Further evidence that prostaglandins inhibit the release of noradrenaline from adrenergic nerve terminals by restriction of availability of calcium. (1/952)

1 Guinea-pig vasa deferentia were continuously superfused after labelling the transmitter stores with [3H](-)-noradrenaline. Release of [3H]-(-)-noradrenaline was induced by transmural nerve stimulation. 2 Prostglandin E2 (14 nM) drastically reduced the release of [3H]-(-)-noradrenaline, while tetraethylammonium (2 mM), rubidium (6 mM), phenoxybenzamine (3 muM) each in the presence or absence of Uptake 1 or 2 blockade, and prolonged pulse duration (from 0.5 to 2.0 ms) all significantly increased the release of [3H]-(-)-noradrenaline per nerve impulse. 3 The inhibitory effect of prostaglandin E2 on evoked release of [3H]-(-)-noradrenaline was significantly reduced by tetraethylammonium, rubidium and prolonged pulse duration, whilst it was actually enhanced by phenoxybenzamine. This indicates that increased release of noradrenaline per nerve impulse does not per se counteract the inhibitory effect of prostaglandin E2. 4 It is concluded that tetraethylammonium, rubidium and prolonged pulse duration counteracted the inhibitory effect of prostaglandin E2 on T3H]-(-)-noradrenaline release by promoting calcium influx during the nerve action potential. The results are consistent with, and add more weight to the view that prostaglandins inhibit the release of noradrenaline by restriction of calcium availability.  (+info)

The interaction of n-tetraalkylammonium compounds with a human organic cation transporter, hOCT1. (2/952)

Polyspecific organic cation transporters in epithelia play an important role in the elimination of many endogenous bioactive amines and therapeutically important drugs. Recently, the first human organic cation transporter (hOCT1) was cloned from liver. The purpose of the current study was to determine the effect of molecular size and hydrophobicity on the transport of organic cations by hOCT1. We studied the interaction of a series of n-tetraalkylammonium (n-TAA) compounds (alkyl chain length, N, ranging from 1 to 6 carbons) with hOCT1 in a transiently transfected human cell line, HeLa. [14C]tetraethylammonium (TEA) uptake was measured under different experimental conditions. Both cis-inhibition and trans-stimulation studies were carried out. With the exception of tetramethylammonium, all of the n-TAAs significantly inhibited [14C]TEA uptake. A reversed correlation of IC50 values (range, 3.0-260 microM) with alkyl chain lengths or partition coefficients (LogP) was observed. trans-Stimulation studies revealed that TEA, tetrapropylammonium, tetrabutylammonium, as well as tributylmethylammonium trans-stimulated TEA uptake mediated by hOCT1. In contrast, tetramethylammonium and tetrapentylammonium did not trans-stimulate [14C]TEA uptake, and tetrahexylammonium demonstrated an apparent "trans-inhibition" effect. These data indicate that with increasing alkyl chain lengths (N >/= 2), n-TAA compounds are more poorly translocated by hOCT1 although their potency of inhibition increases. Similar findings were obtained with nonaliphatic hydrocarbons. These data suggest that a balance between hydrophobic and hydrophilic properties is necessary for binding and subsequent translocation by hOCT1.  (+info)

Arterial flow conditions downregulate thrombomodulin on saphenous vein endothelium. (3/952)

