Effect of nafamostat mesilate on Na+ and K+ transport properties in the rabbit cortical collecting duct.

1. To determine the mechanism(s) of hyperkalemia caused by nafamostat mesilate (NM), a serine-protease inhibitor, we investigated the effects of the drug on Na+ and K+ transport properties of the collecting duct (CD) cell in the isolated and perfused cortical collecting duct from rabbit kidneys. 2. NM at 10(-4) M in the lumen, hyperpolarized the apical membrane in parallel with increases in transepithelial resistance (RT) and fractional apical membrane resistance (fRA). 3. These effects were completely inhibited by pretreatment with 50 microM luminal amiloride, whereas they were not affected by luminal addition of 2 mM Ba2+. 4. NM at 10(-4) M in the bath slightly but significantly depolarized the basolateral membrane without any changes in RT or fRA, although NM at 10(-5) M in the bath had no effect on the electrical parameters. 5. It is concluded that NM mainly acts on the apical membrane of the CD cell and inhibits the amiloride-sensitive Na+ conductance in the apical membrane.     

Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells

AIM: To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. METHODS: Recombinant baculoviruses carrying the beta-galactosidase reporter gene under the control of an early to late (ETL) promoter of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) or a cytomegalovirus immediate early promoter (CMV promoter) were constructed. COS1, HeLa, CHO-K1, hFob1.19, and MCF-7 mammalian cells were tested for the expression of b-galactosidase. RESULTS: ETL promoter activity was higher in bone-derived hFob1.19 than in COS1, HeLa, CHO-K1, or MCF-7 mammalian cells. The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression. CONCLUSION: ETL promoter activity in mammalian cells is baculovirus gene expression-dependent, and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.     

The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors

Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.     

Mechanisms of the hyperkalaemia caused by nafamostat mesilate: effects of its two metabolites on Na+ and K+ transport properties in the rabbit cortical collecting duct.

1. The present experiments were undertaken to determine the mechanism(s) of hyperkalaemia caused by nafamostat mesilate (NM), a serine-protease inhibitor. 2. We investigated the effects of luminal addition of two metabolites of NM, p-guanidinobenzoic acid (PGBA) and 6-amidino-2-naphthol (AN), on Na+ and K+ transport properties of the collecting duct (CD) cell in the isolated perfused cortical collecting duct (CCD) from rabbit kidneys, because these metabolites, but not NM, were mainly excreted into the urine. 3. Addition of PGBA at 10(-5) and 10(-4) M in the lumen resulted in a hyperpolarization of VA in parallel with increases in transepithelial resistance (RT) and fractional apical membrane resistance (fRA). PGBA added to the luminal perfusate at 10(-5) and 10(-4) M changed VA, RT and fRA in a dose-dependent manner. These effects were completely inhibited by pretreatment with luminal amiloride (50 microM). PGBA at 10(-6) M in the lumen had no effect on the electrical parameters. 4. Luminal addition of AN at 10(-4) M also caused the apical membrane to hyperpolarize in parallel with increases in RT and fRA. These effects were also completely inhibited by pretreatment with luminal amiloride (50 microM). AN at 10(-5) M in the lumen had no effect on the electrical parameters. 5. We conclude that two metabolites of NM, PGBA and AN, act on the apical membrane of the CD cell and inhibit the amiloride-sensitive Na+ conductance, resulting in an inhibition of K+ secretion. This direct action of these metabolites, rather than NM, on the CCD might contribute to the NM-induced hyperkalaemia.     

Ambroxol inhibits Na+ absorption by canine airway epithelial cells in culture.

To study the effect of ambroxol on ion transport functions of airway mucosa, we measured bioelectric properties of canine cultured tracheal epithelium under short-circuit conditions in-vitro. Addition of ambroxol to the submucosal but not to the mucosal solution in an Ussing chamber decreased short-circuit current, transepithelial potential difference and cell conductance. The ambroxol-induced decrease in short-circuit current was not affected by bumetanide or diphenylamine-2-carboxylate, but it was abolished by pretreatment of cells with amiloride. These results suggest that ambroxol may selectively inhibit Na+ absorption by airway epithelium, thereby increasing water composition in airway surface fluid and reducing mucus viscosity.     

