Characterization of acid-sensitive ion channels in freshly isolated rat brain neurons

Transient proton-activated currents induced by rapid shifts of the extracellular pH from 7.4 to ≤6.8 were recorded in different neurons freshly isolated from rat brain (hypoglossal motoneurons, cerebellar Purkinje cells, striatal giant cholinergic interneurons, hippocampal interneurons, CA1 pyramidal neurons and cortical pyramidal neurons) using whole-cell patch clamp technique. Responses of hippocampal CA1 pyramidal neurons were weak (100–300 pA) in contrast to other types of neurons (1–3 nA). Sensitivity of neurons to rapid acidification varied from pH₅₀ 6.4 in hypoglossal motoneurons to 4.9 in hippocampal interneurons. Proton-activated currents were blocked by amiloride (IC₅₀ varied from 3.6 to 9.5 μM). Reversal potential of the currents was close to E_Na, indicating that the currents are carried by sodium ions. The data obtained suggest that the proton-activated currents in the neurons studied are mediated by acid-sensitive ion channels. Strong acidification (pH<4) induced biphasic responses in all neuron types: the transient current was followed by a pronounced sustained one. Sustained current was not blocked by amiloride and exhibited low selectivity for sodium and cesium ions. Slow acidification from pH 7.4 to 6.5 did not induce detectable whole-cell currents. At pH 6.5, most of the channels are desensitized and responses to fast pH shifts from this initial level are decreased at least 10 times. This suggests that slow acidification which is well known to accompany some pathological states should rather desensitize than activate acid-sensitive ion channels and depress their function.

Our results provide evidence for a widespread and neuron-specific distribution of acid-sensitive ion channels in the brain. The large amplitudes and transient character of currents mediated by these channels suggest that they could contribute to fast neuronal signaling processes.     

Sodium channel currents in rat hippocampal NG2 glia: characterization and contribution to resting membrane potential

We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I_Na) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7–21 rats express low density I_Na (9.5–15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC₅₀ value of 39.3 nM. The I_Na expression correlated with a 25 mV more depolarized resting membrane potential (RMP) as compared with non-I_Na-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 μM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I_Na-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I_Na expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 μM TTX. Analysis of the I_Na activation/inactivation curves yields an I_Na “window current” at −40±20 mV, implying a persistent I_Na component being active around the NG2 glia RMP of −45 mV. According to the constant-field equation analysis, this active I_Na component leads to a pNa/pK ratio of 0.14 at RMP which is threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I_Na component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.     

Niflumic acid reduces the hyperpolarization-activated current (I(h)) in rod photoreceptor cells.

We examined the effects of niflumic acid (NFA), a chloride channel blocker, on the hyperpolarization-activated current (I_h) in newt rod photoreceptors. At 100 μM, NFA delayed the activation of I_h induced by hyperpolarizing voltage pulses to −83 mV from a holding potential of −43 mV, and reduced the steady-state current. However, reduction by NFA was weakened when I_h was activated by hyperpolarizing steps to −123 mV, suggesting that these effects were voltage-dependent. The suppressive effects of NFA on I_h were accompanied by a negative shift in activation voltage. NFA also delayed the relaxation of I_h tail currents, showing that this drug also inhibited deactivation of the current. The reversal potential and the fully activated conductance were not affected. These observations suggest that NFA reduces I_h by modifying the gating kinetics of the underlying channels. The suppressive actions of NFA remained when intracellular Ca²⁺ was strongly chelated, and the failure of suppression by NFA in inside-out patches suggests that the agent may act on the I_h channel from the extracellular side. These results, obtained in rod photoreceptors, are consistent with similar effects of NFA on I_f in cardiac myocytes, suggesting that both currents share similar pharmacological properties.     

