Habitual exercise enhances neuromuscular transmission efficacy of rat soleus muscle in situ.

Rat motor nerve terminals and the endplates they interact with exhibit changes to varying patterns of use, as when exposed to increased activation in the form of endurance exercise training. The extent to which these changes affect neuromuscular transmission efficacy is uncertain. In this study, the effects of habitual exercise on the electrophysiological properties of neuromuscular transmission in rat soleus muscle were investigated using a novel in situ approach. Consistent with previous reports, miniature endplate potential frequency was enhanced by habitual exercise. Other passive properties, such as resting membrane potential, miniature endplate potential amplitude, and "giant" miniature endplate potential characteristics were unaltered by the training program. Full-size endplate potentials were obtained by blocking soleus muscle action potentials with mu-conotoxin GIIIb. Quantal content values were 91.5 and 119.9 for control and active groups, respectively (P < 0.01). We also measured the rate and extent of endplate potential amplitude rundown during 3-s trains of continuous stimulation at 25, 50, and 75 Hz; at 50 and 75 Hz, we found both the rate and extent of rundown to be significantly attenuated (10--20%) in a specific population of cells from active rats (P < 0.05). The results establish the degree of activity-dependent plasticity as it pertains to neuromuscular transmission in a mammalian slow-twitch muscle.

Effect of antidepressants on ATP-dependent calcium uptake by neuronal endoplasmic reticulum.

This study investigated the effect of tricyclic and atypical antidepressants on adenosine triphosphate (ATP) dependent calcium uptake by the endoplasmic reticulum of lysed synaptosomes from rat brain cortex. Tricyclic antidepressants (imipramine, desipramine, clomipramine, amitriptyline) exhibited no effect in the lower range (0.06 to 2 microM) of drug concentrations, and a concentration-dependent inhibition of calcium uptake in the upper range (6 to 200 microM). A concentration-dependent inhibition was observed for atypical antidepressants (mianserin, desmethylmianserin, venlafaxine, desmethylvenlafaxine, fluoxetine) in both the lower and the upper range of drug concentrations. Since no stimulation of calcium uptake was observed in either concentration range, it appears that the tricyclic and atypical antidepressants tested are not capable of normalizing, through their effect on the endoplasmic reticulum, an overactive calcium signal. which is possibly implicated in the etiology of affective disorders. Also, although only marginal inhibition of calcium uptake is expected at brain concentrations of tricyclics and mianserin-desmethylmianserin that are likely to be encountered during clinical use, a more substantial inhibition could occur with fluoxetine.

Inhibition of K+-induced Ca2+ uptake in rat hippocampus synaptosomes by mianserin enantiomers.

In order to test potential links with other stereospecific neurobiological effects of mianserin, the present study explored the stereospecificity for inhibition of depolarization-induced Ca2+ uptake by mianserin. Synaptosomes from rat hippocampus were incubated with 45Ca2+ in either resting or depolarizing (60 mM K+) choline medium, in the absence or presence (0.6-200 microM) of a mianserin enantiomer. The enantiomers were equipotent (IC50 approximating 50 microM) at inhibiting net depolarization-induced Ca2+ uptake. This finding, therefore, cannot help to explain the stereoselective enhancement of noradrenaline release by S(+)-mianserin; it is also not in keeping with the stereospecificity exhibited by mianserin in acute tests predictive of antidepressant activity, thus suggesting that the calcium-channel blocking activity of mianserin is not linked to its antidepressant effect.

Endurance training increases acetylcholine receptor quantity at neuromuscular junctions of adult rat skeletal muscle.

The aim of the study was to test the hypothesis that a 16 week endurance training program would alter the abundance of endplate-associated nicotinic acetylcholine receptors (nAChR) in various rat skeletal muscles. We found a 20% increase in endplate-specific [125I]alpha-bungarotoxin binding in several muscles of trained rats, accompanied by equal susceptibility of toxin binding to the inhibitory effect of D-tubocurarine in sedentary and trained muscles. We conclude that the neuromuscular junction adaptations that occur with increased chronic activation include an increase in nAChR number. Results of experiments designed to determine nAChR turnover also suggest that this effect is mediated by an alteration in the receptor's metabolic state. The potential implications and mechanisms of this adaptation are discussed.

