Synthesis, antibacterial, and antioxidant studies of 7-amino-3-(4-fluorobenzoyl)indolizine-1-carboxylate derivatives

In the present work, the quaternary salts of 4-aminopyridine, i.e., 4-amino-1-[2-(4-bromophenyl)-2-oxoethyl]pyridin-1-iumbromides were obtained by stirring 4-aminopyridine with phenacyl bromides in acetone at room temperature separately.These quaternary salts of 4-aminopyridine were treated with acetylenes (electron deficient), in the presence of anhydrouspossium carbonate in N,N-dimethylformamide solvent to get indolizine derivatives. The structures of newly synthesisedcompounds have been confirmed by spectroscopic techniques, such as liquid chromatography mass spectrometry,1H-NMR, and elemental analysis. Synthesized all compounds were screened for antibacterial and antioxidant activity.The compounds 2e, 2g, and 2j shows inhibition zone against bacteria and compounds 2a and 2f moderately active againstbacteria. All compounds 2a to 2j show 1,1-diphenyl-2-picrylhydrazide radical free radical scavenging activity, NitricOxide free radical scavenging activity, Reducing power scavenging activity, and Lipid peroxidation inhibition activity.

Detecting the aminopyridine compounds in workplace air by solvent eluting-gas chromatography / 中国职业医学

OBJECTIVE: To establish a method for simultaneous detection of 2-aminopyridine, 3-aminopyridine and 4-aminopyridine in workplace air by solvent eluting-gas chromatography. METHODS: 2-aminopyridine, 3-Aminopyridine and 4-aminopyridine in workplace air were collected by glass fiber filter paper dipped with sulfuric acid solution, and eluted using sodium hydroxide solution. After that, the eluent was separated by capillary column, and finally detected using the nitrogen and phosphorus detector. RESULTS: The quantitative determination ranges of 2-aminopyridine, 3-aminopyridine and 4-aminopyridine were 0.30-800.00, 0.80-800.00 and 2.00-800.00 mg/L respectively, with the correlative coefficients greater than 0.999 9. The minimum detectable concentrations were 4.67, 8.00 and 40.00 μg/m~3 respectively, and the minimum quantification concentrations were 14.00, 28.00 and 133.33 μg/m~3(45.00 L sample, 3.00 mL eluent) respectively. The average elution efficiencies were 79.63%-98.61%, 83.80%-101.42% and 81.60%-100.29% respectively. The within-run relative standard deviations(RSD) were 1.13%-3.65%, 1.47%-4.00% and 1.94%-5.15% respectively, and the between-run RSD were 1.77%-5.30%, 2.06%-4.65% and 2.59%-6.46% respectively. Samples were stable at room temperature for at least 14 days. CONCLUSION: This method is appropriate to be applied for simultaneous detection of 2-aminopyridine, 3-aminopyridine and 4-aminopyridine in workplace air.

Echinacoside, an active constituent of Herba Cistanche, suppresses epileptiform activity in hippocampal CA3 pyramidal neurons

Echinacoside, an active compound in the herb Herba Cistanche, has been reported to inhibit glutamate release. In this study, we investigated the effects of echinacoside on spontaneous excitatory synaptic transmission changes induced by 4-aminopyridine (4-AP), by using the in vitro rat hippocampal slice technique and whole-cell patch clamp recordings from CA3 pyramidal neurons. Perfusion with echinacoside significantly suppressed the 4-AP-induced epileptiform activity in a concentration-dependent manner. Echinacoside reduced 4-AP-induced increase in frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but it did not affect the amplitude of sEPSCs or glutamate-activated currents, implicating a presynaptic mechanism of action. Echinacoside also potently blocked sustained repetitive firing, which is a basic mechanism of antiepileptic drugs. These results suggest that echinacoside exerts an antiepileptic effect on hippocampal CA3 pyramidal neurons by simultaneously decreasing glutamate release and blocking abnormal firing synchronization. Accordingly, our study provides experimental evidence that echinacoside may represent an effective pharmacological agent for treating epilepsy.

