Proton-dependent inhibition of the cardiac sodium channel Nav1.5 by ranolazine.

Ranolazine is clinically approved for treatment of angina pectoris and is a potential candidate for antiarrhythmic, antiepileptic, and analgesic applications. These therapeutic effects of ranolazine hinge on its ability to inhibit persistent or late Na(+) currents in a variety of voltage-gated sodium channels. Extracellular acidosis, typical of ischemic events, may alter the efficiency of drug/channel interactions. In this study, we examined pH modulation of ranolazine's interaction with the cardiac sodium channel, Nav1.5. We performed whole-cell path clamp experiments at extracellular pH 7.4 and 6.0 on Nav1.5 transiently expressed in HEK293 cell line. Consistent with previous studies, we found that ranolazine induced a stable conformational state in the cardiac sodium channel with onset/recovery kinetics and voltage-dependence resembling intrinsic slow inactivation. This interaction diminished the availability of the channels in a voltage- and use-dependent manner. Low extracellular pH impaired inactivation states leading to an increase in late Na(+) currents. Ranolazine interaction with the channel was also slowed 4-5 fold. However, ranolazine restored the voltage-dependent steady-state availability profile, thereby reducing window/persistent currents at pH 6.0 in a manner comparable to pH 7.4. These results suggest that ranolazine is effective at therapeutically relevant concentrations (10 µM), in acidic extracellular pH, where it compensates for impaired native slow inactivation.

Effects of the antianginal drug, ranolazine, on the brain sodium channel Na(V)1.2 and its modulation by extracellular protons.

BACKGROUND AND PURPOSE: Ranolazine is an antianginal drug currently approved for treatment of angina pectoris in the United States. Recent studies have focused on its effects on neuronal channels and its possible therapeutic uses in the nervous system. We characterized how ranolazine affects the brain sodium channel, Na(V)1.2, and how its actions are modulated by low pH. In this way, we further explore ranolazine's potential as an anticonvulsant and its efficacy in conditions like those during an ischaemic stroke. EXPERIMENTAL APPROACH: We performed whole-cell patch-clamp experiments on the voltage-gated sodium channel, Na(V)1.2. Experiments were performed with extracellular solution titrated to either pH 7.4 or pH 6.0 before and after ranolazine perfusion. KEY RESULTS: Ranolazine accelerates onset and slows recovery of fast and slow inactivation. Ranolazine increases the maximum probability of use-dependent inactivation and reduces macroscopic and ramp sodium currents at pH 7.4. pH 6.0 reduced the slowing of fast inactivation recovery and inhibited use-dependent block by ranolazine. In the presence of ranolazine, the time constants of slow inactivation recovery and onset were significantly increased at pH 6.0 relative to pH 7.4 with 100 µM ranolazine. CONCLUSIONS AND IMPLICATIONS: Our work provides novel insights into the modulation of brain sodium channel, Na(V)1.2, by ranolazine. We demonstrate that ranolazine binds Na(V)1.2 in a state-dependent manner, and that the effects of ranolazine are slowed but not abolished by protons. Our results suggest that further research performed on channels with epilepsy-causing mutations may prove ranolazine to be an efficacious therapy.

Effects of late sodium current enhancement during LQT-related arrhythmias. A simulation study.

Long QT syndrome is a repolarization disorder characterized by marked prolongation of QT interval. A clear consequence of long QT syndrome is the occurrence of a polymorphic ventricular tachycardia called Torsade de Pointes, which has been related to early after depolarizations (EADs) formation. This repolarizing disorder has been observed under pathological situations, such as heart failure, oxidative stress, ventricular hypertrophy and/or in the presence of pure class III antiarrhythmics. Under such pathologies electrophysiological changes affect the electrical activity of the cell. Lately, the enhancement of late sodium current (I(NaL)) and its role has become a source of interest. In this work, a mathematical model of I(NaL) has been proposed and incorporated to the ten Tussher model of the human ventricular action potential (AP), specifically in M cells. We simulated and analyzed the effects of I(NaL) enhancement in combination with LQT-related pathologies and administration of I(Kr) blockers, on the AP. This study demonstrates that I(NaL) prolongs AP duration (APD) in a rate-dependent manner. Indeed, a 10-fold increase of I(NaL) prolongs APD in 80% for a stimulation rate of 1 Hz and 100% for 0.25 Hz. Also, intracellular sodium concentration [Na(+)](i) significantly increases in the presence of enhanced I(NaL), increasing the probability of EADs formation through calcium overload in cells prone to develop EADs.

