Dose range evaluation of liposomal nystatin and comparisons with amphotericin B and amphotericin B lipid complex in temporarily neutropenic mice infected with an isolate of Aspergillus fumigatus with reduced susceptibility to amphotericin B.
(9/410)
Using an isolate of Aspergillus fumigatus that is less susceptible in vivo to amphotericin B than most other isolates, we compared different doses of liposomal nystatin (L-nystatin), liposomal amphotericin B (L-amphotericin), and amphotericin B lipid complex (ABLC) with amphotericin B deoxycholate. Four experiments with intravenously infected neutropenic mice were conducted. A dose of L-nystatin at 10 mg/kg of body weight was toxic (the mice had fits or respiratory arrest). The optimal dosage of L-nystatin was 5 mg/kg daily on days 1, 2, 4, and 7 (90% survival). This was superior to L-amphotericin (5 mg/kg [P = 0.24] and 1 mg/kg [P < 0.0001]), ABLC (5 mg/kg [P = 0.014] and 1 mg/kg [P < 0.0001]), and amphotericin B deoxycholate (5 mg/kg [P = 0.008]). In terms of liver and kidney cultures, L-nystatin (5 mg/kg) was superior to all other regimens (P = 0.0032 and <0.0001, respectively). Higher doses of L-amphotericin (25 and 50 mg/kg) in one earlier experiment were more effective (100% survival) than 1 mg of L-amphotericin per kg and amphotericin deoxycholate (5 mg/kg) in terms of mortality and both liver and kidney culture results and to L-amphotericin (5 mg/kg) in terms of liver and kidney culture results only. ABLC (25 mg/kg) given daily for 7 days was superior to ABLC (50 mg/kg [P = 0.03]) but not to ABLC at 5 mg/kg or amphotericin B deoxycholate in terms of mortality, although it was in terms of liver and kidney culture results. No dose-response for amphotericin B (5 and 1 mg/kg) was demonstrable. In conclusion, in this stringent model, high doses of L-amphotericin and ABLC could overcome reduced susceptibility to amphotericin B deoxycholate, but all were inferior to 5- to 10-fold lower doses of L-nystatin. (+info)
Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor.
(10/410)
We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles. (+info)
Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes.
(11/410)
Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 +/- 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 +/- 10% per 100 meq/l). Increases in membrane PP2A activity (270 +/- 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 +/- 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes. (+info)
Stimulation of Cl(-) secretion by chlorzoxazone.
(12/410)
We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K(+) channels (K(Ca)), thereby stimulating Cl(-) secretion across several epithelia. In our pursuit to identify potent modulators of Cl(-) secretion that may be useful to overcome the Cl(-) secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl(-) secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl(-) secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K(Ca). In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I(K), which was inhibited by CTX and also stimulated an apical I(Cl) that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I(Cl) may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated (86)Rb(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K(+) channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K(Ca), whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the DeltaF508 mutation of CFTR, there was no detectable Cl(-) secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl(-) secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia. (+info)
A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta.
(13/410)
OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle. (+info)
Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells.
(14/410)
We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection. (+info)
The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits.
