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The increasing need in the development of storage devices is calling for the formulation of alternative electrolytes, electrochemically stable and safe over a wide range of conditions. To achieve this goal, electrolyte chemistry must be explored to propose alternative solvents and salts to the current acetonitrile (ACN) and tetraethylammonium tetrafluoroborate (Et4NBF4) benchmarks, respectively. Herein, phenylacetonitrile (Ph-ACN) has been proposed as a novel alternative solvent to ACN in supercapacitors. To establish the main advantages and drawbacks of such a substitution, Ph-ACN + Et4NBF4 blends were formulated and characterized prior to being compared with the benchmark electrolyte and another alternative electrolyte based on adiponitrile (ADN). While promising results were obtained, the low Et4NBF4 solubility in Ph-ACN seems to be the main limiting factor. To solve such an issue, an ionic liquid (IL), namely 1-ethyl-3-methylimidazolium bis [(trifluoromethyl)sulfonyl] imide (EmimTFSI), was proposed to replace Et4NBF4. Unsurprisingly, the Ph-ACN + EmimTFSI blend was found to be fully miscible over the whole range of composition giving thus the flexibility to optimize the electrolyte formulation over a large range of IL concentrations up to 4.0 M. The electrolyte containing 2.7 M of EmimTFSI in Ph-ACN was identified as the optimized blend thanks to its interesting transport properties. Furthermore, this blend possesses also the prerequisites of a safe electrolyte, with an operating liquid range from at least &minus, 60 &deg, C to +130 &deg, C, and operating window of 3.0 V and more importantly, a flash point of 125 &deg, C. Finally, excellent electrochemical performances were observed by using this electrolyte in a symmetric supercapacitor configuration, showing another advantage of mixing an ionic liquid with Ph-ACN. We also supported key structural descriptors by density functional theory (DFT) and COnductor-like Screening Model for Real Solvents (COSMO-RS) calculations, which can be associated to physical and electrochemical properties of the resultant electrolytes.

Aquaporin-4 (AQP-4) is the principal brain water channel and is predominantly expressed in astrocytes suggesting its dynamic involvement in water homeostasis in brain tissue. Due to the co-localization of AQP-4 and inward rectifier K+ channels Kir 4.1, a functional coupling between these proteins has been proposed. AQP-4 has a putative role in the physiopathology of brain disorders including epilepsy and trauma. S100B is a calcium-binding protein expressed and secreted by astrocytes, and commonly used as a parameter of astroglial activation. Here, we investigate a possible link between AQP-4 activity (and Kir 4.1) and S100B secretion in hippocampal slices of rats of different ages using non-specific inhibitors of AQP-4 (AZA, acetazolamide and TEA, tetraethylammonium) and Kir 4.1 (barium chloride). We found that blockade of AQP-4 with TEA and AZA produced an increase in S100B secretion in young rats, compatible with an astroglial activation observed in many conditions of brain injury. On the other hand, BaCl2 induced Kir 4.1 inhibition caused a decrease in S100B secretion. Both channels, AQP-4 and Kir 4.1, exhibited a similar ontogenetic profile, in spite of the functional uncoupling, in relation to S100B secretion. Moreover, we found a significant increase in the S100B secretion basal levels with the increasing of animal age and the incubation with high levels of potassium resulted in a decrease of S100B secretion in 30 and 90-day old rats. These data, together with previous observations from gap junctions and glutamate transport of astrocytes, contribute to characterize the operational system involving astroglial activation, particularly on S100B secretion, in brain disorders.

There is a multiplicity of potassium channels in nervous tissue. These have been characterized on the basis of their electrophysiological actions but more information is required on their structures and on the functions of the different subtypes of channel in different parts of the nervous system. We currently also lack drugs which are specific in opening or closing individual subtypes of channel. However, when more is known about the structure and function of these channels and when more specific modulators of their activity are available, it is likely that the use of such compounds may be of great value in the treatment of a variety of conditions affecting the nervous system, including epilepsy, the damage due to cerebral anoxia, neurodegenerative disorders and demyelinating disorders.

The whole-cell configuration of the patch-clamp technique was used to characterize the biophysical and pharmacological properties of an oscillating K(+)-current that can be induced by intracellular application of GTP[gamma S] in mouse pancreatic B cells (Ammälä et al. 1991). These K+ conductance changes are evoked by periodic increases in the cytoplasmic Ca2+ concentration ([Ca2+]i) and transiently repolarize the B cell, thus inhibiting action-potential firing and giving rise to a bursting pattern. GTP[gamma S]-evoked oscillations in K+ conductance were reversibly suppressed by a high (300 microM) concentration of carbamylcholine. By contrast, alpha 2-adrenoreceptor stimulation by 20 microM clonidine did not interfere with the oscillatory behaviour but evoked a small sustained outward current. At 0 mV membrane potential, the oscillating K(+)-current elicited by GTP[gamma S] was highly sensitive to extracellular tetraethylammonium (TEA; 70% block by 1 mM). The TEA-resistant component, which carried approximately 80% of the current at -40 mV, was affected neither by apamin (1 microM) nor by tolbutamide (500 microM). The current evoked by internal GTP[gamma S] was highly selective for K+, as demonstrated by a 51-mV change in the reversal potential for a sevenfold change in [K+]o. Stationary fluctuation analysis indicated a unitary conductance of 0.5 pS when measured with symmetric (approximately 140 mM) KCl solutions. The estimated single-channel conductance with physiological ionic gradients is 0.1 pS. The results indicate the existence of a novel Ca(2+)-gated K+ conductance in pancreatic B cells. Activation of this K+ current may contribute to the generation of the oscillatory electrical activity characterizing the B cell at intermediate glucose concentrations.

The observation of a double-quantum filtered signal of quadrupolar nuclei (e.g. 23 Na) in solution has been traditionally interpreted as a sign for anisotropic reorientational motion. Ling and Jerschow (2007) [23] have found that a 23 Na double-quantum signal is observed also in solutions of TmDOTPNa 5 . Interference effects between the quadrupolar and the paramagnetic interactions have been reported to lead to the appearance of double-quantum coherences even in the absence of a residual quadrupolar interaction. In addition, such processes lead to differential linebroadening effects between the satellite transitions, akin to effects that are well known for dipolar-CSA cross-correlation. Here, we report experiments on sodium in the presence of LnDOTP compounds, where it is shown that these cross-correlation effects correlate well with the pseudo-contact shift. In addition, anisotropic g -values of the lanthanide compounds in question, can also lead to alignment within the magnetic field, and consequently to the appearance of line splitting and double-quantum coherences. The two competing effects are demonstrated and it is concluded that both cross-correlated relaxation and alignment in the magnetic field must be at work in the systems described here.