Serum levels of oestrogens, progesterone, follicle-stimulating hormone and sex-hormone-binding globulin during simultaneous vaginal administration of 17β-oestradiol and progesterone in the pre- and post-menopause

Serum concentrations of 17β-oestradiol (E₂), unconjugated oestrone (E₁), total oestrone (tE₁), progesterone (P), follicle-stimulating hormone (FSH) and sex-hormone-binding globulin (SHBG) were measured before and after daily intravaginal administration of 250 μg micronized E₂ and 10 mg micronized P for 14 days to 12 post-menopausal and for 1 day only (during cycle days 5–8) to 11 pre-menopausal women. In the post-menopausal women the levels of all steroids increased to maximum values on day 1, 8–10 h after administration and fell thereafter. In the pre-menopausal women the steroid concentrations rose slowly to a plateau level 10–15 h after administration. Significantly higher absorption of E₂ and E₁ (area under the curve increments) was noted in the post-menopausal than in the pre-menopausal women.

In the post-menopausal women the steroid levels measured on days 7 and 14 corresponded to those observed in the very early or late luteal phase. Area under the curve increments were usually smaller on days 7 and 14 than on day 1 and the absorption kinetics altered to a ‘pre-menopausal’ pattern. FSH levels were significantly reduced as from 12 h after administration on day 1 and onwards. A slight (10%) but significant increase in SHBG levels was noted on day 14. It was concluded that the combined E₂ and P treatment used in this investigation brings about a physiological response with only minimal side effects on the liver as judged from changes in SHBG concentrations.     

A pH curve of human resting saliva sampled with a small paper slip and its medical application

This paper described physiochemical characteristics of the pH curve of saliva that accompanied escape of CO₂ and that was recorded by a previously reported method. Monthly sample of the resting saliva was collected by a small paper slip from five adults (59.9±16.5 years old) over 36 months. The pH curves were examined to represent differences in secretion characteristics among individuals. The following results were obtained: (1) Three variables, pH₁, Δ pH_I, and Δ pH_L, that represent a saliva pH curve indicated secretory characteristics of each individual since the intra-individual variance of them was significantly smaller than the inter-individual variance. (2) These values (mean±S.D.) obtained from five adults were 7.03±0.54, 0.24±0.21, and −0.10±0.12, and those obtained from 663 young adults (21.7±2.4 years old) were 7.14±0.44, 0.26±0.23, and −0.08±0.13. (3) A characteristic about amplitude of the Δ pH_I of each individual was maintained over 36 months under the healthy condition. Seasonal variation of the three pH variables and their statistical distribution were also investigated.     

Comparison of pharmacokinetic profiles of a 17β-estradiol gel 0.6 mg/g (Gelestra) with a transdermal delivery system (Estraderm TTS 50) in postmenopausal women at steady state

Objectives: to compare the patterns of a 17β-estradiol (E₂) gel containing 0.6 mg/g (1.5 mg E₂ per day, Gelestra); with the transdermal delivery system (Estraderm TTS 50) applied every 3 days over a 14-day period to women in spontaneous or surgical menopause. Methods: a single centre, open, randomised, parallel-group study was conducted. A total number of 33 postmenopausal women were enrolled. In 23 of them the menopause occurred spontaneously, while 10 women were bilaterally ovariectomized. Randomly, the subjects were treated with Estraderm TTS 50 (no. 8) or with Gelestra (no. 14). The pharmacokinetic study of the drugs was performed at the seventh, ninth and 14th day in Gelestra treated women and at the first, third and second day in Estraderm TTS 50 treated women. In fact, the seventh, ninth and 14th day of percutaneous treatment corresponds to the first, third and second day of application of the transdermal system application. Blood samples were taken by each subject at baseline and 1, 2, 3, 4, 8, 12 and 24 h after the gel or transdermal system application. In almost all samples the level of E₂ and estrone (E₁) were evaluated. Statistical analysis was performed by comparing the two groups of treatment. The following parameters were assessed: mean E₂ and E₁ concentrations, E₂ peak serum concentration within interval from 0 to 72 h (C_max), E₂ trough concentration within interval from 0 to 72 h (C_min), area under the E₂ time concentration curve in the interval from 0 to 72 h (AUC_(0–72)), the average E₂ concentration during the measurement interval, calculated by dividing AUC_(0–72) by 72 h (C_av), E₁/E₂ ratio, and percentage fluctuation (%Fluct) which is equal to 100 (C_max−C_min/C_max). Results: there was no significant difference in E₂ C_av between the two treatments. However, significant differences in favour to the gel on the first day (first h) and on third day (72nd h) and in favour to the patch at the second day (48th h) were detected. C_max, E₁/E₂ ratio and AUC_(0–72) were not statistically different, while a significantly higher C_min for the gel was observed. Furthermore, the 90% confidence interval for AUC_(0–72) ratio (0.83–1.10) was within the commonly applied bioequivalence acceptance range (0.80–1.25). The %Fluct was significantly lower for Gelestra than for Estraderm TTS 50. Conclusions: although the mean E₂ and E₁concentrations, C_max, E₁/E₂ ratio and the AUC_(0–72) did not differ between the two E₂ treatments, the Gelestra treatment showed a lower day-to-day variation over the three day application, than the Estraderm TTS 50.     