Atypical antidepressants inhibit depolarization-induced calcium uptake in rat hippocampus synaptosomes.

The effect of the atypical antidepressants mianserin, iprindole, and fluoxetine on synaptosomal calcium uptake was tested under conditions where a selective action on voltage-dependent calcium channels can be documented. Synaptosomes from rat hippocampus were incubated with 45calcium either in choline-rich medium or in depolarizing (60 mM K+) choline-rich medium, and drug effects on calcium uptake in these two conditions, as well as on the net depolarization-induced calcium uptake, were studied in the range of concentrations 0.6-200 microM. A concentration-dependent marked inhibition of uptake in depolarizing choline medium was observed for the three antidepressants, whereas only a minor degree of inhibition of uptake in resting choline medium was present at the highest drug concentration; as a result, the concentration-effect relationships exhibited a strong concentration-dependent inhibition of net depolarization-induced calcium uptake. The IC50 values, calculated by interpolation of the last three or four points of the concentration-effect relationships, were 27, 39, and 68 microM for fluoxetine, iprindole, and mianserin, respectively. Significant degrees of calcium channel inhibition are not expected at brain concentrations of mianserin and iprindole that are likely to be encountered during clinical use; however, the fluoxetine concentration-effect relationship established in the present study, coupled with the published ratio of 20:1 for brain:plasma concentrations of fluoxetine-norfluoxetine in humans, suggests that brain calcium channel function could be appreciably reduced in some patients treated with this atypical antidepressant.

Differential effect of desipramine and 2-hydroxydesipramine on depolarization-induced calcium uptake in synaptosomes from rat limbic sites.

This study was conducted to investigate the inhibition of synaptosomal 45Ca uptake by desipramine and its major metabolite 2-hydroxydesipramine in the rat hippocampus and cingulate cortex, areas associated with emotional control. A concentration-dependent inhibition of net depolarization-induced 45Ca uptake was observed for desipramine (20-200 microM) in synaptosomes from both sites. However, 20 microM 2-hydroxydesipramine failed to inhibit calcium channel function in either of the two limbic sites; higher concentrations (60 or 200 microM) did produce a minor degree of inhibition in hippocampus synaptosomes. Others have shown that the clinically encountered plasma concentrations of 2-hydroxydesipramine are lower than those of desipramine, and the brain concentration of 2-hydroxydesipramine is therefore not expected to surpass or even reach 20 microM. In view of the previously observed clinical activity of 2-hydroxydesipramine, the present results indicate that calcium channel antagonism may not be the basis for the therapeutic effect of tricyclic antidepressants.

Chronic exercise increases SNAP-25 abundance in fast-transported proteins of rat motoneurones.

The aim of the present study was to determine whether endurance exercise training selectively modifies the relative abundance of some of the proteins that are subjected to fast axonal transport. Rats were trained on treadmill for 11-13 weeks. [35S]methionine was injected into the ventral horn of L4-L5 spinal cord segments, and transported [35S]methionine-labelled proteins were analysed on fluorograms of sodium dodecyl sulphate polyacrylamide gels. The proportion of a 28 kDa protein increased significantly after training, from 4.9% in controls of 7.7% in trained animals. Two-dimensional electrophoresis and immunoprecipitation identified it as SNAP-25/SuP. The increased availability of SNAP-25, a synaptic protein, may constitute part of the molecular basis of exercise-induced changes in nerve terminal morphology and physiology.

Absence of stereoselectivity of some tricyclic antidepressants for the inhibition of depolarization-induced calcium uptake in rat cingulate cortex synaptosomes.

This study was conducted in order to investigate the inhibition of synaptosomal 45calcium uptake by oxaprotiline, trimipramine and doxepin stereoisomers in the rat cingulate cortex which is an associative area of the cortex that interacts with the limbic system. A concentration-dependent inhibition of net depolarization-induced 45calcium uptake was observed for all substances tested. No significant difference in potency could be established within any of the pairs of antipodes and the IC50 values obtained were in the 30 microM to 50 microM range. These results are quite similar to those previously obtained with this group of compounds in hippocampal synaptosomes where discrepancies between relative calcium channel antagonism and clinical activity of the antipodes have been identified. Therefore, the present study of stereoisomers of tricyclic antidepressants fails to resolve these discrepancies and does not provide unequivocal support for the hypothesis that calcium channel blockade is somehow responsible for the therapeutic effect of tricyclic antidepressants.