BACKGROUND: The antithrombogenic properties of venous endothelium may be attenuated when vein is implanted in the arterial circulation. Such changes may facilitate thrombosis, which is the final common pathway for saphenous vein arterial bypass graft occlusion. METHODS AND RESULTS: Using human saphenous vein in a validated ex vivo flow circuit, we investigated (1) the possibility that arterial flow conditions (mean pressure, 100 mm Hg, 90 cpm, approximately 200 mL/min) alter the concentration of proteins involved in regulating thrombosis at the vessel wall and (2) the influence of ion channel blockade on such effects. Concentrations of thrombomodulin and tissue factor were quantified by Western blotting (ratio of von Willebrand factor staining) and immunohistochemistry (as a percentage of CD31-staining area). Thrombomodulin concentrations after 90 minutes of venous and arterial flow conditions were quantified by immunostaining (68.9+/-4.8% and 41.0+/-3.0% CD31, respectively; P<0.01) and by Western blotting (1.35+/-0.20 and 0. 15+/-0.03 ratio of von Willebrand factor, respectively; P<0.01). The ability of endothelial cells to generate activated protein C also decreased from 62+/-14 to 19+/-10 ng. min-1. 1000 cells-1 (P=0.01). The significant reduction in thrombomodulin was attenuated if calcium was removed from the perfusate but not by external vein stenting. Inclusion in the vein perfusate of drugs that reduce calcium entry (including Gd3+, to block stretch-activated ion channels, and nifedipine) abolished the reduction in thrombomodulin concentration observed after arterial flow conditions. In freshly excised vein, negligible concentrations of tissue factor were detected on the endothelium and concentrations did not increase after 90 minutes of arterial flow conditions, although the inclusion of nifedipine caused the immunostaining to increase from 3.0+/-0.4% to 8.5+/-0.7% CD31 (P<0.02). CONCLUSIONS: In saphenous vein endothelium exposed to arterial flow conditions, there is rapid downregulation of thrombomodulin, sufficient to limit protein C activation, by a calcium-dependent mechanism.  (+info)

Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander retinal neurons. (4/952)

1. Whole-cell voltage clamp experiments were performed on isolated spiking retinal neurons from the salamander retina. Calcium channel currents were studied using barium as the charge carrier while potassium and sodium currents were suppressed with TEA and TTX, respectively. 2. Baclofen, a metabotropic GABA receptor agonist, both enhanced and suppressed high-voltage-activated calcium channel current. Baclofen facilitated an L-type channel current, and this effect was not voltage dependent. As reported previously, baclofen inhibited an N-type channel current and this action was voltage dependent. 3. While the suppressive effect was mediated by a fast-acting, direct G-protein action, the facilitatory effect was slower and was blocked by inhibitors of protein kinase C (PKC), either GF-109203x or the PKC (19-36) sequence fragment. 4. The pharmacology of the inhibitory and facilitatory responses differed. Commonly used antagonists of metabotropic GABA receptors, CGP35348 and CGP55845, were more potent antagonists of the inhibitory response. Similarly, a selective agonist at the metabotropic GABA receptor, APMPA, was also more effective in eliciting the inhibitory response. 5. These observations indicate that there may be two baclofen-sensitive metabotropic GABA receptors with opposing effects on calcium channel current. This is the first description of a facilitatory action of GABAB receptors and indicates that GABA may not function exclusively as an inhibitory transmitter.  (+info)

Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations. (5/952)

In the present study, functional characteristics of organic cation transporter (OCTN)1, which was cloned as the pH-dependent tetraethylammonium (TEA) transporter when expressed in mammalian human embryonic kidney (HEK)293 cells, were further investigated using Xenopus oocytes as well as HEK293 cells as gene expression systems. When OCTN1-derived complementary RNA was injected into Xenopus oocytes, pH-dependent transport of [14C]TEA was observed as the same in HEK293 cells. In contrast, a replacement of sodium ions with potassium ions in the surrounding medium did not cause any change in [14C]TEA uptake in Xenopus oocytes expressed with OCTN1. In addition, when OCTN1 was expressed in HEK293 cells, efflux of TEA from the cells was pH dependent, with an accelerated rate at acidic external medium pH. Accordingly, membrane potential or sodium ions are suggested to have no influence on [14C]TEA transport and the transport activity of OCTN1 is directly affected by pH itself. Furthermore, addition of the unlabeled TEA in external medium enhanced the efflux of preloaded [14C]TEA. These observations suggest that OCTN1 is a pH-dependent and bidirectional TEA transporter. OCTN1-mediated [14C]TEA uptake was inhibited by various organic cations such as cimetidine, procainamide, pyrilamine, quinidine, quinine, and verapamil. In addition, uptakes of cationic compounds such as [3H]pyrilamine, [3H]quinidine, and [3H]verapamil and zwitterionic L-[3H]carnitine were increased by expression of OCTN1 in Xenopus oocytes. Accordingly, OCTN1 was functionally demonstrated to be a multispecific and pH-dependent organic cation transporter, which presumably functions as a proton/organic cation antiporter at the renal apical membrane and other tissues.  (+info)