Biological and endocrine aspects of transdermal 17β-oestradiol administration in post-menopausal women

The plasma protein distribution of oestradiol (E₂) and oestrone (E₁) during transdermal E₂ administration (100 μg/24 hr) was studied in 12 post-menopausal women. The E₂ and E₁ levels observed were 43–83 pg/ml and 37–73 pg/ml, respectively. The levels of the free, albumin-bound and sex-hormone-binding globulin (SHBG) bound fractions were in the ranges 1.4–1.9%, 60–65% and 35–45%, respectively, in the case of E₂, and 2.8–3.0%, 80–89% and 15–20%, respectively, in that of E₁. The SHBG levels also remained unaltered. It was concluded that transdermal administration of E₂ at the dosage employed produces a physiological plasma protein distribution of E₂ and E₁ and does not affect liver protein production.     

Protein kinase C shifts the voltage dependence of KCNQ/M channels expressed in Xenopus oocytes

It is well established that stimulation of G_q-coupled receptors such as the M1 muscarinic acetylcholine receptor inhibits KCNQ/M currents. While it is generally accepted that this muscarinic inhibition is mainly caused by the breakdown of PIP₂, the role of the subsequent activation of protein kinase C (PKC) is not well understood. By reconstituting M currents in Xenopus oocytes, we observed that stimulation of coexpressed M1 receptors with 10 μ m oxotremorine M (oxo-M) induces a positive shift (4–30 mV, depending on which KCNQ channels are expressed) in the conductance–voltage relationship ( G – V ) of KCNQ channels. When we applied phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, we observed a large positive shift (17.8 ± 1.6 mV) in the G – V curve for KCNQ2, while chelerythrine, a PKC inhibitor, attenuated the shift caused by the stimulation of M1 receptors. By contrast, reducing PIP₂ had little effect on the G – V curve for KCNQ2 channels; although pretreating cells with 10 μ m wortmannin for 30 min reduced KCNQ2 current amplitude by 80%, the G – V curve was shifted only slightly (5 mV). Apparently, the shift induced by muscarinic stimulation in Xenopus oocytes was mainly caused by PKC activation. When KCNQ2/3 channels were expressed in HEK 293T cells, the G – V curve seemed already to be shifted in a positive direction, even before activation of PKC, and PMA failed to shift the curve any further. That alkaline phosphatase in the patch pipette shifted the G – V curve in a negative direction suggests KCNQ2/3 channels are constitutively phosphorylated in HEK 293T cells.     

Modulation of the gating of the transient outward potassium current of rat isolated cerebellar granule neurons by lanthanum

The effects of the trivalent cation, lanthanum (La³⁺) on voltage-dependent K⁺ conductances were studied in rat isolated cerebellar granule neurons under whole-cell voltage-clamp conditions. La³⁺ at low micromolar concentrations caused a pronounced enhancement in the outward current evoked by depolarising steps from –50 mV, with the apparent recruitment of an inactivating component. The steady-state inactivation curve for the transient outward current, evoked by depolarising steps from –140 mV, was shifted by approximately 40 mV in the depolarising direction by 10 M La³⁺, with a slight increase in the slope factor. The kinetics of activation and inactivation were slowed in the presence of La³⁺. A shift of 10 mV in the depolarising direction was seen for the activation curve of the delayed rectifier current in the presence of 10 M La³⁺. These results indicate that La³⁺ has a potent effect on the gating characteristics of voltage-activated K⁺ currents. This effect cannot be explained by surface charge considerations.     