Recent studies provided evidence that trans-resveratrol (3,4',5-trihydroxystilbene, found in high concentrations in some red wines, may possibly decrease the risk of coronary heart disease mortality. The aim of this study, performed with large epicardial porcine coronary arteries (PCA) strips, was to investigate the relaxant effect of trans-resveratrol on these main conductance vessels, which have been described to be pathologically prone for vasospastic contractions. The data show that the tonic component of the biphasic contractions induced by histamine, as well as the contractions induced by F- ions (10 mmol/l), which activate G proteins downstream of the receptors, could dose-dependently be inhibited by trans-resveratrol (0.1-100 mumol/l). The EC50 values of the dose-response curves established for the inhibition of the sustained component of histamine-induced contractions were very similar to those obtained for the relaxations of fluoride-induced contractions: 0.45 +/- 0.08 and 0.29 +/- 0.05 mumol/l, resp. (n = 6). Ouabain (10 mumol/l)-induced contractions and rhythmic contractions elicited by tetraethylammonium (12 mmol/l) were also strongly inhibited by trans-resveratrol (20 mumol/l). It may be inferred from the results obtained in this study, that the relaxation of the coronary conductance vessels induced by trans-resveratrol is possibly based on a nongenomic interaction with steroid-like receptors located on the cell membrane.

The operation of neuronal networks depends on the firing patterns of the network's neurons. When sustained current is injected, some neurons in the central nervous system fire a single action potential and others fire repetitively. For example, in Xenopus laevis tadpoles, primary-sensory Rohon-Beard (RB) neurons fired a single action potential in response to 300-ms rheobase current injections, whereas dorsolateral (DL) interneurons fired repetitively at 10–20 Hz. To investigate the basis for these differences in vivo, we examined drug-induced changes in the firing patterns of Xenopus spinal neurons using whole cell current-clamp recordings. Neuron types were initially separated through cluster analysis, and we compared results produced using different clustering algorithms. We used these results to develop a predictive function to classify subsequently recorded neurons. The potassium channel blocker tetraethylammonium (TEA) converted single-firing RB neurons to low-frequency repetitive firing but reduced the firing frequency of repetitive-firing DL interneurons. Firing frequency in DL interneurons was also reduced by the potassium channel blockers 4-aminopyridine (4-AP), catechol, and margatoxin; 4-AP had the greatest effect. The calcium channel blockers amiloride and nimodipine had few effects on firing in either neuron type but reduced action potential duration in DL interneurons. Muscarine, which blocks M-currents, did not affect RB neurons but reduced firing frequency in DL interneurons. These results suggest that potassium currents may control neuron firing patterns: a TEA-sensitive current prevents repetitive firing in RB neurons, whereas a 4-AP-sensitive current underlies repetitive firing in DL interneurons. The cluster and discriminant analysis described could help to classify neurons in other systems.

An investigation was performed to determine whether decamethonium and carbamoylcholine share a common transport mechanism with tetraethyl-ammonium and N1-methylnicotinamide in mouse kidney slices. Slices were incubated (1 hour) in Krebs-Ringer bicarbonate medium (37°, pH 7.4) containing 2 × 10-6M 14C-labelled decamethonium or carbamoylcholine both in the absence and presence of the drugs. Tetraethylammonium and N1-methylnicotinamide stimulated decamethonium uptake at relatively low concentrations (5 × 10-5M and 10-4M tetraethylammonium, 10-4M and 5 × 10-4M N1-methylnicotinamide), whereas both agents at higher concentrations (10-3M and 10-2M tetraethylammonium, 2 × 10-2M N1-methylnicotinamide) depressed the uptake. Tetraethylammonium (10-4M, 10-3M) and N1-methylnicotinamide (10-3M) also inhibited the uptake of carbamoylcholine. The initial decamethonium influx (3 minutes incubation) could be stimulated by pre-incubating slices (1 hour) with 3 × 10-4M tetraethylammonium or 5 × 10-4M N1-methylnicotinamide (in the absence of decamethonium) before being transferred to a final medium containing only decamethonium. Stimulation can thus be interpreted as an example of accelerative exchange diffusion, which should indicate that efflux of tetraethylammonium or N1-methylnicotinamide accumulated by the slices accelerates the influx of decamethonium. It is concluded that decamethonium and carbamoylcholine use a carrier mechanism which is also involved in tetraethylammonium and N1-methylnicotinamide transport. This transport mechanism is most likely in part at least, identical with that responsible for the secretion of tetraethylammonium and N1-methylnicotinamide (and other organic cations) by the intact kidney.

Transungual transport is hindered by the inherent small effective pore size of the nail even when it is fully hydrated. The objectives of this study were to determine the effects of chemical enhancers thioglycolic acid (TGA), glycolic acid (GA), and urea (UR) on transungual transport and iontophoresis efficiency. In vitro passive and iontophoretic transport experiments of model permeants mannitol (MA), UR, and tetraethylammonium (TEA) ion across the fully hydrated, enhancer-treated and untreated human nail plates were performed in phosphate-buffered saline. The transport experiments consisted of several stages, alternating between passive and anodal iontophoretic transport at 0.1 mA. Nail water uptake experiments were conducted to determine the water content of the enhancer-treated nails. The effects of the enhancers on transungual electroosmosis were also evaluated. Nails treated with GA and UR did not show any transport enhancement. Treatment with TGA at 0.5 M enhanced passive and iontophoretic transungual transport of MA, UR, and TEA. Increasing the TGA concentration to 1.8 M did not further increase TEA iontophoresis efficiency. The effect of TGA on the nail plates was irreversible. The present study shows the possibility of using a chemical enhancer to reduce transport hindrance in the nail plate and thus enhance passive and iontophoretic transungual transport.