(+/-)-Pindolol acts as a partial agonist at atypical beta-adrenoceptors in the guinea pig duodenum

The agonistic and antagonistic effects of (+/-)-pindolol (1-(1H-indol-4-yloxy)-3-[(1-methylethyl)amino]-2-propanol) were estimated to clarify whether (+/-)-pindolol acts as a partial agonist on atypical beta-adrenoceptors in the guinea pig duodenum. (+/-)-Pindolol induced concentration-dependent relaxation with a pD2 value of 5.10 +/- 0.03 and an intrinsic activity of 0.83 +/- 0.03. However, the relaxations to (+/-)-pindolol were not antagonized by the non-selective beta1- and beta2-adrenoceptor antagonist (+/-)-propranolol (1 microM). In the presence of (+/-)-propranolol (1 microM), the non-selective beta1-, beta2- and beta3-adrenoceptor antagonist (+/-)-bupranolol (30 microM) induced a rightward shift of the concentration-response curves for (+/-)-pindolol (apparent pA2 = 5.41 +/- 0.06). In the presence of (+/-)-propranolol, (+/-)-pindolol (10 microM) weakly but significantly antagonized the relaxant effects to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta3-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl) amino]propyl]phenoxyacetic acid sodium salt) and a non-conventional partial beta3-adrenoceptor agonist (+/-)-CGP12177A([4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride). These results demonstrate that (+/-)-pindolol possesses both agonistic and antagonistic effects on atypical beta-adrenoceptors in the guinea pig duodenum.     

TPP+ accumulation in rat brain synaptosomes as a probe for Na+ channels

Tetraphenylphosphonium (TPP+) accumulation in rat brain synaptosomes was measured in the presence of various stimuli. TPP+ accumulation was sensitive to an increase of extracellular K+ or to the presence of Na+ channel neurotoxins. Examination of the effects of drugs with different physico-chemical properties on TPP+ accumulation showed that there was no effect under low and high K+ conditions; pimozide, flunarizine and buterizine were active in the presence of veratridine or scorpion venom. These results were compared to data obtained for the specific binding of [3H]BTX-B, a selective marker for the Na+ channel. It can be concluded that certain drugs, although active on [3H]BTX-B binding, are not necessarily identified when TPP+ is used as probe.     

A conformationally-biased, response-selective agonist of C5a acts as a molecular adjuvant by modulating antigen processing and presentation activities of human dendritic cells

A partial mechanism by which a conformationally-biased, response-selective agonist of complement component C5a, Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg or YSFKPMPLaR (EP54), acts as a molecular adjuvant is presented by showing the manner in which this peptide engages human dendritic cells (DC). Confocal microscopy was used to show that fluorescent-labeled EP54 (0.2 microM) and fluorescent-labeled B and T cell epitopes attached to EP54 (i.e., EP54-containing vaccines, 0.2 microM) were internalized by human DCs well within 30 min of exposure. After 24 h of exposure, EP54 and the B and T cell epitopes of the EP54-containing vaccines (20 microM) were presented on the DC surface in the context of HLA-ABC and HLA-DR determinants. Also, exposure of DCs to EP54 (50 microg/ml) induced the activation of genes specific for the Th1 cytokines IL-6, IL-12, INFgamma, and TNFalpha as well as the Th2 cytokine IL-4. Internalization, HLA expression, and cytokine gene activation were not observed in the presence of the inactive, scrambled EP54 constructs arguing that these effects of EP54 are mediated predominately via C5a receptors on the DC surface.     

Pipequaline acts as a partial agonist of benzodiazepine receptors: An electrophysiological study in the hippocampus of the rat

Pipequaline (PK 8165), a quinoline derivative and a ligand of the benzodiazepine binding site, is a clinically-effective anxiolytic, which is devoid of sedative and anticonvulsant properties. Several biochemical and behavioral studies have indicated that this molecule shares some properties with both agonists and antagonists of benzodiazepine receptors. The present in vivo electrophysiological studies were undertaken to determine the effects of microiontophoretic applications and of intravenous injections of pipequaline on hippocampal pyramidal neurons, activated by kainate, glutamate or acetylcholine and to characterize the effects of pipequaline on the action of benzodiazepines. Intravenously administered pipequaline exerted a partial suppression of activations by kainate, glutamate and acetylcholine. Microiontophoretic applications of pipequaline reduced the neuronal activation by kainate. This effect was blocked by RO 15-1788. In small intravenous doses, pipequaline potentiated the effect of microiontophoretically-applied flurazepam whereas, in larger doses, it suppressed the effects of microiontophoretically-applied flurazepam and of intravenously administered lorazepam on kainate-induced activation. Similarly, microiontophoretic applications of pipequaline blocked the suppressant effect of microiontophoretically-applied flurazepam on kainate-induced activation. These results constitute further evidence that the selective anxiolytic activity of pipequaline might be ascribed to its partial agonistic action on benzodiazepine receptors.     