Back extensor muscle oxygenation and fatigability in healthy subjects and low back pain patients during dynamic back extension exertion

The purpose of this study was to assess if chronic low back pain patients have impaired paraspinal muscle O₂ turnover and endurance capacity as compared to healthy control subjects during dynamic exercise. Middle-aged healthy male subjects (n = 12, control) and male patients with chronic low back pain (n = 17, CLBP) participated in the study. L4–L5 level paraspinal muscle fatigue was objectively assessed during earlier validated 90 s dynamic back endurance test (spectral EMG, MPF_slope). Also EMG amplitude (EMG_amplitude) and initial MPF (MPF_initial) were assessed from the initial 5 s of the endurance contraction. Simultaneously near infrared spectroscopy (NIRS) was used for quantitative measurement of local L4–L5 paraspinal muscle O₂ consumption. Subcutaneous tissue thickness (ATT) was measured from the EMG and NIRS recording sites. The results indicated that control and CLBP groups were compatible as regarding anthropometric variables, paraspinal muscle activation levels (EMG_amplitude), initial MPF (MPF_initial) and ATT. When the ATT was used as a covariate in the ANOVA analysis, CLBP group did not show significantly greater paraspinal muscle fatigability (right MPF_slope – 12.2 ± 10.7%/min, left right MPF_slope – 12.6 ± 13.3%/min) or O₂ consumption (right NIRS_slope – 52.8 ± 79.6 μM/l/s) as compared to healthy controls (right MPF_slope – 11.9 ± 7.6%/min, left MPF_slope – 12.7 ± 8.6%/min, right NIRS_slope – 53.7 ± 95.2 μM/l/s). As a conclusion, these CLBP male patients did not show any impaired rate of paraspinal muscle oxygen consumption or excessive paraspinal muscle fatigability during dynamic exercise as compared with healthy controls. Subcutaneous tissue thickness has a strong influence on the NIRS and EMG amplitude measurements and, if unchecked, it could result in the false interpretation of the results.     

Effects of magnetic fields on the accumulation of thiobarbituric acid reactive substances induced by iron salt and H2O2 in mouse brain homogenates or phosphotidylcholine

In this study, we examined the effects of magnetic fields (MFs) on the generation of thiobarbituric acid reactive substances (TBARS) in the mouse brain homogenates or phosphotidylcholine (PC) solution, incubated with FeCl₃ and/or H₂O₂. Active oxygen species were generated and lipid peroxidation was induced in mouse brain homogenates by incubation with iron ions, resulting in the accumulation of TBARS. Lipid peroxidation was induced in PC by incubation with iron ions and H₂O₂. Exposure to sinusoidal MFs (60 Hz, 0.2–1.2 mT), symmetric sawtooth-waveform MFs (50 Hz, 25–600 mT/s), rectangular MFs (1/0.4–1/16 Hz, 3.3 mT) and static MFs (1, 5–300 mT) had no effect on the accumulation of TBARS in brain homogenates induced by FeCl₃. In contrast, when the homogenates were incubated with FeCl₃ in static MFs (2–4 mT), the accumulation of TBARS was decreased. However, this inhibitory effect disappeared when EDTA was added to the homogenate and incubated with H₂O₂. The accumulation of TBARS in PC solution incubated with FeCl₃ and H₂O₂ was also inhibited by the static MF. These results indicate that only static MFs had an inhibitory effect on iron-induced lipid peroxidation and the effectiveness of this magnetic field on iron ion-induced active oxygen species generation is restricted to a so called ‘window’ of field intensity of 2–4 mT.     