Robust anti-arrhythmic efficacy of verapamil and flunarizine against dofetilide-induced TdP arrhythmias is based upon a shared and a different mode of action.

BACKGROUND AND PURPOSE: The high predisposition to Torsade de Pointes (TdP) in dogs with chronic AV-block (CAVB) is well documented. The anti-arrhythmic efficacy and mode of action of Ca(2+) channel antagonists, flunarizine and verapamil against TdP were investigated. EXPERIMENTAL APPROACH: Mongrel dogs with CAVB were selected based on the inducibility of TdP with dofetilide. The effects of flunarizine and verapamil were assessed after TdP and in different experiments to prevent dofetilide-induced TdP. Electrocardiogram and ventricular monophasic action potentials were recorded. Electrophysiological parameters and short-term variability of repolarization (STV) were determined. In vitro, flunarizine and verapamil were added to determine their effect on (i) dofetilide-induced early after depolarizations (EADs) in canine ventricular myocytes (VM); (ii) diastolic Ca(2+) sparks in RyR2(R4496+/+) mouse myocytes; and (iii) peak and late I(Na) in SCN5A-HEK 293 cells. KEY RESULTS: Dofetilide increased STV prior to TdP and in VM prior to EADs. Both flunarizine and verapamil completely suppressed TdP and reversed STV to baseline values. Complete prevention of TdP was achieved with both drugs, accompanied by the prevention of an increase in STV. Suppression of EADs was confirmed after flunarizine. Only flunarizine blocked late I(Na). Ca(2+) sparks were reduced with verapamil. CONCLUSIONS AND IMPLICATIONS: Robust anti-arrhythmic efficacy was seen with both Ca(2+) channel antagonists. Their divergent electrophysiological actions may be related to different additional effects of the two drugs.

Oligomerization of thioglutamic acid: encapsulated reactions and lipid catalysis.

For cellular life to begin on the early Earth, a permeation mechanism would be required to allow polar solutes to enter a membrane-bounded compartment. A second process--internal polymerization of peptides from amino acid--would also be an essential step toward the first compartmented metabolic pathways leading to biosynthesis. Here we report a study of amino acid permeation into lipid vesicles, in which thioglutamic acid penetrated lipid bilayer membranes at rates sufficient to support internal polymerization reactions. We also investigated spontaneous non-enzymatic polymerization reactions of thioglutamic acid to form oligopeptides. We found that oligomers up to 11mers are produced in the reaction mixture, and conclude that certain lipid surfaces can act as catalysts in promoting an oligomerization reaction. These observations are pertinent to understanding processes by which peptide bond synthesis could take place in primitive cellular life on the early Earth.

Effect of food seasoning spices mixture on biomarkers of oxidative stress in tissues of fructose-fed insulin-resistant rats.

High fructose feeding in normal rats induces insulin resistance and also facilitates oxidative damage. The present study examines the effects of a spices mixture (SM) on oxidative stress markers and antioxidant potential in tissues of high fructose-fed insulin-resistant rats. Male Wistar rats received a semisynthetic diet containing either 60% fructose or 60% starch. SM administration at three different doses (10, 30, and 50 mg/day per rat) was initiated orally 15 days later and continued for the next 30 days. After the total experimental period of 45 days, peroxidation of lipids and antioxidant status in liver and kidney were quantified. Fructose-treated rats showed increased levels of peroxidation indices such as thiobarbituric acid-reactive substances and lipid hydroperoxides in tissues. The condition was associated with an inadequate antioxidant system. Administration of SM along with fructose diet reduced the levels of peroxidation markers in tissues and improved the antioxidant status. The positive effect of SM on the oxidant-antioxidant balance could be attributed to the active constituents of the different spices present in the mixture.

