Contraluminal transport of organic cations in the proximal tubule of the rat kidney

In order to study the quantitative structure/activity relationship of organic cation transport across the contraluminal side of the proximal renal tubule cell, the stopped-flow capillary microperfusion method was applied and the inhibitory potency (apparent K _i values) of different homologous series of substrates against N ¹-[³H]methylnicotinamide (NMeN⁺) transport was evaluated. Aniline and its ring- or N-substituted analogues as well as the aminonaphthalines do not interact with the contraluminal NMeN⁺ transporter except for the quaternary trimethylphenylammonium and pararosaniline, which bear a permanent positive charge, and for 1,8-bis-(dimethylamino)naphthaline, which forms an intramolecular hydrogen bond. If, however, one or more than one methylene group is interposed between the benzene ring and the amino group, the compounds interact with the contraluminal NMeN⁺ transporter in proportion to their hydrophobicity parameter, i.e. the octanol/water partition coefficient (log octanol). The catecholamines and other hydroxyl-substituted phenylethyl analogues also follow this rule. In addition, the N-heterocyclic pyridine, quinoline, isoquinoline and acridine analogues also interact with the contraluminal NMeN⁺ transporter, when their pK _a values are higher than 5.0, and, an inverse correlation between pK _a and log K _{i, NMeN} was observed. An exception to this rule are those hydroxy compounds of pyridine, quinoline and isoquinoline that show tautomerism. These compounds slightly inhibit NMeN⁺ transport despite low pK _a values. The quaternary nitrogen compounds of aniline and the N-heterocyclic analogues, as far as tested, all interact with the contraluminal NMeN⁺ transporter in relation to their hydrophobicity. The data indicate that the contraluminal NMeN⁺ transporter interacts with N-compounds according to their hydrophobicity and/or according to their basicity (affinity to protons). The reason for deviation of the aniline analogues and the OH-tautomeric heterocyclic N-compounds from this behaviour is discussed.     

The effectiveness of different isomers of octanol as blockers of harmaline-induced tremor

Intracellular recording in the guinea-pig brainstem slice has demonstrated that high molecular weight alcohols block the low threshold calcium channel (LTCC) in the inferior olive (IO). These alcohols thus provide a tool for understanding the function of the pacemaking cellular networks of the olivo-cerebellar system, since the LTCC has been implicated in the oscillatory behavior of these neurons. Aspects of normal and pathological tremor are also believed to be mediated by these circuits, and thus development of effective ways of blocking the LTCC in vivo may eventually lead to novel treatments for essential tremor. The present experiments evaluated the effectiveness of the isomers of octanol in decreasing harmaline-induced tremor in vivo in the rat. Harmaline was used in this study because its tremorgenic action is mediated at the level of IO; octanol was found to be a potent antagonist of harmaline-induced tremor. Significant differences between the isomers further suggested conformational differences. This, taken in conjunction with the lack of effect of octanol in both IO lesioned rats and oxotremorine-induced tremor, implied that the action of the alcohol may be mediated at a specific binding site. These findings thus support the conclusions that the antagonism of harmaline-induced tremor by octanol occurs in the IO, and, in view of the previously reported in vitro data, that octanol may be an effective blocker of the LTCC in vivo.     

