Ion channels as targets for neuroprotective agents.
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Animals↗
Biomedical subjects
C S Huang
Publications and source records attributed to C S Huang.
Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels.
Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 microM carbachol was enhanced by the first co-application with 10 or 100 microM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 microM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3-3 microM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.
Acetylcholine↗
G-proteins are involved in riluzole inhibition of high voltage-activated calcium channels in rat dorsal root ganglion neurons.
Effects of riluzole on high voltage-activated (HVA) calcium channels of rat dorsal root ganglion neurons were studied using the whole-cell patch-clamp technique. Riluzole at 30 microM inhibited the HVA currents. The onset and offset of riluzole inhibitory effect were slow usually taking more than 3 min. Riluzole inhibition of the HVA currents was abolished and partially reduced by addition of 500 microM GDP-beta-S and 1 mM N-ethylmaleimide, respectively, to the pipette solution. Pre-treatment with pertussis toxin or application of depolarizing pre-pulses did not affect riluzole's inhibitory effect on the HVA currents. Riluzole inhibition of the HVA currents was also blocked by internal application of 50 microg/ml protein kinase A inhibitory peptide. It was concluded that pertussis toxin-insensitive G-proteins and protein kinase A may be involved in riluzole inhibition of the HVA currents.
Animals↗
Lead modulation of the neuronal nicotinic acetylcholine receptor in PC12 cells.
Lead is known to modulate several ligand- and voltage-gated ion channels, including the nicotinic acetylcholine receptor (AChR) channel. We examined the effects of lead on the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using whole-cell and single-channel patch-clamp techniques to clarify the detailed mechanism of action. Lead suppressed acetylcholine-induced currents in a dose-dependent manner with an EC50 value of 37 microM and a Hill coefficient of 0.82. At the single-channel level, 1-10 microM lead shortened the opening and burst durations, and increased the duration of mean closed time. The open probability was significantly decreased by lead. These changes of single-channel kinetics result in a significant decrease in the total charge carried through the open AChR channels explaining the suppressive effect of lead on acetylcholine-induced whole-cell currents.
Acetylcholine↗
Differential action of riluzole on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels.
The effects of riluzole, a neuroprotective drug, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. At the resting potential, riluzole preferentially blocked TTX-S sodium channels, whereas at more negative potentials, it blocked both types of sodium channels almost equally. The apparent dissociation constants for riluzole to block TTX-S and TTX-R sodium channels in their resting state were 90 and 143 microM, respectively. Riluzole shifted the voltage dependence of activation of TTX-R sodium channels in the depolarizing direction more than that of TTX-S sodium channels. The voltage dependence of the fast inactivation of both types of sodium channels was shifted in the hyperpolarizing direction in a dose-dependent manner, and the apparent dissociation constants for riluzole to block the inactivated channels were estimated to be 2 and 3 microM for the TTX-S and TTX-R sodium channels, respectively, indicating a much higher affinity for the inactivated channels than for the resting channels. Riluzole was equally effective in blocking both types of sodium channels in their slow inactivated state. Since more TTX-S channels are inactivated than TTX-R channels at the resting potential, riluzole blocks TTX-S sodium channels more potently than TTX-R sodium channels. It was concluded that one of the mechanisms by which riluzole exerts its neuroprotective action is to preferentially block the inactivated sodium channel of damaged or depolarized neurons under ischemic conditions, thereby suppressing excess stimulation of the glutamatergic receptors and massive influx of Ca++.
Animals↗
Effects of the neuroprotective agent riluzole on the high voltage-activated calcium channels of rat dorsal root ganglion neurons.
The effects of riluzole, a neuroprotective drug, on high voltage-activated (HVA) calcium channels of rat dorsal root ganglion neurons were studied using the whole-cell patch-clamp technique. Riluzole inhibited HVA calcium channel currents in a dose-dependent, time-dependent and reversible manner. The apparent dissociation constants for riluzole inhibition of the transient and sustained components of the current were 42.6 and 39.5 microM, respectively. Riluzole accelerated the activation kinetics of calcium channels without affecting the voltage dependence of activation. It accelerated the fast component of deactivation kinetics without affecting the slow component. It also accelerated fast and slow inactivation kinetics of the HVA channels. However, only one of the two components in the steady-state inactivation curve for the HVA channels was shifted in the hyperpolarizing direction by riluzole, which indicates differential block of the multiple-type HVA channels. By use of the specific blockers nimodipine, omega-conotoxin GVIA and omega-agatoxin IVA, the HVA calcium channels were found to comprise L-type (10%), N-type (63%), P/Q-type (23%) and R-type (9%). Riluzole blocked N- and P/Q-type channels, but not L-type channel, with the order of efficacy of P/Q- > N- >> L-type channels. Riluzole inhibition of N- and P/Q-type calcium channels may result in reduced calcium influx at presynaptic terminals, which thereby decreases excessive excitatory neurotransmitter release, especially glutamate, a mechanism known to cause neuronal death in ischemic conditions.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Two novel glucose 6-phosphate dehydrogenase deficiency mutations and association of such mutations with F8C/G6PD haplotype in Chinese.