Effect of some stereoisomeric tricyclic antidepressants on 45Ca uptake in synaptosomes from rat hippocampus.

The present study has examined the inhibition of synaptosomal 45calcium uptake by trimipramine, oxaprotiline and doxepin, and their stereoisomers, in synaptosomes from the rat hippocampus. No significant difference in potency could be established for inhibition of net depolarization-induced 45calcium uptake for any pair of antipodes, and the IC50 values for calcium channel blockade were in the vicinity of 30 microM for this group of compounds. Trimipramine, doxepin and oxaprotiline also inhibited the 45calcium uptake mediated by Na(+)-Ca2+ exchange, with IC50 values of 71 microM, 110 microM, and 100 microM, respectively. The similar potency of doxepin isomers for inhibition of voltage-dependent calcium channels is in harmony with their reported similar potency in the clinic. A slight difference in potency is reported between the isomers of oxaprotiline in the behavioral despair test in rats, and the dextrorotatory isomer of trimipramine is reported to be a much more potent antidepressant than the levorotatory isomer: these order of potencies do not correspond perfectly with the similar potency of the antipodes against voltage-dependent calcium channels. The present study of stereoisomeric tricyclic antidepressants therefore fails to provide unequivocal support for the hypothesis that calcium channel blockade by tricyclic antidepressants is involved in their therapeutic effect.

Effect of trimipramine on depolarization-induced and Na(+)-Ca2+ exchange-induced 45calcium uptake in synaptosomes from the cortex of the rat brain.

The present study examined the inhibition of synaptosomal uptake of 45calcium by racemic trimipramine and nortrimipramine and by enantiomers of trimipramine. Trimipramine, nortrimipramine, (+)-trimipramine and (-)-trimipramine inhibited the net K(+)-induced uptake of 45calcium with IC50 values of 31, 39, 17 and 95 microM, respectively. No significant difference could be detected between the parent compound trimipramine and the metabolite nortrimipramine; however, the levorotatory isomer had an IC50 value significantly larger than the dextrorotatory isomer. At normal therapeutic doses, a 25-40% inhibition of net K(+)-induced uptake of 45calcium, could be expected with trimipramine or 30-50% inhibition for trimipramine and nortrimipramine combined; these data, therefore, do not exclude the possibility that inhibition of voltage-dependent calcium channels could contribute to the therapeutic effect of trimipramine. The order of potency of stereoisomers of trimipramine, for inhibition of calcium channels, was the same as their reported order of potency in the clinic; this parallelism adds support to the possible involvement of blockade of calcium channels in the antidepressant effect. With respect to uptake of 45calcium induced by the Na(+)-Ca2+ exchange process, all drugs inhibited this mechanism with a similar potency (IC50 74-91 microM); the drugs are not expected to have a significant effect on this exchange process, at therapeutic antidepressant doses.

Mechanism of aminopyridine-induced release of [3H]dopamine from rat brain synaptosomes.

1. Aminopyridines (APs) induced the release of [3H]dopamine (3H-DA) from rat synaptosomal preparations. 2. 4-AP and 3,4-DAP were of equal efficacy in inducing release of 3H-DA; 3-AP, 2-AP and 2,6-AP were less active; pyridine and pyridine-4-carboxylamide were inactive. 3. Cd2+ was more effective in inhibiting 4-AP-induced release of 3H-DA (IC50 approximately 4 microM) than Co2+ and Ni2+ (IC50s approximately 500 microM). 4. While 4-AP increased the 45Ca2+ content of whole synaptosomal preparations, no effect of 4-AP on 45Ca2+ content was observed in lysed synaptosomal preparations. 5. 4-AP-induced 45Ca2+ uptake was inhibited by Cd2+, Ni2+ and Co2+ in concentration ranges similar to those inhibiting 3H-DA release.

Tricyclic antidepressants inhibit voltage-dependent calcium channels and Na(+)-Ca2+ exchange in rat brain cortex synaptosomes.

