K+ channels and the microglial respiratory burst.
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Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K+ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K+ currents had properties of Kv1.3 and the Ca2+/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2. (+info)
Substrate binding is the rate-limiting step in thromboxane synthase catalysis.
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Thromboxane synthase (TXAS) is a "non-classical" cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105-130 s(-1); 2) binding of cyanide, a carbon-based ligand, is a one-step process with k(on) of 2.4 M(-1) s(-1) and k(off) of 0.112 s(-1); and 3) both imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 x 10(4) M(-1) s(-1) and 1.5 x 10(5) M(-1) s(-1) for imidazole and clotrimazole, respectively, followed by a slow conformational change with off-rate constants of 8.8 s(-1) and 0.53 s(-1), respectively. The results of our binding study indicate that the TXAS active site is hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min(-1) and that the k(cat)/K(m) for PGH2 consumption was 3 x 10(6) M(-1) s(-1). Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, indicating minimal accumulation of any heme-derived intermediates. Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state. Further kinetic analysis using a combination of rapid-mixing chemical quench and computer simulation showed that the kinetic parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2-2.0 x 10(7) M(-1) s(-1); the rate of catalytic conversion of PGH2 to TXA2 or MDA was at least 15,000 s(-1) and the lower limit of the rates for products release was 4,000-6,000 s(-1). Given that the cellular PGH2 concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with "classical" P450 enzymes. (+info)
Clotrimazole is a selective and potent inhibitor of rat cytochrome P450 3A subfamily-related testosterone metabolism.
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In this study, clotrimazole (CTZ) and ketoconazole (KTZ) were evaluated for their inhibition of testosterone metabolism catalyzed by rat hepatic microsomes differentially expressing certain cytochrome P450 enzymes. The objective was to compare the inhibitory potencies using hepatic microsomes from adult female rats treated with dexamethasone (F-DEX) and hepatic microsomes from vehicle-treated adult male rats (M-VEH), which are known to contain high levels of isozymes CYP3A1 (3A23) and 3A2, respectively. The results demonstrate that CTZ is a very potent and selective inhibitor of the 6beta-hydroxylation of testosterone, a CYP3A-mediated reaction, in all rat metabolic systems tested. The IC(50) value was 9.7 nM in F-DEX, and 6.7 nM in M-VEH for CTZ. The in vitro inhibitory potency for CTZ significantly exceeds the same parameters for KTZ, a well established specific inhibitor of human CYP3A-mediated reactions. It was found that the IC(50) values of KTZ in F-DEX and M-VEH were 69 and 780 nM, respectively. These values for KTZ are 10-fold and 100-fold higher, respectively, than for CTZ. CTZ, at the concentration that inhibits 90% and more of CYP3A-mediated reactions (40 nM), has less than a 10% inhibitory effect on the activities of other rat liver enzymes, such as CYP1A1, -1A2, -2A1, -2B1, -2B2, -2C11, and -2E1. In summary, CTZ is a more potent and selective inhibitor of all CYP3A-mediated reactions than KTZ in rat hepatic microsomes. (+info)
A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion.
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Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting beta-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the beta-cell response to arginine and glucose after nNOS blockade with N(G)-nitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that beta-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal beta-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in beta-cell dysregulation involved in certain pathological hyperinsulinic states. (+info)
An investigation into the pathogenesis of vulvo-vaginal candidosis.
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OBJECTIVE: To monitor yeasts isolated from women during and between episodes of recurrent vulvo-vaginal candidosis (VVC) to determine whether vaginal relapse or re-infection occurred. METHODS: Women presenting at the genitourinary medicine clinic with signs and symptoms of VVC were recruited to the study (n = 121). A vaginal washing, high vaginal swab (HVS) and rectal swab were taken and the women treated with a single 500 mg clotrimazole pessary. Women were asked to re-attend after 1, 4, and 12 weeks, or when the VVC recurred, when vaginal washings and HVS were repeated. Candida isolates recovered were strain typed using the Ca3 probe and their similarity assessed. Antifungal susceptibility to fluconazole and clotrimazole were determined. RESULTS: Of the women recruited, 47 completed the study, either returning for four visits or suffering a recurrence during the study period. Of the 22 women who experienced recurrence, the same strain was responsible for the initial and recurrent episode in 17 women. For the remaining five women, four had strain replacement and one had a change of species. None of the isolates recovered from the women demonstrated resistance to either clotrimazole or fluconazole. CONCLUSIONS: Our findings support the theory of vaginal relapse and thus may support the use of more prolonged courses of antifungal therapy initially to increase the chances of eradication of the yeast. (+info)
Effect of clotrimazole on Naegleria fowleri.
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The sensitivity of 18 strains of Naegleria fowleri to clotrimazole (Bay b5097) was tested. They showed minimal inhibitory concentrations in the range 0-03-0-125 mug/ml, and minimal amoebicidal concentrations in the range 0-125-0-25 mug/ml. Mice inoculated with N. fowleri were not protected from infection by doses of 100 mg clotrimazole/kg per day given for five days after inoculation. Mice had serum levels of up to 6 mug/ml in the first 32 hours after inoculation. Therefore clotrimazole appears to be ineffective in protecting against infection with N. fowleri. (+info)
Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1.
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Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr(250) or Val(275) in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(250) and Val(275) line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobic interactions with the pi-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside. (+info)
Suppression of transient outward potassium currents in mouse ventricular myocytes by imidazole antimycotics and by glybenclamide.
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The whole-cell patch-clamp technique was used in adult mouse ventricular myocytes at 22 degrees C to study the transient outward current (I(to)) and its sensitivity to the antimycotics miconazole and clotrimazole, as well as to glybenclamide. I(to) elicited by depolarizing steps from a holding potential of -80 mV consisted of a fast inactivating component and a slowly inactivating component. In the presence of miconazole (IC50 of approximately 8 microM) or clotrimazole, I(to) peak amplitude was reduced and its inactivation accelerated, due to a selective suppression of the slow component, without an effect on the fast component or on the noninactivating current. The effect did not reverse upon washout, was not induced by intracellular drug application, and occurred without a change of the steady-state inactivation. In the presence of glybenclamide I(to) peak amplitude was reduced and its inactivation accelerated. In contrast to the antimycotics, glybenclamide suppressed both the fast and the slow components (IC50 of approximately 50 microM), its effect was reversible, and was associated with a negative shift of the steady-state inactivation. These data demonstrate a pharmacological separation of I(to) components using antimycotic drugs but not glybenclamide. (+info)