Transungual iontophoretic transport of model neutral permeants mannitol (MA), urea (UR), and positively charged permeant tetraethylammonium ion (TEA) across fully hydrated human nail plates at pH 7.4 were investigated in vitro. Four protocols were involved in the transport experiments with each protocol divided into stages including passive and iontophoresis transport of 0.1 and 0.3 mA. Water and permeant uptake experiments of nail clippings were also conducted to characterize the hydration and binding effects of the permeants to the nails. Iontophoresis enhanced the transport of MA and UR from anode to cathode, but this effect (electroosmosis) was marginal. The transport of TEA was significantly enhanced by anodal iontophoresis and the experimental enhancement factors were consistent with the Nernst–Planck theory predictions. Hindered transport was also observed and believed to be critical in transungual delivery. The barrier of the nail plates was stable over the time course of the study, and no significant electric field-induced alteration of the barrier was observed. The present results with hydrated nail plates are consistent with electrophoresis-dominant (the direct field effect) transungual iontophoretic transport of small ionic permeants with small contribution from electroosmosis. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:893–905, 2008

Projection neurons of the substantia nigra reticulata (SNr) convey basal ganglia (BG) processing to thalamocortical and brain stem circuits responsible for movement. Two models try to explain pathological BG performance during Parkinson disease (PD): the rate model, which posits an overexcitation of SNr neurons due to hyperactivity in the indirect pathway and hypoactivity of the direct pathway, and the oscillatory model, which explains PD as the product of pathological pattern generators disclosed by dopamine reduction. These models are, apparently, incompatible. We tested the predictions of the rate model by increasing the excitatory drive and reducing the inhibition on SNr neurons in vitro. This was done pharmacologically with bath application of glutamate agonist N-methyl-d-aspartate and GABAA receptor blockers, respectively. Both maneuvers induced bursting behavior in SNr neurons. Therefore synaptic changes forecasted by the rate model induce the electrical behavior predicted by the oscillatory model. In addition, we found evidence that CaV3.2 Ca2+ channels are a critical step in generating the bursting firing pattern in SNr neurons. Other ion channels involved are: hyperpolarization-activated cation channels, high-voltage-activated Ca2+ channels, and Ca2+-activated K+ channels. However, although these channels shape the temporal structure of bursting, only CaV3.2 Ca2+ channels are indispensable for the initiation of the bursting pattern.

The sex steroid hormone estrogen down-regulates renal organic cation (OC) transport in animals, and it may contribute to sex-related differences in xenobiotic accumulation and excretion. Also, the presence of various endocrine-disrupting chemicals, i.e., environmental chemicals that possess estrogenic activity (e.g., xenoestrogens) may down-regulate various transporters involved in renal accumulation and excretion of xenobiotics. The present study characterizes the mechanism by which long-term (6-day) incubation with physiological concentrations of 17beta-estradiol (E(2)) or the xenoestrogens diethylstilbestrol (DES) and bisphenol A (BPA) regulates the basolateral membrane transport of the OC tetraethylammonium (TEA) in opossum kidney (OK) cell renal cultures. Both 17beta-E(2) and the xenoestrogen DES produced a dose- and time-dependent inhibition of basolateral TEA uptake in OK cell cultures, whereas the weakly estrogenic BPA had no effect on TEA uptake. Treatment for 6 days with either 1 nM 17beta-E(2) or DES reduced TEA uptake by approximately 30 and 40%, respectively. These effects were blocked completely by the estrogen receptor antagonist ICI 182780 (Faslodex, fulvestrant), suggesting that these estrogens regulate OC transport through the estrogen receptor, which was detected (estrogen receptor alpha) in OK cell cultures by reverse transcription-polymerase chain reaction. The J(max) value for TEA uptake in 17beta-E(2)- and DES-treated OK cell cultures was approximately 40 to 50% lower than for ethanol-treated cultures, whereas K(t) was unaffected. This reduction in transport capacity was correlated with a reduction in OC transporter OCT1 protein expression following treatment with both agents.

The substrate specificities of human (h) multidrug and toxin extrusion (MATE) 1 and hMATE2-K were examined to find functional differences between these two transporters by the transfection of the cDNA of hMATE1 and hMATE2-K into HEK293 cells. Western blotting revealed specific signals for hMATE1 and hMATE2-K consistent with a size of 50 and 40 kDa, respectively, in the transfectants as well as human renal brush-border membranes under reducing conditions. In the presence of oppositely directed H + -gradient, the transport activities of various compounds such as tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, creatinine, guanidine, procainamide, and topotecan were stimulated in hMATE1- and hMATE2-K-expressing cells. In addition to cationic compounds, anionic estrone sulfate, acyclovir, and ganciclovir were also recognized as substrates of these transporters. Kinetic analyses demonstrated the Michaelis–Menten constants for the hMATE1-mediated transport of tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, guanidine, procainamide, topotecan, estrone sulfate, acycrovir, and ganciclovir to be (in mM) 0.38, 0.10, 0.17, 0.78, 2.10, 1.23, 0.07, 0.47, 2.64, and 5.12, respectively. Those for hMATE2-K were 0.76, 0.11, 0.12, 1.98, 4.20, 1.58, 0.06, 0.85, 4.32, and 4.28, respectively. Although their affinity for hMATE1 and hMATE2-K was similar, the zwitterionic cephalexin and cephradine were revealed to be specific substrates of hMATE1, but not of hMATE2-K. Levofloxacin and ciprofloxacin were not transported, but were demonstrated to be potent inhibitors of these transporters. These results suggest that hMATE1 and hMATE2-K function together as a detoxication system, by mediating the tubular secretion of intracellular ionic compounds across the brush-border membranes of the kidney.

It is known that myogenic reactivity is a fundamental determinant of the relative constancy of blood flow through the cerebral artery. It is also known that acute alteration of pH significantly affects the cerebral circulation and, therefore, we investigated the effect of mechanism of action of intracellular acidosis on myogenic tone in rabbit basilar artery. Myogenic tone was developed by imposed stretch of basilar artery and intracellular acidosis induced by the bath application of 20 mmol/L sodium acetate. Sodium acetate caused a biphasic increase in myogenic tone. The initial component reached a peak quickly and then fell slowly to a lower steady-state significantly above basal tone. The sodium acetate-induced increase in myogenic tone was completely inhibited by elimination of external Ca2+, or treatment of nifedipine, but not with gadolinium or NPPB. TEA (5 mmol/L) and iberiotoxin (100 nmol/L) inhibited the sodium acetate-induced increase in myogenic tone. In inside-out patch-clamp recordings, decreasing pH of the mock intracellular solution from 7.4 to 6.9 markedly inhibited BKCa currents. Several inhibitors involved in Ca2+ sensitization pathways, 10(-6) mol/L Y-27632, 5 x 10(-7) mol/L calphostin C and 10(-5) mol/L PD98059 had no effect on the sodium acetate-induced increase in myogenic tone. These results suggest that intracellular acidosis increases stretch-induced myogenic tone in rabbit basilar artery. Furthermore, voltage-dependent Ca2+ influx plays a key role in intracellular acidosis-induced increase in myogenic tone and may be mediated, at least in part, by inhibition of BKCa.