Na+K+-ATPase activity as a biomarker of toxaphene toxicity in Unio tumidus.

In this study, the effect of toxaphene (camphechlor) on ATPase activity in the microsomal fraction of the Unio tumidus's digestive gland was determined. Toxaphene is a man-made mixture consisting of polychlorinated monoterpens, predominantly bornanes. This compound was primarily used as an insecticide, but in 1982 was officially banned because of its destructive effects on human and animal health. Toxaphene can be transported in the air at long distances and can persist in air, soil and water for years revealing acute and chronic toxicity towards aquatic organisms and wildlife, the increasing risk of cancer in both humans and animals. The microsomal fraction isolated from digestive glands was exposed to 1 x 10(-3) M, 1 x 10(-5) M and 1 x 10(-7) M of toxaphene. The obtained data showed that toxaphene induced a loss of ATPase activity in all used concentrations. The Lineweaver-Burk plots for microsomal Na+K+-ATPase in the presence or the absence of toxaphene as an inhibitor indicated a competitive type of inhibition.     

The interaction of polypeptide neurotoxins with tetrodotoxin-resistant Na+ channels in mammalian cardiac cells. Correlation with inotropic and arrhythmic effects

This paper describes the interaction of several polypeptide neurotoxins isolated from sea anemone toxins and scorpion venom with the tetrodotoxin-resistant Na+ channel of rat cardiac cells. The 22Na+ flux and tension development were measured to examine in parallel the cardiotonic and cardiotoxic effects of these polypeptides. Inotropic effects and arrhythmias were seen in the concentration range in which an action of the toxins on the Na+ channel was observed. The maximal inotropic effect was systematically observed at toxin concentrations below the concentration value observed for half-maximal stimulation of 22Na+ flux through the Na+ channel. Arrhythmias began at concentrations near the value for half-maximal stimulation of 22Na+ flux by the toxins. Toxins extracted from the sea anemones Anemonia sulcata and Anthopleura xanthogrammica were more active than scorpion toxins and sea anemone Radianthus paumotensis toxins. The most interesting among all the toxins tested for potential use in cardiotherapy was toxin II from Anthopleura xanthogrammica.     

Baculovirus as a highly efficient expression vector in insect and mammalian cells

Baculovirus has been widely used for the production of recombinant proteins in insect cells. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of baculovirus have been greatly expanded. The prospects and drawbacks of baculovirus-mediated gene expression, either in insect or in mammalian cells, are reviewed. Recent progresses in expanding the applications to studies of gene regulation, viral vector preparation, in vivo and ex vivo gene therapy studies, generation of vaccine vectors, etc are discussed and the efforts directed towards overcoming the existing bottlenecks are particularly emphasized.     

Dexamethasone increases fluid absorption via Na+/H+ exchanger (NHE) 3 activation in normal human middle ear epithelial cells

The proper homeostasis of the liquid lining the surface of the middle ear cavity is vitally important for maintaining a fluid-free middle ear cavity. Disruption of this homeostasis leads to fluid collection in the middle ear cavity and results in otitis media with effusion. We demonstrated the molecular and functional expression of the Na+/H+ exchanger (NHE)s in normal human middle ear epithelial (NHMEE) cells. We also evaluated the role of NHEs in fluid absorption and the effect of dexamethasone on NHE function and NHE-dependent fluid absorption in NHMEE cells. Western blot analysis was performed for NHE1, -2, and -3 in NHMEE cells. The fluid absorption rate was measured after liquid application on the luminal surface of the cells. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent probe bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. NHE activity was determined as Na+-induced pHi recovery from an acid load achieved by luminal exposure to 40 mmol/l NH4Cl. NHE1, -2 and -3 were all expressed in the NHMEE cells. The pHi recovery rate was suppressed by inhibition of NHE2 and -3 with HOE694 at concentrations greater than 50 microM. Inhibition of NHE3 with 650 microM of HOE694 or S3226 significantly decreased the fluid absorption rate. Dexamethasone increased the Na+-induced pHi recovery rate which was reversed by the inhibition of NHE3 with 650 microM of HOE694. Dexamethasone treatment up-regulated NHE3 expression in a dose-dependent manner. The fluid absorption rate was increased by treatment with dexamethasone (10(-7) M) and reversed by the inhibition of NHE3. In summary, we have shown that NHE3 are involved in the regulation of both pHi and fluid absorption on the luminal surface of NHMEE cells. Dexamethasone stimulates NHE3 expression and NHE3-dependent fluid absorption in NHMEE cells. These findings provide a new insight into mechanisms that regulate periciliary fluid and the therapeutic mechanisms behind steroid treatment of otitis media with effusion.     

