State-dependent block of rabbit vascular smooth muscle delayed rectifier and Kv1.5 channels by inhibitors of cytochrome P450-dependent enzymes.
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The effects of the cytochrome P450 inhibitors clotrimazole, ketoconazole, and 1-aminobenzotriazole (1-ABT) on native delayed rectifier (K(DR)) and cloned Kv1.5 (RPV Kv1.5) K+ channels of rabbit portal vein (RPV) myocytes were determined using whole-cell and single channel patch-clamp analysis. Clotrimazole reduced K(DR) and RPV Kv1.5 whole-cell current with respective Kd values of 1.15 +/- 0.39 and 1.99 +/- 0.6 microM. Clotrimazole acted via an open state blocking mechanism based on the following: 1) the early time course of K(DR) current activation was not affected, but inhibition developed with time during depolarizing steps and increased the rate of decay in current amplitude; 2) the inhibition was voltage-dependent, increasing steeply over the voltage range of K(DR) activation; and 3) mean open time of RPV Kv1.5 channels in inside-out patches was decreased significantly. Ketoconazole reduced K(DR) current amplitude with a Kd value of 38 +/- 3.2 microM. However, ketoconazole acted via a closed (resting) state blocking mechanism: 1) K(DR) amplitude was reduced throughout the duration of depolarizing steps and the rate of decay of current was unaffected, 2) there was no voltage dependence to the block by ketoconazole over the K(DR) activation range, and 3) ketoconazole did not affect mean open time of RPV Kv1.5 channels in inside-out membrane patches. 1-ABT between 0.5 and 3 mM did not affect native K(DR) or RPV Kv1.5 current of rabbit portal vein myocytes. Clotrimazole and ketoconazole, but not 1-ABT, suppress vascular K(DR) channels by direct, state-dependent block mechanisms not involving the modulation of cytochrome P450 enzyme activity. (+info)
Angiotensin II type AT(2) receptor mRNA expression and renal vasodilatation are increased in renal failure.
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Kidney failure is associated with changes in renal vascular responses to angiotensin (Ang) II. We characterized expression of Ang II receptors and the renal vasoconstrictor and vasodilator responses to Ang II in kidneys from sham-operated and kidney failure rats. In the isolated perfused kidney of sham-operated rats, Ang II (1, 2, 4, and 8 ng) increased perfusion pressure by 27+/-6, 41+/-10, 54+/-11, and 74+/-12 mm Hg, respectively. These responses were amplified by 62+/-10% (P<0.05) in kidney failure rats. Losartan (1 micromol/L), an angiotensin type 1 (AT(1)) receptor blocker, abolished renal vasoconstriction induced by Ang II, unmasking a renal vasodilatation that was greater in kidney failure rats. CGP-42112 (1 micromol/L) or PD 123,319 (1 micromol/L), angiotensin type 2 (AT(2)) receptor ligands, blunted Ang II-induced renal vasodilatation. In the renal tissue of kidney failure rats, there was a marked increase in expression of AT(1) and AT(2) mRNA receptor. Ang II-induced vasodilatation was blunted by eicosatetraynoic acid (1 micromol/L), the all-purpose inhibitor of arachidonic acid metabolism; clotrimazole (1 micromol/L), an inhibitor of epoxygenase-dependent arachidonic acid metabolism; or Nomega-nitro-L-arginine methyl ester (L-NAME; 1 micromol/L), an inhibitor of NO synthesis. On stimulation with Ang II, 20-HETE was the predominant product released into the renal effluent of sham-operated rats, whereas epoxy-eicosatrienoic acids were the predominant products released into the effluent of kidney failure rats. These data suggest that during development of kidney failure, there is induction of the AT(2) receptors, which may account for increased Ang II-dependent vasodilatation through the predominant release of epoxyeicosatrienoic acids. (+info)
Regulation of K(+) current in human airway epithelial cells by exogenous and autocrine adenosine.
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The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K(+) channels, most likely Ca(2+)-dependent intermediate-conductance K(+) (I(K)) channels. A(1) but not A(2) receptor antagonists blocked the effects of adenosine. RT-PCR studies showed that A549 cells expressed mRNA for I(K)-1 channels as well as A(1), A(2A), and A(2B) but not A(3) receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of K(+) channels in A549 cells and that hENTs play a crucial role in this process. (+info)
Clotrimazole binds to heme and enhances heme-dependent hemolysis: proposed antimalarial mechanism of clotrimazole.
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Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K(a) = 6.54 x 10(8) m(-2)). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = 1/2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite. (+info)
Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells.
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Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter. This paper shows that transport of the model substrate, 3 microm dinitrophenyl S-glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2-20 microm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action. (+info)
Adenovirus vector-mediated reporter system for in vivo analyses of human CYP3A4 gene activation.
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The use of cultured mammalian cells and artificial promoters for analyses of gene regulation gives results that are sometimes inconsistent with in vivo events and thus inconclusive. To understand the in vivo mechanism of chemically mediated CYP3A4 gene activation, we have used a natural promoter of the CYP3A4 gene and an adenovirus as a reporter vector. The adenovirus reporter vector (AdCYP3A4-362) was constructed with a proximal promoter region (-362 to +11 nt) of the CYP3A4 gene and a luciferase-reporter gene. AdCYP3A4-362 was then infected into mice, and both the reporter and mouse CYP3A activities were measured. Clear increases in the reporter activity were observed in livers of all mice treated with chemicals. The profile of the CYP3A4 gene activation with chemicals was in good agreement with that of endogenous mouse CYP3A-mediated testosterone 6beta-hydroxylase. Introduction of nucleotide mutations in the receptor-binding region (ER-6) of the CYP3A4 promoter resulted in diminished reporter activity. These results indicate the advantage of the adenovirus-mediated in vivo system over the currently available in vitro systems for gene transcriptional activation. (+info)
Characterization of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3.
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Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K+ channels involved in basal, cAMP- and Ca2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (I(sc)). The non-specific K+ channel inhibitor Ba2+ inhibited the basal I(sc) by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K+ conductance is required for maintenance of basal anion secretion. Using the K+ channel blockers clofilium and clotrimazole, we found basal I(sc) to be sensitive to clofilium, with a small clotrimazole-sensitive component. By stimulating the cAMP and Ca2+ pathways, we determined that cAMP-stimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K+ channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K+ conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated I(sc). Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K+ channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K+ channel types, possibly involving one or more class of KCNQ1-containing channel complexes. (+info)
Topical antifungal drug products for over-the-counter human use; amendment of final monograph. Final rule.
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The Food and Drug Administration (FDA) is issuing a final rule amending the final monograph for over-the-counter (OTC) topical antifungal drug products to add the ingredient clotrimazole as generally recognized as safe and effective for the treatment of athlete's foot, jock itch, and ringworm. This final rule is part of FDA's ongoing review of OTC drug products. (+info)