The allosteric potentiation of nicotinic acetylcholine receptors by galantamine is transduced into cellular responses in neurons: Ca2+ signals and neurotransmitter release

Neuronal nicotinic acetylcholine receptors (nAChR) modulate a variety of cellular responses, including Ca2+ signals and neurotransmitter release, which can influence neuronal processes such as synaptic efficacy and neuroprotection. In addition to receptor activation through the agonist binding site, an allosteric modulation of nAChR has also been described for a novel class of allosteric ligands. Of these, the acetylcholinesterase inhibitor and Alzheimer drug galantamine represents the prototypical allosteric ligand, based on its potentiation of nAChR-evoked single-channel and whole-cell currents. The aim of this study was to establish whether the allosteric potentiation of nAChR currents is transduced in downstream cellular responses to nAChR activation, namely increases in intracellular Ca2+ and [3H]noradrenaline release. In SH-SY5Y cells, galantamine potentiated nicotine-evoked increases in intracellular Ca2+ and [3H]noradrenaline release with a bell-shaped concentration-response profile; maximum enhancement of nicotine-evoked responses occurred at 1 muM galantamine. This potentiation was blocked by mecamylamine, whereas galantamine had no effect on these measures in the absence of nicotine. Galantamine did not compete for radioligand binding to the agonist binding sites of several nAChR subtypes, consistent with an allosteric mode of action. Unlike galantamine, the acetylcholinesterase inhibitors rivastigmine and donepezil did not potentiate nAChR-mediated responses, whereas donepezil was a reasonably potent inhibitor of nicotine- and KCl-evoked increases in Ca2+. nAChR-mediated [3H]noradrenaline release from hippocampal slices was also potentiated by galantamine, with an additional component attributable to acetylcholinesterase inhibition and subsequent increase in acetylcholine. These results indicate that the allosteric regulation of nAChR results in the potentiation of receptor-dependent cellular processes relevant to many of the physiological consequences of nAChR activation.     

Blockade of Ca2+ -activated K+ channels by galantamine can also contribute to the potentiation of catecholamine secretion from chromaffin cells

Galantamine is a drug in clinical use for the treatment of Alzheimer's disease, but its mechanism(s) of action remains controversial. Here we addressed the question whether galantamine could potentiate neurotransmitter release by inhibiting small conductance Ca2+ -activated K+ channels (KCa2). Galantamine potentiated catecholamine secretory responses induced by 10 s pulses of acetylcholine and high [K+]o applied to fast-superfused bovine adrenal chromaffin cell populations. Catecholamine release was significantly enhanced by galantamine although we did not find concentration dependence in the range 0.1-1 microM. The KCa2 channel blocker apamin (0.3 microM) occluded the potentiating effects of galantamine on acetylcholine-evoked secretion. Like apamin, galantamine also modified the firing of action potentials, but to a lesser extent. In addition, 1 microM galantamine reduced by 41% the KCa2 current without modifying the voltage-dependent Ca2+ currents. These results constitute the first direct evidence that galantamine can potentiate neurotransmitter release by blocking KCa2 channels, in addition to its already demonstrated capacity to mildly block acetylcholinesterase or potentiate allosterically nicotinic receptors.     

Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between pharmacological effects and clinical efficacy