The purported dopamine agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) acts as a nonselective α-adrenoceptor agonist in inducing hypertension,hypomotility and hypothermia in the rat

Following peripheral administration the purpoted dopamine (DA) agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) was shown to increase the diastolic blood pressure of pithed rats and to decrease rat motility and rectal temperature. Dose-effect relationships were established and half-maximal effective doses for the hypertensive and hypothermic response to DPI were calculated to be 4·4 nmol/kg i.v. and 2.0 μmol/kg i.p., respectively. Pretreatment with various antagonists revealed that both α₁- and α₂-adrenergic mechanisms were responsible for the DPI-induced hypertension, hypomotility and hypothermia, indicating that DPI acts as a non-selective α-adrenoceptor agonist. Qualitatively the DPI-induced effects were found to correlate well with those reported for its structural analogue clonidine, thus suggesting a similar mechanism of action for these agents. DA receptor antagonists appeared to lack inhibitory potency towards any of DPI-elicited responses. The results do not therefore support the designation of DPI as a DA receptor agonist.     

The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation

Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E₂ enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE₂ on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE₂ reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE₂ increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE₂ in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-pt-PGE₂ enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-pt-PGE₂ on pulmonary inflammatory diseases.     

Inhibition by SEA0400, a selective inhibitor of Na+/Ca2+ exchanger, of Na+ -dependent Ca2+ uptake and catecholamine release in bovine adrenal chromaffin cells

The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger (NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na(+) by treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent (45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM, respectively. The IC(50) values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 microM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC(50) values of SEA0400 were 5.1 and 4.5 microM and the values of KB-R7943 were 2.6 and 2.1 microM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [(3)H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC(50) = 90 microM) and KB-R7943 (IC(50) = 12 microM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.     

Demonstration of a Na+: Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs

Summary Iminodibenzyl-, iminostilbene-, dibenzocycloheptadiene-, dibenzooxepine- and dibenzothiepine-derivatives of tricyclic antidepressant drugs were able to inhibit Na⁺-stimulated Mg²⁺ efflux in human erythrocytes at concentrations of 10^–5–10^–3 mol/l. Tricyclic antidepressant drugs belonging to other chemical groups, non-tricyclic antidepressant drugs and phenothiazines were less potent inhibitors (IC₅₀ of 10^–4 mol/l or higher).Imipramine and dothiepine, the most potent compounds, inhibited the Mg" carrier with IC₅₀ of 2.5 and 4 × 10^–5 mol/1 respectively. These IC₅₀ are of similar order of magnitude to those of some classical transport inhibitors (such as furosemide for the [Na⁺K⁺,Cl^–]-cotransport system). In addition, these concentrations of imipramine and dothiepine were free of: i) side effects on other erythrocyte Na and K⁺ transport pathways (with the exception of a slight inhibition of Ca²⁺-sensitive K⁺-channels and [Na⁺,K⁺,Cl^–]- and [K⁺,Cl^–]-cotransport systems) and ii) toxic effects on the membrane leak for divalent or monovalent cations. Therefore, we selected imipramine as an useful tool for investigating fluxes catalyzed by the Na⁺-stimulated Mg²⁺ carrier.Imipramine was tested on the initial rate of ouabain and bumetanide-resistant net Na⁺ influx in Na⁺-depleted, Mg²⁺-loaded erythrocytes. The compound was able to inhibit a Na⁺ influx of about 300–500 mol (l · cells × h)^–1 with an IC₅₀ of about 3 x 10^–5 mol/1. This imipramine-sensitive Na⁺ influx was coupled with an imipramine-sensitive Mg²⁺ efflux in a stoichiometry of 3.03±0.34 (mean±SEM of 7 experiments).Abbreviations MOPS 4-morpholinopropanesulfonic acid - PCMBS p-chloromercuribenzenesulfonate - EGTA ethylene glycol bis-(beta-aminoethyl ether)N,NNN-tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Send offprint requests to R. Garay at the above address