Protective role of curcumin on renal ischemia reperfusion injury via attenuating the inflammatory mediators and Caspase-3.

Renal ischemia/reperfusion (I/R) damage may arise due to nephron sparing surgery in patient with a solitary kidney of restricted renal parenchymas. Apoptosis, inflammation and oxidative stress play a significant role in the expansion of renal dysfunction following renal I/R. The aim of the current investigation was to particularize the potential effect of curcumin against hypoxia induced renal injury. The albino Wistar rats divided into groups and each group contains six rats. They groups are normal control; disease control; curcumin (5 mg/kg per day) and another group orally treated with curcumin (10 mg/kg per day) for two weeks before induction of renal I/R. The renal and serum samples were collected and used for the biochemical estimation. The renal tissue was further used for the histopathological estimation. The result of the current investigation demonstrated that the curcumin significantly (P<0.01) attenuated I/R induced renal injury in a dose-dependent way. It also causes significant (P<0.01) reduction in the serum creatinine, blood urea nitrogen level and also suppressed the kidney injury molecules-1. Additionally, it also causes significant inhibition of the malonaldehyde, caspase-3, myeloperoxide, lactose dehydrogenase and interferon-gamma together with enhanced interlukin-10 content.

Comparison of dual-source parallel radio frequency transmission liver MRI at 3.0 T with conventional MRI.

AIM: Aim of the present study was to investigate the role of dual-source parallel Radio frequency (RF) and single-source excitation in liver imaging at 3.0 T MR. METHODS: This study was a retrospective analysis. One hundred and seven subjects underwent a 3.0 T TX MR scanning including axial spectrally selective attenuated inversion recovery (SPAIR) T2WI, axial DWI and coronal balanced-fast field echo (Balanced FFE). Each sequence was carried out with both single-source and dual-source RF excitation. Student's t test was used to compare whether there was difference between single-source and dual-source RF excitation in the image uniformity, single-to-noise ratio (SNR) and contrast-to-noise ratio (CNR). Wilcoxon signed rank test was used to determine whether there was difference between conventional and parallel transmission in the score of image quality. Reader agreement was assessed using the Cohen's Kappa test. RESULTS: For the image uniformity, there was significant difference between single-source and dual-source excitation (418.40±66.75 for single-source vs. 416.26±50.61 for dual-source, t=2.524, P<0.05). There also existed significant difference between single-source and dual-source excitation in SNR and CNR, respectively. The SNR and CNR of parallel transmission (22.03±12.16 and 18.33±10.01, respectively) were both higher than those of single transmission (20.36±11.21 and 15.22±8.95, respectively) (t=-2.630, P<0.05 for SNR and t=-4.238, P<0.05 for CNR). Image quality comparisons revealed significantly better results with dual-source than single-source RF excitation at SPAIR T2WI (1.4±0.42 vs. 1.81±0.27), DWI (1.08±0.46 vs. 1.63±0.36) and balanced FFE sequence (0.95±0.45 vs. 1.65±0.37, Z=-5.894, -5.801 and -6.985, respectively, P<0.01). In the comparison of image quality, the agreement between the two readers was very good (Kappa>0.8, P<0.05). CONCLUSION: Dual-source parallel RF excitation MR imaging in liver enables reducing dielectric shading, improving homogeneity of the RF magnetic induction field, and increasing SNR and CNR at 3.0 T.

[Clinical management and prognosis for descending necrotizing mediastinitis].