Establishment and evaluation of a guinea pig model of itching / 中国实验动物学报

Objective To optimize the optimal doses of histamine and 4-aminopyridine (4-AP) in the establish-ment of guinea pigs models of itching, and to establish a new guinea pig model of itching. Methods The central composite design-response surface method was used to arrange the experiment. In the experiment different pruritus agents were hypo-dermically injection of 0. 5 mL in the depilated area, and the scratching incubation period and scratching number in 30 mi-nutes were counted after the injection. The guinea pig itching model was evaluated by observing the behavioral changes of guinea pigs and measuring the levels of histamine and interleukin-6 in the blood. Results The behavioral experiments found that the scratching frequency in the the combination group was significantly higher than the histamine group and 4-AP group (P<0. 01). The itching latency of the combination group was significantly shorter than that of the histamine group and 4-AP group (P<0. 01). Compared with the control group, the histamine concentrations of the combination group and histamine group were significantly increased ( P<0. 05 or P<0. 01 ) , and the level of the combination group was lower than that of the histamine group (P<0. 05). Compared with the control group, the serum IL-6 concentrations of histamine group, 4-AP group and combination group were significantly higher (P<0. 01 or P<0. 05), and those in the combination group were significantly higher than the histamine and 4-AP groups. Compared with the control group, pathologic examina-tion showed proliferation of inflammatory cells in all model groups, and the reaction of the combination group was more ob-vious. Conclusions The optimal conditions used in this experiment are easy to achieve and have good reproducibility in the establishment of a guinea pig model of itching.

Establishment and evaluation of a guinea pig model of itching / 中国实验动物学报

Objective To optimize the optimal doses of histamine and 4-aminopyridine (4-AP) in the establish-ment of guinea pigs models of itching, and to establish a new guinea pig model of itching. Methods The central composite design-response surface method was used to arrange the experiment. In the experiment different pruritus agents were hypo-dermically injection of 0. 5 mL in the depilated area, and the scratching incubation period and scratching number in 30 mi-nutes were counted after the injection. The guinea pig itching model was evaluated by observing the behavioral changes of guinea pigs and measuring the levels of histamine and interleukin-6 in the blood. Results The behavioral experiments found that the scratching frequency in the the combination group was significantly higher than the histamine group and 4-AP group (P<0. 01). The itching latency of the combination group was significantly shorter than that of the histamine group and 4-AP group (P<0. 01). Compared with the control group, the histamine concentrations of the combination group and histamine group were significantly increased ( P<0. 05 or P<0. 01 ) , and the level of the combination group was lower than that of the histamine group (P<0. 05). Compared with the control group, the serum IL-6 concentrations of histamine group, 4-AP group and combination group were significantly higher (P<0. 01 or P<0. 05), and those in the combination group were significantly higher than the histamine and 4-AP groups. Compared with the control group, pathologic examina-tion showed proliferation of inflammatory cells in all model groups, and the reaction of the combination group was more ob-vious. Conclusions The optimal conditions used in this experiment are easy to achieve and have good reproducibility in the establishment of a guinea pig model of itching.

Anticonvulsant and in vitro antioxidant activities of Momordica cissoides L. (Cucurbitaceae).

The present study investigated the anticonvulsant effect of an herbal medicine candidate Momordica cissoides, by using two animal models of epilepsy; Pilocarpine-induced status epilepticus (SE) and seizures and the 4-aminopyridine-induced (4-AMP) seizures methods using albino mice. Four doses (425, 212.5, 106.25 and 42.5 mg/kg) of the plant extract were prepared and administered to four groups of mice respectively. for each group test M. cissoides protected 100% of mice at the doses 106.25 and 212.5 mg/kg and 80% at the doses of 42.5 and 425 mg/kg against generalized convulsions induced by pilocarpine (PILO) and 100% at the doses 106.25 and 212.5 mg/kg against tonic and clonic convulsions induced by 4-aminopyridine (4-AMP). Moreover, for the test of induction of convulsions by PILO, M. cissoides protected 100% of mice at doses 106.25 and 212.5 mg/kg and 80% at the doses of 42.5 and 425 mg/kg against death after 1 h and 24 h respectively. In addition, study evaluated the free radical scavenging of the decoction extracts of M. cissoides leaf in vitro. The antioxidant activity of M. cissoides were evaluated using the free radical scavenging activity assay (DPPH), total phenolic (TPC) and total flavonoids content (TFC) and ferric reducing antioxidant potential. Attained results show that the extract of M. cissoides leaf can be used in SE, generalized seizure, tonic and clonic seizures treatment. Moreover, the antiepileptic effect of this extract is probably caused by its antioxidant properties.