A METHOD FOR MEASURING OCTANOL:WATER PARTITION COEFFICIENTS OF HIGHLY TOXIC ORGANOPHOSPHORUS COMPOUNDS

Octanol:water partition coefficients (log P) can be used to provide invaluable information for the overall understanding of the uptake, distribution, biotransformation, and elimination of a wide variety of chemicals. This is especially important in the study of certain classes of organophosphorus compounds, e.g., chemical warfare agents. Although several analytical methods currently exist for providing the necessary data for determination of log P values, their use in the study of chemical warfare agents has been limited due to the rapid hydrolysis and biolethality of this class of compounds. To overcome those limitations a rapid, convenient, and safe method for generating the required data was developed using a computer-controlled gas chromatograph (GC) equipped with a nitrogen phosphorus detector. For method development four organophosphorus compounds were used:diisopropyl methyl phosphonate (DIMP), diethyl p -nitrophenyl phosphate (paraoxon), diisopropyl fluorophosphate (DFP), and pinacolyl methylphosphonofluoridate (soman). The method was capable of determining the concentration of DIMP in hexane over the concentration range of 1 to 1 × 10-4 mg / m L by comparing the GC peak areas to analytic standards (r2=0.999). The latter concentration was found to be the limiting concentration for qualitative analysis. A concentration of analyte of 10-2 mg / m L was found to provide excellent quantitative results. The intra- and interanalysis coefficient of variation (CV) for DIMP, over the concentration range 10-1 to 10-2 mg / m L in octanol subsequently diluted 1:100 in hexane or in water subsequently extracted with an equal volume of octanol followed by a 1:100 dilution in hexane, were 7.3 and 3.5%, respectively. The intra- and interanalysis CV for paraoxon, DFP, or soman, each at a fixed concentration of 10-2mg / m L in the same two solvents and treated in the same manner as DIMP, were 4.0 and 5.6% for paraoxon, 3.3 and 3.6% for DFP, and 0.7 and 1.1% for soman, respectively. Log P values were determined and found to be 0.478 for DIMP, 1.600 for paraoxon, 1.173 for DFP, and 1.824 for soman. All results were in excellent agreement with previously determined theoretical or experimental values. Based on the log P determined for DFP and soman it was possible to estimate the fluorophosphate and fluorophosphonate fragment values to be 2.14 and 1.97, respectively. Using this method toxic organophosphorus compounds at a concentration of 10-2 mg / m L could be analyzed directly in octanol, the organic solvent used for log P determinations. This analytical method was reproducible, safe, and sensitive where determination of the presence of organophosphorus compounds is a desired endpoint for comparative in vitro analytical studies.     

Octanol modulation of neuronal nicotinic acetylcholine receptor single channels

BACKGROUND: We have previously shown that alcohols exert a dual action on neuronal nicotinic acetylcholine receptors (AChRs), with short-chain alcohols potentiating and long-chain alcohols inhibiting acetylcholine (ACh)-induced whole-cell currents. At the single-channel level, ethanol increased the channel open probability and prolonged the channel open time and burst duration. In this study, we examined the detailed mechanism of the inhibitory action of the long-chain alcohol n-octanol on the neuronal nicotinic AChR. METHODS: Single-channel currents induced by application of 30 nm ACh were recorded with the patch-clamp technique from human embryonic kidney cells stably expressing the human alpha4beta2 AChR. RESULTS: Several single-channel parameters were markedly changed by octanol. At least two conductance-state currents were induced by low concentrations of ACh, and octanol increased the proportion of the low-conductance-state current relative to the high-conductance-state current without changing the current amplitude. Major analyses of temporal properties of single-channel currents were performed on the high-conductance-state currents. Octanol decreased the burst duration and duration of openings within burst and prolonged the mean closed time. All of these changes contributed to the decrease in the open probability in a concentration-dependent manner. CONCLUSIONS: Several aspects of octanol action on neuronal AChRs at the single-channel level are compatible with an atypical open channel block model reported with muscle nicotinic AChRs. The potentiating action of short-chain alcohols and the inhibitory action of long-chain alcohols on the neuronal nicotinic AChR are mediated through different mechanisms.     

A liquid ion-exchanger alternative to KCl for filling intracellular reference microelectrodes

We have developed a filling solution for silanised microelectrodes consisting of potassium tetrakis (p-chlorophenyl) borate in octanol. Microelectrodes filled with this reference liquid ion-exchanger (RLIE) have equal selectivities to Na and K, and give the same membrane potential as do KCl-filled microelectrodes. The RLIE microelectrodes are more stable, less damaging to the cell membrane and do not leak Cl^– ions. Their high resistance, however, makes them unsuitable for recording rapid potential changes or for passing current.     