A transfected cell model for the renal toxin transporter, rOCT2. (6/952)

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.  (+info)

The effect of deep pore mutations on the action of phenylalkylamines on the Kv1.3 potassium channel. (7/952)

We investigated the action of the phenylalkylamines verapamil and N-methyl-verapamil on the Kv1.3 potassium channel using the whole-cell configuration of the patch-clamp technique. Our goal was to identify their binding as a prerequisite for using the phenylalkylamines as small, well-defined molecular probes, not only to expand the structural findings made with peptide toxins or by crystallization, but also to use them as lead compounds for the generation of more potent and therefore more specific K+ channel modulators. Competition experiments with charybdotoxin, known to interact with external residues of Kv1.3, showed no interaction with verapamil. The internal application of quarternary N-methyl-verapamil in combination with verapamil suggested competition for the same internal binding site. Verapamil affinity was decreased 6 fold by a mutation (M395V) in a region of the internal pore which forms part of the internal tetraethylammonium (TEA+) binding site, although mutations at neighbouring residues (T396 and T397) were without effect. Modification of C-type inactivation by mutations in the internal pore suggest that this region participates in the inactivation process. The action of phenylalkylamines and local anaesthetics on L-type Ca2+ channels and Na channels, respectively, and verapamil on Kv1.3 indicate very similar blocking mechanisms. This might allow the use of these compounds as molecular probes to map the internal vestibule of all three channel types.  (+info)

Activity-dependent modulation of K+ currents at presynaptic terminals of mammalian central synapses. (8/952)

1. The activity-dependent regulation of presynaptic K+ currents at the CA3-CA1 synapse in the rat hippocampus was investigated during a train of evoked afferent action potentials. The waveforms of presynaptic compound action potentials (cAPs) and presynaptic Ca2+ transients ([Ca2+]pre,t) were measured with fluorescent voltage-sensitive and Ca2+-sensitive indicators in rat brain slices. 2. Under control conditions, presynaptic cAPs and the accompanying [Ca2+]pre,t displayed similar amplitudes for each stimulus, suggesting that there was no cumulative change of K+ and Ca2+ currents during the test train. However, when a subgroup of presynaptic K+ channels was blocked by a low concentration of 4-aminopyridine (4-AP, 40 microM), a significant facilitation of the [Ca2+]pre,t was observed. 3. This phenomenon was not due to a direct action of 4-AP on presynaptic Ca2+ channels, but to cumulative suppression of the K+ conductance as indicated by the corresponding change in waveforms of the cAP and presynaptic fibre volley. The observed facilitation was not an artifact by virtue of increased fibre recruitment, nor was it related to the accumulation of extracellular K+; rather, it was dependent on Ca2+ influx and stimulation frequency. The time course of recovery from facilitation was closely related to the decay of the intracellular Ca2+ concentration. 4. The facilitation was not blocked by a saturating concentration of 4-AP (8 mM) but was reduced during the application of the K+ channel blocker tetraethylammonium (TEA, 10 mM), implicating the involvement of TEA-sensitive K+ channels. Such activity-dependent suppression of presynaptic K+ conductance could lead to excessive transmitter release and might explain the hippocampal epileptiform activity that can be induced by application of 4-AP.  (+info)