Linkage of low and high affinity anti-fluorescein idiotype families

Data presented in this study describes the isolation and characterization of two anti-fluorescein (Fl) hybridoma proteins 3–24 and 12–40, both IgG₁, with a K_a = 2.8 and 3.4 × 10⁶ M⁻¹, respectively, at 37°C. These clones inhibited (6.8 ± 2.8 − 20.8 ± 0.6% at l μg/well) the idiotype-anti-idiotype interactions (IAII) of anti-Fl clones 3–13 and 3–17, which define a previously described low affinity idiotype family. Antibodies 3–24 and 12–40 also inhibited (45.0 ± 3.0 and 61.3 ± 5.6%, respectively, at 1 μg/well) an IAII denfied by a high affinity (K_a = 5.2 ± 1.5 × 10⁹ M⁻¹ at 37°C) anti-Fl clone, 4-4. Hybridoma proteins 3–13 and 3–17 possess similar affinities for Fl (K_a = 3.8 ± 5.1 and 5.9 ± 4.0 × 10⁴ M⁻¹) and are known to be idiotypically unrelated to clone 4-4. While 3–24 and 12–40 appeared very similar, non-identity of their active sites was established by heterologous idiotypic inhibitions, fine specificity of binding and spectral measurements (Q_max and λ_max) of bound Fl. All IAII (3–13, 3–17, 9–40 and 4-4) were inhibited>80% by the presence of 10⁻⁴M F1 or F1-BSA, In addition, four intermediate affinity (6.0 × 10⁶ K_a 5.3 × 10⁸ M⁻¹) anti-FI clones, comprising a second previously described idiotype family (designated the 9–40 family) were further analyzed. Inhibition of the 9–40 IAII by all heterologous proteins in the 9–40 family (except clone 5–27), and clones 3–24, 12–40 and 4-4 ranged from 87.7 ± 1.3 to 95.4 ± 1.0% at 1μg/well. Titration of the 9–40 IAII inhibition by antibodies 9–40, 3–24, 12–40 or 4-4 generated essentially superimposable profiles. In reciprocal inhibition experiments, using the 4-4 IAII, clones 3–24, 12–40, 9–40 and 4-4 gave distinct idiotypic titration patterns. Thus, members of the 9–40 family, 3–24 and 12–40 were more closely related to intermediate affinity clone 9–40 than high affinity clone 4-4. Finally all members of the 9–40 family also significantly inhibited both the 3–13 and 3–17 IAII (11.8 ± 3.1 − 32.9 ± 6.1 at 1 μg/well) and gave distinct idiotypic inhibition profiles. Clones 3–24 and 12–40, characterized in this report, and the 9–40 family provide linkage between idiotypically distinct anti-Fl hybridoma proteins differing in affinity by> 20,000-fold. This linkage provides a greater span in affinity, than in all previously reported idiotypic families, within restricted or unrestricted systems.     

Activation of the hyperpolarization-activated current (if) in sino-atrial node myocytes of the rabbit by vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in extrinsic and intrinsic nerves of the heart. VIP can be released by vagal stimulation but, contrary to ACh, causes positive chronotropic effects as a result of binding to cardiac receptors which stimulate adenylate cyclase, and thus has been implicated in vagal tachycardias. Since the rate of diastolic depolarization of sinoatrial (SA) node myocytes depends on the hyperpolarization-activated current (i_f), which is directly activated by cytoplasmic cAMP, we studied the action of VIP on i f in myocytes isolated from the SA node of the rabbit. VIP (0.65 M) reversibly increased i_f at –65 mV but had no effect at –115 mV suggesting that its primary effect was to shift the activation curve to more positive voltages. Hyperpolarizing ramp and voltage compensation protocols indicated that VIP shifts the activation curve of i_f by approximately 5–6 mV in the positive direction with no change in maximal conductance. This shift may be the mechanism by which VIP produces its positive chronotropic effect and supports a negative feedback role for this peptide during elevated vagal activity.     

The effect of substance P and related peptides on the guinea-pig lung strip

Substance P is present in sensory nerves in the lung and we report here its actions on the lung strip. Substance P was shown to produce rapid, small contractions of the lung strip at doses from 10^–9 to 10^–5 M, and there was no apparent dose-response relationship. In the presence of a mixture of three inhibitors of substance P breakdown: bacitracin, 1,4-dithio-l-threitol and ethylenediaminetetraacetic acid all at 10^–4 M, the responses to substance P were greatly increased and dose-response curves could be established. The concentrations producing half maximal effect in the presence of these inhibitors were 6×10^–6 M ± 4×10^–6 M. The inhibitors of substance P breakdown were found to have no effect on the histamine dose-response curve: any small shifts obtained were not significant.Chlorpheniramine 10^–6 M and 10^–5 M shifted the histamine dose-response curve to the right producing dose ratios of 100 and 900 respectively. At these doses chlorpheniramine had no effect on the dose-response curve to substance P. A dose of substance P not itself producing a response (5×10^–8 M) caused an increase in the size of the responses to histamine. The histamine dose-response curve was shifted to the left in the presence of substance P producing an average dose ratio of 1.4±0.2. In the presence of the peptidase inhibitors, dose-response curves have also been produced for physalaemin (10^–7–10^–5 M) and eledoisin (10^–7–10^–5 M).     

