Effect of neomycin on post-tetanic twitch tension of the mouse diaphragm preparation.

The effect of calcium (0.5-6 mM) and neomycin (0.1-0.2 mM) on the maximum post-tetanic twitch tension (MTT) and post-tetanic depression (PTD) of the indirectly elicited twitch tension was studied on the mouse isolated phrenic nerve-diaphragm preparation. The effect of neomycin on MTT of directly stimulated twitch tension was also tested in (+)-tubocurarine pretreated preparations. Three-dimensional plots between MTT and frequency and duration of indirect tetanic stimulation revealed that the frequencies and durations inducing maximal MTT were 500 Hz for 20 s in 0.5 mM CaCl2, 100 Hz for 5 s in 2 mM CaCl2 and 100 Hz for 10 s in 6 mM CaCl2. The frequency and duration inducing maximal PTD was 100 Hz for 20 s in 0.5 mM CaCl2, but there was no PTD in 2 mM or 6 mM CaCl2. Neomycin was associated with significantly greater MTT than in control if the duration of tetanic stimulation was 1 or 2 s, while it was associated with less MTT if the duration of tetanic stimulation was 10 or 20 s. Neomycin caused PTD in 2 mM CaCl2; sometimes the depressive effect was so severe that twitch tension was abolished. The maximal depression effect was found after 100 Hz tetanic stimulation for 20 s. Increasing the extracellular calcium concentration to 6 mM antagonized the effects of neomycin on MTT and PTD, whereas neostigmine (1.6 microM) antagonized the effect partially. Neomycin had no effect on the MTT or PTD of the directly stimulated twitch tension. It is concluded that neomycin alters the conditions of tetanic stimulation inducing MTT.     

Effects of memantine on the twitch tension of mouse diaphragm

The effects of memantine (50-175 microM) on the post-tetanic potentiation of the twitch tension were studied on the isolated mouse nerve diaphragm preparation. Memantine completely abolished the twitch tension elicited indirectly while it had no effect on the directly elicited twitch tension. Memantine also decreased the post-tetanic potentiation of amplitude of endplate potential and twitch tension. The duration of tetanic stimulation that induced a maximal decrease of twitch tension was 10-20 s. It is suggested that the effect of memantine on post-tetanic potentiation may be due to its voltage- and time-dependent effect on the ion channel-acetylcholine receptor complex.     

The effect of catecholamines on the influx of calcium and the development of tension in denervated mouse diaphragm muscle.

1 The nature of the catecholamine-induced contracture of chronically denervated mouse diaphragm muscle has been investigated and compared with the contractural response evoked by acetylcholine. 2 The time course of onset of catecholamine-sensitivity in denervated diaphragm muscles was similar to the development of acetylcholine sensitivity. However, catecholamine contractures were absent in tissues denervated for periods longer than 90 days whereas acetylcholine-sensitivity was still evident several months after denervation. 3 The catecholamine-induced contracture of the denervated muscle was inhibited specifically by beta-receptor blocking drugs and was unaffected by alpha-receptor blocking drugs and cholinoceptor antagonists. 4 Catecholamine-induced contractures of denervated muscles, unlike contractures to acetylcholine, were dependent upon the presence of spontaneous fibrillation and the amplitude of spontaneous fibrillation was increased by catecholamines. Fibrillation was absent in the presence of tetrodotoxin (1 muM), 2,4-dinitrophenol (10 muM), potassium cyanide (10 muM), ouabain (100 muM), in lithium chloride Ringer solution and at low temperature. Under these conditions catecholamine-induced contractures, but not those to acetylcholine, were abolished. 5 Labelled calcium was found progressively to enter denervated muscle fibres and this entry of calcium was increased by catecholamines. It is suggested that this calcium entry may represent either an increased calcium permeability of denervated muscle fibres which is increased further by catecholamines or the presence of a calcium current that occurs during the fibrillatory potentials of denervated muscle.     