Recently, we have isolated the rat (r) H+/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H+ gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [14C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [14C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H+ gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [14C]tetraethylammonium uptake by an H+ gradient [intravesicular H+ concentration ([H+]in) > extravesicular H+ concentration ([H+]out)] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [14C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H+ gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.

The previous data indicated that the testosterone (Tes)-induced relaxation of thoracic aorta is greater in spontaneously hypertensive rats (SHR) than in normotensive rats (Wistar-Kyoto rats; WKY) and that there were differences between SHR and WKY in the functions of K(ATP), K(v), and K(Ca) channels. The present study was carried out to ascertain the mechanisms of the Tes-induced relaxation. Indomethacin (30 muM) pretreatment suppressed the Tes-induced relaxation. Following noradrenalin (NA)-induced vasoconstriction, the relaxation induced by Tes was significantly attenuated by endothelium removal in SHR (not in WKY), but the dilatory effect of Tes following KCl-induced vasoconstriction was not attenuated by endothelium removal. After tetraethylammonium (K(Ca) channel inhibitor) or iberiotoxin (large conductance, Ca(2+) activated BK channel inhibitor) pretreatment, the Tes-induced relaxation was attenuated in SHR, but not in WKY. This attenuation in SHR was not observed after endothelium removal. The above results suggest that the relaxation induced by Tes following NA-induced vasoconstriction in SHR results from hyperpolarization due to BK channel opening.

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices.

We have examined the role of the human organic cation transporters [hOCTs and human novel organic cation transporter (hOCTN); SLC22A1-5] and apical multidrug and toxin extrusion (hMATE) in the cellular accumulation and cytotoxicity of platinum agents using the human embryonic kidney (HEK) 293 cells transiently transfected with the transporter cDNAs. Both the cytotoxicity and accumulation of cisplatin were enhanced by the expression of hOCT2 and weakly by hOCT1, and those of oxaliplatin were also enhanced by the expression of hOCT2 and weakly by hOCT3. The hOCT-mediated uptake of tetraethylammonium (TEA) was markedly decreased in the presence of cisplatin in a concentration-dependent manner. However, oxaliplatin showed almost no influence on the TEA uptakes in the HEK293 cells expressing hOCT1, hOCT2, and hOCT3. The hMATE1 and hMATE2-K, but not hOCTN1 and OCTN2, mediated the cellular accumulation of cisplatin and oxaliplatin without a marked release of lactate dehydrogenase. Oxaliplatin, but not cisplatin, markedly decreased the hMATE2-K-mediated TEA uptake. However, the inhibitory effect of cisplatin and oxaliplatin against the hMATE1-mediated TEA uptake was similar. The release of lactate dehydrogenase and the cellular accumulation of carboplatin and nedaplatin were not found in the HEK293 cells transiently expressing these seven organic cation transporters. These results indicate that cisplatin is a relatively good substrate of hOCT1, hOCT2, and hMATE1, and oxaliplatin is of hOCT2, hOCT3, hMATE1, and hMATE2-K. These transporters could play predominant roles in the tissue distribution and anticancer effects and/or adverse effects of platinum agent-based chemotherapy.

The epoxyeicosatrienoic acids (EETs) have been identified as endothelium-derived hyperpolarizing factors. Metabolism of the EETs to the dihydroxyeicosatrienoic acids is catalyzed by soluble epoxide hydrolase (sEH). Administration of urea-based sEH inhibitors provides protection from hypertension-induced renal injury at least in part by lowering blood pressure. Here, we investigated the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their action on the vasculature. Mesenteric resistance arteries were isolated from Sprague-Dawley rats, pressurized, and constricted with the thromboxane A2 agonist U46619 (9,11-dideoxy-11,9-epoxymethano-prostaglandin F2alpha). Mesenteric arteries were then incubated with increasing concentrations of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). AUDA resulted in a concentration-dependent relaxation of mesenteric arteries, with 10 microM resulting in a 48 +/- 7% relaxation. Chain-shortened analogs of AUDA had an attenuated vasodilatory response. Interestingly, at 10 microM, the sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexylureido)dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active, resulting in a 25 +/- 8%, 10 +/- 9%, and -8 +/- 3% relaxation, respectively. Treatment of mesenteric arteries with tetraethylammonium, iberiotoxin, ouabain, or glibenclamide did not alter AUDA-induced relaxation. The AUDA-induced relaxation was completely inhibited when constricted with KCl. In separate experiments, denuding mesenteric resistance vessels did not alter AUDA-induced relaxation. Taken together, these data demonstrate that adamantyl-urea inhibitors have unique dilator actions on vascular smooth muscle compared with other sEH inhibitors and that these dilator actions depend on the adamantyl group and carbon chain length.

This study investigated the relaxant actions of danshen (Salvia miltiorrhiza) and its lipid-soluble- and water-soluble-fractions on endothelium-denuded rat isolated femoral artery rings. Danshen, its water-soluble fraction and its lipid-soluble fraction produced relaxation of the phenylephrine-precontracted artery rings with IC50 values of 149 +/- 20 microg/mL, 160 +/- 25 microg/mL, and 23 +/- 6 microg/mL, respectively. Pretreatment of the artery rings with a non-selective potassium channel inhibitor tetraethylammonium (TEA, 10 mM) produced a significant two-fold rightward shift of the concentration-response curve to danshen and a four-fold shift to its water-soluble fraction, but had no effect on the lipid-soluble fraction. A 3.3-fold shift was produced on the concentration-response curve of danshen when the artery rings were pretreated with a mixture of 10 mM TEA, 1 mM 4-aminopyridine (K(V) blocker), 1 microM glibenclamide (K(ATP) blocker), 100 nM iberiotoxin (BK(Ca) blocker), and 100 microM barium chloride (K(IR) blocker). Involvement of Ca2+ channels was investigated in endothelium-denuded artery rings incubated with Ca2+-free buffer and primed with 1 microM phenylephrine or 60 mM KCl for 5 minutes prior to adding CaCl2 to elicit contraction. In artery rings primed with phenylephrine, pretreatment with 1 mg/mL danshen, 1 mg/mL water-soluble fraction of danshen, 0.1 mg/mL lipid-soluble fraction of danshen, and 100 nM nifedipine abrogated the CaCl2-induced contraction. On the other hand, in artery rings primed with KCl, these agents produced 40%, 25%, 53%, and 92% inhibition on the maximum contraction induced by CaCl2, respectively. Increasing the concentrations of danshen and its water-soluble fraction to 3 mg/mL, and the lipid-soluble fraction to 0.3 mg/mL further reduced the maximum contraction to 92%, 93%, and 83%, respectively. Taken together, these findings suggested the vasorelaxant actions of danshen and its fractions were produced primarily by inhibition of Ca2+ influx in the vascular smooth muscle cells and a small component was mediated by the opening of K+ channels.