The deficiency in cholinergic neurotransmission in Alzheimer's disease has led to the development of cholinesterase inhibitors as the first-line treatment for symptoms of this disease. The clinical benefits of these agents include improvements, stabilisation or less than expected decline in cognition, function and behaviour. The common mechanism of action underlying this class of agents is an increase in available acetylcholine through inhibition of the catabolic enzyme, acetylcholinesterase. There is substantial evidence that the cholinesterase inhibitors, including donepezil, galantamine and rivastigmine, decrease acetylcholinesterase activity in a number of brain regions in patients with Alzheimer's disease. There is also a significant correlation between acetylcholinesterase inhibition and observed cognitive improvement. However, the cholinesterase inhibitors are reported to have additional pharmacological actions. Rivastigmine inhibits butyrylcholinesterase with a similar affinity to acetylcholinesterase, although it is not clear whether the inhibition of butyrylcholinesterase contributes to the therapeutic effect of rivastigmine. Based on data from preclinical studies, it has been proposed that galantamine also potentiates the action of acetylcholine on nicotinic receptors via allosteric modulation; however, the effects appear to be highly dependent on the concentrations of agonist and galantamine. It is not yet clear whether these concentrations are related to those achieved in the brain of patients with Alzheimer's disease within therapeutic dose ranges. Preclinical studies have shown that donepezil and galantamine also significantly increase nicotinic receptor density, and increased receptor density may be associated with enhanced synaptic strengthening through long-term potentiation, which is related to cognitive function. Despite these differences in pharmacology, a review of clinical data, including head-to-head studies, has not demonstrated differences in efficacy, although they may have an impact on tolerability. It seems clear that whatever the subsidiary modes of action, clinical evidence supporting acetylcholinesterase inhibition as the mechanism by which cholinesterase inhibitors treat the symptoms of Alzheimer's disease is accumulating. Certainly, as a class, the currently approved cholinesterase inhibitors (donepezil, galantamine, rivastigmine and tacrine) provide important benefits in patients with Alzheimer's disease and these drugs offer a significant advance in the management of dementia.     

Cellular responses to nicotinic receptor activation are decreased after prolonged exposure to galantamine in human neuroblastoma cells

In this study, we have examined cellular responses of neuroblastoma SH-SY5Y cells after chronic treatment with galantamine, a drug used to treat Alzheimer's disease that has a dual mechanism of action: inhibition of acetylcholinesterase and allosteric potentiation of nicotinic acetylcholine receptors (nAChR). Acute experiments confirmed that maximum potentiation of nicotinic responses occurs at 1 microM galantamine; hence this concentration was chosen for chronic treatment. Exposure to 1 microM galantamine for 4 days decreased Ca(2+) responses (by 19.8+/-3.6%) or [(3)H]noradrenaline ([(3)H]NA) release (by 23.9+/-3.3%) elicited by acute application of nicotine. KCl-evoked increases in intracellular Ca(2+) were also inhibited by 10.0+/-1.9% after 4 days' treatment with galantamine. These diminished responses are consistent with the downregulation of downstream cellular processes. Ca(2+) responses evoked by activation of muscarinic acetylcholine receptors were unaffected by chronic galantamine treatment. Exposure to the more potent acetylcholinesterase inhibitor rivastigmine (1 microM) for 4 days failed to alter nicotine-, KCl-, or muscarinic receptor-evoked increases in intracellular Ca(2+). These observations support the hypothesis that chronic galantamine exerts its effects through interaction with nAChR in this cell line. Exposure to 10 microM nicotine for 4 days produced decreases in acute nicotine- (18.0+/-3.5%) and KCl-evoked Ca(2+) responses (10.6+/-2.5%) and nicotine-evoked [(3)H]NA release (26.0+/-3.3%) that are comparable to the effects of a corresponding exposure to galantamine. Treatment with 1 microM galantamine did not alter numbers of [(3)H]epibatidine-binding sites in SH-SY5Y cells, in contrast to 62% upregulation of these sites in response to 10 microM nicotine. Thus, chronic galantamine acts at nAChR to decrease subsequent functional responses to acute stimulation with nicotine or KCl. This effect appears to be independent of the upregulation of nAChR-binding sites.     

Interactions of calcium antagonists and the calcium channel agonist Bay K 8644 on neurotransmission of the mouse isolated vas deferens.