A pharmacological study of the effect of carbamazepine on neuromuscular transmission in the rat diaphragm

The effects of carbamazepine (0.042-0.42 mM) on neuromuscular transmission were studied on the isolated rat phrenic nerve diaphragm preparation using standard pharmacological and electrophysiological methods. Carbamazepine decreased (1) the antidromic activity of the phrenic nerve, (2) the amplitude of the endplate potential (EPP) and miniature endplate potential (MEPP), (3) the quantal content of the endplate potential, (4) the indirectly-elicited twitch tension, (5) the muscle contracture in chronically denervated muscle induced by acetylcholine (ACh) and (6) the amplitude of the compound phrenic nerve action potential, in a concentration-dependent manner. The antidromic activity of the phrenic nerve was the most affected, while the phrenic nerve compound action potential was least affected. However, the IC50 for carbamazepine (the concentration of carbamazepine that inhibited 50% of the response) was in the same order of concentration, i.e. 0.11-0.3 mM. Compared with the effect of carbamazepine on the indirectly-elicited twitch tension with its actions described above, it is concluded that carbamazepine interfered with the neuromuscular activity by inhibiting pre- and postsynaptic process and conduction in the phrenic nerve.     

The effect of temperature on the direct muscle twitch response and the action of drugs on the isolated denervated rat diaphragm

The effect of temperature on the direct twitch response of the normal and denervated rat diaphragm, and the sensitivity of the contractural response of the denervated muscle to acetylcholine, suxamethonium, decamethonium, and potassium salts, was studied. Cooling the normal diaphragm from 39 to 20° increased the size of the direct twitch, but below this temperature the twitch response of the muscle was progressively depressed. The denervated muscle twitch was, by contrast, maximal at 39° and decreases as the temperature was lowered. This property of the denervated muscle first appeared about the third day after denervation. The contractural response of the denervated diaphragm to applied acetylcholine was enhanced by cooling, while the response to potassium salts was depressed. The depression of the contracture induced by potassium followed closely the depression of the direct muscle twitch of the denervated muscle over the same temperature range.     

Verruculotoxin potentiation of twitch tension in skeletal muscle.

Verruculotoxin [1-oxo-3-benzyl-octahydro-2H-pyrido(1,2-α)pyrazinel, a mycotoxin, was shown to act directly on both isolated mammalian and amphibian skeletal muscles to potentiate twitch tension. After 10 min exposure to a concentration of 0.4 mm, the twitch tensions of the rat diaphragm and frog sartorius muscles were enhanced to 152 and 150% of controls, respectively. In the isolated frog sartorius muscle, 10 min exposure to verruculotoxin (VTX, 0.4 mm) significantly increased the rate of contraction ($\text{dP}\text{dt}$, 120%), contraction time (CT, 127%), and half-relaxation time ($\text{1}\text{2}\text{RT}$, 126%) without significantly changing the twitch duration; in the rat diaphragm preparation, 10 min exposure to VTX (0.4 mm) significantly increased both $\text{dP}\text{dt}$ (141%) and CT (112%) without altering $\text{1}\text{2}\text{RT}$ and twitch duration. Of the C-15 substituted analogs tested, two analogs, fluoroverruculotoxin and methoxyverruculotoxin, enhanced twitch tension with a potency comparable to that of VTX, while chloroverruculotoxin increased twitch tension to only 120% of control. Hydroxyverruculotoxin and analogs lacking the C-3 benzyl group did not alter the twitch amplitude from control. VTX (0.4 mm, 10 min exposure) reduced twitch tension in the isolated cat papillary muscle to 33% of control. VTX (0.4 mm) was nondepolarizing in endplate and nonendplate regions. Thirty minute exposure to VTX irreversibly increased both the rising phase (sodium influx, 115% of control) and the repolarization phase (115% of control) of the muscle action potential. The results of this study indicate that VTX is a new muscle potentiator with properties similar to caffeine and the other Type A muscle potentiators.     

The effects of tetraethylammonium during twitch and tetanic stimulation of the phrenic nerve diaphragm preparation in the rat

Tetraethylammonium (TEA) (2.6 x 10(-3) M) potentiated the twitches of the indirectly- or directly-stimulated phrenic nerve diaphragm of the rat at 37 degrees C by prolonging the action potential of the sarcolemma, due to an inhibition of the repolarizing K+ current. With indirect stimulation, TEA caused a use-dependent inhibition of tetanic contractions, induced every 10 min by 10 sec of 50 Hz stimulation, and a post-tetanic depression of the twitches was observed after about 40 min. Recording of the electromyogram (EMG) and compound action potentials of the phrenic nerve, localized the two inhibitory effects to the neuromuscular junction. They were caused by different mechanisms of action. Choline (3.6 x 10(-4) M) antagonized the depression of the twitch but not the use-dependent inhibition. Lowering the temperature to 20 degrees C reduced the depression of the twitch, whereas the use-dependent inhibition was enhanced. The release of transmitter was probably normal during tetanic stimulation; a post-synaptic desensitization of acetylcholine (ACh) receptors caused the inhibition. Microelectrode recordings of endplate potentials supported this conclusion. The depression of the twitch was due to a presynaptic depletion of transmitter. This was confirmed by inducing an additional depletion and depression of the twitch with N-ethyl-maleimide (2.5 x 10(-5) M). Since the depression of the twitch was antagonized by choline, the depletion was probably due to an inhibited uptake of choline into the nerve terminals.     