The goal of this study was to determine the effects of peroxynitrite (ONOO−) on smooth muscle membrane potential and vasomotor function in rabbit carotid arteries. ONOO− is known to affect vascular tone by several mechanisms, including effects on K+ channels. Xanthine (X, 0.1 mM), xanthine oxidase (XO, 0.01 U/ml), and a low concentration of sodium nitroprusside (SNP, 10 nM) were used to generate ONOO−. In the common carotid artery, X and XO (X/XO) in the presence of SNP tended to increase tension. In contrast, in the internal carotid artery, X/XO in the presence of SNP transiently hyperpolarized the membrane (−8.5 ± 1.8 mV, mean ± SE) and decreased tension (by 85 ± 5.6%). In internal carotid arteries, in the absence of SNP, X/XO did not hyperpolarize the membrane and produced much less relaxation (by 23 ± 5.6%) than X/XO and SNP. Ebselen (50 μM) inhibited both hyperpolarization and relaxation to X/XO and SNP, and uric acid (100 μM) inhibited relaxation. Glibenclamide (1 μM) abolished hyperpolarization and inhibited relaxation during X/XO and SNP. Charybdotoxin (100 nM) or tetraethylammonium (1 mM) did not affect hyperpolarization or relaxation, respectively. These results suggest that ONOO− hyperpolarizes and relaxes smooth muscle in rabbit internal carotid artery but not in common carotid artery through activation of KATP channels.

The neuroendocrine Type 1 Dahlgren cells of the caudal neurosecretory system of the flounder display characteristic bursting activity, which may increase secretion efficiency. The firing activity pattern in these cells was voltage-dependent; when progressively depolarized, cells moved from silent (approximately -70 mV), through bursting and phasic to tonic firing (< -65 mV). Brief (10 s) evoked bursts of spikes were followed by a slow after-depolarization (ADP; amplitude up to 10 mV, duration 10-200 s), which was also voltage-dependent and could trigger a prolonged burst. The ADP was significantly reduced in the absence of external Ca(2+) ions or the presence of the L-type Ca(2+) channel blocker, nifedipine. BayK 8644 (which increases L-type channel open times) significantly increased ADP duration, whereas the Ca(2+)-activated nonselective cation channel blocker, flufenamic acid, had no effect. Pharmacological blockade of Ca(2+)-activated K(+) channels, using apamin and charybdotoxin, increased the duration of both ADP and evoked bursts. However, action potential waveform was unaffected by either apamin/charybdotoxin, nifedipine, BayK 8644 or removal of external Ca(2+). The short duration (approximately 100 ms), hyperpolarization-activated, postspike depolarizing afterpotentials (DAP), were significantly reduced by nifedipine. We propose that long duration ADPs underlie bursts and that short duration DAPs play a role in modulation of spike frequency.

K + channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na + ,K + -ATPase (Na + ,K + -pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na + ,K + -pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca 2+ chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na + ,K + -ATPase and an inducer of the hybrid death of central neurons.

The secretory transport of the H(2)-antagonists, ranitidine and famotidine, across Caco-2 cell monolayers was found to be a saturable process. Both drugs exhibited greater permeability in the basolateral (BL) to apical (AP) direction than in the AP to BL direction, indicating apically directed secretion; BL to AP transport was inhibited by P-glycoprotein (P-gp) inhibitors verapamil and cyclosporin A. The cellular uptake of ranitidine across the BL membrane was saturable and temperature dependent, indicative of carrier-mediated transport. The K(m) and V(max) for the uptake process were estimated to be 66.9 mM and 20.9 nmol/mg of protein/min, respectively. The uptake of [(14)C]ranitidine across the BL membrane was inhibited by unlabeled ranitidine and structurally diverse organic cations. The tetraethylammonium (TEA)-sensitive organic cation transporters are not involved in the uptake of ranitidine and famotidine across the BL membrane of Caco-2. This conclusion was based on the evidence that functionally active TEA-sensitive organic cation transporters did not exist in the BL membranes of the Caco-2 cells, whereas the functionally active TEA-sensitive organic cation transporter(s) in LLC-PK(1) cells did not contribute to the transport of ranitidine or famotidine across the cell monolayers. Thus, we conclude that the secretory transport of ranitidine and famotidine across Caco-2 cell monolayers is mediated by 1) a carrier in the BL membrane that is distinct from the TEA-sensitive organic cation transporter(s) and 2) P-gp in the apical membrane.

Whether cGMP and cytosolic guanylate cyclase (cGC) mediate responses of canine lower esophageal sphincter (LES) to nitric oxide (NO) released from nerves, produced in muscle, or added exogenously was evaluated in vitro. 1-H-(1,2,4)oxadiazole(4,3-alpha)quinoxalin-1-1 (ODQ), inhibitor of cGC, reduced relaxations to nerve stimulation and sodium nitroprusside but not to nitric-oxide synthase activity-dependent outward K(+)-currents in isolated muscle cells. ODQ also failed to increase tone after nerve blockade. Nonspecific K(+) channel blocker, TEA ion at 20 mM was previously shown to increase tone, occlude NO-mediated modulation of tone, and inhibit NO-dependent outward currents but not neural relaxation in LES cells. In this study, TEA abolished neural relaxation and nearly abolished relaxation to sodium nitroprusside when present with ODQ. We conclude that mechanisms coupling NO in canine LES to responses vary with the source of NO. ODQ-dependent mechanisms, presumably involving cGC, mediate actions of NO from nerves, but NO from muscle utilizes TEA-sensitive but not ODQ-dependent mechanisms to modulate tone and outward currents. Exogenous NO utilizes both TEA- and ODQ-dependent mechanisms.