The effects of potassium channel blocker 4-AP on the proliferation of human tongue squamous cell carcino-ma Tca8113 cells / 实用口腔医学杂志

Objective:To study the effects of a potassium channel blocker 4-Amino pyridine(4-AP)on the proliferation of human tongue squamous cell carcinoma Tca8113 cells.Methods:CCK-8 assay was used to detect the proliferation of Tca8113 cells cultured with 4-AP at the concentration of 1,5,10,20,50 and 100 mmol/L for 12,24 and 48 h respectively,the cell proliferation inhibition rate was calculated,the cell cycle distribution was examined by flow cytometry.Data were analyzed by SPSS19.0 software.Results:With the increase of 4-AP concentration and culture time,cells showed some morphologic changes.4-AP at 5 -100 mmol/L dose and time dependently inhibited the proliferation,with 24 h exposure dose dependenty decreased S-phase population and increased G0 /G1 phase population of Tca8113 cells.Conclusion:4-AP may inhibit Tca8113 cell proliferation by regulation of the cell cycle distribution.

4-Aminopyridine Improves Lower Urinary Tract Symptoms in a Patient With Benign Prostatic Hyperplasia and Downbeat Nystagmus Syndrome / 대한배뇨장애요실금학회지

Aminopyridines are potassium channel blockers that increase the excitability of nerve cells and axons; therefore, they are widely used to treat different neurological disorders. Here we present a patient with idiopathic downbeat nystagmus and lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia who was treated with the sustained-release form of 4-aminopyridine (4-AP). During treatment with 4-AP, the LUTS improved. This improvement was monitored by using uroflowmetry and the International Prostate Symptom Score. A significant improvement of symptoms was observed in relation to the voided volume. This included an improved emptying of the bladder without an increase in residual urine. In animal studies, both nonselective K+ channel blockade and selective voltage-sensitive potassium blockade by 4-AP resulted in increased contraction on rat detrusor strips. To our knowledge, this is the first clinical observation of the mode of action of 4-AP in urological symptoms in humans.

The effects of irinotecan combined with 4-amion pyridine on the proliferation of human colorectal cancer cell / 中华消化杂志

Objective To investigate the effects and potential mechanism of irinotecan (CPT-11), an antitumor drug, on human colorectal cancer cell line HT-29 and its impact on 4-amion pyridine (4-AP), a kalium ion channel blocker. Methods The effects of CPT-11, 4-AP and combination of two drugs on proliferation and invasion of HT-29 cells were measured by MTT and Transwell assay respectively. The impact of CPT-11 or 4-AP on cell apoptosis was determined by flow cytometry with Annexin-V and PI staining. The current of ATP sensitive potassium ion (IKATP) was measured by patch clamp. Results The CPT-11 could inhibit proliferation of HT-29 cells at dose from 1.0 to 64.0 μg/ml in dose-and time-dependent manners. Whereas the above effect was enhanced when CPT-11 combined with 4-AP (1.0 mmol/L). The administration of CPT-11 (16.0 μg/ml) or 4-AP (1.0 mmol/L) significantly induced the cell apoptosis and inhibited the invasion of HT-29 cells, furthermore, these effects could be enhanced by combination of two drugs. And the different concentrations of CPT-11 reduced the IKATP of cell membrane in negative dose-dependent manner. Conclusions The effects of CPT-11 on HT-29 cells, such as reducing proliferation and invasion as well as inducing the apoptosis, can be enhanced by 4-AP, which may be related to inhibition of ATP-sensitive potassium channels.

Influence of 4-aminopyridine on human ovarian luteinized granulosa cell proliferation, production, and apoptosis through inhibiting voltage-gated K+ channel / 中华妇产科杂志

