Functional state of the plasma membrane Ca2+ pump in Plasmodium falciparum-infected human red blood cells.
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The active Ca2+ transport properties of malaria-infected, intact red blood cells are unknown. We report here the first direct measurements of Ca2+ pump activity in human red cells infected with Plasmodium falciparum, at the mature, late trophozoite stage. Ca2+ pump activity was measured by the Co2+-exposure method adapted for use in low-K+ media, optimal for parasitised cells. This required a preliminary study in normal, uninfected red cells of the effects of cell volume, membrane potential and external Na+/K+ concentrations on Ca2+ pump performance. Pump-mediated Ca2+ extrusion in normal red cells was only slightly lower in low-K+ media relative to high-K+ media despite the large differences in membrane potential predicted by the Lew-Bookchin red cell model. The effect was prevented by clotrimazole, an inhibitor of the Ca2+-sensitive K+ (KCa) channel, suggesting that it was due to minor cell dehydration. The Ca2+-saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax) of parasitised red cells was marginally inhibited (2-27 %) relative to that of both uninfected red cells from the malaria-infected culture (cohorts), and uninfected red cells from the same donor kept under identical conditions (co-culture). Thus, Ca2+ pump function is largely conserved in parasitised cells up to the mature, late trophozoite stage. A high proportion of the ionophore-induced Ca2+ load in parasitised red cells is taken up by cytoplasmic Ca2+ buffers within the parasite. Following pump-mediated Ca2+ removal from the host, there remained a large residual Ca2+ pool within the parasite which slowly leaked to the host cell, from which it was pumped out. (+info)
Treatment of recurrent vulvovaginal candidiasis.
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Vulvovaginal candidiasis is considered recurrent when at least four specific episodes occur in one year or at least three episodes unrelated to antibiotic therapy occur within one year. Although greater than 50 percent of women more than 25 years of age develop vulvovaginal candidiasis at some time, fewer than 5 percent of these women experience recurrences. Clinical evaluation of recurrent episodes is essential. Patients who self-diagnose may miss other causes or concurrent infections. Known etiologies of recurrent vulvovaginal candidiasis include treatment-resistant Candida species other than Candida albicans, frequent antibiotic therapy, contraceptive use, compromise of the immune system, sexual activity and hyperglycemia. If microscopic examination of vaginal secretions in a potassium hydroxide preparation is negative but clinical suspicion is high, fungal cultures should be obtained. After the acute episode has been treated, subsequent prophylaxis (maintenance therapy) is important. Because many patients experience recurrences once prophylaxis is discontinued, long-term therapy may be warranted. Patients are more likely to comply when antifungal therapy is administered orally, but oral treatment carries a greater potential for systemic toxicity and drug interactions. (+info)
Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.
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The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K(+) channel, IKCa1, is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The "IKCa1-pharmacophore" consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar pi-electron-rich heterocycle (pyrazole or tetrazole) or a -CN group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively block IKCa1. TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) inhibits the cloned and the native IKCa1 channel in human T lymphocytes with a K(d) of 20-25 nM and is 200- to 1,500-fold selective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [(3)H]thymidine incorporation of preactivated lymphocytes with EC(50)-values of 100 nM-1 microM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants. (+info)
Effect of cytochrome P-450 inhibitors econazole, bifonazole and clotrimazole on prostanoid formation.
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1. The present study was carried out to clarify the effect of the imidazole antimycotics econazole, bifonazole and clotrimazole on prostanoid biosynthesis. Osteoblast-like MC3T3-E1 cells stimulated by endothelin-1, melittin, ionomycin or arachidonic acid showed diminished prostaglandin E(2) (PGE(2)) production upon pretreatment with econazole. Following pretreatment with bifonazole, stimulation with ionomycin or arachidonic acid also resulted in decreased PGE(2) formation. Clotrimazole inhibited ionomycin but not arachidonic acid stimulated PGE(2) synthesis in MC3T3-E1 cells. 2. The results observed in osteoblast-like UMR-106 cells pretreated with econazole, bifonazole or clotrimazole and stimulated by arachidonic acid were similar with the exception of clotrimazole which was a more effective inhibitor of PGE(2) biosynthesis than in MC3T3-E1 cells. 3. Upon treatment with arachidonic acid thromboxane B(2) (TXB(2)) production in human platelets was abolished completely at concentrations of the three imidazole antimycotics higher than 5 microM (IC(50)<1 microM). 4. These data were confirmed by a direct assay using purified ram seminal vesicle prostaglandin H(2) synthase-1 (PGHS-1), which clearly showed inhibitory properties of econazole (IC(50) 4.7+/-2.3 microM), bifonazole (IC(50) 9.4+/-0.8 microM) and clotrimazole (IC(50) 4.4+/-0.6 microM). 5. Summarizing, these results indicate an inhibitory effect of econazole, bifonazole and clotrimazole on PGHS-1, varying in its potency dependent on the cell system used. In addition TXB(2) formation is affected at doses even lower than those needed to suppress PGE(2) biosynthesis. (+info)
NH(4)Cl inhibition of acid secretion: possible involvement of an apical K(+) channel in bullfrog oxyntic cells.