We have used a resting (5 mM K+) or depolarizing (60 mM K+) choline-based medium, and a nondepolarizing sodium-based or choline-based medium, to characterize the inhibitory potential of tricyclic antidepressants against the voltage-dependent calcium channels or the Na(+)-Ca2+ exchange process, respectively, in synaptosomes from rat brain cortex. Imipramine, desipramine, amitriptyline, and clomipramine inhibited net K(+)-induced 45Ca uptake with similar IC50 values (26-31 microM), and this uptake was also inhibited by diltiazem with an IC50 of 36 microM; these results indicate an inhibition of voltage-dependent calcium channels by tricyclic antidepressants. The net uptake of 45Ca induced by Na(+)-Ca2+ exchange was also inhibited by the four tricyclic antidepressants tested, but not by diltiazem; imipramine (IC50 = 94 microM) was a more potent inhibitor of this process than desipramine (IC50 = 151 microM), and the IC50 values of amitriptyline (107 microM) and clomipramine (97 microM) were similar to that of imipramine. Some degree (approximately 25%) of brain calcium channel blockade could be present at the steady-state concentrations of tricyclic antidepressants expected to occur therapeutic use of these compounds to treat depression or panic disorder.

Mechanisms of the inhibition of fast axonal transport by local anesthetics.

The present study attempted to clarify the mechanism(s) by which local anesthetics inhibit fast axonal transport. Spinal nerves of the bullfrog were incubated with local anesthetics under conditions known to inhibit transport and the effects of these exposures to local anesthetics on the content of adenosine triphosphate and creatine phosphate in nerves and on the density of microtubules in unmyelinated axons were examined. Lidocaine, at concentrations of 14 or 20 mM, did not reduce significantly the content of adenosine triphosphate (although significant reductions in creatine phosphate were observed); the density of microtubules was also not affected by 14 mM lidocaine. Some mechanism other than inhibition of oxidative metabolism or disruption of microtubules must therefore be responsible for the inhibition of fast axonal transport by 14 mM lidocaine. Significant reductions in the content of adenosine triphosphate were observed with 1 or 2 mM tetracaine and with 0.5 or 1 mM dibucaine (this latter concentration of dibucaine also reduced the content of creatine phosphate); however, comparison with the effects of 2,4-dinitrophenol indicated that these inhibitions of oxidative metabolism were insufficient to inhibit transport in the case of 0.5 mM dibucaine or could at best only partly explain the inhibition of transport in the other cases. Since the density of microtubules was not affected by 1 mM tetracaine and was not sufficiently reduced by 0.5 mM dibucaine to inhibit transport, some other effect must again largely contribute to or be solely responsible for the inhibition of fast axonal transport by these concentrations of dibucaine and tetracaine.

Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats.

In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins.

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules.

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.

Effects of caffeine and cyclic adenosine 3',5'-monophosphate on adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes.

The adenosine triphosphate-dependent calcium uptake by endoplasmic reticulum elements of lysed synaptosomes from rat brain cortex was studied. Caffeine exhibited a biphasic effect on this calcium uptake activity: concentrations of 1, 2, 5, 10 or 30 mM caffeine stimulated calcium uptake by 62, 111, 73, 88 and 60% respectively, whereas calcium uptake was inhibited by 55% at a 60-mM concentration of caffeine. Calcium release from endoplasmic reticulum elements of lysed brain synaptosomes was stimulated by 10 mM caffeine. Cyclic adenosine 3',5'-monophosphate stimulated calcium uptake in the lysed synaptosome preparation: exogenous concentrations of 0.05, 0.5, 5, 50, or 500 microM stimulated uptake by 67, 67, 95, 38 or 67% respectively. To explore the possibility that caffeine stimulated calcium uptake through inhibition of phosphodiesterase and consequent preservation of cyclic adenosine 3',5'-monophosphate, we have tested whether caffeine retained its ability to stimulate calcium uptake under conditions of maximal stimulation by cyclic adenosine 3',5'-monophosphate. The combined presence of 10 mM caffeine and 5 microM cyclic adenosine 3',5'-monophosphate resulted in an approximate doubling of the calcium uptake as compared to the uptake in the presence of the cyclic nucleotide alone, indicating that the stimulation due to caffeine does not occur via cyclic adenosine 3',5'-monophosphate.