1. The present study compares the effects of verapamil and Bay K 8644 on twitches of the mouse vas deferens induced by field stimulation at 0.1 Hz. The influence of interactions between these drugs and nifedipine on neurotransmission was also investigated. 2. Bay K 8644 (0.1 nM-3 microM) and verapamil (1-100 microM) potentiated twitches maximally by about 1000% (EC50 17.3 nM) and 300% (EC50 17.5 microM), respectively. Nifedipine (0.1 nM-1 microM) only reduced twitch magnitude (IC50 7.7 nM). All effects were reversed following washout. 3. Yohimbine (1-100 microM) reversed twitch potentiation caused by verapamil but not by Bay K 8644. Prazosin (1 microM) did not reduce basal twitch tension nor antagonize twitch potentiation by verapamil. 4. Twitch inhibition by nifedipine was unaltered by previous incubation with verapamil (30 microM), but Bay K 8644 (1 microM) shifted the curve to nifedipine 120 fold to the right. Previous incubation with nifedipine (1 microM) blocked potentiation induced by verapamil but did not modify responsiveness to Bay K 8644. 5. Previous addition of verapamil (30 microM) markedly enhanced twitch potentiation caused by Bay K 8644 in a supra-additive fashion. In experiments conducted in the reversed condition, Bay K 8644 (1 nM but not 10 nM) potentiated the effect of verapamil in a similar manner but to a lesser extent. 6. It is concluded that verapamil, in contrast to nifedipine, markedly enhances neurally-evoked twitches of the mouse vas deferens. Bay K 8644 produces essentially the same effect as verapamil, but its potency is 1000 fold and its maximal effect about 3 fold greater than that observed for verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)     

An update on the pharmacology of galantamine

Alzheimer's disease (AD) is associated with a gradual loss of attention and memory that has been related to impairment of brain cholinergic neurotransmission, particularly a deficit of cholinergic neurons. The first therapeutic target that has demonstrated therapeutic efficacy on cognition, behaviour and functional daily activities has been the inhibition of acetylcholinesterase. The acetylcholinesterase inhibitors used to treat AD patients at present are donepezil, rivastigmine and galantamine. This review summarises the current state of the art concerning the pharmacology of galantamine, focusing on the most important details of its possibilities as an acetylcholinesterase inhibitor, an allosteric potentiator of neuronal nicotinic receptors for acetylcholine, a modulator of neurotransmitter release, and an agent causing neuroprotection through an antiapoptotic action. In so doing, galantamine will be discussed in the context of the treatment of dementia, both of AD type and of mixed vascular-Alzheimer type.     

糖尿病大鼠输精管平滑肌收缩超反应性及钙通道特性(英文)

为了解糖尿病对输精管平滑肌功能的影响及其机理,我们比较了链脲菌素(streptozotocin)所致糖尿病大鼠及共同龄对照组大鼠输精管平滑肌对电场刺激,氯化钾,去氧肾上腺素的反应及钙通道的改变。电场刺激引起的收缩反应是单收缩之后继以连续性收缩。糖尿病组单收缩幅度较大,两组连续收缩几乎相同。1μmol/L Bay K8644使糖尿病鼠输精管产生单收缩,对照组无影响。氯化钾引起的收缩和钙内流在两组都增强,但在1 μmol/L Bay K8644存在时,糖尿病组比对照组增加明显。10μmol/L去氧肾上腺素引起的收缩可完全被0.1μmol/L硝苯啶阻断。100μmol/L新霉素在对照组可完全抑制去氧肾上腺素引起的收缩,但在糖尿病组只能部分抑制,并使单收缩增强。用钙通道拮抗剂配体[~3H]PN200-110所做的结合实验表明,对照或糖尿病组钙通道的亲和力和结合位点的数量都没有明显的不同。在输精管可溶性成分中,蛋白激酶C含量在糖尿病组是对照组的两倍。这些结果提示在没有神经病变及钙通道数量不变的情况下,氯化钾增强链脲菌素所致糖尿病大鼠输精管的收缩反应部分是由于钙通道开放的概率和蛋白激酶C活性增强所致。     

Paradoxical effects of thioridazine on electromechanical coupling in the human and rat vas deferens

The effects of thioridazine on the responses of isolated human and rat vas deferens to high [K+]0, A23187 and caffeine were examined. In the presence of Ca2+ (2.5 mM), thioridazine (1-10 microM) induced spontaneous contractions but caused a dose-related inhibition of the phasic and secondary parts of the response to high [K+]0 (136 mM). The relaxation phase of the high [K+]0 response of the human vas deferens was unaffected by thioridazine (up to 10 microM). In Ca2(+)-free/EGTA (0.5 mM) media, thioridazine caused a dose-related potentiation, shortened the latency and prolonged the duration of high [K+]0 responses. Contractions to caffeine (20 mM) and A23187 (20-50 microM) were relatively unchanged by thioridazine (10 microM). The spontaneous activity caused by thioridazine (10 microM) was sensitive to the Ca2(+)-channel blockers nifedipine (10 microM) or verapamil (10 microM). These results indicate that the action of thioridazine during electromechanical coupling in the human and rat vas deferens may involve more than its blockade of voltage gated Ca2+ channels.     

