The cholinergic hypofunction in Alzheimer’s disease(AD)appears to be linked with two other major hallmarks of this disease, β−amyloid and hyperphosphorylated tau protein.Formation of β−amyloids might impair the coupling of M1 muscarinic acetylcholine receptors(mAChR)with G−proteins.This can lead to decreased signal transduction, a decrease of trophic and non−amyloidogenic amyloid precursor protein(APPs)and generation of more β−amyloids, aggravating further the cholinergic deficiency.This review is an attempt to explore the M1 mAChR regulation of β−amyloid metabolism, tau hyperphosphorylation and cognitive functions.The therapeutic potential of M1−selective muscarinic agonists including AF102B, AF150(S), AF267B(the AF series)is evaluated and compared, when possible, with several FDA−approved acetylcholinesterase inhibitors.These M1 agonists can elevate APPs, decrease tau protein phosphorylation/hyperphosphorylation in vitro and in vivo and restore cognitive impairments in several animal models for AD.Except for the M1 agonists, no other compounds were reported yet with combined effects;e.g., amelioration of cognition dysfunction and beneficial modulation of APPs/β−amyloid together with tau hyperphosphorylation/phosphorylation.This property of M1 agonists to alter different aspects associated with AD pathogenesis could represent the most remarkable clinical value of such drugs.
We studied the effect of pioglitazone on the transcription of 42 genes associated with diabetes to examine the relationship between the antidiabetic action of thiazolidinediones(TZDs)and their ability to modulate transcription through their peroxisome proliferater−activated receptor(PPAR)−agonistic activity.Diabetic(db/db)mice were orally administered with pioglitazone for two weeks.Total RNA was prepared from liver, muscle and adipocytes and the quantity of mRNA was determined by comparative RT−PCR.The expression of diabetes−related genes was compared between lean and untreated db/db mice and between untreated and drug−treated db/db mice.The onset of diabetes was associated with a considerable alteration in the expression of a large number of diabetes−related genes.Treatment of db/db mice with pioglitazone modulated the expression of genes involved in the metabolism of glucose, lipids and lipoproteins.This included genes for phosphoenolpyruvate carboxykinase, β−oxidation enzymes, lipoprotein lipase, apolipoprotein AI and uncoupling proteins.Most of the genes responsible for insulin signaling were unaffected.Administration of pioglitazone was also shown to induce PPARγ expression in liver and muscle.It is therefore possible to hypothesize that TZDs may ameliorate diabetes through a mechanism of action involving a direct decrease in plasma glucose and triglyceride levels and improvements in free fatty acid−induced insulin resistance.
We previously showed that blood acetylcholine(ACh)originates mainly from T−lymphocytes, and that stimulation of muscarinic ACh receptors(mAChRs)induces Ca2+ oscillations and up−regulates c−fos gene expression in both T− and B−lymphocytes.In the present study, we investigated which mAChR subtypes are involved in Ca2+ signaling and c−fos gene expression in human T−(CEM)and B−(Daudi)cells.Stimulation of mAChRs with 100μM oxotremorine−M, an M1/M3 agonist, increased levels of intracellular free Ca2+([Ca2+]i)and c−fos mRNA expression in both cell lines.4−DAMP, an M3 antagonist, more effectively blocked the oxotremorine−M−induced increase in [Ca2+]i than pirenzepine and telenzepine, M1−receptor antagonists;AF−DX 116, an M2 antagonist;hexahydrosiladifenidol, a weak M3 antagonist;or hexamethonium and d−tubocurarine, nicotinic receptor antagonists.McN−A−343(100μM), a partial M1−receptor agonist, had no apparent effect on [Ca2+]i in either cell line.The oxotremorine−M−induced up−regulation of c−fos transcription was inhibited by 4−DAMP, but not by pirenzepine or AF−DX 116.Our findings thus suggest that ACh released from T−lymphocytes acts as an autocrine/paracrine factor, transmitting a Ca2+−dependent signal to the nuclei of T− and B−lymphocytes via M3 receptors.