Objective: To investigate the clinical characteristics, treatment experiences and prognostic factors for descending necrotizing mediastinitis (DNM). Methods: A retrospective analysis was performed on the data of 22 patients with DNM diagnosed and treated in Henan Provincial People's Hospital from January 2016 to August 2022, including 16 males and 6 females, aged 29-79 years. After admission, all patients underwent CT scanning of the maxillofacial, cervical, and thoracic regions to confirm their diagnoses. Emergency incision and drainage were performed. The neck incision was treated with continuous vacuum sealing drainage. According to the prognoses, the patients were divided into cure group and death group, and the prognostic factors were analyzed. SPSS 25.0 software was used to analyze the clinical data. Rusults: The main complaints were dysphagia (45.5%, 10/22) and dyspnea (50.0%, 11/22). Odontogenic infection accounted for 45.5% (10/22) and oropharyngeal infection accounted for 54.5% (12/22). There were 16 cases in the cured group and 6 cases in the death group, with a total mortality rate of 27.3%. The mortality rates of DNM typeⅠand typeⅡwere respectively 16.7% and 40%. Compared with the cured group, the death group had higher incidences for diabetes, coronary heart disease and septic shock (all P<0.05). There were statistically significant differences between the cure group and the death group in procalcitonin level (50.43 (137.64) ng/ml vs 2.92 (6.33) ng/ml, M(IQR), Z=3.023, P<0.05) and acute physiology and chronic health evaluation Ⅱ(APACHEⅡ) score (16.10±2.40 vs 6.75±3.19, t=6.524, P<0.05). Conclution: DNM is rare, with high mortality, high incidence of septic shock, and the increased procalcitonin level and APACHE Ⅱ score combined diabetes and coronary heart disease are the poor prognostic factors for DNM. Early incision and drainage combined with continuous vacuum sealing drainage technique is a better way to treat DNM.

[Incidence and prognosis of olfactory and gustatory dysfunctions related to infection of SARS-CoV-2 Omicron strain: a national multi-center survey of 35 566 population].

Objective: This cross-sectional investigation aimed to determine the incidence, clinical characteristics, prognosis, and related risk factors of olfactory and gustatory dysfunctions related to infection with the SARS-CoV-2 Omicron strain in mainland China. Methods: Data of patients with SARS-CoV-2 from December 28, 2022, to February 21, 2023, were collected through online and offline questionnaires from 45 tertiary hospitals and one center for disease control and prevention in mainland China. The questionnaire included demographic information, previous health history, smoking and alcohol drinking, SARS-CoV-2 vaccination, olfactory and gustatory function before and after infection, other symptoms after infection, as well as the duration and improvement of olfactory and gustatory dysfunction. The self-reported olfactory and gustatory functions of patients were evaluated using the Olfactory VAS scale and Gustatory VAS scale. Results: A total of 35 566 valid questionnaires were obtained, revealing a high incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain (67.75%). Females(χ2=367.013, P<0.001) and young people(χ2=120.210, P<0.001) were more likely to develop these dysfunctions. Gender(OR=1.564, 95%CI: 1.487-1.645), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), oral health status (OR=0.881, 95%CI: 0.839-0.926), smoking history (OR=1.152, 95%CI=1.080-1.229), and drinking history (OR=0.854, 95%CI: 0.785-0.928) were correlated with the occurrence of olfactory and taste dysfunctions related to SARS-CoV-2(above P<0.001). 44.62% (4 391/9 840) of the patients who had not recovered their sense of smell and taste also suffered from nasal congestion, runny nose, and 32.62% (3 210/9 840) suffered from dry mouth and sore throat. The improvement of olfactory and taste functions was correlated with the persistence of accompanying symptoms(χ2=10.873, P=0.001). The average score of olfactory and taste VAS scale was 8.41 and 8.51 respectively before SARS-CoV-2 infection, but decreased to3.69 and 4.29 respectively after SARS-CoV-2 infection, and recovered to 5.83and 6.55 respectively at the time of the survey. The median duration of olfactory and gustatory dysfunctions was 15 days and 12 days, respectively, with 0.5% (121/24 096) of patients experiencing these dysfunctions for more than 28 days. The overall self-reported improvement rate of smell and taste dysfunctions was 59.16% (14 256/24 096). Gender(OR=0.893, 95%CI: 0.839-0.951), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), history of head and facial trauma(OR=1.180, 95%CI: 1.036-1.344, P=0.013), nose (OR=1.104, 95%CI: 1.042-1.171, P=0.001) and oral (OR=1.162, 95%CI: 1.096-1.233) health status, smoking history(OR=0.765, 95%CI: 0.709-0.825), and the persistence of accompanying symptoms (OR=0.359, 95%CI: 0.332-0.388) were correlated with the recovery of olfactory and taste dysfunctions related to SARS-CoV-2 (above P<0.001 except for the indicated values). Conclusion: The incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain is high in mainland China, with females and young people more likely to develop these dysfunctions. Active and effective intervention measures may be required for cases that persist for a long time. The recovery of olfactory and taste functions is influenced by several factors, including gender, SARS-CoV-2 vaccination status, history of head and facial trauma, nasal and oral health status, smoking history, and persistence of accompanying symptoms.