Studies on cadmium-induced myotonia in the mouse diaphragm

Summary The purpose of this investigation was to study the possible mechanism of the potentiation of the contractile response and myotonia caused by Cd²⁺ in the mouse diaphragm. Cd²⁺ increased both amplitude and duration of the contractile response to direct stimulation in either 0.25 mM Ca²⁺ Krebs or 2.5 mM Ca²⁺ Krebs containing the K⁺-channel blockers, 4-aminopyridine, uranyl nitrate or tetraethylammonium ion. High K⁺ and tetrodotoxin inhibited these effects of Cd²⁺. Electrophysiological studies revealed that only one or two action potentials were triggered by passing a short depolarizing current across the muscle fibre membrane in 0.25 mM Ca²⁺ Krebs, but in the presence of Cd²⁺, a train of action potentials (153 ± 21 Hz) which lasted for 0.7 ± 0.2 s was induced. Furthermore, Cd²⁺ triggered a train of action potentials evoked by a single extracellular direct stimulation on the muscle fibre in 2.5 mM Ca²⁺ Krebs solution containing either 4-aminopyridine or uranyl nitrate. The membrane depolarized during the repetitive firing and then repolarized immediately after the cessation of repetitive firing. Cd²⁺ (0.1 mM) increased the input resistance of the muscle fibre by 53 ± 7% and this effect was inhibited in low [CI^–]_o. These findings suggest that the contractile potentiation and myotonia induced by Cd²⁺ in the mouse diaphragm are mediated by lowering the Cl^– conductance of the membrane. Send offprint requests to S. Y. Lin-Shiau at the above address     

The effect of carbamazepine on the 2-hydroxylation of desipramine

The effect of carbamazepine (CBZ, 200 mg twice daily for 28 days) on the kinetics of a single oral dose of desipramine (DMI, 100 mg) was investigated in six healthy volunteers. Compared with a control session, treatment with CBZ caused a marked increase in DMI apparent oral clearance (from 1.05 ± 0.40 to 1.38 ± 0.52 1 h per kg, means ± SD,P<0.01) and a significant shortening in DMI half-life (from 22.1 ± 3.5 to 17.8 ± 3.5 h,P<0.01). The amount of 2-hydroxy-desipramine (2-OH-DMI) excreted in urine over a 24-h period was significantly increased during CBZ intake (from 75 ± 15 to 92 ± 16 µmol,P<0.01). These findings suggest that CBZ induces the 2-hydroxylation of DMI, a reaction primarily catalyzed by the polymorphic CYP2D6 isozyme. This interaction may have considerable practical significance.     

The effect of enzyme induction on the cytochrome P450-mediated bioactivation of carbamazepine by mouse liver microsomes.

Predisposition to idiosyncratic toxicity with carbamazepine is thought to be due to a deficiency of the detoxication enzyme, microsomal epoxide hydrolase, although in some cases, concurrent administration of enzyme inducers might be a contributory risk factor, by altering the critical balance between bioactivation and detoxication. In this study, a mouse model has been used to determine the factors affecting carbamazepine bioactivation, using covalent binding and cytotoxicity as markers of bioactivation in vitro. Microsomes prepared from mice pre-treated with phenobarbitone increased (relative to the control microsomes) the formation of cytotoxic (12.3% vs 3.2%), protein-reactive (3.0% vs 2.0%) and stable (33.8% vs 18.1%) metabolites of carbamazepine. Similarly, pre-treatment with dexamethasone also increased the formation of the cytotoxic (24.8% vs 6.7%), protein-reactive (2.8% vs 1.5%) and stable (38% vs 19.8%) metabolites of carbamazepine, while beta-naphthoflavone pretreatment did not increase the formation of either the toxic or stable metabolites of carbamazepine when compared with its control microsomes. Co-incubation with gestodene (10-250 microM) resulted in a dose-dependent inhibition of both the bioactivation of carbamazepine and the formation of its stable 10,11-epoxide. SDS-PAGE and immunoblotting of the microsomes with anti-CYP3A antibody revealed the presence of a 52 kDa protein band in each preparation of microsomes, but the relative intensities of the bands, as measured by laser densitometry, were highest with the phenobarbitone and dexamethasone microsomes. The microsomal oxidation of cortisol to 6 beta-hydroxycortisol was also enhanced by pretreatment of mice with phenobarbitone (6.5% vs 2.7%) and dexamethasone (8.2% vs 4.3%), but not beta-naphthoflavone (2.2% vs 1.6%), when compared with their respective control microsomes, and was inhibited (range 25-68% inhibition), with all the microsomes by gestodene (50 microM). Taken collectively, the data in this study demonstrate that in the mouse, induction of the CYP3A subfamily significantly increases carbamazepine bioactivation. It is likely that in humans inducers of the orthologous form of this enzyme, most notably anticonvulsants, may increase the bioactivation of carbamazepine.     