Dopamine modulates peripheral purinergic neurotransmission through multiple presynaptic receptors: tissue-dependent effects.

This study investigated the identity of presynaptic receptors involved in dopaminergic modulation of purinergic transmission in peripheral tissues including isolated rat vas deferens and urinary bladder. Isometric muscle twitches were established in the two tissues by low frequency electric field-stimulation (0.05 Hz, 1-ms duration, and supramaximal voltage). Exposure to prazosin, 50 nmol l-1 (vas deferens), or atropine, 3 micromol l-1 (urinary bladder), had no effect on the developed twitches. In contrast, desensitisation of P2X-purinoceptors by alpha,beta-methylene ATP (alpha,beta-mATP, 30 micromol l-1) abolished the twitches in both tissues, confirming their purinergic origin. Dopamine (1.8x10(-7) to 4.2x10(-5) mol l-1) reduced the twitch response in a concentration-related manner. Yohimbine (alpha2-adrenoceptor antagonist, 0.3 micromol l-1) significantly (P<0.05) attenuated the inhibitory effects of dopamine and caused an upward shift in the concentration-response curves in the vas deferens and the urinary bladder. On the other hand, a blockade of DA2-dopaminoceptors by domperidone (1 micromol l-1) produced significant (P<0.05) reductions in dopamine responses only in rat vas deferens, with no effect in the urinary bladder. These data suggest that dopamine exerts inhibitory influences on purinergically-mediated muscle twitches in rat vas deferens and urinary bladder. More importantly, the nature of presynaptic receptors (alpha2-adrenergic and/or DA2-dopaminergic) involved in mediating dopamine effects is dependent on the tissue under investigation.     

Inhibition of field stimulation-induced contractions of rabbit vas deferens by muscarinic receptor agonists: selectivity of McN-A-343 for M1 receptors

Inhibition of the field stimulation-induced twitch responses of the rabbit vas deferens by the muscarinic receptor agonist, McN-A-343, has been attributed to presynaptic muscarinic receptors of the M1 subtype located on noradrenergic nerve terminals. Stimulation of these receptors causes inhibition of transmitter release and inhibition of the contractile response. However, the selectivity of McN-A-343 for M1 receptors has been questioned and this throws doubt on whether the prejunctional receptors of the rabbit vas deferens are of the M1 subtype. In this study we have undertaken a comprehensive re-evaluation of the inhibition of prostatic and epididymal portions of the rabbit isolated field-stimulated vas deferens by several agonists, including McN-A-343, and quantified the antagonism by M1-selective antagonists, pirenzepine and telenzepine. Prostatic and epididymal portions of vasa deferentia from New Zealand White rabbits were immersed in a low Ca2+ Krebs solution at 32+/-0.5 degrees C gassed with 5% CO2 in oxygen. Yohimbine (1.0mM) was present throughout to block prejunctional alpha2-adrenoceptors. Field stimulation was applied by repeated application of single pulses (30 V, 0.05 Hz, 0.5 ms) and isometric contractions recorded. Carbachol and oxotremorine initially potentiated the epididymal contractions but at higher concentrations there was inhibition. In the prostatic portion, oxotremorine only inhibited. McN-A-343 produced inhibitory responses only in both epididymal and prostatic portions. Pirenzepine shifted the concentration-response curves forthe inhibitory responses to oxotremorine to the right. However, the potentiation of the twitches also became more apparent with the lower concentrations of oxotremorine. Schild plots for the antagonism by pirenzepine yielded pA2 values of 7.96+/-0.004 and 7.7+/-0.02 for the epididymal and prostatic portions, respectively. The concentration-response curves for the inhibition of twitches by McN-A-343 were displaced to the right in a parallel manner by pirenzepine in both prostatic and epididymal portions with no potentiation of the twitches. The Schild plot for this antagonism generated pA2 values of 7.68+/-0.01 and 8.07+/-0.01, respectively. Telenzepine caused parallel shifts of the McN-A-343 concentration-response curves to the right in prostatic portions, the pA2 value being 8.70+/-0.13. Telenzepine (10(-7) M) abolished the inhibitory effect of carbachol to reveal only concentration-dependent potentiation of the contractions. The Schild plot for antagonism of this contractile effect yielded a pA2 value (7.07+/-0.09) that was significantly less by almost two orders of magnitude (1.70) than the value for the antagonism by telenzepine of the McN-A-343-induced inhibitory response. The pA2 values of pirenzepine and telenzepine against the inhibitory responses of the rabbit vas deferens are consistent with the involvement of M1 receptors. This leads to the conclusion that McN-A-343 causes inhibition through this receptor type. The doubts concerning the selectivity of McN-A-343 for M1 receptors are therefore unfounded. The fact that McN-A-343 does not display a selective binding profile suggests that its selectivity does not arise from affinity differences but probably resides in its intrinsic efficacy.     