Glucose 6-phosphate dehydrogenase (G6PD) deficiency is a common genetic disease affecting 3% of the total Chinese population in Taiwan. To investigate the molecular basis of this deficiency, we analyzed blood samples from G6PD-deficient newborns using a nonradioactive polymerase chain reaction coupled with single-stranded conformation polymorphism (PCR-SSCP) analysis. We identified two novel G6PD mutations in Chinese. The first, G6PD Miaoli, involved a C-->G substitution at nucleotide (nt) 519, producing a Phe173 to Leu change in the protein. The second mutation (G6PD Keelung) involved a C-->T change at nt 1387, resulting in an Arg463 to Cys substitution. The F8C/G6PD (coagulation factor VIIIc) haplotype that spans the Xq28 region from the gene for coagulation factor VIIIc to the gene for G6PD was also investigated in Chinese using PCR and restriction enzyme digestion. Of the 16 possible haplotypes, only four were found, which suggests that these four polymorphic sites are in strong linkage disequilibrium. Analysis of the association of G6PD mutations with F8C/G6PD haplotype revealed that nt 517, 592, 835, and 1387 mutations are linked to haplotype VI+VII, whereas the nt 519 mutation is linked to haplotype III. The finding that some G6PD mutations are associated with a particular F8C/G6PD haplotype may be useful for future population studies.
Female↗
Differential block of two types of sodium channels by anticonvulsants.
The differential effects of the anticonvulsants phenytoin and carbamazepine on the sodium channels of rat dorsal root ganglion (DRG) neurons were studied using the patch clamp technique. The action potentials from tetrodotoxin-resistant (TTX-R) cells were less sensitive to phenytoin and carbamazepine than those from tetrodotoxin-sensitive (TTX-S) cells. The steady-state inactivation curve for TTX-S sodium channels was shifted by as much as 20.6 mV and 11.4 mV by phenytoin and carbamazepine at 300 microM, respectively, yet the curve for TTX-R sodium channels was shifted only by 6.0 mV and 6.9 mV, respectively. Thus, the differential action potential block of TTX-S and TTX-R cells by phenytoin and carbamazepine is due to different voltage dependence of and differential drug effects on the inactivation kinetics of two types of sodium channels.
Action Potentials↗
Differential effects of hexachlorocyclohexane isomers on the GABA receptor subunits expressed in human embryonic kidney cell line.
We have recently demonstrated by patch clamp experiments that the four isomers of hexachlorocyclohexane (HCH), alpha-, beta-, gamma- and delta-HCH insecticides, modulated the kinetics of the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The present paper reports the differential effects of HCH isomers of the GABA-induced chloride currents in three combinations of alpha, beta and gamma subunits of GABAA receptor expressed in human embryonic kidney cells. When co-applied with GABA, gamma-HCH strongly suppressed the peak amplitude of GABA-induced current, and delta-HCH strongly enhanced it in the alpha 1 beta 2 gamma 2s, alpha 1 beta 2, alpha 6 beta 2 gamma 2s combinations in a dose-dependent manner. There was little or no difference in the dose dependence of the effects between gamma- and delta-HCH in any of the three subunit combinations. However, alpha- and beta-HCH showed differential effects on GABA-induced chloride currents in the three subunit combinations tested. alpha-HCH showed enhancing effects on the peak current in alpha 1 beta 2 gamma 2s, small enhancing effects on alpha 1 beta 2, and biphasic effects on alpha 6 beta 2 gamma 2s subunit combinations. beta-HCH had little or no effect on the peak current in alpha 1 beta 2 gamma 2s and alpha 1 beta 2 combinations, but suppressed currents in the alpha 6 beta2 gamma 2s subunit combination in a dose-dependent manner. The differential actions of HCH isomers may produce variable effects on different regions of the nervous systems and in different species of animals.
Cell Line↗
Potent modulation of neuronal nicotinic acetylcholine receptor-channel by ethanol.
Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC50 of 88.5 microM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.
Alcoholic Intoxication↗
Neonatal jaundice and molecular mutations in glucose-6-phosphate dehydrogenase deficient newborn infants.