Drug-induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine.

BACKGROUND AND PURPOSE: Fluoxetine (Prozac) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. EXPERIMENTAL APPROACH: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells. KEY RESULTS: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Food seasoning spices mixture improves glucose metabolism and lipid profile in fructose-fed hyperinsulinemic rats.

Fructose feeding has been shown to induce insulin resistance in rats, associated with hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. We have investigated the effect of administering food seasoning spices mixture (SM) on glucose, insulin, and lipids in circulation and carbohydrate enzymes in the erythrocytes of high fructose-fed rats. Additionally, we also measured the protein glycation status by assaying the levels of glycated hemoglobin, fructosamine, and plasma protein glycation. Male Wistar rats received a daily diet containing either 60% fructose or 60% starch (control). The rats were administered SM at three different doses (10, 30, or 50 mg/day per rat) orally 15 days later. At the end of the 45-day experimental period, fructose-fed rats showed significantly higher levels of plasma glucose and insulin, dyslipidemia, and alterations in enzyme activities. Treatment with SM significantly reduced plasma glucose and insulin levels and brought about a favorable lipid profile. In these rats, the activities of enzymes of glucose metabolism were normal. These effects were observed at all three doses of SM. High homeostasis model assessment (HOMA) values indicated insulin resistance in fructose-fed rats, while the HOMA values in SM-treated fructose-fed rats were comparable to those of control rats. We conclude that administration of SM improves glucose metabolism and plasma lipid profile in fructose-fed rats, possibly through improved insulin-sensitizing actions of the active constituents.

Issues of environmental compliance in developing countries.

Environmental laws define the scarcity of environmental resources as they affect the factor endowment of a country and therefore its position in the international division of labour. There is now also a general agreement that applying the "polluter pays" principle should solve environmental problems. As the burden of abatement increases, as measured by the ratio of abatement expenditure to sales, there is definitely an incentive for firms to either invest in cleaner technology or more efficient abatement technology. There is also evidence that taxes and charges, designed to internalise externalities, can actually affect trade. It is interesting to know if the developing countries face particular market access problems in the face of stringent environmental standards and regulations. While it is true that stringent measures impose market access restrictions and cause limitations on competitiveness, this is much more widely felt by the developing countries because of lack of infrastructure and monitoring facilities, limited technology choices, inadequate access to environment-friendly raw materials, lack of complete information, presence of small-scale exporters and emergence of environmental standards in sectors of export interest to developing countries. The small and medium enterprises often divert sales either to the domestic market or to external markets where environmental requirements are less stringent, in order to save on their costs. In developing countries, 80% of the tanning industry is comprised of small and medium enterprises (SMEs) processing raw to semi-finished leather, usually less than 2 tons per day. In Europe and other developed countries the SMEs in the leather sector have vanished due to strict environmental legislation and this will likely occur in developing countries also. The environmental legislation has not always been practical, either because the laws are too ambitious or unrealistic in certain parameters, or because they have lacked effective instrumentation and institutional support. Some environmental regulations have not succeeded as they do not match the technical requirements and economic reality of the country or region, or because they do not take the institutional capabilities of the society that has to implement them into consideration. For the survival and sustenance of the SMEs in the leather industry, it may be a viable alternative to carry out the tanning process in a decentralized fashion such that the raw to semi-finished process is carried out in the large scale sector while the semi-finished to finished process could either be reserved or open to competition as per the countries' requirements. But the issue of concern is whether it is fair that the raw to semi-finished tanning process, containing 70% of the pollution discharge should be undertaken by developing countries alone, especially if it is at the cost of their survival! However, the game analysed in the paper reveals that tanning units in developing countries would prefer to comply with the regulations and stay in the industry, the alternatives being to collude or to compete!