[Clinical analysis of 5 cases of dural arteriovenous fistulas complained of pulsatile tinnitus].

Objective: The clinical characteristics of dural arteriovenous fistula with pulsatile tinnitus were analyzed to deepen the understanding of the disease. Methods: The clinical data of five patients complained of pulsatile tinnitus and diagnosed dural arteriovenous fistula in Henan People's Hospital from May 2013 to June 2018 were retrospectively analyzed, including 3 males and 2 females, aged 27-65 years. Results: The main clinical symptoms of the five patients were continuous pulsatile tinnitus, accompanied/not accompanied by headache, memory decline, etc., with a course of three months to 20 years. They were diagnosed as dural arteriovenous fistula by digital subtraction angiography, and three cases of tinnitus disappeared and two cases of tinnitus were relieved after embolization. Conclusions: The dural arteriovenous fistula is a rare and complicated disease. When the patient complain of the pulsatile tinnitus, the related etiology should be considered and managed properly.

[Analyses of diagnosis and treatment of foreign body aspiration in children with tracheobronchial variations].

Objective: To investigate the key issues in the diagnosis and treatment of foreign body aspiration in children with tracheobronchial variations. Methods: A retrospective study was performed for 11 pediatric patients who were treated in Department of Otorhinolaryngology and Head and Neck Surgery, Henan Province People's Hospital after a diagnosis of foreign body aspiration with tracheobronchial variations between January 2015 and December 2017. There were 7 males and 4 females among the 11 cases of foreign body aspiration with tracheobronchial variations, ranging between 9 months and 11 years of age. Results: Among 11 cases, the types of variationswere tracheal bronchus in 9 cases, bridging bronchus in 1 case and simple tracheal stenosis in 1 case. All of the pediatric patients were under general anesthesia, and the foreign bodies were removed by bronchoscopy successfully with no significant complications. Conclusions: The possibility of tracheobronchial variations should be considered in children with recurrent wheezing and poor efficacy of regular treatment before foreign body aspiration. Removal of foreign body via rigid bronchoscope under general anesthesia is a safe and effective treatment. These children are needed to combine the situation oftracheobronchial variations and the location of foreign bodies to guide the operation, and strengthened the perioperative treatment.

A Fuzzy Reasoning Design for Fault Detection and Diagnosis of a Computer-Controlled System.

A Fuzzy Reasoning and Verification Petri Nets (FRVPNs) model is established for an error detection and diagnosis mechanism (EDDM) applied to a complex fault-tolerant PC-controlled system. The inference accuracy can be improved through the hierarchical design of a two-level fuzzy rule decision tree (FRDT) and a Petri nets (PNs) technique to transform the fuzzy rule into the FRVPNs model. Several simulation examples of the assumed failure events were carried out by using the FRVPNs and the Mamdani fuzzy method with MATLAB tools. The reasoning performance of the developed FRVPNs was verified by comparing the inference outcome to that of the Mamdani method. Both methods result in the same conclusions. Thus, the present study demonstratrates that the proposed FRVPNs model is able to achieve the purpose of reasoning, and furthermore, determining of the failure event of the monitored application program.