This study examined whether the renal kallikrein−kinin system(KKS)is involved with furosemide−induced natriuresis in rats.Intravenous administration of furosemide(10 mg/kg)to anesthetized rats infused with physiological saline(saline)increased renal KK excretion as well as urine volume and urinary excretions of sodium, chloride and potassium.The change in the increase of renal KK excretion by furosemide at a dose of 1.0mg/kg relative to the control was larger than that of urine volume.Pretreatment with a B2−receptor antagonist, 8−[3−[N−[(E)−3−(6−acetamidopyridin−3−yl)acryloylglycyl]−N−methylamino]−2, 6−dichloro−benzyloxy]−2−methylquinoline(FR173657, 100mg/kg), significantly inhibited the furosemide−induced natriuresis by 58.6%.The effect of FR173657 on the furosemide−induced natriuresis was also examined in hypotonic saline−loading rats.Similar to the saline−loading rats, urinary excretion of sodium collected during the first 8 h in metabolic cages significantly reduced by 22.4% when FR173657(100mg/kg)was given concurrently with furosemide(100mg/kg)and hypotonic saline(5% of body wt.).These results indicate that furosemide increased renal KK excretion through a mechanism different from a washout mechanism and induced natriuresis partly through an augmentation of the renal KKS following the increase in renal KK excretion in both the saline− and hypotonic saline−loading rats.
Murine neuroblastoma cell line Neuro−2A cells and rat brain astrocytes showed a dose−dependent increase in intracellular Ca2+ in response to bradykinin, when assessed by a single cell image analyzing system.The Ca2+ increase in Neuro−2A cells by bradykinin was also examined by a suspension fluorescent assay using fura−2 loading.The Ca2+ increase in both cases was suppressed by a bradykinin B2 receptor antagonist, Hoe 140, but not by a B1 receptor antagonist, des−Arg−Hoe 140, suggesting that the effect occurred via specific B2 receptor activation.RT−PCR for bradykinin B2 receptor mRNA showed that both Neuro−2A cells and the astrocytes expressed B2 receptor mRNA.Binding of [3H]bradykinin to Neuro−2A cells was assessed, and a specific binding constant of 0.75 nM was determined.Furthermore, the increase in [Ca2+]i by bradykinin could be caused by a release of Ca2+ from storage sites in the endoplasmic reticulum, since thapsigargin and U−73122 attenuated the effect of bradykinin in Neuro−2A as well as in astrocytes.These results indicate that both astrocytes and neuroblastoma Neuro−2A cells stimulated by bradykinin could express a bradykinin B2 receptor−mediated intracellular Ca2+ increase leading to signal transduction.
It was previously found that human chymase cleaves big endothelins(ETs)at the Tyr31−Gly32 bond and produces 31−amino acid ETs(1−31).In the present study, human plasma concentrations of ET−1(1−31)and ET−1 were examined and the effect of synthetic ET−1(1−31)on the proliferation of cultured human mesangial cells(HMCs)was investigated.The proliferative effect of ET−1(1−31)was evaluated from the [3H]−thymidine uptake.The activity of extracellular signal−regulated kinase(ERK)and DNA binding activity of activator protein−1 were determined by using an in−gel kinase assay and gel mobility shift assay, respectively.Immunoreactive ET−1(1−31)was detectable in plasma, but the level was slightly lower than that of ET−1.ET−1(1−31)increased [3H]−thymidine incorporation in HMCs to a degree similar to that induced by ET−1.ET−1(1−31)also activated ERK1/2.Inhibition of protein kinase C and ERK kinase caused a reduction of ET−1(1−31)−induced ERK1/2 activation.The ERK1/2 activation was followed by an increase in transcription factor activator protein−1 DNA binding activity.These findings suggest that ET−1(1−31)is a bioactive peptide in humans and ET−1(1−31)itself stimulates HMC proliferation.