Molecular mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene and clinical manifestations of neonatal jaundice in 112 male and 50 female Chinese neonates with G6PD deficiency were studied. In the 112 males, the nucleotide (nt) 1376 (G-->T) mutation was the dominant type (50.0%), followed by nt 1388 (G-->A) (16.1%), nt 493 (A-->G) (8.0%), nt 1024 (C-->T) (6.2%), nt 95 (A-->G) (5.4%), nt 392 (G-->T) (1.8%), nt 487 (G-->A) (1.8%), nt 871 (G-->A) (0.9%), and nt 1360 (C-->T) (0.9%). The nt 871 variant has not been reported in Taiwan before. The occurrence rates for nt 1376, nt 1388, nt 493, nt 95, and nt 1024 mutations in the 50 females were 44.0%, 18.0%, 12.0%, 6.0%, and 6.0%, respectively. The type of G6PD mutation in 10 male and 7 female neonates has not been identified yet. Although G6PD deficient neonates had higher frequency of phototherapy than G6PD normal neonates in both sexes, a significant difference in the prevalence of hyperbilirubinemia (peak bilirubin > or = 15.0 mg/dl) between G6PD deficient and normal neonates was found only in males. Further analysis showed that duration of phototherapy was longer in G6PD deficient male neonates than in the control group, while the outcome of phototherapy was better in subjects with non-nt 1376 mutations than subjects with the nt 1376 mutation. Most (78.3%) of the 23 G6PD deficient neonates who subsequently suffered from neonatal hyperbilirubinemia carried the nt 1376 mutation. The results of this study indicate that the nucleotide substitution at 1376 is the most common and important mutation for G6PD deficiency in Chinese neonates in Taiwan.
Base Sequence↗
Mercury chloride modulation of the GABAA receptor-channel complex in rat dorsal root ganglion neurons.
Mercury compounds affect synaptic transmission through multiple actions on various receptors and ion channels. One of their target sites is the GABAA receptor-channel complex, which is stimulated by mercury chloride (1-10 microM) in a potent and efficacious manner. We studied the mechanisms by which mercury chloride modulates the activity of the GABA system of rat dorsal root ganglion neurons in primary culture using the whole-cell patch clamp technique. The active form of mercury chloride was determined by measuring mercury potentiation of GABA-induced currents as a function of extracellular pH and chloride concentration. Experiments with various chloride concentrations indicated that Hg2+ was far less potent than HgCl2 and HgCl4(2-). Experiments with pH changes indicated that the mercury chloride-hydroxyl complex, predominantly HgCl4(OH)3-, was equally potent to non-complexed mercury chloride, predominantly HgCl4(2-). Mercury chloride potentiation of GABA-induced currents was use dependent, increasing with the frequency of channel openings. However, the potentiation was independent of membrane potential suggesting that mercury chloride binds to an external site of the receptor. Also supporting an external binding site is the observation that preapplication of mercury chloride alone for up to 60-sec potentiated GABA-induced currents. This site appears to be distinct from the zinc binding site. Desensitization of GABA-induced currents was accelerated by mercury chloride. Mercury chloride potentiation of the currents was blocked by cysteine and iodoacetamide suggesting involvement of sulfhydryl groups in this action.
Animals↗
Breast cancer vascularity: color Doppler sonography and histopathology study.
In this prospective study, the authors examined 50 patients with breast tumors (malignant, n = 32; benign, n = 18) to investigate the correlation between color Doppler flow mapping and histopathological findings and to evaluate the clinical significance of color Doppler mapping. Among the 32 patients with breast cancer, color Doppler signals were detected in 24 patients (75%). The maximum flow velocities varied from 5 to 34 cm/sec, with 16 (67%) of them above 15 cm/sec. Among the 18 patients with benign tumors, color Doppler signals could be detected in 7 (39%). The maximum flow velocity varied from 3 to 30 cm/sec but was over 15 cm/sec in only two patients (28%). Histological studies revealed that color Doppler signals detected by Doppler sonography correlated with disordered neovascularization penetrating the lesion from its periphery, consisting of thin-walled blood vessels and large arteriovenous shunts. Although large tumors tend to have high Doppler flow, there is no significant correlation between the maximum flow velocity and tumor size. There is also no significant correlation between the detection of high flow color Doppler signals and the age, receptor status, tumor size, lymph node metastases, or clinical stage of patients with breast cancer. However, there is a positive association (p < 0.05) between nodal metastases and higher tumor flow velocity in T1 (< or = 2 cm) breast tumors, but not in larger tumors. It is concluded that color Doppler is useful in the assessment of tumor vascularity but is of limited value in the differentiation of benign from malignant lesions. However, the presence of color Doppler signals in T1 breast cancer suggesting early dissemination of the cancer might be of important clinical significance in detecting those small, apparently early, but aggressive tumors with poor prognosis.
Adult↗
Diagnostic value of C-reactive protein in children with perforated appendicitis.