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This study was undertaken to determine the mechanism by which ammonium chloride (NH(4)Cl) inhibits stimulated acid secretion in the bullfrog gastric mucosa. To this end, four possible pathways of inhibition were studied: 1) blockade of basolateral K(+) channel, 2) blockade of ion transport activity, 3) neutralization of secreted H(+) in the luminal solution, or 4) ATP depletion. Addition of nutrient 10 mM NH(4)Cl (calculated NH(3) concentration = 92.5 microM and NH(4)(+) concentration = 9.91 mM) inhibited acid secretion within 30 min. Inhibition of acid secretion did not occur by blockade of basolateral K(+) channel activity or ion transport activity or by neutralization of the luminal solution. Although ATP depletion occurred in the presence of NH(4)Cl, the magnitude of ATP depletion in 30 min was not sufficient to inhibit stimulated acid secretion. By comparing the effect of NH(4)Cl on the resistance of inhibited or stimulated tissues, we demonstrate that NH(4)Cl acts specifically on stimulated tissues. We propose that NH(4)Cl blocks activity of an apical K(+) channel present in stimulated oxyntic cells. Our data suggest that the activity of this channel is important for the regulation of acid secretion in bullfrog oxyntic cells. (+info)
Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.
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ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy. (+info)
Expression and biological significance of Ca2+-activated ion channels in human keratinocytes.
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In whole-cell recordings from HaCaT keratinocytes, ATP, bradykinin, and histamine caused a biphasic change of the membrane potential consisting of an initial transient depolarization, followed by a pronounced and long-lasting hyperpolarization. Flash photolysis of caged IP3 mimicked the agonist-induced voltage response, suggesting that intracellular Ca2+ release and subsequent opening of Ca2+-activated ion channels serve as the common transduction mechanism. In contrast, cAMP- and PKC-dependent pathways were not involved in the electrophysiological effects of the extracellular signaling molecules. The depolarization was predominantly mediated by a DIDS- and niflumic acid-sensitive Cl- current, whereas a charybdotoxin- and clotrimazole-sensitive K+ current underlay the prominent hyperpolarization. Consistent with the electrophysiological data, RT-PCR showed that HaCaT keratinocytes express two types of Ca2+-activated Cl- channels, CaCC2 and CaCC3 (CLCA2), as well as the Ca2+-activated K+ channel hSK4. That the pronounced hSK4-mediated hyperpolarization bears significance on the growth and differentiation properties of keratinocytes is suggested by RNase protection assays showing that hSK4 mRNA expression is strongly down-regulated under conditions that allow keratinocyte differentiation. hSK4 might thus play a role in linking changes in membrane potential to the biological fate of keratinocytes. (+info)
Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones.
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1. The pharmacology of the slow afterhyperpolarization (sAHP) was studied in cultured rat hippocampal pyramidal neurones. 2. Clotrimazole, its in vivo metabolite, 2-chlorophenyl-bisphenyl-methanol (CBM) and the novel analogues, UCL 1880 and UCL 2027, inhibited the sI(AHP) with similar IC50s (1-2 microM). 3. Clotrimazole and CBM also inhibited the high voltage-activated (HVA) Ca2+ current in pyramidal neurones with IC50s of 4.7 microM and 2.2 microM respectively. UCL 1880 was a less effective Ca2+ channel blocker, reducing the HVA Ca2+ current by 50% at 10 microM. At concentrations up to 10 microM, UCL 2027 had no effect on the Ca2+ current, indicating that its effects on the sI(AHP) were independent of Ca2+ channel block. 4. Clotrimazole also inhibited both the outward holding current (IC50=2.8 microM) present at a potential of -50 mV and the apamin-sensitive medium AHP (mAHP; IC50 approximately amp;10 microM). The other clotrimazole analogues tested had smaller effects on these two currents. The present work also shows that 100 nM UCL 1848, an inhibitor of apamin-sensitive conductances, abolishes the mAHP. 5. Currents were recorded from HEK293 cells transfected with hSK1 and rSK2. The SK currents were very sensitive to inhibition by UCL 1848 but were not significantly reduced by the sI(AHP) inhibitor, UCL 2027 (10 microM). 10 microM UCL 1880 reduced the hSK1 current by 40%. 6. UCL 2027 appears to be the first relatively selective blocker of the sAHP to be described. Furthermore, the ability of UCL 2027 to block the sAHP with minimal effect on SK1 channel activity questions the role of this channel in the sAHP. (+info)