Insulinotropic effect of new glibenclamide isosteres.
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The aim of the present study was to characterize the effects of BM 208 (N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano- N"- cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene), two newly synthesized isosteres of glibenclamide, on ionic and secretory events in rat pancreatic islet cells. Both compounds inhibited 86Rb (42K substitute) outflow from rat pancreatic islets perifused throughout at low (2.8 mM) D-glucose concentration. In excised inside-out membrane patches, BM 208 and BM 225 reduced the frequency of KATP+ channel openings. The inhibition of 86Rb outflow induced by BM 208 and BM 225 coincided with an increase in 45Ca outflow. The latter phenomenon was abolished in islets exposed to Ca2+-free media. Both isosteres of glibenclamide increased the [Ca2+]i in single pancreatic islet cells. This effect was counteracted by verapamil, a Ca2+ entry blocker. In islets exposed to 2.8 mM glucose and extracellular Ca2+, BM 208 and BM 225 stimulated insulin output. The secretory capacity of BM 225 was more marked than that of BM 208, but the time courses of the cationic and secretory responses exhibited obvious dissociations. These data suggest that the secretory capacity of BM 208 and BM 225 results, at least in part, from the inhibition of ATP-sensitive K+ channels with subsequent increase in Ca2+ inflow. The dissociation between cationic and secretory variables further suggests that the modifications in Ca2+ handling are not solely attributable to a primary inhibition of the ATP-sensitive K+ channels. (+info)
Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit.
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Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit. (+info)
Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle.
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BACKGROUND: In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. METHODS: Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. RESULTS: cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. CONCLUSION: In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage. (+info)
Genistein inhibits the regulation of active sodium-potassium transport by dopaminergic agonists in nonpigmented ciliary epithelium.
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PURPOSE: To determine whether dopamine receptor stimulation regulates Na,K-ATPase-mediated ion transport in cultured nonpigmented ciliary epithelium (NPE). METHODS: Using a rabbit NPE cell line, active Na-K transport activity was determined by measuring ouabain-sensitive potassium (86Rb) uptake in cell monolayers. Western blot analysis of membrane material obtained from cell homogenates was conducted to examine tyrosine phosphorylation of membrane proteins. RESULTS: Ouabain-sensitive potassium (86Rb) uptake was inhibited in the presence of either dopamine or the D1-selective agonist SKF82958. The response was suppressed by SCH23390, a D1 antagonist, but not by sulpiride, a D2-selective antagonist. Quinpirole, a D2-selective agonist, did not cause inhibition of ouabain-sensitive potassium (86Rb) uptake. Cyclic adenosine monophosphate (cAMP) was detectably increased in SKF82958-treated cells, although the concentration of SKF required to elevate cell cAMP was higher than the concentration needed to inhibit ouabain-sensitive potassium (86Rb) uptake. The protein kinase A inhibitor H89 prevented the 86Rb uptake response to SKF82958. Genistein, an inhibitor of tyrosine kinases, also prevented the 86Rb uptake response to SKF82958. Membrane material isolated from cells exposed to SKF82958 showed an increase in the density of several phosphotyrosine bands. These changes in phosphotyrosine immunoblot density were not observed in material isolated from cells that received either genistein or SCH23390 before SKF82958 treatment. CONCLUSIONS: The results of this study suggest D1 agonists cause a reduction of Na,K-ATPase-mediated ion transport by a mechanism that could involve a tyrosine kinase step. (+info)
Minor structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors.
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The major nitroimine insecticide imidacloprid (IMI) and the nicotinic analgesics epibatidine and ABT-594 contain the 6-chloro-3-pyridinyl moiety important for high activity and/or selectivity. ABT-594 has considerable nicotinic acetylcholine receptor (AChR) subtype specificity which might carry over to the chloropyridinyl insecticides. This study considers nine IMI analogues for selectivity in binding to immuno-isolated alpha1, alpha3 and alpha7 containing nicotinic AChRs and to purported alpha4beta2 nicotinic AChRs. Alpha1- and alpha3-containing nicotinic AChRs (both immuno-isolated by mAb 35, from Torpedo and human neuroblastoma SH-SY5Y cells, respectively) are between two and four times more sensitive to DN-IMI than to (-)-nicotine. With immuno-isolated alpha3 nicotinic AChRs, the tetrahydropyrimidine analogues of IMI with imine or nitromethylene substituents are 3-4 fold less active than (-)-nicotine. The structure-activity profile with alpha3 nicotinic AChRs from binding assays is faithfully reproduced in agonist potency as induction of 86rubidium ion efflux in intact cells. Alpha7-containing nicotinic AChRs of SH-SY5Y cells (immuno-isolated by mAb 306) and rat brain membranes show maximum sensitivity to the tetrahydropyrimidine analogue of IMI with the nitromethylene substituent. The purported alpha4beta2 nicotinic AChRs [mouse (Chao & Casida, 1997) and rat brain] are similar in sensitivity to DN-IMI, the tetrahydropyrimidine nitromethylene and nicotine. The commercial insecticides (IMI, acetamiprid and nitenpyram) have low to moderate potency at the alpha3 and purported alpha4beta2 nicotinic AChRs and are essentially inactive at alpha1 and alpha7 nicotinic AChRs. In conclusion, the toxicity of the analogues and metabolites of nicotinoid insecticides in mammals may involve action at multiple receptor subtypes with selectivity conferred by minor structural changes. (+info)
UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation.