The effect of long−term treatment with dihydropyridine calcium antagonists(amlodipine, pranidipine, nicardipine)on the periarterial nerve function was investigated in the perfused mesenteric vascular bed isolated from spontaneously hypertensive rat(SHR).Male 8−week−old SHR received amlodipine(0.01% and 0.02%)and nicardipine(0.1%)in drinking water and pranidipine(0.0035% and 0.035%)in rat chow for 7 weeks.Mean blood pressure in SHR was significantly lowered by long−term treatment with each calcium antagonist.In mesenteric vascular preparations treated with each calcium antagonist, vasoconstriction induced by periarterial nerve stimulation(PNS;4, 8 and 12 Hz)was significantly smaller than that in non−treated SHR.The PNS(8 Hz)−evoked norepinephrine(NE)overflow in the perfusate was significantly decreased by amlodipine and pranidipine treatment, whereas nicardipine−treatment significantly enhanced the overflow of NE.In preparations with active tone produced by methoxamine and guanethidine, the PNS−induced vasodilation mediated by calcitonin gene−related peptide(CGRP)−containing(CGRPergic)vasodilator nerves was not affected by these drugs.These results suggest that long−term treatment of SHR with long−acting drugs, amlodipine and pranidipine, reduces sympathetic adrenergic nerve function but calcium antagonists have no effect on CGRPergic nerve function.
We investigated the relationship between the induction of spatial cognition impairment in the 8−arm radial maze task and regional changes(ventral hippocampus(VH), dorsal hippocampus, frontal cortex, and basolateral amygdala nucleus)in brain acetylcholine(ACh)release using microdialysis in rats treated with muscarinic(M)receptor antagonists.In a behavioral study, two M1 antagonists, scopolamine(0.5 mg/kg, i.p.and 20 μg, i.c.v.)and pirenzepine(80 μg, i.c.v.), but not an M2 antagonist, AF−DX116(40−80 μg, i.c.v.), disrupted spatial cognition in the 8−arm radial maze task.In brain microdialysis with Ringer’s solution containing 0.1 mM eserine sulfate, scopolamine and AF−DX116, but not pirenzepine, increased ACh release in the VH.Moreover, in the bilateral injection of scopolamine(2 μg/side), the VH and dorsomedial thalamus nucleus were important regions for scopolamine−induced impairment of spatial cognition.A simultaneous determination of the behavioral changes revealed that scopolamine(0.5 mg/kg, i.p.)markedly decreased the ACh contents and also increased the ACh release in all regions tested.Especially, the changes in the ACh release of the VH closely paralleled the induction of the scopolamine−induced impairment of spatial cognition.These results suggest that the blocking balance between M1 and M2 muscarinic receptor in the VH therefore plays a major role in the spatial cognition impairment induced by scopolamine in the 8−arm radial maze task.
The effect of an endogenous 5−hydroxytryptamine(5−HT)precursor, 5−hydroxytryptophan(5−HTP), on the luminal outflow of 5−HT was examined using the luminally perfused isolated colon of the guinea pig, a model that would facilitate the pharmacological analysis of luminal 5−HT release from enterochromaffin cells(EC cells).5−HTP(1−10μM)concentration−dependently caused an increase of the luminal outflow of 5−HT.Either tetrodotoxin(0.3μM)or atropine(0.2μM)did not affect the 5−HTP−evoked increase in luminal 5−HT outflow, while the L−type calcium channel blocker, nicardipine(1μM)or diltiazem(1μM)reduced the 5−HTP−evoked 5−HT outflow by 47% and 61%, respectively.SB203186(1μM), a 5−HT4−receptor antagonist, enhanced the 5−HTP−evoked 5−HT outflow, while ramosetron(1μM), a 5−HT3−receptor antagonist reduced the stimulating effect of 5−HTP by 66%.Ketanserin(0.1μM), a 5−HT2A−receptor antagonist did not modify the stimulatory effect of 5−HTP.It is concluded that in the guinea pig colon, 5−HTP facilitates the luminal 5−HT release from EC cells, with no involvement of neuronal mechanisms and a non−neuronal cholinergic system.Furthermore, non−neuronal 5−HT3 and 5−HT4 receptors appear to contribute to the regulation of the luminal 5−HT release evoked by 5−HTP.This new bioassay of the guinea pig colon allows the pharmacological characterization of uncomplicated luminal 5−HT release from EC cells.