The effect of body size on the metabolic clearance of carbamazepine

OBJECTIVES: To examine the profile of the known pathways of carbamazepine (CBZ) metabolism in a group of children and adolescents, and to test for associations with physical measurements, age and plasma hormonal levels. STUDY DESIGN: Cross-sectional study of children and adolescents attending a neurological outpatients department who were medicated with CBZ. Partial clearances of CBZ to CBZ-epoxide (CBZ-ep), CBZ-10,11-trans-diol (CBZ-diol), 2-hydroxy-CBZ (CBZ-2-OH), 3-hydroxy-CBZ (CBZ-3-OH), CBZ-acridan (CBZ-acr) and their respective glucuronides were calculated by relating 24-h recovery of these metabolites from urine to trough steady-state serum CBZ levels. CBZ and its metabolites were measured by a gradient high performance liquid chromatography (HPLC) method. Serum CBZ-ep, LH, FSH, prolactin, IGF-I, and testosterone or oestradiol and progesterone were also measured. Surface area (SA) and liver volume (LV) were calculated from height and weight. RESULTS: Twelve males and nine females with an age range of 6-17 years participated in the study. Partial clearance to each of the metabolites was most strongly correlated with the calculated size of the liver relative to body weight. These associations persisted when corrected for potential confounders using multiple regression analysis. CONCLUSION: In the age group studied, urinary clearance of CBZ to its known metabolites is proportional to the size of the liver relative to body weight.     

Effects of papaverine on twitches in mouse diaphragm

This study examined the inhibitory effects of papaverine on twitches directly elicited by electrical stimulation of the mouse diaphragm. Papaverine (3-100 μM) inhibited twitches in a dose-dependent manner. Papaverine increased the cyclic adenosine monophosphate (cAMP) but not cyclic guanosine monophosphate (cGMP) content. IBMX, Db-cAMP and 8-br-cGMP did not affect twitches, whereas verapamil and NaCN inhibited twitches. Increases in extracellular Ca2+ removed the twitch inhibition caused by verapamil but not that caused by papaverine. Papaverine (30 and 100 μM) and NaCN (1 mM) decreased creatine phosphate and ATP contents. These results suggest that the relaxing effects of papaverine on mouse diaphragm are mainly due to inhibition of aerobic energy metabolism.     

The effect of prednisolone on neuromuscular transmission in the rat diaphragm.

Prednisolone, in concentrations of 0.004--0.032 mmol/l, increased the amplitude of the miniature end-plate potentials (MEPPs). A maximum increase to 134% of the control values was seen at 0.016 mmol/l. At higher prednisolone concentrations the MEPP amplitude gradually decreased to reach 77% of the control value at 0.62 mmol/l. The MEPP frequency was increased to twice the control value at 0.62 mmol/l. The quantal content of the end-plate potential (EPP), however, was not influenced by prednisolone. The response to iontophoretically applied acetylcholine was diminished, especially at 0.62 mmol/l prednisolone. Prednisolone, therefore, caused presynaptic effects as was shown by an increase in unitary MEPP amplitude and by a considerable number of giant MEPPs, which at increasing prednisolone concentrations was antagonized increasingly by a postsynaptic depressant effect. These results also provide an explanation for the adverse effects of prednisolone on the end-plate potential and on neuromuscular transmission.     