ACCURACY OF SERUM - ASCITES ALBUMIN GRADIENT IN THE AETIOLOGICAL DIAGNOSIS OF ASCITES.

50 adults with ascites admitted to our hospital were studied. Simultaneous samples of ascitic fluid and blood were collected and subjected to analysis including ascitic fluid total protein and serum ascites albumin gradient The cut off value of serum-ascites albumin gradient for differentiating between high and low gradient was taken as 1.1 gm % and of ascitic fluid protein for differentiating exudate and transudate as 2.5 gm%. The sensitivity, specificity, positive predictive value and negative predictive value of high gradient and transudative ascites in diagnosing portal hypertension were 943%, 60%, 84.6%, 81.8% and 62.9%, 133%, 91.7% and 50% respectively. High gradient ascites is a sensitive test in the diagnosis of portal hypertension as a cause of ascites. The exudate-transudate approach has severe limitations in the differential diagnosis of ascites.

Cocaine blocks HERG, but not KvLQT1+minK, potassium channels.

Cocaine causes cardiac arrhythmias, sudden death, and occasionally long QT syndrome in humans. We investigated the effect of cocaine on the human K(+) channels HERG and KvLQT1+minK that encode native rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier K(+) channels in the heart. HERG and KvLQT1+minK channels were heterologously expressed in human embryonic kidney 293 cells, and whole-cell currents were recorded. Cocaine had no effect on KvLQT1+minK current in concentrations up to 200 microM. In contrast, cocaine reversibly blocked HERG current with half-maximal block of peak tail current of 7.2 microM. By using a protocol to quickly activate HERG channels, we found that cocaine block developed rapidly after channel activation. At 0 mV, the time constants for the development of block were 38.2 +/- 2.1, 15.2 +/- 0.8, and 6.9 +/- 1.1 ms in 10, 50 and 200 microM cocaine, respectively. Cocaine-blocked channels also recovered rapidly from block after repolarization. At -100 mV, recovery from block followed a biphasic time course with fast and slow time constants of 3.5 +/- 0.7 and 100.3 +/- 15.4 ms, respectively. Using N-methyl-cocaine, a permanently charged, membrane-impermeable cocaine analog, block of HERG channels rapidly developed when the drug was applied intracellularly through the patch pipette, suggesting that the cocaine binding site on the HERG protein is located on a cytoplasmic accessible domain. These results indicate that cocaine suppresses HERG, but not KvLQT1+minK, channels by preferentially blocking activated channels, that it unblocks upon repolarization, and does so with unique ultrarapid kinetics. Because the cocaine concentration range we studied is achieved in humans, HERG block may provide an additional mechanism for cocaine-induced arrhythmias and sudden death.

Increasing rates of hospitalization due to septicemia in the US elderly population, 1986-1997.

Rates of hospitalization due to septicemia (International Classification of Diseases, Ninth Revision, Clinical Modification, code 038) in the US elderly population for 1986-1997 were examined, using Medicare administrative data. Age group-, sex-, and race-adjusted rates more than doubled from 1986 through 1997, from 3.42 to 7.42 per 1000 beneficiaries. The 1997 rates of septicemia increased with age, from 4.47 per 1000 beneficiaries among persons 65-74 years old to 18.1 per 1000 beneficiaries among persons > or =85 years old. The rates of septicemia were slightly greater among men (7.46 per 1000 beneficiaries) than among women (7.39 per 1000 beneficiaries) and were higher among blacks (13.61 per 1000 beneficiaries) than among whites (6.89 per 1000 beneficiaries). The most likely sites of the origin of the septicemia were the urinary tract (40.1%) and lungs (15.3%). Escherichia coli and Staphylococcus species were the most frequently reported organisms. Diabetes was listed as a comorbidity in 24.5% of the hospitalizations. We estimate that the cost to Medicare for septicemia hospitalizations in 1997 was >$1.8 billion.

