Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity. (1/14)

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.  (+info)

Mechanism underlying flow stimulation of sodium absorption in the mammalian collecting duct. (2/14)

Vectorial Na(+) absorption across the aldosterone-sensitive distal nephron plays a key role in the regulation of extracellular fluid volume and blood pressure. Within this nephron segment, Na(+) diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial Na(+) channel (ENaC), which is considered to be the rate-limiting step for Na(+) absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net Na(+) absorption and that increases in laminar shear stress activate ENaC expressed in oocytes by increasing channel open probability. We therefore examined whether flow stimulates net Na(+) absorption (J(Na)) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated J(Na) in CCDs were mediated by ENaC, as J(Na) was inhibited by benzamil. Flow-dependent increases in J(Na) were observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal Ca(2+) concentration ([Ca(2+)]) or chelating intracellular [Ca(2+)] with BAPTA did not prevent the flow-dependent increase in J(Na). Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced J(Na) when tubules were perfused at a slow flow rate. However, trypsin did not further enhance J(Na) in CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive J(Na) in oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in J(Na) accompanying increases in luminal flow rates reflects an increase in channel open probability.  (+info)

Cytoplasmic microtubules are essential for the formation of membrane-bound polyribosomes. (3/14)

Colchicine, at low intracellular concentrations, causes a rapid depolymerization of membrane-associated polyribosomes. Poly(A+) mRNA is rapidly lost from these polysomes, and 80 S monomers are left attached to the membranes of the endoplasmic reticulum. Binding studies and measurements of intracellular colchicine concentrations indicate that the drug is acting via depolymerization of cytoplasmic microtubules. The vinca alkaloids, vincristine and vinblastine, have the same effect on polyribosomes, whereas lumicolchicine is ineffective. Furthermore, cordycepin and actinomycin D are without effect on polyribosomes indicating that colchicine is not simply inhibiting the production or transport of new mRNA. It appears that disruption of the cytoplasmic microtubule network prevents membrane-associated ribosomes from reinitiating protein synthesis resulting in the rapid loss of mRNA.  (+info)

Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells. (4/14)

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.  (+info)

Pharmacological characterization of the slow component of deactivation of guinea-pig isolated ileum to the spasmogenic action of C5adesArg. (5/14)

The slow component of deactivation of guinea-pig isolated ileum to C5adesArg was studied to analyse the mechanism of loss and subsequent recovery of sensitivity. Neither cycloheximide (10(-3) M) nor colchicine (5 X 10(-5) M), vinblastine, lumicolchicine, or cytochalasin B (each 2 X 10(-5) M) affected significantly the spasmogenic effect of C5adesArg or the course of deactivation produced by repeated applications; chloroquine (2 X 10(-4) M) inhibited the spasmogenic effect unspecifically without interfering with deactivation. Recovery from slow deactivation was totally blocked by chloroquine and considerably diminished by colchicine and vinblastine, but was not affected by the other agents. It is proposed that recovery involves lysosomal processing of C5a receptors (occupied by the peptide) but does not require biosynthesis of new receptors.  (+info)

Effect of colchicine on the uptake of prolactin and insulin into Golgi fractions of rat liver. (6/14)

In previous studies we have shown that 125I-labeled prolactin is taken up by a receptor-dependent process and concentrated in an intact form in Golgi elements from female rat liver (J. Biol. Chem., 1979, 254:209-214). In this study we have examined the effect of colchicine on this uptake process into Golgi elements. Colchicine [25 mumol (10 mg)/100 gm body wt] was injected intraperitoneally in adult female rats, and hepatic Golgi fractions were prepared at 1, 2, and 3 h postinjection. The enzyme recoveries and morphological appearance of fractions from colchicine-treated and control (alcohol alone) animals were similar. At times greater than 1 h after colchicine there was a marked (greater than 60%) inhibition of uptake of 125I-ovine prolactin (125I-oPRL) into Golgi light and intermediate fractions but no inhibition of uptake into Golgi heavy and plasmalemma elements. At times from 2 to 45 min postinjection, 125I-oPRL was extracted from Golgi elements and found to be largely intact as judged by rebinding to receptors. The inhibitory effect of colchicine was seen at doses ranging from 0.25 mumol to 25 mumol/100 g body wt. Vincristine also inhibited 125I-oPRL uptake into the Golgi light and intermediate fractions but lumicolchicine had no inhibitory effect. There was a smaller effect of colchicine both at early (1 h) and later (3 h) times on the extent and pattern of 125I-insulin uptake. Colchicine treatment did not produce a significant change in lactogen receptor levels in the Golgi fractions. These results demonstrate that colchicine treatment inhibited the transfer of prolactin into Golgi vesicular elements. The much smaller effect on insulin uptake suggests that there may be differences in the manner in which the two hormones are handled in the course of internalization.  (+info)

A colchicine-sensitive uptake system in Morris hepatomas. (7/14)

The interference of microtubular disruptors with the uptake of amino acids and other low molecular weight substrates has been studied in Morris hepatomas, host liver, and regenerating liver. Colchicine inhibits amino acid transport (alpha-aminoisobutyric acid, L-methionine, and L-leucine) in hepatomas by 59-98% whereas transport in host and regenerating liver is not impeded but increased. In hepatomas, treatment urea, and carbonate. Vinblastine, but not lumicolchicine or cytochalasin B, is an effective inhibitor. The inhibition of uptake is not linked to a decrease of cellular ATP and UTP. The data suggest that the transport of low molecular weight substrates in hepatomas is related to microtubules or other colchicine-binding structures, e.g., of the plasma membrane. This colchicine-sensitive uptake system in hepatomas may be due to the malignant transformation of hepatocytes.  (+info)

Effect of colchicine on drug-induced changes in plasma renin concentration in rats. (8/14)

Cytoplasmic microtubules appear to play a role in the secretion of a variety of protein and protein hormones. Involvement of microtubules in renin secretion has been hypothesized but not established. The present studies were designed to determine: 1) if the antimicrotubule drug, colchicine, would alter plasma renin concentration (PRC); and 2) if changes in PRC could be related to an effect on cytoplasmic microtubules. Dose response experiments in Sprague-Dawley rats showed that 0.4 or 0.8 mg/kg/day i.p. of colchicine for 3 days significantly increased PRC while a dose of 0.2 mg/kg/day was without effect. The increase in PRC at the higher doses was associated with toxicity of the drug. In other experiments, rats pretreated with colchicine (0.2 mg/kg/day) or saline received either furosemide (5 mg/kg) or isoproterenol (25 micrograms/rat) i.p. to stimulate renin secretion. Colchicine at a dose that did not alter basal PRC significantly inhibited an increase in PRC after stimulation with either isoproterenol or furosemide. Lumicolchicine, a structural isomer of colchicine without antimicrotubule activity, did not alter the response to isoproterenol stimulation. These data suggest that microtubules play a role in the increase in renin secretion following stimulation.  (+info)