuscular effect of antibiotics.

The relative contributions of the pre- and postsynaptic components of the myoneural blocking effect of different antibiotics were studied using: (a) a radio-active method that measures selectively the Ca(2+)-dependent, stimulation evoked, quantally released, (3)H-acetylcholine ((3)H-ACh) from the mouse in vitro phrenic nerve-hemidiaphragm preparation without cholinesterase inhibition; (b) measurement of the force of contraction of the indirectly or directly stimulated muscle. The antibiotics studied (neomycin, polymyxin B and lincomycin), reduced the release of (3)H-ACh evoked by stimulation (18 trains of 40 shocks at 50 Hz) in a concentration dependent manner. While the inhibitory effect of neomycin was inversely related to [Ca(2+)](o), that of lincomycin was moderately and that of polymyxin B was not affected by increasing [Ca(2+)](o) from 0.75 to 5.0 mM. Similarly, the d-tubocurarine (d-Tc)-induced inhibition of the release of (3)H-ACh was independent of [Ca(2+)](o). The K-channel blocking agent, 4-aminopyridine (4-AP), enhanced the release of ACh in a concentration dependent manner and prevented the neuromuscular effect of neomycin. However, the neuromuscular effect of polymyxin B and of lincomycin was not affected by 4-AP. Atropine, enhanced the release of (3)H-ACh. Antibiotics, however, were still able to reduce the release of ACh when the negative muscarinic feedback mechanism of ACh release was eliminated by atropine. Our findings indicate that the antibiotics studied possess both pre- and postsynaptic effects. Presynaptically they reduce the evoked release of ACh; postsynaptically they inhibit muscle contractility. The rank order of presynaptic action is neomycin >polymyxin B >lincomycin.