An electrophysiological study was performed with mice lacking complexin II, a presynaptic protein.The long−term potentiation(LTP)by high−frequency stimulation, recorded in the hippocampal CA1 area, was decreased in complexin II−lacking mice(CPXII KO mice).The overall postsynaptic currents elicited by low frequency stimulation on the Schaffer collateral/commissural fibers in the hippocampal CA1 pyramidal cells were not different between wild−type and mutant mice.Excitatory postsynaptic currents(EPSCs)recorded in the presence of 50μM bicuculline and inhibitory postsynaptic currents(IPSCs)recorded in the presence of 50μM AP−5(DL−2−amino−5−phosphonopentanoic acid)+ 30μM CNQX(6−cyano−7−nitroquinoxaline−2, 3−dione)were also identical between wild−types and mutants.Furthermore, the EPSCs following repetitive stimulation(10 Hz)in CPXII KO mice did not show any difference with wild−types.These findings suggest that complexin II does not play a crucial role in ordinary neural transmission, short−term synaptic plasticity or synaptic transmission during high−frequency repetitive stimulation.Therefore, the protein is thought to be involved in the LTP process following tetanic stimulation, including the induction and /or maintenance of the LTP.
A large number of fentanyl analogues have been synthesized so far, both to establish the structure−activity−relationship(SAR)and to find novel, clinically useful antinociceptive drugs.In this study, the newly synthesized fentanyl analogue 3−carbomethoxy fentanyl(iso−carfentanil)was compared to fentanyl for its antinociceptive activity(tail−immersion test)in rats.It was revealed that the introduction of a 3−carbomethoxy group in the piperidine ring of fentanyl skeleton reduced the potency and shortened the duration of action of the parent compound, i.e., fentanyl.The antinociceptive potency of 3−carbomethoxy fentanyl is influenced mainly by the steric factor(voluminosity of the carbomethoxy group and the cis/trans isomerism), while the chemical nature of the group is probably irrelevant.This is in agreement with SAR studies of other 3−substituted fentanyl analogues.In contrast to potency, the duration of action is not affected by cis/trans isomerism.It is assumed that the time course of action of 3−carbomethoxy fentanyl is influenced by the nature of the carbomethoxy group.Since the potency and the duration of action of this novel antinociceptive compound are interesting from the aspect of SAR studies and have potential promise for clinical application, 3−carbomethoxy fentanyl deserves to be extensively evaluated.
We evaluated the effects of SMP−300(N−(aminoiminomethyl)−11−chloro−5, 6, 7, 8−tetrahydro−8−oxo−4H−pyrrolo[3, 2, 1−kl]benzazocine−2−carboxamide monomethanesulfonate monohydrate), a newly synthesized compound, on Na+/H+ exchange activity in rat cardiomyocytes and on other ion transporters, channels and receptors.We also investigated the protective effects of SMP−300 in isolated ischemic rat hearts and rat isoproterenol− or vasopressin−induced experimental angina models.SMP−300 concentration−dependently inhibited recovery from acidosis in rat myocytes, and its IC50 for Na+/H+ exchange was 6 nM.In comparison, its IC50s for Na+/Ca2+ exchange and for the Na+ channel were >1000 nM, and those for other channels or receptors tested were >10, 000 nM.In rat isolated perfused hearts, SMP−300(10-8−10-7M), administered only at preischemia and not during reperfusion, significantly improved the postischemic recovery of cardiac function.SMP−300(0.03−0.3 mg/kg, i.v.)or 5−(N−ethyl−N−isopropyl)−amiloride(1 mg/kg, i.v.)prevented the isoproterenol−induced ST−segment depression in the ECG of anesthetized rats, in a dose−dependent manner.SMP−300(0.1 mg/kg, i.v.)and 5−(N−ethyl−N−isopropyl)−amiloride(1 mg/kg, i.v.)also inhibited the vasopressin−induced ST−segment depression in the ECG of anesthetized rats.This is the first report presenting the protective effect of Na+/H+ exchange inhibitors on isoproterenol− or vasopressin−induced ECG changes in rats, providing the future perspective of SMP−300, a potent Na+/H+ exchange inhibitor, as an anti−anginal drug.