[Diagnosis and therapy strategy of acute invasive fungal rhino-sinisitis].

Summary Acute invasive fungal rhino-sinisitis(AIFRS) is a kind of acute fungus infectious disease.It ofen deteriorates rapidly and abruptly,which occurs in the nose and sinuses with somepeople who have low immunological function.This paper reviewed the progress about the acute invasive fungal rhino-sinisitis's clinical characteristics,diagnosis,treatment and prognosis to improve the level of early diagnosis and reduce the mortality.

[Endoscopic sinus surgery for the treatment of invasive fungal sinusitis].

Objective:To explore the early diagnosis, the treatment and theprognosis of invasive fungal rhinosinusitis.Method:By summarizing the clinical data of 18 patients, CT and MRI images, pathological diagnosis and follow-up results to analysis the early diagnosis and the treatment.Result:All 5 patients with invasive fungal sinusitis were infected with mucor, 3 of whom died of intracranial complications. Among 13 patients with chronic invasive fungal rhinosinusitis, 9 were aspergillus, 3 were mucor, and 1 was negative. There were 9 cases of diabetes, 1 cases of ankylosing spondylitis, 3 cases of hypoproteinemia, 1 cases of organ transplantation, and 1 cases of leukemia. There were 3 cases of death who unenforced the operation, 15 patients received enlarged endoscopic surgery, postoperative antifungal therapy. Followed up 1-3 years, relapse in 3 cases, 1 case of death.Conclusion:The diagnosis of FRS needs to be combined with clinical manifestations, imaging features and pathological findings. The treatment requires surgery to completely remove diseased tissue (enlarged sinus open surgery) combined with antifungal use in sufficient quantities.

[Voice acoustic study of plasma radiofrequency ablation for the treatment of laryngeal premalignant lesions].

Objective:To study the voice function effect of low temperature plasma radiofrequency ablation in the treatment of patients with laryngeal premalignant lesions. Method:Fifty cases of laryngeal premalignant lesions were treated with low temperature plasma radiofrequency ablation. All of the patients were examined by electronic laryngoscopy and acoustic analysis(F0，Jitter，Shimmer，NNE，HNR) in 2 weeks,1 month,3 months after surgery. Voice acoustic results were compared with a control group of 50 normal adults for the further analysis. Result:Fifty patients with laryngeal premalignant lesions were treated by low temperature plasma radiofrequency ablation.The result showed that 47 patients(94%)were successfully decannulated without serious complications, such as dyspnea, aphonia and anterior glottic stenosis. Acoustic analysis showed that F0,Jitter,Shimmer and NNE were significantly different from normal 2 weeks after surgery（P<0.01）.Voice function recovered weakly 1 month after operation（P<0.05）.There were no significant differences in the vocal parameters between plasma radiofrequency ablation group and control group 3 months after surgery(P>0.05). Conclusion:Radiofrequency coblation was a safe,minimally invasive and effective surgical method and can be widely used to treat laryngeal premalignant lesions．.

Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers.

Batrachotoxin (BTX)-activated Na+ channels from rabbit skeletal muscle were incorporated into planar lipid bilayers. These channels appear to open most of the time at voltages greater than -60 mV. Local anesthetics, including QX-314, bupivacaine, and cocaine when applied internally, induce different durations of channel closures and can be characterized as "fast" (mean closed duration less than 10 ms at +50 mV), "intermediate" (approximately 80 ms), and "slow" (approximately 400 ms) blockers, respectively. The action of these local anesthetics on the Na+ channel is voltage dependent; larger depolarizations give rise to stronger binding interactions. Both the dose-response curve and the kinetics of the cocaine-induced closures indicate that there is a single class of cocaine-binding site. QX-314, though a quaternary-amine local anesthetic, apparently competes with the same binding site. External cocaine or bupivacaine application is almost as effective as internal application, whereas external QX-314 is ineffective. Interestingly, external Na+ ions reduce the cocaine binding affinity drastically, whereas internal Na+ ions have little effect. Both the cocaine association and dissociation rate constants are altered when external Na+ ion concentrations are raised. We conclude that (a) one cocaine molecule closes one BTX-activated Na+ channel in an all-or-none manner, (b) the binding affinity of cocaine is voltage sensitive, (c) this cocaine binding site can be reached by a hydrophilic pathway through internal surface and by a hydrophobic pathway through bilayer membrane, and (d) that this binding site interacts indirectly with the Na+ ions. A direct interaction between the receptor and Na+ ions seems minimal.