Ion channels were studied using the whole-cell patch clamp technique in bovine retinal and choroidal microvascular endothelial cells (MVEC) cultured under the same conditions. The two types of MVEC expressed inward currents at hyperpolarizing voltage steps and showed small outward currents at depolarizing steps. The extrapolated reversal potentials of the inward currents were near to the potassium equilibrium potential. Cs+ and the K+ channel blocker TEA reduced the amplitudes of the currents indicating the selectivity and permeability for potassium. This was confirmed by changes of outside K+ concentration shifting the I-V curves to the right. RT-PCR studies revealed the presence of mRNA of Kir2.1, an inwardly rectifying K+ channel, in retinal and choroidal MVEC. The profile of the small outward currents is related to the Kv family but not identical with the Kv1.4 subtype.

Abnormalities in nitric oxide (NO) bioavailability have been reported in blacks. Whether there are differences in endothelium-derived hyperpolarizing factor (EDHF) in addition to NO between blacks and whites and how these affect physiological vasodilation remain unknown. We hypothesized that the bioavailability of vascular NO and EDHF, at rest and with pharmacological and physiological vasodilation, varies between whites and blacks.In 74 white and 86 black subjects without known cardiovascular disease risk factors, forearm blood flow was measured using plethysmography at rest and during inhibition of NO with N(G)-monomethyl-L-arginine and of K(+) Ca channels (EDHF) with tetraethylammonium. The reduction in resting forearm blood flow was greater with N(G)-monomethyl-L-arginine (P=0.019) and similar with tetraethylammonium in whites compared with blacks. Vasodilation with bradykinin, acetylcholine, and sodium nitroprusside was lower in blacks compared with whites (all P0.0001). Inhibition with N(G)-monomethyl-L-arginine was greater in whites compared with blacks with bradykinin, acetylcholine, and exercise. Inhibition with tetraethylammonium was lower in blacks with bradykinin, but greater during exercise and with acetylcholine.The contribution to both resting and stimulus-mediated vasodilator tone of NO is greater in whites compared with blacks. EDHF partly compensates for the reduced NO release in exercise and acetylcholine-mediated vasodilation in blacks. Preserved EDHF but reduced NO bioavailability and sensitivity characterizes the vasculature in healthy blacks.http://clinicaltrials.gov/. Unique identifier: NCT00166166.

Aims Fibroblasts, which play an important role in cardiac function/dysfunction, including arrhythmogenesis, have voltage-dependent (Kv) currents of unknown importance. Here, we assessed the differential expression of Kv currents between atrial and ventricular fibroblasts from control dogs and dogs with an atrial arrhythmogenic substrate caused by congestive heart failure (CHF). Methods and results Left atrial (LA) and ventricular (LV) fibroblasts were freshly isolated from control and CHF dogs (2-week ventricular tachypacing, 240 bpm). Kv currents were measured with whole-cell voltage-clamp, mRNA by quantitative polymerase chain reaction (qPCR) and fibroblast proliferation by 3H-thymidine incorporation. Robust voltage-dependent tetraethylammonium (TEA)-sensitive K+ currents (IC50 ∼1 mM) were recorded. The morphologies and TEA responses of LA and LV fibroblast Kv currents were similar. LV fibroblast Kv-current densities were significantly greater than LA, and Kv-current densities were significantly less in CHF than control. The mRNA expression of Kv-channel subunits Kv1.5 and Kv4.3 was less in LA vs. LV fibroblasts and was down-regulated in CHF, consistent with K+-current recordings. Ca2+-dependent K+-channel subunit (KCa1.1) mRNA and currents were less expressed in LV vs. LA fibroblasts. Inhibiting LA fibroblast K+ current with 1 mmol/L of TEA or KCa1.1 current with paxilline increased proliferation. Conclusions Fibroblast Kv-current expression is smaller in CHF vs. control, as well as LA vs. LV. KCa1.1 current is greater in LA vs. LV. Suppressing Kv current with TEA enhances fibroblast proliferation, suggesting that Kv current might act to check fibroblast proliferation and that reduced Kv current in CHF may contribute to fibrosis. Fibroblast Kv-current remodelling may play a role in the atrial fibrillation (AF) substrate; modulating fibroblast K+ channels may present a novel strategy to prevent fibrosis and AF.

Estrogen (17beta-estradiol; 17betaE) and xenoestrogens, estrogenic compounds that are not steroid hormones, have non-genomic actions at plasma membrane receptors unrelated to the nuclear estrogen receptor. The open probability (P(o)) of large conductance Ca(2+)/voltage-sensitive k(+)(BK) channels is increased by 17betaE through the regulatory beta1 subunit. The pharmacological nature of the putative membrane binding site is unclear. We probed the site by determining whether tamoxifen ((Z)-1-(p-dimethylaminoethoxy-phenyl)-1,2-diphenyl-1-butene; Tx), a chemotherapeutic xenoestrogen, increased P(o) in clinically relevant concentrations (0.1-10 microm). In whole cell patch clamp recordings on canine colonic myocytes, which express the beta1 subunit, Tx activated charybdotoxin-sensitive K(+) current. In single channel experiments, Tx increased the NP(o) (P(o) x number channels; N) and decreased the unitary conductance (gamma) of BK channels. Tx increased NP(o) (EC(50) = 0.65 microm) in excised membrane patches independent of Ca(2+) changes. The Tx mechanism of action requires the beta1 subunit, as Tx increased the NP(o) of Slo alpha expressed in human embryonic kidney cells only in the presence of the beta1 subunit. Tx decreased gamma of the alpha subunit expressed alone, without effect on NP(o). Our data indicate that Tx increases BK channel activity in therapeutic concentrations and reveal novel pharmacological properties attributable to the alpha and beta1 subunits. These data shed light on BK channel structure and function, non-genomic mechanisms of regulation, and physiologically and therapeutically relevant effects of xenoestrogens.