Objective To study the influence of 4-aminopyridine(4-AP)on proliferation,production,and apoptosis through inhibiting voltage-gated K+channel(Kv)in ovarian luteinized granulosa cells.nethods Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programme.The cultured granulosa cells were divided into 4 groups:blank group,4-AP treated group,human chorionic gonadotropin(hCG)-induced group and hCG+4-AP cotreated group.The final concentrations of hCG and 4-AP were 1250 U/L and 5 nmol/L respectively.The progesterone production WaS detected by the chemoluminescence method.The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by RT.PCR The influence on the early apoptosis of gTanulosa cells bv 4-AP was observed by flow eytometry.Cellular caSpage-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium(MTT)method.Results(1)Kv mRNA wag expressed in granulosa cell.(2)The progesterone production64),(206±32),(1991±172)and(763±79)nmol/L,respectively after24 hours culture.Exposure of the(3)The flow cytometry analysis and the cellular caapase-3 A405 showed that 4-AP increased the percentage ofearly phase apoptosis(P<0.01):4-AP treated group VS blank group[(40±5)%and 0.049 ±0.009]VS[(17±4)% and 0.029±0.008],hCG+4-AP CO-treated group VS hCG-induced group[(25±4)%and0.039 ±0.0081 VS[(15±3)%and 0.022 ±0.007].(4)24 hours after treated with 4-AP and hCG,theinhibitory rate of cultured granulosa cells of 4-AP treated group was higher than the blank group(19.7%VS0).and that of hCG+4-AP co-treated group was obviously higher than hCG-induced group(34.6% VS O,P<0.01).Conclusions The voltage-gated K+ channels expressed by ovarian luteinized granulosa cellplay an important role in cell proliferation,production,and apoptosis.4-AP may inhibit differentiation ofprogesterone in granulosa ceHs through the inhibition of proliferation and induction of apoptosis.

The Effects of Lamotrigine on Epileptiform Discharges Induced by Mg2+ -free Medium and 4-aminopyridine in Hippocampal Slices of Immature Rats / 대한소아신경학회지

PURPOSE: In order to elucidate the actual mechanism and the optimal concentration of Lamotrigine(LTG) that suppresses epileptiform discharges, we observed epileptiform discharges from hippocampal slices of immature rat in 4-aminopyridine(4-AP) added Mg2+ - free medium of artificial cerebrospinal fluid(aCSF) with various LTG concentrations. METHODS: We divided 19-23 day-old Sprague-Dawley rats into 4 groups; control group(n=12) and 3 LTG groups depending on the concentrations of LTG such as 400 (n=9), 800(n=7), and 1,000(n=8) microM. The rats were anesthetized and their brains were taken, soaked in aCSF(NaCl 125 mM, KCl 2.5 mM, NaH2PO4 2 mM, MgSO4 1.25 mM NaHCO3 25 mM, CaCl2 2 mM, Glucose 10 mM, pH 7.3-7.4). And then the brains were cut into 400 microm hippocampal slices by a vibratome. The slices of control group were soaked in 200 microM 4-AP added Mg2+ -free medium of aCSF for 1 hour, and then extracellular recordings were performed in hippocampal CA1 pyramidal region. The slices of LTG groups were soaked in the solution containing 400, 800, and 1,000 microM LTG, then extracellular recordings were performed. RESULTS: Interictal discharges were observed in all the control and the LTG groups. The latency to the first interictal discharges after 4-AP addition was 52.7+/-26.9 sec in control group, but was 225.0+/-28.2 sec in 800 microM and 322.1+/-116.4 sec in 1,000 microM group of LTG(P<0.05). The duration of interictal discharges was 64.6+/-35.6 sec in control group, but was the shortest in 800 microM group of LTG at 39.3+/-12.6 sec. Ictal discharges were observed in all of control and 400 microM group, but the frequency was decreased as the concentration of LTG increases, 57.1% in 800 microM, 12.5% in 1,000 microM group. The latency to ictal discharge after 4-AP addition was 142.1+/-52.6 sec in control group, but increased as the concentration of LTG increases, 304.4+/-84.5 sec in 400 microM group and 689.8+/-213.1 sec in 800 microM group(P<0.05). The duration of ictal discharges was 1,534.7/-339.3 sec in control group, but decreased as the concentration of LTG increases, it was 126.5+/-76.1 sec in 800 microM group(P <0.05) and 42 sec in 1,000 microM group. CONCLUSION: The antiepileptic effects of LTG were most significant when the concentration, inhibiting epileptiform discharges induced by 4-AP and Mg2+ -free medium in hippocampal slices of immature rats, was 800 microM or higher. Although the basic pharmacologic mechanism of LTG is the inhibition of sodium channel, it may also work on potassium channel at higher concentrations.