Binding affinity and stereoselectivity of local anesthetics in single batrachotoxin-activated Na+ channels.

Several local anesthetics (LA) have been previously shown to block muscle batrachotoxin (BTX)-activated Na+ channels in planar bilayers. The mean dwell time of different LA drugs, however, varies widely, from less than 10 ms to longer than several seconds. In this study, we have examined the structural determinants that govern the dwell time, the binding affinity, and the stereoselectivity of LA drugs using cocaine and bupivacaine homologues, RAC compounds, and their available stereoisomers. Our results from the structure-activity experiments reveal that (a) there are two apparent hydrophobic binding domains present in the LA binding site; one interacts with the aromatic moiety of the LA drugs, and the other interacts with the alkyl group attached to the tertiary amine of the LA drugs; (b) the LA mean dwell time and the binding affinity are largely determined by the hydrophobic interactions; (c) the LA binding site is highly stereoselective, with a difference in KD values over 50- and 6-fold for (+/-) cocaine and (+/-) bupivacaine, respectively; (d) the cocaine stereoselectivity is comparable among muscle, brain, and heart BTX-activated Na+ channels; and finally and most unexpectedly, (e) the stereoselectivity of LA drugs in BTX-activated Na+ channels appears greatly different from that reported in normal Na+ channels. Possible explanations for this difference are discussed.

Rapid voltage-dependent dissociation of scorpion alpha-toxins coupled to Na channel inactivation in amphibian myelinated nerves.

The voltage-dependent action of several scorpion alpha-toxins on Na channels was studied in toad myelinated nerve under voltage clamp. These toxins slow the declining phase of macroscopic Na current, apparently by inhibiting an irreversible channel inactivation step and thus permitting channels to reopen from a closed state in depolarized membranes. In this article, we describe the rapid reversal of alpha-toxin action by membrane depolarizations more positive than +20 mV, an effect not achieved by extensive washing. Depolarizations that were increasingly positive and of longer duration caused the toxin to dissociate faster and more completely, but only up to a limiting extent. Repetitive pulses had a cumulative effect equal to that of a single pulse lasting as long as their combined duration. When the membrane of a nonperfused fiber was repolarized, the effects of the toxin returned completely, but if the fiber was perfused during the conditioning procedure, recovery was incomplete and occurred more slowly, as it did at lower applied toxin concentrations. Other alpha-type toxins, from the scorpion Centruroides sculpturatus (IVa) and the sea anemone Anemonia sulcata (ATXII), exhibited similar voltage-dependent binding, though each had its own voltage range and dissociation rate. We suggest that the dissociation of the toxin molecule from the Na channel is coupled to the inactivation process. An equivalent valence for inactivation gating, of less than 1 e per channel, is calculated from the voltage-dependent change in toxin affinity.

Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification.

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.

Altered stereoselectivity of cocaine and bupivacaine isomers in normal and batrachotoxin-modified Na+ channels.