Because the features and kinetics of adeno−associated virus(AAV)−mediated gene transfer to endothelial cells(EC)are yet to be ultimately determined, we tested variables pertinent to the efficiency of AAV−mediated gene transfer to bovine aortic endothelial cells(BAEC).The variables with AAV vectors were compared with the better characterized adenovirus(Ad)vectors.There is a dose−response relationship between multiplicity of infection(moi)of AAV or Ad vectors and transduction efficiency in BAEC.The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured BAEC.AAV and Ad vectors showed an incubation−time−dependent increase in transduction efficiency of LacZ gene to the BAEC up to 12 h of vector exposure.Although the similar kinetics of transduction efficiency of LacZ gene to BAEC was found in both vectors, the duration of gene expression was longer in AAV vector than that in Ad vectors in vitro.These results indicate that AAV−vector is efficient for gene transfer to EC, and higher moi of vectors or a longer period exposure of vectors to EC can facilitate efficient transduction of a foreign gene into cultured EC.For the duration of gene expression, the AAV vectors may be better than Ad vectors.
Rat and human liver microsomes oxidized ranitidine to its N−oxide(66−76%)and S−oxide(13−18%)and desmethylranitidine(12−16%).N− and S−oxidations of ranitidine were inhibited by metimazole [flavin−containing monooxygenase(FMO)inhibitor] to 96−97% and 71−85%, respectively, and desmethylation of ranitidine was inhibited by SKF525A [cytochrome P450(CYP)inhibitor] by 71−95%.Recombinant FMO isozymes like FMO1, FMO2, FMO3 and FMO5 produced 39, 79, 2180 and 4 ranitinine N−oxide and 45, 0, 580 and 280 ranitinine S−oxide pmol·min-1·nmol-1 FMO, respectively.Desmethyranitinine was not produced by recombinant FMOs.Production of desmethylranitidine by rat and human liver microsomes was inhibited by tranylcypromine, α−naphthoflavon and quinidine, which are known to inhibit CYP2C19, 1A2 and 2D6, repectively.FMO3, the major form in adult liver, produced both ranitidine N− and S−oxides at a 4 to 1 ratio.FMO1, expressed primarily in human kidney, was 55− and 13−fold less efficient than the hepatic FMO3 in producing ranitidine N− and S−oxides, respectively.FMO2 and FMO5, although expressed slightly in human liver, kidney and lung, were not efficient producers of ranitidine N− and S−oxides.Thus, urinary contents of ranitidine N−oxide can be used as the in vivo probe to determine the hepatic FMO3 activity.
Smooth muscle responsiveness of intrapulmonary small bronchi obtained from repeatedly antigen−challenged rats was compared with that from control animals to determine whether smooth muscle contractility of peripheral airways is augmented by such repeated challenge.In intact(non−permeabilized)smooth muscles of intrapulmonary bronchi, the acetylcholine(ACh)−induced contractile response was significantly augmented in the repeated challenge group, although 60−mM K+−induced contraction was within the normal level.In β−escin−permeabilized muscles, no significant difference between groups was observed in the Ca2+−induced contractile responses.Thus, augmented ACh−induced contraction of intact intrapulmonary small bronchial smooth muscle might be, at least in part, due to an enhanced ACh−mediated Ca2+−sensitizing signal.
The effect of a synthetic estrogen, diethylstilbestrol(DES), on kainate−induced currents was investigated in the hippocampal CA1 pyramidal neurons acutely dissociated from the mice using the nystatin−perforated patch−clamp recording configuration under voltage−clamp conditions.DES inhibited the current evoked by 100μM kainate in a concentration−dependent manner with a half−maximum inhibitory concentration of 8.8μM.The action of DES was voltage−independent.Since DES produced a suppression of the maximum response of the kainate concentration−response curve, the inhibition by DES of the kainate−induced current appears to be non−competitive.