Electrophysiological properties of Xenopus oocytes exposed to alkaline extracellular pH (pHo) were investigated by measuring whole-cell currents using the two-electrode voltage-clamp method. Alkaline pHo (8.5 - 10.5) elicited an outward current in a pHo-dependent manner with a concomitant increase in the membrane conductance. This outward-current response was dependent on K+ because it was suppressed by a K+ channel blocker tetraethy lammonium+ (20 mM), and the reversal potential of the response was in good agreement with the Nernst equation for K+. The response was not affected by pretreatment of oocytes with the acetoxymethyl ester of bis-(o-aminophenoxy)-ethane-N,N,N’N’-tetraacetic acid (10 μM), a membrane-permeant intracellular Ca2+ chelator, but it was augmented by forskolin (0.4 μM), a stimulant of adenylate cyclase. The outward-current response originates in the oocyte but not in the surrounding follicle cells because the current could still be evoked when follicle cells were removed by collagenase or when gap junctions connecting the oocyte membrane and follicle cells were blocked by 1-octanol (1 mM). It is concluded that the outward current elicited by alkaline pHo in Xenopus oocytes is dependent on the activation of K+ channels via the cAMP pathway and that the outward current originates in the oocyte rather than the surrounding follicle cells.

The effect of pH on the secretory transport of Cd through the intestinal brush border membrane was investigated using isolated rat intestinal brush border membrane vesicles (BBMV) and the Caco-2 intestinal epithelial cell line. BBMV equilibrated at pH 5.5 or 7.5 (pH(in)) were mixed with an experimental buffer at pH 5.5 or 7.5 (pH(out)) containing CdCl(2). The initial accumulation of Cd in BBMV incubated for 1 or 3 min at pH(in) 5.5 and pH(out) 7.5 (outwardly directed H(+)-gradient) was significantly higher than that at pH(in)=pH(out)=7.5, but the equilibrated Cd accumulation incubated for 30 min was marginally lower. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore, diminished the increasing effect of the H(+)-gradient on the initial Cd accumulation. Caco-2 cell monolayers cultured on permeable membranes were incubated with CdCl(2) from the basolateral medium, and the transport of Cd from the basolateral to apical medium and the accumulation of Cd in the monolayers were measured. Cd transport was increased by lowering the pH of the apical medium, and was accompanied by a decrease in the Cd accumulation. Coincubation with CdCl(2) and tetraethylammonium, a typical substrate for H(+)-antiport of the renal organic cation transporter, from the basolateral medium slightly but significantly decreased the basolateral-to-apical transport of Cd, with a concomitant increase in the Cd accumulation. These findings suggest the secretory transport of Cd through the intestinal brush border membrane not only via passive diffusion but also via H(+)-antiport of the putative organic cation transporter.

Aminoguanidine has potential pharmacologic utility for diabetes and nitric oxide - mediated inflammation. Because aminoguanidine is positively charged at physiologic pH (pK(a) approximately 10), it is unlikely that simple diffusion is a predominant mechanism for cellular penetration. This study sought to determine the transport processes by which aminoguanidine, a cationic compound, traverses across cellular membranes. In cultured opossum kidney (OK) cell monolayers, aminoguanidine transport involved both saturable and non-saturable diffusion processes. At passage numbers below 67, the observed V(max) and K(m) for saturable influx were significantly lower than that observed at passages greater than 79 (V(max): low passage, 21.2+/-7.8 pmol/(min*mg protein), n=3; versus high passage, 129.7+/-24.3 pmol/(min*mg protein), n=3, P

P2X(2) purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG(+), Tris(+), TMA(+), and TEA(+). The selectivity sequence for currents carried by alkali metal ions is: K(+)Rb(+)Cs(+)Na(+)Li(+), which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG(+), Tris(+), TMA(+) and TEA(+) were virtually impermeant. The divalent ions (Mn(2+), Mg(2+), Ca(2+) and Ba(2+)) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn(2+)Mg(2+)Ca(2+)Ba(2+).

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity as its major toxicity in rats. Previous studies have shown that NDPS induces nephrotoxicity following oxidation of the succinimide ring to form N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and the hydrolysis product of NDHS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). Our recent work found that sodium sulfate potentiated NDPS nephrotoxicity, suggesting that sulfate conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. The purpose of this study was to determine if sodium sulfate also potentiated the nephrotoxicity of the two nephrotoxic metabolites of NDPS and further to see if sodium sulfate potentiated NDHS and 2-NDHSA nephrotoxicity to the same degree. Male Fischer 344 rats (4-16 rats/group) received an intraperitoneal (ip) injection of sodium sulfate (10 mg/kg) 20 min before a non-nephrotoxic dose (0.05 mmol/kg, ip) of NDHS or 2-NDHSA, or vehicle (12.5% dimethyl sulfoxide in sesame oil). Renal function was then monitored over 48 h. Sodium sulfate pretreatment potentiated the renal effects of a non-nephrotoxic dose of NDHS and 2-NDHSA to induce nephrotoxicity. Nephrotoxicity was characterized by diuresis, increased proteinuria, elevated blood urea nitrogen (BUN) concentration, increased kidney weight and proximal tubular necrosis. Differences in the potentiation of NDHS and 2-NDHSA nephrotoxicity by sodium sulfate were also observed as NDHS nephrotoxicity was potentiated to a lesser degree than 2-NDHSA-induced nephrotoxicity. These results support the likelihood that one or more sulfate conjugate(s) of NDPS metabolites contribute to NDPS nephrotoxicity.