Arsenic trioxide-induced HeLa cell death is partially prevented by K + channel blockers / 药学学报

Aim To investigate the effects of K + channel blockers on arsenic trioxide-induced HeLa cell death. Methods Viability of HeLa cells was assessed by mitochondrial dehydrogenase activity using colorimetric MTT assay and the voltage-dependent K+ currents were recorded by using patch-clamp rest living cells after As2 O3 24 h-incubation showed significant increase of K + currents densities. At + 80mV, the densities of K+ currents (61 ± 18) pA/10 pF (n = 8) in As2O3 24 h-incubation group were significantly more than that in the control group (38 ± 10) pA/10 pF (n = 8, P ＜ 0. 05 ). The HeLa cells were prevented partially from As2 O3-induced cell death by co-application for 24 h with typical voltageeffects on HeLa cells. Conclusion Chronic treatment with As2 O3 increased voltage-dependent K+currents in HeLa cells and the cell death induced by As2O3 was reduced partially by voltage-dependent K +channel blockers, 4-aminopyridine or tetraethylammonium.

4 Aminopyridine Induced Epileptiform Activity / 대한소아신경학회지

PURPOSE: The goal of the present study was to investigate the effects of 4-aminopyridine(4-AP) on the excitability of visual cortex, observe the induction of epileptiform activity and define the characteristics of spontaneous activity. METHODS: We divided 19 to 23-day-old Sprague-Dawley rats into 3 groups by the concentration of 4-AP:5(n=10), 50(n=11), and 100(n=12) microM. The slices from their brains were incubated in artificial CSF for 1 hour, and then extracellular recordings were performed. RESULTS: Spontaneous epileptiform activities were observed in 50 and 100 microM 4-AP groups. The latencies of interictal epileptiform activity were 7.8+/-1.1 and 5.8+/-0.9 min, the frequencies 1.8+/-0.2 and 24.1+/-6.6 min-1, the amplitudes 0.7+/-0.1 and 2.8+/-0.5 mV, and the durations 238.0+/-57.8 and 242.2+/-70.0 ms in 50 and 100 microM 4-AP groups respectively. The latencies of ictal epileptiform activity were 21.0+/-9.8 and 6.7+/-2.3 min, the frequencies 116.2+/-46.7 and 193.7+/-26.4/event, the amplitudes 3.1+/-0.8 and 2.8+/-0.9 mV, and the durations 26.9+/-27.6, 35.2+/-12.6 s in 50 and 100 microM 4-AP groups respectively. CONCLUSION: 4-AP showed increased excitability in the visual cortex and induced interictal and ictal spontaneous epileptiform activity. This induction was decreased by D- AP5 or CNQX. Those results suggest that both types of inotropic excitatory amino acid receptors are overactivated and contribute to seizure initiation and propagation.

The Effects of Topiramate on Epileptiform Discharges Induced by Mg(2+)- Free Medium and 4-Aminopyridine in Hippocampal Slices of Immature Rats / 대한소아신경학회지