The effect of phenobarbital and carbamazepine on the ethanol withdrawal reaction in the rat

The effects of phenobarbital (PB) and carbamazepine (CZ) on the ethanol withdrawal reaction in the rat were investigated in a blind study including an untreated control group. Physical ethanol dependence was established by intragastric intubation during a 4-day period. Both the degree of intoxication and the withdrawal reaction were assessed by standardised assessment instruments. Treatment with PB (40–60 mg/kg) and CZ (80–120 mg/kg) was initiated 10 h after the last ethanol dose and continued during the first 24 h of withdrawal. Serum concentrations of the drugs were measured.Both PB and CZ significantly reduced the ethanol withdrawal reaction compared to controls, and PB was significantly more effective than CZ. The degree of drug intoxication signs assessed by the same rating scale as the degree of ethanol intoxication indicated that maximum tolerable drug doses were used.PB probably exerts its treatment effect through the mechanism of cross dependence with ethanol, while CZ may exert a more specific effect on limbic structures responsible for central nervous system excitability.Abbreviations ANOVA analysis of variance - BEC blood ethanol concentration - CIS cumulated intoxication score - CZ carbamazepine - ICS incomplete clonic seizure - PB phenobarbital     

Modification by dantrolene, procaine and suxamethonium of caffeine-induced changes in aequorin luminescence transients and twitch tensions of directly-stimulated diaphragm muscle of mouse.

A convenient method is described for measuring simultaneously Ca2+-related aequorin luminescence and twitch tension in the isolated diaphragm muscle of the mouse. Forty to fifty fibres were injected intracellularly with aequorin solution and the mechanical and luminescence responses to direct stimulation were recorded. The replacement of Na+ by K+ (to obtain 59 or 143.4 mM K+) in the nutrient solution decreased both aequorin luminescence and twitch tensions, but after a time lag, it produced a contracture. Caffeine (5 or 10 mM) increased both aequorin luminescence and twitch tensions, and after a time lag, it also produced a contracture. Dantrolene (1 and 30 microM) and procaine (10 microM, 300 microM and 1 mM) decreased aequorin luminescence transients and twitch tension. In addition procaine inhibited the caffeine-induced increase of aequorin luminescence, but dantrolene did not have this effect. At concentrations causing neuromuscular block, suxamethonium (130 microM) decreased aequorin luminescence transients and twitch tension. By contrast, (+)-tubocurarine (6.5 microM) did not affect the aequorin luminescence in directly stimulated muscles. These results suggest that Ca+-related aequorin luminescence transients accompanied by twitch tensions reflect the intracellular fast mobilization of compartmentalized Ca2+ from plasma membrane or sarcoplasmic reticulum, and that the increase in resting luminescence caused by a K+- or caffeine-induced contracture may be produced by the slow mobilization of Ca2+ from sarcoplasmic reticulum.     

The effect of carbamazepine on valproic acid disposition in adult volunteers.

1. Pharmacokinetic parameters for valproic acid (VPA) were determined before and following 2 weeks of carbamazepine (CBZ) administration in five healthy male volunteers. Mean VPA dosage was 16.4 mg kg-1 day-1. CBZ dosage was started at 100 mg twice daily and increased after 1 week to a total daily dose of 300 mg. 2. After CBZ administration, mean VPA plasma clearance increased from 0.90 +/- 0.18 s.d. to 1.26 +/- 0.24 l h-1 (P less than 0.05) as did clearance of free VPA (20.8 +/- 7.6 to 37.0 +/- 13.6 l h-1). Mean VPA elimination rate constant increased from 0.051 +/- 0.011 to 0.067 +/- 0.011 h-1 (P less than 0.05) after CBZ administration. 3. Mean area under the serum concentration vs time curve decreased from 675.0 +/- 130.5 to 475.7 +/- 75.7 mg l-1 h (P less than 0.05) after CBZ administration. Mean serum VPA half-life decreased from 14.0 +/- 2.4 to 10.6 +/- 1.4 h (P less than 0.05). Mean serum VPA trough concentrations decreased from 44.0 +/- 16.7 to 27.0 +/- 10.4 micrograms ml-1 (P less than 0.05). 4. A significant change was not observed in the mean VPA volume of distribution after CBZ coadministration suggesting that enzyme induction rather than a competition for plasma protein binding sites was involved in this interaction. 5. Despite the increased clearance of VPA, the urinary recovery of VPA or conjugate did not increase after CBZ administration.