Penfluridol, chlorprothixene and haloperidol block fast axonal transport in an order of potency consistent with a mechanism related to inhibition of calmodulin.

The effects of the inhibitors of calmodulin penfluridol, chlorprothixene and haloperidol on fast axonal transport, the content of adenosine triphosphate and creatine phosphate and the density of axonal microtubules, were measured in spinal nerves of the bullfrog in vitro. These drugs inhibited the fast orthograde transport of [3H]leucine-labelled proteins: 35 microM penfluridol, 70 microM cis-chlorprothixene, and 200 microM haloperidol were needed to produce and approximately 50% inhibition of transport, and the order of potency was, therefore, penfluridol greater than cis-chlorprothixene greater than haloperidol; the trans isomer of chlorprothixene was as effective as the cis isomer-of chlorprothixene in inhibiting fast axonal transport. None of these drugs significantly reduced the density of microtubules in unmyelinated axons of nerves, incubated as for a transport experiment. Exposure to the concentration of these drugs which inhibited transport did not reduce significantly the content of adenosine triphosphate of the nerves, except for a 22% reduction by trans-chlorprothixene, and they had no significant effect on the content of creatine phosphate except for a 27% reduction by penfluridol and a 20% reduction by trans-chlorprothixene. The inhibition of axonal transport by these drugs can therefore not be explained either by an interference with oxidative metabolism or by disruption of microtubules. The order of potency of penfluridol, chlorprothixene and haloperidol as inhibitors of fast axonal transport parallels their known order of potency as antagonists of calmodulin; inhibition of axonal transport may therefore be related to inhibition of the function of calmodulin by these drugs.

Fast axonal transport of acetylcholinesterase in rat sciatic motoneurons is enhanced following prolonged daily running, but not following swimming.

The effects of increases in neuronal activity on fast axonal transport of acetylcholinesterase (AChE) in sciatic motoneurons were studied by subjecting rats to daily running or swimming training (8 weeks). Net accumulation of AChE activity proximal and distal to a ligature served to evaluate orthograde and retrograde transport. Results showed that runners had greater orthograde and retrograde transport of AChE as compared to control animals, while no changes were found in swimmers. These adaptations in the runners were caused by the long-term nature of the training regimen since an acute exercise session had no effect on AChE transport. The observed changes may be attributed to an increase in the mobile fraction of AChE in the motoneurons. Since swimming training had no effect on transport but entails a high level of neuronal activity, it is suggested that increased impulse activity is not the factor mediating the adaptations in axonal transport of AChE which resulted from running training.

Presence of glycoproteins in the cell nucleus as shown by radioautographic studies after administration of [3H]fucose and [3H]galactose.

Dorsal root ganglia were removed from adult bullfrogs and incubated with [3H]fucose for intervals from 15 min to 1 h, followed by fixation. Some ganglia were post-incubated in the absence of [3H]fucose for up to 17 h. In additional in vivo experiments, young frogs were injected with [3H]fucose, and killed 30 min or 1 h later, and then ganglia were removed and fixed. Electron microscope radioautographs of the ganglia revealed an intense radioautographic reaction over the nuclei of Schwann and satellite cells as early as 5 min after initial exposure to [3H]fucose. At time intervals up to 2 h after initial exposure to [3H]fucose, the silver grains were evenly distributed over both the periphery and internal regions of the nucleus, while at 18 h they were localized to the cell periphery. In occasional cells, the perinuclear space was expanded in some areas and was the site of reaction. In young rats, injected with [3H]galactose and killed 15 min to 5 h later, electron microscope radioautographs revealed heavy reaction over the nuclei of duodenal villous and crypt columnar cells, in which the grains were evenly distributed over both the peripheral and internal regions. In mitotic cells, grains appeared to be associated with the condensed chromatin of forming chromosomes. These results provide strong evidence that glycoproteins exist in the nuclei of the above cell types and that they are actively renewed. The rapid appearance of nuclear reaction after initial exposure to [3H]fucose or [3H]galactose indicates that either these sugars are added to glycoproteins within the nucleus itself or that they migrate rapidly to this site after having been glycosylated elsewhere.