PURPOSE: Topiramate(TPM), one of the newest antiepileptic drugs, has been prescribed not only to refractory partial seizures but to generalized tonic-clonic seizures. However, its action mechanisms are not well understood and the optimal dose of antiepileptic efficacy in animal seizure models is not determined yet. In order to elucidate the action mechanisms and the optimal concentration of TPM that suppresses epileptic discharges, we observed ictal and interictal discharges from immature rat hippocampal slices in Mg(2+)-free, and 4-aminopyridine(AP) added artificial CSF with various TPM concentrations. METHODS: We divided Sprague-Dawley rats of 19 to 23 days old into 5 groups; namely, a control group(n=12) and 4 TPM groups according to the concentration of TPM, 6 (n=11), 20(n=7), 60(n=10), and 200(n=14) micrometer. The rats were anesthetized and their brains were taken, and soaked in artificial CSF(NaCl 125 mM; KCl 2.5 mM; NaH2PO4 2 mM; MgSO4 1.25 mM; NaHCO3 25 mM; CaCl2 2 mM, Glucose 10 mM, and pH 7.3-7.4). Then the brains were cut into 400 micrometer hippocampal slices by a vibratome. The slices of the control group were soaked in 200 micrometer 4-AP added Mg(2+)-free medium for 1 hour, and then extracellular recordings were performed in the hippocampal CA1 pyramidal region. The slices of TPM groups were soaked in solutions containing 6, 20, 60, 200 micrometer TPM, and then extracellular recordings were performed. RESULTS: Interictal discharges were observed in the control group and 6, 20 micrometer groups but the frequency decreased as the concentration of TPM increased:90% in 60 micrometer group, and 35.7% in 200 micrometer group. And the amplitude of TPM groups was much smaller than that of the control group. The latency to the first interictal discharge after 4-AP addition was 52.7+/-7.5 sec in the control group, 290.2+/-78 sec in 60 micrometer group, and 568+/-113.1 sec in 200 micrometer group. Duration of the interictal discharge was 64.6+/-10.3 sec in the control group, but was prolonged to 141+/-38.1 sec in 60 micrometer group(P<0.05). Ictal discharges were observed in all of the control and 6 micrometer groups, but the frequency decreased as the concentration of TPM increased:55.6% in 60 micrometer, and 28.6% in 200 micrometer groups. The amplitude of the TPM groups was much smaller than that of the control group. The latency to ictal discharges after 4-AP addition was 141+/-15.2 sec in the control group, but increased as the concentration of TPM increased:431.8+/-57.4 sec in 60 micrometer, and 627.8+/-143.5 sec in 200 micrometer group(P<0.05). The duration of ictal discharges was 1,534.7+/-97.9 sec in the control group, but decreased as the concentration of TPM increased, the shortest in 60 micrometer group, 155.2+/-65.5 sec(P<0.05). Status epilepticus was seen in 58.3% of the control and 27.2% of 6 microM groups. CONCLUSION: TPM suppresses the frequency, latency, and duration of epileptiform discharges induced by Mg(2+)-free, and 4-AP added artificial CSF in immature rat hippocampal slices, starting from 20 micrometer and reaching the maximal effect at over 60 micrometereter. This finding is presumably due to TPM enhancing of GABA receptor currents and/or K+ channel conductance in response to TPM.

Effects of 4-aminopyridine on the gastrointestinal function of murine / 中国药理学通报

Aim To investigate the effects of 4-aminopyridine (4-AP),K+ channel blocker, on the gastrointestinal function of murine. Methods Charcoal suspension was used to detect the effects of 4-AP on gastrointestinal motility of mice in vivo, contraction of isolated fundus longitudinal strips and duodenum of rats were studied in vitro,gastric acid secretion was measured by Gastric Secretion Test. Results 4-AP(5 mg?kg -1,ig)inhibited the gastrointestinal motility of mice. 4-AP(5 mmol?L -1) increased the maximum contractive force and minimum relaxation force, decreased the amplitude and frequency of the isolated duodenum peristaltic contraction. 4-AP(2.5 mg?kg -1,ip) significantly enhanced gastric acid secretion of rats. Conclusion 4-AP inhibited gastrointestinal motility and enhanced gastric acid secretion in murine.

4-Aminopyridine Inhibits the Large-conductance Ca2+ -activated K+ Channel (BKCa) Currents in Rabbit Pulmonary Arterial Smooth Muscle Cells

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance Ca2+ -activated K+ channel (BKCa) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of BKCa. The BKCa currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single BKCa currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit BKCa currents in the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent K+ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.

Inhibitory Effects of Potassium Channel Blockers on Carbachol-induced Contraction in Rat Detrusor Muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 micrometer to 5 micrometer. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 micrometer thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potasssium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.

Testosterone Causes Simultaneous Decrease of Ca2+i and Tension in Rabbit Coronary Arteries: by Opening Voltage Dependent Potassium Channels

The relationship between the level of testosterone and the incidence of coronary heart disease is still controversial in the view of the results of clinical and epidemiologic studies. This uncertainty might be partly due to relatively small number of experimental studies undertaken to investigate the cellular mechanism underlying the vascular responses to testosterone. To further investigate the cellular mechanisms of testosterone with respect to vascular response, we investigated the effect of testosterone on contractility and intracellular Ca2+ regulation in a rabbit coronary artery and evaluated the underlying mechanism of testosterone-induced changes of coronary vascular tone by using various pharmacological blockers. Testosterone was found to relax rabbit coronary arteries in a dose-dependent manner, and no significant difference was found in the relaxation response to testosterone with or without endothelium. Similar results were obtained in male and non-pregnant female rabbit coronary arteries. The relaxation response of rabbit coronary arteries to testosterone was greater for PGF2alpha-contracted rings than for KCl contracted rings, which suggest the involvement of K+ channels. Furthermore, the relaxation response to testosterone was significantly reduced by 4-aminopyridine, a sensitive blocker of voltage dependent K+ channels, but not by low doses of tetraethylammonium or iberiotoxin, a Ca2+ activated K+ channel blocker. Testosterone simultaneously reduced the intracellular Ca2+ concentration ([Ca2+]i) and tension, and 4-AP effectively antagonized the testosterone-induced change of [Ca2+]i and tension. Therefore, it may be concluded that the stimulation of voltage dependent K channels is responsible, at least in part, for the testosterone-induced relaxation of rabbit coronary arteries.