K+ channels play an important role in renal electrolyte transport. They provide the negative membrane potential, are involved in cell volume regulation, recycle K+, which is transported into the different renal cells by the Na+-2Cl−-K+ transporter and Na+-K+-ATPases, and regulate K+ homeostasis via K+ secretion in the cortical collecting duct (Lang & Rehwald, 1992; Wang et al. 1992, 1997; Giebisch, 1995; Greger, 1996). Several different K+ channels have been reported for luminal and basolateral membranes of renal epithelia. In patch clamp experiments on the luminal membrane of the proximal tubule at least four different K+ channels were identified: a 33 pS inwardly rectifying K+ channel in the S3 segment of rabbit and mouse (Gogelein & Greger, 1984), a 60 pS linear K+ channel in the same segment of mouse (Gogelein, 1990), a 200 pS maxi-K channel and a 42 pS K+ channel in primary cultures of rabbit proximal tubules (Merot et al. 1989; Dubeet al. 1990). In the basolateral membrane of the S2 segment of rabbit a 50 pS inwardly rectifying K+ channel has been identified (Gogelein & Greger, 1987; Parent et al. 1988). Not much is known about the regulation of these ion channels. The K+ channel of the basolateral membrane of proximal tubules as well as the maxi-K channel are activated by depolarization, while the former is also pH and ATP dependent. It has been speculated that they might be involved in the repolarization of the membrane potential after the occurrence of Na+-dependent transport (Greger & Gogelein, 1987; Lang & Rehwald, 1992). Furthermore, it was shown that the maxi-K channel of primary cultures of proximal tubules is involved in cell volume regulation (Dubeet al. 1990). The basolateral K+ channel is Ca2+ independent, but seems to be coupled to the function of the Na+-K+-ATPase and can be regulated by cytosolic ATP (Tsuchiya et al. 1992; Hurst et al. 1993). Most K+ channels are downregulated by intracellular acidification like the 50 pS K+ channel of the basolateral membrane of rabbit and mouse (Gogelein & Greger, 1984) and the 42 pS K+ channel of the luminal membrane from primary cultured proximal tubule cells (Merot et al. 1989). The proximal tubule is the most important location in the kidney for the Na+-coupled uptake of substrates such as glucose, phosphate, sulphate and amino acids. Since the driving force for Na+-coupled substrate transport is provided by the chemical gradient for Na+ and the negative membrane potential (Lang et al. 1986), K+ channels are essential in this part of the nephron to guarantee the maintenance of this negative membrane potential. In this study we provide evidence for a novel Ca2+-dependent, intermediate conductance, depolarization-activated K+ channel that is directly regulated via cGMP in human proximal tubule cells (IHKE-1).

This study was to investigate possible mechanisms associated with vascular hyporeactivity to vasoconstrictor agents in rats with endotoxaemia. Wistar-Kyoto rats were anaesthetised and injected with endotoxin [E. coli lipopolysaccharide (LPS); 10 mg/kg, i.v.] for 4 h. Pressor responses to noradrenaline (NA; 1 microg/kg, i.v.) were determined prior to and at every hour after LPS injection. After the in vivo experiment, rat thoracic aortas were excised and prepared as rings 3-4 mm in width. The endothelium was mechanically removed to evaluate K(+)-channel activity and the effects of nitric oxide (NO) on the vascular smooth muscle. Our results demonstrated that: (1) injection of LPS caused a significant fall in blood pressure and a severe vascular hyporeactivity to NA in the anaesthetised rat, (2) the relaxation induced by the K(+)channel opener cromakalim was greater in rings obtained from endotoxaemic rats and this enhanced relaxation was partially inhibited by pretreatment of these rings with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of the NO/cGMP pathway, (3) endotoxaemia for 4 h was also associated with a profound vascular hyporeactivity to NA ex vivo and this vascular hyporesponsiveness was partially inhibited by ODQ, tetraethylammonium (TEA, a non-selective inhibitor of K(+)-channels) and charybdotoxin [CTX, a selective inhibitor of large conductance calcium-activated K(+)- channels (BK(Ca))], but not by apamin, and (4) the combination of TEA or CTX with ODQ completely restored that vascular responsiveness to normal. These results suggest that activation of BK(Ca) and overproduction of NO in the vascular smooth muscle simultaneously contribute to vascular hyporeactivity to vasoconstrictor agents in endotoxaemia.

Growing body of evidence indicates that the functional responses of cells to muscarinic acetylcholine receptors (mAChRs) are mediated by multiple receptor subtypes. It is commonly thought that the M2 receptor is the only functional mAChR subtype in the heart and little data regarding the potential roles of other subtypes in cardiac tissues has been reported. In the present study, we provide functional evidence for the presence and physiological function of an M3 receptor in canine atrial myocytes. Using whole-cell patch-clamp techniques, we consistently found that pilocarpine, an mAChR agonist, induced a K+ current similar to but distinct from the classical delayed rectifier K+ current. Same observations were obtained when choline or tetramethylammonium (TMA) was applied to the bath. The currents were abolished by 1 microM atropine. Antagonists selective to M1 (pirenzepine, 100 nM), M2 (methoctramine 100 nM), or M4 (tropicamide 200 nM) receptors failed to alter the currents. Conversely, three different M3-selective inhibitors, p-F-HHSiD (20-200 nM), 4-DAMP methiodide (2-10 nM) and 4-DAMP mustard (4-20 nM), all produced concentration-dependent suppression of the currents. A cDNA fragment representing the M3 receptor was isolated from dog atrial RNA and the mRNA level of this construct was 0.7 +/- 0.1 pg/microg total RNA, as quantified by the competitive RT-PCR methods. Our data strongly suggested that an M3 receptor exists and is coupled to a K+ channel in the heart.

1. Using the perforated patch-clamp or whole-cell clamp technique, we investigated the contribution of Ca2+-activated K+ current (IK(Ca)) and non-selective cation currents (INSC) to the membrane potential in small pulmonary arterial smooth muscle cells of the rabbit. 2. The resting membrane potential (Vm) was -39.2 +/- 0.9 mV (n = 72). It did not stay at a constant level, but hyperpolarized irregularly, showing spontaneous transient hyperpolarizations (STHPs). The mean frequency and amplitude of the STHPs was 5.6 +/- 1. 1 Hz and -7.7 +/- 0.7 mV (n = 12), respectively. In the voltage-clamp mode, spontaneous transient outward currents (STOCs) were recorded with similar frequency and irregularity. 3. Intracellular application of BAPTA or extracellular application of TEA or charybdotoxin suppressed both the STHPs and STOCs. The depletion of intracellular Ca2+ stores by caffeine or ryanodine, and the removal of extracellular Ca2+ also abolished STHPs and STOCs. 4. Replacement of extracellular Na+ with NMDG+ caused hyperpolarization Vm of without affecting STHPs. Removal of extracellular Ca2+ induced a marked depolarization of Vm along with the disappearance of STHPs. 5. The ionic nature of the background inward current was identified. The permeability ratio of K+ : Cs+ : Na+ : Li+ was 1.7 : 1.3 : 1 : 0. 9, indicating that it is a non-selective cation current (INSC). The reversal potential of this current in control conditions was calculated to be -13.9 mV. The current was blocked by millimolar concentrations of extracellular Ca2+ and Mg2+. 6. From these results, it was concluded that (i) hyperpolarizing currents are mainly contributed by Ca2+-activated K+ (KCa) channels, and thus STOCs result in transient membrane hyperpolarization, and (ii) depolarizing currents are carried through NSC channels.

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.