Cx36在癫持续状态大鼠海马神经元的表达

目的观察连接蛋白36（Cx36）在癫持续状态大鼠海马神经元的表达。方法建立36只氯化锂-匹罗卡品诱导的癫持续状态大鼠模型,随机分为生理盐水组、喹啉组、辛醇组（每组12只）,分别予以腹腔注射生理盐水、喹啉、辛醇,通过Racine评分判断大鼠给药前后癫发作情况,利用免疫荧光染色法、Western-blot法检测各组大鼠海马Cx36的表达。结果喹啉组和辛醇组给药前后大鼠行为学评分比较差异有统计学意义（P〈0.01）;而生理盐水组差异无统计学意义（P〉0.05）。喹啉组和辛醇组大鼠海马神经元Cx36的表达明显低于生理盐水组（P〈0.01）,但2组Cx36的表达量差异无统计学意义（P〉0.05）。结论 Cx36在癫发作中起着重要作用,可能成为潜在的治疗靶点。     

Classification Analysis of P-Glycoprotein Substrate Specificity

Prediction of P-glycoprotein substrate specificity (S_PGP) can be viewed as a constituent part of a compound's “pharmaceutical profiling” in drug design. This task is difficult to achieve due to several factors that raised many contradictory opinions: (i) the disparity between the S_PGP values obtained in different assays, (ii) the confusion between Pgp substrates and inhibitors, (iii) the confusion between lipophilicity and amphiphilicity of Pgp substrates, and (iv) the dilemma of describing class-specific relationships when Pgp has no binding sites of high ligand specificity. In this work, we compiled S_PGP data for 1000 compounds. All data were represented in a binary format, assigning S_PGP=1 for substrates and S_PGP=0 for non-substrates. Each value was ranked according to the reliability of experimental assay. Two data sets were considered. Set 1 included 220 compounds with S_PGP from polarized transport across MDR1 transfected cell monolayers. Set 2 included the entire list of 1000 compounds, with S_PGP values of generally lower reliability. Both sets were analysed using a stepwise classification structure–activity relationship (C-SAR) method, leading to derivation of simple rules for crude estimation of S_PGP values. The obtained rules are based on the following factors: (i) compound's size expressed through molar weight or volume, (ii) H-accepting given by the Abraham's β (that can be crudely approximated by the sum of O and N atoms), and (iii) ionization given by the acid and base pK_a values. Very roughly, S_PGP can be estimated by the “rule of fours”. Compounds with (N+O)≥8, MW>400 and acid pK_a > 4 are likely to be Pgp substrates, whereas compounds with (N+O)≤4, MW<400 and base pK_a<8 are likely to be non-substrates. The obtained results support the view that Pgp functioning can be compared to a complex “mini-pharmacokinetic” system with fuzzy specificity. This system can be described by a probabilistic version of Abraham's solvation equation, suggesting a certain similarity between Pgp transport and chromatographic retention. The chromatographic model does not work in the case of “marginal” compounds with properties close to the “global” physicochemical cut-offs. In the latter case various class-specific rules must be considered. These can be associated with the “amphiphilicity” and “biological similarity” of compounds. The definition of class-specific effects entails construction of the knowledge base that can be very useful in ADME profiling of new drugs.     