The inhibitory effects of local anesthetics (LAs) of cocaine and bupivacaine optical isomers on Na+ currents were studied in clonal GH3 cells under whole-cell patch clamp conditions. At holding potential of -100 mV, all four isomers inhibited peak Na+ currents when the cell was stimulated infrequently. The dose-response curves of this tonic block of peak Na+ currents by (-)/(+) cocaine and (-)/(+) bupivacaine were well fitted by the Langmuir isotherm, suggesting that one LA isomer blocked one Na+ channel. Each pair of isomers showed no greater than a twofold difference in stereoselectivity toward Na+ channels. Additional block of Na+ currents occurred when the cell was stimulated at 2 Hz. This use-dependent block was also observed in all four isomers, which again displayed little stereoselectivity. The voltage dependence of the use-dependent block produced by cocaine isomers did not overlap with the activation of Na+ channels but did overlap with the steady-state inactivation (h infinity), indicating that cocaine can bind directly to the inactivated state of Na+ channels before channel opening. In comparison, the peak batrachotoxin (BTX)-modified Na+ currents were little inhibited by cocaine and bupivacaine isomers. However, the maintained BTX-modified Na+ currents were highly sensitive toward the (-) form of cocaine and bupivacaine isomers during a prolonged depolarization. As a result, a profound time-dependent block of BTX-modified Na+ currents was evident in the presence of these LA isomers. The estimated values of the equilibrium dissociation constant (KD in micromolar) at +50 mV were 35.8, 661, 7.0, and 222 for (-)/(+) cocaine and (-)/(+) bupivacaine, respectively. Although chloramine-T (CT) also modified the fast inactivation of Na+ channels and gave rise to a maintained Na+ current during a prolonged depolarization, LA isomers showed no greater stereoselectivity in blocking this maintained current than in blocking the normal transient Na+ current. We conclude that (a) cocaine and bupivacaine isomers exhibit only weak stereoselectivity toward the LA receptor in normal and CT-treated Na+ channels, (b) BTX drastically modifies the configuration of the LA binding site so that the LA stereoselectivity of the open Na+ channels is altered by an order of magnitude, and (c) the (-) forms of cocaine and bupivacaine interact strongly with the open state of BTX-modified Na+ channels but only weakly, if at all, with the closed state. The last finding may explain why most LA drugs were reported to be less effective toward BTX-modified Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

Binding of benzocaine in batrachotoxin-modified Na+ channels. State-dependent interactions.

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

Quaternary ammonium compounds as structural probes of single batrachotoxin-activated Na+ channels.

Quaternary ammonium (QA) blockers are well-known structural probes for studying the permeation pathway of voltage-gated K+ channels. In this study we have examined the effects of a series of n-alkyl-trimethylammonium compounds (Cn-QA) on batrachotoxin (BTX)-activated Na+ channels from skeletal muscle incorporated into planar lipid bilayers. We found that these amphipathic QA compounds (Cn-QA where n = 10-18) block single Na+ channels preferentially from the internal side with equilibrium dissociation constants (KD) in the submicromolar to micromolar range. External application of amphipathic QA compounds is far less effective, by a factor of greater than 200. The block can be described by a QA molecule binding to a single site in the Na+ channel permeation pathway. QA binding affinity is dependent on transmembrane voltage with an effective valence (delta) of approximately 0.5. QA dwell times (given as mean closed times, tau c) increase as a function of n-alkyl chain length, ranging from approximately 13 ms for C10-QA to 500 ms for C18-QA at +50 mV. The results imply that there is a large hydrophobic region within the Na+ channel pore which accepts up to 18 methylene groups of the Cn-QA cation. This hydrophobic domain may be of clinical significance since it also interacts with local anesthetics such as cocaine and mepivacaine. Finally, like BTX-activated Na+ channels in bilayers, unmodified Na+ channels in GH3 cells are also susceptible to QA block. Amphipathic QA cations elicit both tonic and use-dependent inhibitions of normal Na+ currents in a manner similar to that of local anesthetic cocaine. We conclude that amphipathic QA compounds are valuable structural probes to study the permeation pathway of both normal and BTX-activated Na+ channels.

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Inhibition of sodium currents by local anesthetics in chloramine-T-treated squid axons. The role of channel activation.

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.