Transbronchial needle aspiration of central and peripheral nodules.

Wang and Terry first described the technique of sampling of mediastinal lymph nodes using a flexible bronchoscope (FB) in 1983. Since then, the scope of transbronchial needle aspiration (TBNA) has increased enormously, and its value for diagnosis of central and peripheral lung lesions, even in the absence of endobronchial disease, is now recognized. Improvements in the diagnostic yield of TBNA aspirates, and increasing knowledge of predictors of a positive aspirate, has reduced the need for mediastinoscopy, and occasionally thoracotomy, with benefits in terms of reduced healthcare costs and improved patient welfare. Despite the fact that TBNA is a minimally invasive procedure, also for the staging of lung cancer, it unfortunately remains underutilized. This review aims to give the reader an overview of the indications, outcome data, technical considerations, and advantages of routinely performing TBNA. The technical suggestions to improve the yield from TBNA will hopefully provide greater understanding of this modality and help the pulmonologist to incorporate it in daily practice with more confidence.

Cardiotoxic effects of fenfluramine hydrochloride on isolated cardiac preparations and ventricular myocytes of guinea-pigs.

The cardiotoxic effects of fenfluramine hydrochloride on mechanical and electrical activity were studied in papillary muscles, Purkinje fibres, left atria and ventricular myocytes of guinea-pigs. Force of contraction (f(c)) was measured isometrically, action potentials and maximum rate of rise of the action potential (V(max)) were recorded by means of the intracellular microelectrode technique and the sodium current (I(Na)) with patch-clamp technique in the cell-attached mode. For kinetic analysis (S)-DPI-201-106-modified Na(+) channels from isolated guinea-pig ventricular heart cells were used. Fenfluramine (1 - 300 microM) produced negative chronotropic and inotropic effects; additional extracellular Ca(2+) competitively antagonized the negative inotropic effect. Fenfluramine concentration-dependently reduced V(max) and showed tonic blockade of sodium channels, shortened the action potential duration in papillary muscles and Purkinje fibres. In cell-attached patches, fenfluramine decreased I(Na) concentration-dependently (10 - 100 microM), frequency-independently (0.1 - 3 Hz; 30 microM). The h(infinity) curve was shifted towards hyperpolarizing direction. At 30 microM, fenfluramine blocked the sodium channel at all test potentials to the same degree, and neither changed the threshold and reversal potentials nor the peak of the curve. No effect on single channel availability, but a significant decrease in mean open times and increase in mean closed times was observed. Mean duration of the bursts decreased and number of openings per record increased with increasing drug concentration. It is concluded that the effect on I(Na) plays an important role in the cardiotoxicity of fenfluramine in addition to primary pulmonary hypertension and valvular disorders.

A case of ochronosis: MRI of the lumbar spine.

We present the MRI features of the lumbar spine in a patient with ochronosis.

Differentiation of Asian rice gall midge, Orseolia oryzae (Wood-Mason), biotypes by sequence characterized amplified regions (SCARs).

We developed a polymerase chain reaction (PCR)-based assay that distinguished five different biotypes of the Asian gall midge (Orseolia oryzae), a major insect pest of rice. A total of 400 random primers were screened using random amplified polymorphic DNAs (RAPDs). Five diagnostic PCR products were isolated, cloned, sequenced and converted to sequence characterized amplified regions (SCARs). Primers specific to these SCARs were able to amplify specific DNA fragments from genomic DNAs of five biotypes of gall midge in a multiplexed-PCR-based assay. The amplified DNA fragments were used as diagnostic markers to identify different biotypes of gall midge. The SCAR primers were also capable of differentiating the Asian from the African rice gall midge (Orseolia oryzivora) as well as detecting a variant of biotype 5 which caused an outbreak in Kerala, India. Unlike the use of plant host differentials and midge feeding behaviour for identifying biotypes, this assay is fast, reliable and unaffected by environmental factors.