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1. UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of approximately 200 nM and 1:1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at - 50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to approximately 160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between tau(h) and IC50 values. 3. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. 5. UK-78,282 effectively suppresses human T-lymphocyte activation. (+info)
[Ca2+]i determines the effects of protein kinases A and C on activity of rat renal Na+,K+-ATPase.
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1. It is well established that the activity of Na+,K+-ATPase (NKA) is regulated by protein kinases A (PKA) and C (PKC), but results on their effects have been conflicting. The aim of this study was to examine if this is ascribed to the intracellular concentration of Ca2+ ([Ca2+]i). 2. Rat renal NKA was stably expressed in COS cells (green monkey kidney cells). Increases in [Ca2+]i were achieved with the Ca2+ ionophore A23187 and verified by direct measurements of [Ca2+]i using fura-2 AM as an indicator. The activity of NKA was measured as ouabain-sensitive 86Rb+ uptake and the state of phosphorylation of NKA was monitored with two site-directed phosphorylation state-specific antibodies. 3. Activation of PKA with forskolin decreased NKA activity by 45.5 +/- 8.9 % at low [Ca2+]i (120 nM) and increased it by 40.5 +/- 6.4 % at high [Ca2+]i (420 nM). The change in NKA activity by forskolin correlated with the level of increase in [Ca2+]i. 4. The effect of 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a specific PKC activator, on the activity of NKA was also Ca2+ dependent, being inhibitory when [Ca2+]i was low (29.3 +/- 3.6 % decrease at 120 nM Ca2+) and stimulatory when [Ca2+]i was high (36.6 +/- 10.1 % increase at 420 nM Ca2+). 5. The alpha subunit of NKA was phosphorylated under both low and high [Ca2+]i conditions upon PKA or PKC activation. PKA phosphorylates Ser943. PKC phosphorylates Ser23. 6. To see if the observed effects on NKA activity are secondary to changes in Na+ entry, we measured NKA hydrolytic activity using permeabilized membranes isolated from cells under controlled Na+ conditions. A decreased activity at low [Ca2+]i and no change in activity at high [Ca2+]i were observed following forskolin or OAG treatment. 7. Purified NKA from rat renal cortex was phosphorylated and inhibited by PKC. This phosphorylation-associated inhibition of NKA was neither affected by Ca2+ nor by calmodulin, tested alone or together. 8. We conclude that effect of PKA/PKC on NKA activity is dependent on [Ca2+]i. This Ca2+ dependence may provide an explanation for the diversity of responses of NKA to activation of either PKA or PKC. (+info)
pH regulation of K(+) efflux from myocytes in isolated rat hearts: (87)Rb, (7)Li, and (31)P NMR studies.
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This study investigates the effects of intracellular (pH(i)) and extracellular pH (pH(e)) on the efflux of Rb(+) and Li(+) in isolated rat hearts. (87)Rb and (7)Li NMR were used to measure Rb(+) and Li(+) content, respectively, of hearts, and (31)P NMR was used to monitor pH(i), pH(e), and phosphate levels. After 30-min equilibration with Rb(+) or Li(+), effluxes were initiated by switching perfusion to a Rb(+)- or Li(+)-free, high-K(+) (20.7 mM) Krebs-Henseleit buffer with 15 microM bumetanide. Monensin (2 microM) increased pH(i) from 7.10 +/- 0.05 to 7.32 +/- 0.07 and resulted in activation of Rb(+) efflux; the first-order rate constant (k x 10(3), in min(-1)) increased from 42 +/- 2 to 116 +/- 16. Glibenclamide (4 microM) did not inhibit monensin-activated Rb(+) efflux (k = 110 +/- 17), whereas quinine (0.2 mM) slightly inhibited it by 19 +/- 9%. Infusion of 15 mM NH(4)Cl during Rb(+) washout increased k for Rb(+) efflux by 93% (81 +/- 8), which was glibenclamide and quinine insensitive, and caused a transient increase in pH(i) to 7.25 +/- 0.08. Intracellular Li(+) inhibited NH(4)Cl-stimulated Rb(+) efflux by 55%. Monensin and NH(4)Cl stimulated Li(+) efflux by 40%, increasing k from 29 +/- 3 to 43 +/- 7 and 41 +/- 3, respectively. The stimulation was not sensitive to 10 microM dimethylamiloride. Intracellular acidosis that resulted from the washout of NH(4)Cl (pH 6.86 +/- 0.2) slightly inhibited Rb(+) efflux (k = 36 +/- 5), whereas NH(4)Cl itself in the absence of pH(i) changes did not markedly affect Rb(+) efflux. A moderate increase in pH(i) (7.17 +/- 0.06) produced by washout of 15 mM 2, 2-dimethylpropionate (DMP)-Tris from hearts preequilibrated with DMP did not markedly affect Rb(+) efflux. Neither global alkalosis (pH(i) 7.4, pH(e) 7.55) nor acidosis (pH(i) approximately pH(e) 6.8) produced by 3 mM Tris base or 5 mM MES, respectively, affected Rb(+) efflux. We suggest that intracellular alkalosis stimulates Rb(+) (K(+)) and Li(+) effluxes by activating a nonselective sarcolemmal K(+) (Li(+))/cation exchanger or a K(+) (Li(+))-anion symporter. (+info)