Ammonium production during hypo-osmotic stress leads to alkalinization of acidocalcisomes and cytosolic acidification in Trypanosoma cruzi

Osmotic swelling of Trypanosoma cruzi epimastigotes resulted in alkalinization of acidocalcisomes, as revealed by changes in acridine orange fluorescence of intact cells. Concomitant with these changes, intracellular ammonium levels increased while extracellular ammonium levels decreased significantly. Hypo-osmotic stress also resulted in cytosolic acidification. The observed changes in intracellular pH (pH_i) were independent of extracellular calcium, and other ions concentration. Taken together, these results are consistent with a stimulation of ammonium production upon hypo-osmotic stress and its accumulation in acidocalcisomes resulting in their alkalinization, which might be responsible for polyphosphate hydrolysis and osmotic changes in the organelles.     

Binding and GTPgammaS autoradiographic analysis of preproorphanin precursor peptide products at the ORL1 and opioid receptors

Utilizing agonist-stimulated GTPγS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [³⁵S]-GTPγS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [³⁵S]-GTPγS binding in a pattern similar to that described for [¹²⁵I]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II_1–17) had no effect, suggesting that they do not mediate their reported analgesic effects via a G_i/o-coupled receptor (i.e. opioid or ORL1). Unlike OFQ II_1–17, high concentrations of its C-terminal extension, OFQ II_1–28, stimulated [³⁵S]-GTPγS binding in a mu (μ) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II_1–28 at the cloned ORL1 and μ opioid receptors. OFQ II_1–28 had no specific binding at either ORL1 or μ opioid receptors at concentrations up to 50 μM. This lack of affinity was not consistent with a μ-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II_1–28 possesses analgesic activity, this effect does not appear to be mediated via direct binding at the μ opioid receptor. Taken together, these findings support the view that (1) OFQ is the only ppOFQ peptide that binds to and activates the ORL1 receptor and (2) OFQ II_1–28 does not bind or stimulate [³⁵S]-GTPγS binding in cells expressing the μ opioid receptor.     

胡椒碱减轻H2O2 引起的兔心房肌细胞内向整流钾电流及超速激活延迟整流钾电流的异常改变

目的: 研究胡椒碱对H₂O₂引起的兔单个心房肌细胞内向整流钾电流(I_K1)及超速激活的延迟整流钾电流(I_KUr)异常的影响。方法: 采用全细胞膜片钳技术分析50 μmol/L H₂O₂对兔单个心房肌细胞I_K1和I_KUr的影响,并研究预先应用7 μmol/L胡椒碱对其的保护作用。结果: 7 μmol/L胡椒碱对正常兔心房肌细胞I_K1和I_KUr及其通道动力学无显著影响。在50 μmol/L H₂O₂作用下,兔心房肌细胞I_K1峰值由(-148.2±16.7)pA/pF降低至(-64.2±9.8)pA/pF (P<0.05),电流-电压曲线上移;而I_KUr峰值由(16.0±2.1)pA/pF降低至(6.1±1.4)pA/pF (P<0.05),电流-电压曲线下移,通道稳态激活曲线右移,通道稳态失活曲线左移及恢复时间减慢,而且存在频率依赖性特征。预先给予7 μmol/L胡椒碱,明显减轻H₂O₂对I_K1和I_KUr的抑制作用(P<0.01),并可减少H₂O₂对超速激活延迟整流钾通道动力学的异常影响。结论: 胡椒碱可减轻氧化应激对心房肌细胞I_K1和I_KUr的影响。     