Competitive antagonism by glibenclamide of cromakalim inhibition of twitch contractions in rabbit vas deferens

Electrically induced contractions of the rabbit isolated vas deferens were potentiated by carbachol but inhibited by the K+ channel opener cromakalim. The inhibition by cromakalim could be competitively antagonized by 10(-7)-10(-6) M glibenclamide (pA2 = 7.17) and was also reversed by carbachol, extra K+ or Bay k 8644. The data support the view that smooth muscle membrane depolarization is the cause for carbachol potentiation in rabbit vas deferens and that hyperpolarization by cromakalim is susceptible to blockade by the inhibitor of ATP-sensitive K+ channels, glibenclamide.     

Protective effect of donepezil in a primary culture of rat cortical neurons exposed to oxygen-glucose deprivation

Donepezil hydrochloride (donepezil: (+/-)-2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-indan-1-one monohydrochloride) is a potent acetylcholinesterase inhibitor used for treatment of Alzheimer's disease. Although acetylcholinesterase inhibitors are used as a symptomatic treatment for Alzheimer's disease, it is not clear whether or not they are effective against progressive degeneration of neuronal cells. In this study, we investigated the neuroprotective effects of donepezil and other acetylcholinesterase inhibitors used for treatment of Alzheimer's disease, i.e., galantamine, rivastigmine, and tacrine. As a neurodegenerative model, we used rat cortical neurons exposed to oxygen-glucose deprivation. Lactate dehydrogenase (LDH) released into the culture medium was measured as a marker of neuronal cell damage. First, the effects of donepezil (10 microM) on three different treatment schedules (from 12 h before to 24 h after oxygen-glucose deprivation (pre-12 h), from 1 h before to 24 h after oxygen-glucose deprivation (pre-1 h) and from 1 h after to 24 h after oxygen-glucose deprivation (post-1 h)) were compared. The pre-12-h treatment most effectively inhibited LDH release. The protective effect of donepezil was confirmed morphologically. Next, the effects of donepezil and the other three acetylcholinesterase inhibitors were compared under the pre-12-h treatment condition. Donepezil (0.1, 1, and 10 microM) significantly decreased LDH release in a concentration-dependent manner. However, galantamine (1, 10, and 100 microM), tacrine (0.1, 1, and 10 microM), and rivastigmine (0.1, 1, and 10 microM) did not significantly decrease LDH release. The neuroprotective effect of donepezil was not antagonized by scopolamine or mecamylamine. These results demonstrate that donepezil has a protective effect against oxygen-glucose deprivation-induced injury to rat primary cultured cerebral cortical neurons. Besides, it is suggested that this effect of donepezil is independent of muscarinic cholinergic system and nicotinic cholinergic system. Thus, donepezil is expected to have a protective effect against progressive degeneration of brain neuronal cells in ischemic cerebrovascular disease and Alzheimer's disease.     