缝隙连接胞间通讯对大鼠局灶性脑梗死体积的影响

目的探讨缝隙连接胞间通讯变化对大鼠局灶性脑梗死体积的影响。方法Wistar大鼠随机分为4组,干预组术前腹腔注射缝隙连接特异性阻滞剂辛醇,对照组及假手术组给予等量生理盐水,溶剂对照组给予等量溶剂。线栓法建立大脑中动脉闭塞／再灌注模型。采用Longa5分制评分法评价动物神经功能缺损程度、TTC染色考察脑梗死体积变化并在光镜下观察其病理变化。结果在缺血24h和缺血6h／再灌注6h条件下,辛醇干预组脑梗死体积百分比与手术对照组比较明显减小(P<0．05)。镜下可见辛醇组缺血中心区及半影区面积均明显缩小,半影区病理改变较轻,相对面积比显著减少。两组动物神经功能缺损程度虽有差异,但不具有统计学意义(P>0．05)。结论星形胶质细胞的缝隙连接在缺血性卒中半影区的病变进展中扮演重要角色。抑制缝隙连接胞间通讯对半影区神经元起到了保护作用,减小了梗死体积。探索下调缝隙连接胞间通讯功能的方法可能成为治疗脑梗死的新思路。     

Influence of polarity on dose-response relationships of intrathecal opioids in rats

Dose-response curves were constructed for intrathecal morphine (M), oxymorphone (OM), hydromorphone (HM), diamorphine (DM), 14-hydroxydihydromorphine (OHM), oxycodone (OC), hydrocodone (HC) and fentanyl (F). Intrathecal catheters were placed in 69 rats under halothane/N₂O anaesthesia. After recovery, baseline hot plate and tail flick latencies were measured, and a dose of opioid was given. Hot plate and tail flick latencies were assessed at 5, 15, 30, 60, 90, 120 min and then hourly until they returned to within 25% of baseline. Response latencies were converted to per cent of maximum possible effect (% MPE) and the area under the % MPE × time curve was taken as the response. This measure includes information about both potency and duration of action. Each rat received 3 opioids and saline at intervals of 2–3 days. On a fifth occasion, the animal's first treatment was repeated. Each opioid was studied over an 8-fold dose range.

Results of both hot plate and tail flick were best described by a model including log(dose), a component due to development of tolerance over the 5 experimental days, and an among-rat variation term. In the hot plate test, doses equieffective in producing a response (AUC) over the dose range studied were in the order OHM < OM < HM < M < F < DM < HC < OC. Slopes of the log(dose)-response curves were similar for all drugs except OHM, which had a steeper slope.

A model is proposed in which hot plate and tail flick latencies are prolonged while CSF concentrations of a drug are above its minimum effective concentration, and drug is cleared from the CSF by a first-order process, possibly uptake into the spinal cord and removal via the blood. This model predicts that log(dose)-response curves will be linear, as was observed, with slopes inversely proportional to the rate constant for clearance from CSF. According to this model the steeper slope of the OHM log(dose)-response may be interpreted as indicating slower clearance from CSF. OHM has the lowest octanol/pH 7.4 buffer distribution coefficient (0.34) of all opioids studied, possibly leading to a lower rate of uptake into the spinal cord.     

Mercury compounds: lipophilicity and toxic effects on isolated myocardial tissue

Lipophilicity is suggested to modulate the diffusion and the cytotoxic effects of mercury compounds. To investigate this, the positive inotropic effect of four Hg compounds (HgCl₂, CH₃HgCl, chlormerodrin, bromomercurihydroxypropane) was studied in catecholamine-depleted isolated heart muscle preparations. The rate of development of the positive effect was inversely correlated to the concentration in the case of HgCl₂ and chlormerodrin, i.e. the product of concentration (c) and time to halfmaximal effect (t₅₀) remained constant. This was in accordance with the assumption of a permeation-controlled rate of action, as was shown earlier forp-chloromercuriphenylsulfonic acid. In addition, the c×t₅₀ values of the individual mercurials decreased hyperbolically with the increase in lipophilicity as measured by the octanol/water partition. The results support the view that the toxicity of mercurials increases with their lipid solubility. In conjunction with the previously reported negative inotropic effect of Hg compounds, a model is proposed allocating thiol groups responsible for the negative inotropic action to lipid compartments within the cell membrane, while SH groups conveying the increase in contraction force are thought to be situated at the internal surface of the sarcolemma.