Callosal agenesis and absence of primary visual cortex induced by prenatal X rays impair navigation's strategy and learning in tasks involving visuo-spatial working but not reference memory in mice

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive and memory deterioration. Production and accumulation of β-amyloid peptide (Aβ) is central to the pathogenesis of AD. Recent studies have demonstrated that PKA/CREB-dependent signaling pathway and long-term potentiation are inhibited by sublethal concentrations of Aβ_1–42 in cultured hippocampus neurons. Here, we examined the effects of nobiletin on the Aβ-induced inhibition of CREB phosphorylation in cultured rat hippocampus neurons. A sublethal concentration of Aβ_1–42 or Aβ_1–40 decreased glutamate-induced CREB phosphorylation, whereas pretreatment with nobiletin reversed the Aβ-induced decrease in CREB phosphorylation. The effects of nobiletin on impairment of learning ability were also examined in chronically Aβ_1–40 infused AD model rats using the eight-arm radial maze. In the AD model rats, nobiletin showed protective effects on Aβ_1–40-induced impairment of learning ability. These results suggest that nobiletin has the potential for becoming a novel lead compound for drug development for AD.     

GABAA, GABAC and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells

Rod bipolar cells relay visual signals evoked by dim illumination from the outer to the inner retina. GABAergic and glycinergic amacrine cells contact rod bipolar cell terminals, where they modulate transmitter release and contribute to the receptive field properties of third order neurones. However, it is not known how these distinct inhibitory inputs affect rod bipolar cell output and subsequent retinal processing. To determine whether GABA_A, GABA_C and glycine receptors made different contributions to light-evoked inhibition, we recorded light-evoked inhibitory postsynaptic currents (L-IPSCs) from rod bipolar cells mediated by each pharmacologically isolated receptor. All three receptors contributed to L-IPSCs, but their relative roles differed; GABA_C receptors transferred significantly more charge than GABA_A and glycine receptors. We determined how these distinct inhibitory inputs affected rod bipolar cell output by recording light-evoked excitatory postsynaptic currents (L-EPSCs) from postsynaptic AII and A17 amacrine cells. Consistent with their relative contributions to L-IPSCs, GABA_C receptor activation most effectively reduced the L-EPSCs, while glycine and GABA_A receptor activation reduced the L-EPSCs to a lesser extent. We also found that GABAergic L-IPSCs in rod bipolar cells were limited by GABA_A receptor-mediated inhibition between amacrine cells. We show that GABA_A, GABA_C and glycine receptors mediate functionally distinct inhibition to rod bipolar cells, which differentially modulated light-evoked rod bipolar cell output. Our findings suggest that modulating the relative proportions of these inhibitory inputs could change the characteristics of rod bipolar cell output.     

Mechanisms of formation of IgE-binding factors (soluble CD23)—I. Fcε R II bearing B cells generate IgE-binding factors of different molecular weights

IgE-binding factors (soluble CD23) are generally considered to have an M_r of 25,000–27,000. The present study first indicates that IgE-BFs with an M_r of 33,000 or 37,000 may also be produced by Fcε R II bearing B cells, depending upon the culture conditions and the nature of the Fcε R II bearing cells. Extending our previous observations that the M_r 25,000–27,000 IgE-BFs are derived from the cleavage of soluble M_r 37,000 precursors, we show here that this cleavage is specifically inhibited by iodoacetamide but not by several other protease inhibitors. The proteolytic enzyme involved in the cleavage of M_r 33,000–37,000 precursors into M_r 25,000–27,000 IgE-BFs is cell-associated and is specifically expressed on Fcε R II bearing cells. As expected, these M_r 33,000 and 37,000 fragments of Fcε R II are capable of binding to IgE. The site at which these molecules are cleaved from Fcε R II was located by determining their amino-terminal sequence. The M_r 37,000 IgE-BFs start at position 81 (glutamine) and the M_r 33,000 IgE-BFs start at position 102 (leucine) of the Fcε R II sequence. Taken collectively, the present study not only contributes to our understanding of the mechanisms of formation of IgE-BFs, but also provides a means to prepare different molecular forms of IgE-BFs which may display different biological activity.