UNLABELLED: The diagnostic value of serum C-reactive protein (CRP) levels in children with perforated appendicitis was prospectively studied in 78 consecutive patients with histologically confirmed appendicitis. The patients were divided into two groups: group A included 56 patients with perforated appendicitis and group B consisted of 22 patients with simple appendicitis. Serum CRP level and leucocyte count were assayed in all and abdominal ultrasonography was performed in 75. The mean age group A patient was significantly lower than that of group B patients (7.5 vs. 10.4 years, P < 0.001). Group A patients had a significantly higher mean serum CRP levels than group B patients (92 vs. 31 mg/l, P < 0.001), while the mean leucocyte count was comparable in the two groups. Of 75 examined patients, 73 (97%) had a pre-operative sonographic diagnosis of appendicitis. CONCLUSION: Perforation is a common complication in children with appendicitis, especially in those of young age and with prolonged pain duration. Greatly increased serum CRP levels (> or = 50 mg/l) and abdominal ultrasonography are important diagnostic aids in such patients.
Adolescent↗
Radioimmunoassay for ziovudine in rat placenta and fetus.
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Animals↗
Ileal perforation caused by congenital or perinatal cytomegalovirus infection.
Enteritis developed in a 42-day-old female infant, and during hospitalization ileal perforation occurred unexpectedly. Serologic data, urinary viral culture, and pathologic studies of the resected ileal segment all suggested cytomegalovirus infection. Gastrointestinal involvement in congenital or perinatal infection with this virus may be relatively more common than previously recognized.
Cytomegalovirus↗
Effects of the nootropic drug nefiracetam on the GABAA receptor-channel complex in dorsal root ganglion neurons.
The effects of nefiracetam on GABA-induced chloride currents were studied with rat dorsal root ganglion neurons in primary culture using the whole-cell patch-clamp technique. The dose-response curve for GABA-induced currents was shifted by 16 microM to lower concentrations by 10 microM nefiracetam while the maximal response was reduced by 22.84 +/- 0.68%. Thus at a low concentration (10 microM) of GABA, the chloride currents were potentiated by nefiracetam in a concentration-dependent manner. With 10 microM nefiracetam, the potentiation occurred slowly and the recovery after washout was also slow. The desensitization of the GABAA receptor at high concentration (100 microM) of GABA was accelerated by nefiracetam. The recovery process of chloride currents from desensitization was not affected by nefiracetam. KT 5720 (0.56 microm), a specific protein kinase A (PKA) inhibitor, blocked the transient potentiation of GABA-activated currents by nefiracetam, but did not affect the acceleration of desensitization. Nefiracetam suppression of GABA-induced currents was also abolished by KT 5720 or the pertussis toxin. Thus, nefiracetam may inhibit Gi/G(o) proteins leading to a cascade of events that increase the intracellular cAMP level, activate the PKA system, and suppress GABA-induced currents. Nefiracetam-induced transient potentiation and acceleration of desensitization of GABA-induced currents may involve other pathways. The nefiracetam modulation of the GABAA receptor function will result in a nootropic effect on the central nervous system through modification of synaptic transmission.
Animals↗
Maternal-fetal pharmacokinetics of zidovudine in rats.
The disposition of zidovudine (AZT) was investigated in near-term (day 20) pregnant rats after intravenous bolus administration of AZT at 50 mg/kg. A compartmental pharmacokinetic model was developed to describe AZT concentrations in maternal plasma (1), placenta (2), fetus (3), amniotic fluid (4), and the maternal tissue compartment (5). Model equations were fitted simultaneously to all concentration data by NONLIN least-squares regression. The model that best described the AZT concentration data (F test, AIC, sum of weighted squared residuals) incorporated bidirectional transfer between maternal plasma reversible placenta, placenta reversible fetus, placenta reversible amniotic fluid, and maternal plasma reversible tissue compartment. Transfer rate constants (1/h) were as follows: k12, 0.58 +/- 0.41; k21, 47.64 +/- 46.61; k23, 67.50 +/- 42.03; k32, 13.09 +/- 8.80; k24, 0.62 +/- 0.03; k42, 0.32 +/- 0.06; k15, 5.75 +/- 7.00; k51, 4.12 +/- 1.01; and k10, 1.51 +/- 0.80. AZT rapidly distributed into tissue and placenta compartments. However, AZT accumulated more slowly into amniotic fluid. Intercompartmental distributional clearances suggest that the mechanism of maternal-placental, placental-fetal, and fetal-amniotic fluid transfer of AZT was by passive diffusion. This maternal-fetal model for AZT may offer a useful approach for describing the placental transfer kinetics of other antiviral nucleosides as well.
Amniotic Fluid↗