Comparison of the effects of apamin, a Ca2+-dependent K+ channel blocker, and arylazido aminopropionyl ATP (ANAPP3), a P2-purinergic receptor antagonist, in the guinea-pig vas deferens

Apamin, which blocks Ca2+-dependent increases in K+ permeability, antagonizes ATP-induced relaxation of several smooth muscles. The ATP photoaffinity label arylazido aminopropionyl ATP (ANAPP3), following its photolysis in the presence of the guinea-pig vas deferens, antagonizes contractile responses to ATP. This study was conducted to determine whether apamin antagonizes ATP-induced responses in the guinea-pig vas deferens, and also to evaluate whether ANAPP3 antagonizes responses to ATP by interfering with Ca2+-dependent K+ permeability changes. Apamin (10(-6) M) potentiated ATP-induced contractions. This potentiation was nonspecific in that responses to norepinephrine, histamine and acetylcholine also were enhanced; responses to KCl were unaffected. To evaluate the possible interactions between the two agents at the same cellular site, the effect of apamin was examined in ANAPP3-treated tissues. In such tissues apamin did not potentiate the residual responses to ATP; however, apamin was nevertheless able to potentiate responses of ANAPP3-treated tissues to norepinephrine, histamine and acetylcholine, and responses to KCl remained unaffected. These studies provide additional support for the view that ANAPP3 antagonizes ATP-induced responses of the guinea-pig vas deferens by blocking P2-purinergic receptors. The antagonism by ANAPP3 is not attributable to a blockade of Ca2+-dependent K+ permeability changes.     

An update on the pharmacology of galantamine

Alzheimer's disease (AD) is associated with a gradual loss of attention and memory that has been related to impairment of brain cholinergic neurotransmission, particularly a deficit of cholinergic neurons. The first therapeutic target that has demonstrated therapeutic efficacy on cognition, behaviour and functional daily activities has been the inhibition of acetylcholinesterase. The acetylcholinesterase inhibitors used to treat AD patients at present are donepezil, rivastigmine and galantamine. This review summarises the current state of the art concerning the pharmacology of galantamine, focusing on the most important details of its possibilities as an acetylcholinesterase inhibitor, an allosteric potentiator of neuronal nicotinic receptors for acetylcholine, a modulator of neurotransmitter release, and an agent causing neuroprotection through an antiapoptotic action. In so doing, galantamine will be discussed in the context of the treatment of dementia, both of AD type and of mixed vascular-Alzheimer type.     

Newly synthesized alpha 1-adrenoceptor blockers (SM911 and SM2470) and characterization of alpha-adrenoceptors in rat aortic strips, rat vas deferens preparations and rabbit aortic strips

Alpha 1-adrenergic potencies of SM911 and SM2470, whose chemical structures are similar to that of prazosin, a selective alpha 1-adrenoceptor blocker, were tested in rabbit aortic strips, rat aortic strips and rat vas deferens preparations. SM2470 was as potent as prazosin in alpha 1-adrenoceptor blocking effects, though SM911 was 0.5-0.1 as potent as prazosin. The pA2-values for prazosin, SM911 and SM2470 were approximately one order of magnitude lower in rabbit aortic strips and rat vas deferens preparations than in rat aortic strips, suggesting that alpha 1-adrenoceptors in these tissues may not be identical. SM911 and SM2470 as well as prazosin did not interact with alpha 2- and beta-adrenoceptors, muscarinic and nicotinic cholinoceptors, and histamine and serotonin receptors in doses up to 10(-5) M.     

Adjunctive galantamine, but not donepezil, enhances the antipsychotic-like effect of raclopride in rats

Acetylcholine (ACh) esterase inhibitors like galantamine and donepezil have been tested as adjunct treatment in schizophrenia. Although ACh esterase inhibition might confer some antipsychotic activity, the role of allosteric potentiation of nicotinic ACh receptors (nAChRs), which is an additional mechanism of galantamine, remains elusive. Therefore, the potential antipsychotic-like effects of galantamine and donepezil, respectively, alone, and in combination with the dopamine D2/3 receptor antagonist, raclopride, were tested in the conditioned avoidance response (CAR) test and extrapyramidal side-effect liability was assessed with the catalepsy test. Neither galantamine nor donepezil alone suppressed CAR selectively. Galantamine, but not donepezil, enhanced the raclopride-induced suppression of CAR, predicting augmentation of antipsychotic activity. In contrast to donepezil, galantamine did not increase catalepsy, alone or combined with raclopride. These data suggest that allosteric potentiation of nAChRs may mediate the antipsychotic-like effect of adjunctive galantamine and provide support for the development of alpha7 nAChR-selective allosteric potentiators for schizophrenia.     