Neuromuscular Blocking Properties of Dantrolene at the Neuromuscular Junction and Its Recovery with Various Antagonists / 대한마취과학회지

BACKGROUND: Dantrolene produces skeletal muscle relaxation by a direct action on excitation-contraction coupling, presumably by decreasing the amount of calcium released from the sarcoplasmic reticulum. The mechanism underlying this action is extrajunctional. The aim of this study was to evaluate the pharmacodynamic properties of dantrolene at the neuromuscular junction and the reversal effects of substances as possible dantrolene antagonists in vitro. METHODS: The effects of evoked twitch tension response have been studied on the isolated phrenic nerve hemidiaphragm muscle strips of the rat, using a single twitch (0.1 Hz) and the train of four (TOF; 2 Hz for 2s) stimulation. The maximum effect (E(max)) and TOF ratio at each point of twitch depression after cumulative doses of dantrolene were measured mechanomyographically. The EC(50) and EC(95) of dantrolene were calculated using an inhibitory sigmoid E(max) model. The reversal effect to E(max) after administration of 10 mM of dantrolene was determined by various doses of neostigmine, pyridostigmine or 4-aminopyridine respectively. RESULTS: The E(max) was 76.14% of the initial twitch tension, but the residual twitch tension was remained until five times (10 mM) of the dose for the E(max) was administered. TOF stimulation to the residual twitch tension did not demonstrate any fade. The EC(50) and EC(95) of dantrolene were 0.379 and 3.177 mM respectively. Neostigmine and pyridostigmine produced a transient but incomplete recovery of twitch tension, which rapidly fell to the level of the twitch response before the drugs were given. However, 4-aminopyridine produced a dose-dependent recovery of the twitch response. The addition of neostigmine (0.5 mg/ml) or pyridostigmine (2.5 mg/ml) did not decrease the EC(50) and EC(95) of 4-aminopyridine in reversing the effect of dantrolene. CONCLUSIONS: These RESULTS have demonstrated the evidence that dantrolene did not completely depress the twitch tension, leaving if at nearly 25%, and accompanying TOF response without fade, and that anticholinesterases were ineffective in antagonizing its blockade. However, 4-aminopyridine was effective and may not be related to the propensity for pre- and postjunctional cholinergic receptor blockade at the neuromuscular junction.

The Effect of 4-Aminopyridine with Anticholinesterases on MgSO4-Rocuronium-Induced Neuromuscular Blockade in Vitro / 대한마취과학회지

BACKGROUND: The aim of this study was to evaluate the effect of 4-aminopyridine (4-AP) combined with anticholiesterase (antiChE) in antagonizing MgSO4-rocuronium-induced neuromuscualr blockade using a rat hemidiaphragm. METHODS: A hemidiaphragm with phrenic nerve was dissected and was mounted in a bath containing oxygenated Krebs solution. The phrenic nerve was stimulated supramaximally and the twitch response (0.1 Hz) was stabilized for at least 30 minutes. After maximal twitch inhibition by IC95 (concentration of 95% twitch inhibition) of rocuronium and MgSO4 20 mg was achieved, antagonistic effects of 1.6, 16 microgram/ml of edrophonium, 0.1, 1.0 microgram/ml of neostigmine, 0.5, 5.0 microgram/ml of pyridostigmine, and 0.8 microgram/ml of 4-AP combined with each of the above mentioned antiChEs were investigated. RESULTS: Whereas antiChE alone at low concentration partially recovered only the twitch response, 4-AP combined with antiChE recovered both the twitch and train-of-four responses significantly. CONCLUSIONS: 4-AP enhances antagonism of a magnesium-rocuronium induced neuromuscular blockade by edrophonium, neostigmine or pyridostigmine in vitro.