(15/410)
Nyotran is a liposomally encapsulated i.v. formulation of the antifungal polyene nystatin. This drug was evaluated in a series of reproductive toxicity studies, according to the guidelines outlined by the International Conference on Harmonization (ICH). A fertility and early embryonic development study (SEG I) and a prenatal and postnatal development (SEG III) study were conducted in rats, and embryo-fetal development (SEG II) studies were conducted in rats and rabbits. Nyotran was administered iv in all studies. In SEG I and SEG III, rats were administered daily doses of 0.5, 1.5, or 3.0 mg/kg Nyotran. In both studies, parental mortality and toxicity in the 3.0 mg/kg dose group necessitated the lowering of the high dose to 2.0 mg/kg/day. Parental toxicity, in the form of decreased body weights, decreased food consumption, and piloerection were also observed at the 1.5 mg/kg/day dose level in the SEG I and SEG III studies. Despite the parentally toxic doses in the SEG I study, there was no effect of Nyotran on F0 male or female fertility or early embryonic development of F1 offspring. In the SEG III study, lactational body weights of the F1 generation were decreased at all Nyotran dose levels. There was no effect on pre-wean developmental landmarks, but post-wean development was affected by Nyotran administration at all dosage levels. Preputional separation was delayed in the 1.5 and 3.0/2.0 mg/kg/day F1 offspring, auditory startle function was decreased in F1 females at all dose levels, and motor activity was decreased in male F1 offspring at all dose levels. However, there were no treatment-related effects on the subsequent mating of the F1 generation and resulting F2 offspring. In SEG II studies, rats and rabbits were also administered 0.5, 1.5, or 3.0 mg/kg/day of Nyotran during gestation. The high dose in these SEG II studies was not lowered, as the maternal animals were able to tolerate the shorter duration of dosing. Maternal effects in rabbits were observed only in the high-dose group and were limited to decreased food consumption and decreased absolute and relative liver weight. Decreased food consumption in high-dose dams and clinical weight loss in some animals at the mid- and high-dose levels evidenced maternal toxicity in rats. Nyotran did not have any effect on Caesarian section parameters in either rats or rabbits and no effect on the incidence of fetal malformations in rabbits. A statistically significant increase in mild hydrocephaly, observed in 4 rat fetuses, was seen at the highest dose level of 3.0 mg/kg/day. The biological significance and relationship to Nyotran treatment of this finding is not clear. This finding may represent a change in the background incidence or a change in the pattern of responsiveness of this strain of rat fetus to the test chemical. Toxicokinetic data were also collected in the SEG II rabbit and rat studies for comparison to human exposures. In both species, systemic exposure to the nystatin at effective antifungal concentrations was demonstrated. The systemic exposures in rats and rabbits were, however, considerably less than have been reported in humans administered clinical doses of 2 or 4 mg/kg/day Nyotran. Thus, humans tolerate higher dosages and systemic exposures of Nyotran relative to rats and rabbits and there is no margin of safety in either dosage level or systemic exposure to drug. Given this lack of a margin of safety and the effects on postnatal development in F1 rats, caution should be exercised when using this drug in females of childbearing potential. (+info)
Compartmental pharmacokinetics and tissue distribution of multilamellar liposomal nystatin in rabbits.
(16/410)
The plasma pharmacokinetics of multilamellar liposomal nystatin were studied in normal, catheterized rabbits after single and multiple daily intravenous administration of dosages of 2, 4, and 6 mg/kg of body weight, and drug levels in tissues were assessed after multiple dosing. Concentrations of liposomal nystatin were measured as those of nystatin by a validated high-performance liquid chromatography method, and plasma concentration data were fitted into a two-compartment open model. Across the investigated dosage range, liposomal nystatin demonstrated nonlinear kinetics with more than proportional increases in the AUC(0-24) and decreasing clearance, consistent with dose-dependent tissue distribution and/or a dose-dependent elimination process. After single-dose administration, the mean C(max) increased from 13.07 microg/ml at 2 mg/kg to 41.91 microg/ml at 6 mg/kg (P < 0.001); the AUC(0-24) changed from 11.65 to 67.44 microg. h/ml (P < 0.001), the V(d) changed from 0.205 to 0. 184 liters/kg (not significant), the CL(t) from 0.173 to 0.101 liters/kg. h (P < 0.05), and terminal half-life from 0.96 to 1.51 h (P < 0.05). There were no significant changes in pharmacokinetic parameters after multiple dosing over 14 days. Assessment of tissue concentrations of nystatin near peak plasma levels after multiple dosing over 15 days revealed preferential distribution to the lungs, liver, and spleen at that time point. Substantial levels were also found in the urine, raising the possibility that renal excretion may play a significant role in drug elimination. Liposomal nystatin administered to rabbits was well tolerated and displayed nonlinear pharmacokinetics, potentially therapeutic peak plasma concentrations, and substantial penetration into tissues. Pharmacokinetic parameters were very similar to those observed in patients, thus validating results derived from infection models in the rabbit and allowing inferences to be made about the treatment of invasive fungal infections in humans. (+info)