The influence of ouabain on twitch contractions in the presence of veratridine

1. Direct stimulation evoked twitches in mouse diaphragm muscles in presence of 10 microM D-tubocurarine in vitro. Effects of ouabain and their dependence on K+ were examined on the twitch responses and action potentials in the presence and absence of twitch potentiators. 2. Ouabain inhibited twitch contractions only in the presence of veratridine, aconitine and monensin while it had no inhibitory effect on control twitches. The interactions between ouabain and these twitch potentiators depended on the presence of external K+, except in the case of monensin. 3. Removal of Ca2+ from a bathing solution accelerated the potentiating effect of veratridine and the antagonizing effect of ouabain. 4. Caffeine further potentiated the twitches which had been attenuated by ouabain combined with veratridine. 5. Ouabain combined with veratridine consistently decreased resting membrane potentials, action potentials and overshoot potentials and prolonged time to peak of and duration of the muscle action potentials. 6. Tetraethylammonium, 4-aminopyridine, and caffeine produced twitch potentiation which was insensitive to ouabain or the removal of K+. 7. These results suggest that twitch contractions in the presence of activators of sodium channels link with activation of Na+-K+-ATPase. Accumulation of Na+ inside the muscle fibres may uncouple the excitation-contraction system. 8. This uncoupling may not include the caffeine-sensitive process that controls the release of Ca2+ from the sarcoplasmic reticulum. Na+ accumulation may decrease transmembraneous gradient of this ions, thereby causing a reduction in excitation coupled with twitch contraction.     

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine,donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A (K(i) 7 x 10(-9) M) and donepezil (K(i) 4 x 10(-9) M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil (K(i) 3.5 x 10(-9) M) and tacrine (K(i) 2.3 x 10(-8) M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants (K(i)) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.     

The involvement of fast calcium channel activity in the selective activation of phasic contractions by partial depolarization in rat vas deferens smooth muscle

In the prostatic portion of the rat vas deferens, 65% of the preparations studied developed pronounced rapid twitch activity in response to slight depolarization by 15 mM K+ salines. The mechanism underlying this response was studied using treatments designed to inhibit the influence of endogenous transmitters and using recognized calcium antagonist drugs. Although the action of phentolamine was inconclusive, experiments employing guanethidine, reserpine, 6-hydroxydopamine, atropine and alpha,beta-methylene ATP suggest that endogenous transmitter release was not responsible for the observed twitch activity. Twitch activity was strongly dependent upon [Ca]0. The 15 mM K+ twitch activity was inhibited by verapamil (5 X 10(-5) M) but was resistant to 10(-3) M lanthanum. Twitch activity was, however, abolished by 10(-3) M Mn2+ ions and was markedly potentiated by 2 X 10(-3) M TEA. The rapid twitch activity exhibited a strong voltage-dependency, being abolished by [K]0 elevations of 25 mM and above. It is concluded that this phasic activity of the vas deferens smooth muscle may depend upon fast calcium channel activity which, in contrast to voltage-dependent slow calcium channel activity, shows ready voltage-inactivation on substantial depolarization.     

Pharmacological profiles of galantamine: the involvement of muscarinic receptor

Galantamine, currently used in Japan for the treatment of Alzheimer's disease, may improve cognitive dysfunction and psychiatric illness in schizophrenia, major depression, bipolar disorder and alcohol abuse. It is a rather weak acetylcholinesterase inhibitor in vitro, but has additional allosteric potentiating effects at nicotinic receptors. We have found that galantamine increased acetylcholine levels in the brain. This suggests that the pharmacological effects of galantamine are mediated by not only nicotinic receptors but also muscarinic receptors. We found that galantamine, but not donepezil, improved prepulse inhibition (PPI) deficits in isolation-reared mice, although both drugs improved PPI deficits in an apomorphine model. The difference in the effects on PPI deficits between galantamine and donepezil may be explained by the effects on muscarinic receptors. This review summarizes the pharmacological profiles of galantamine, focusing on the importance of muscarinic receptors.