Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin-induced intestinal damage.
(25/2012)
1. The relative contributions of superoxide anion (O2-) and peroxynitrite (PN) were evaluated in the pathogenesis of intestinal microvascular damage caused by the intravenous injection of E. coli lipopolysaccharide (LPS) in rats. The superoxide dismutase mimetic (SODm) SC-55858 and the active peroxynitrite decomposition catalysts 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulphonatophenyl)-por phyrinato iron (III) and 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)-porphyrinato iron (III) (FeTMPS, FeTMPyP respectively) were used to assess the roles of O2- and PN respectively. 2. The intravenous injection of LPS elicited an inflammatory response that was characterized by a time-dependent infiltration of neutrophils, lipid peroxidation, microvascular leakage (indicative of microvascular damage), and epithelial cell injury in both the duodenum and jejunum. 3. Administration of the SODm SC-55858, FeTMPS or FeTMPyP at 3 h post LPS reduced the subsequent increase in microvascular leakage, lipid peroxidation and epithelial cell injury. Inactive peroxynitrite decomposition catalysts exhibited no protective effects. Only, SC-55858 inhibited neutrophil infiltration. 4. Our results suggest that O2 and peroxynitrite play a significant role in the pathogenesis of duodenal and intestinal injury during endotoxaemia and that their remoyal by SODm and peroxynitrite decomposition catalysts offers a novel approach to the treatment of septic shock or clinical conditions of gastrointestinal inflammation. Furthermore, the remarkable protection of the intestinal epithelium by these agents suggests their use during chemo- and radiation therapy, cancer treatments characterized by gastrointestinal damage. Potential mechanisms through which these radicals evoke damage are discussed. (+info)
Nucleotide-dependent bisANS binding to tubulin.
(26/2012)
Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes. (+info)
Alteration of microtubule polymerization modulates arteriolar vasomotor tone.
(27/2012)
Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 microM), nocodazole (10 microM), or demecolcine (10 microM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30-170 cmH(2)O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca(2+)](i). (+info)
Induced differentiation in HT29, a human colon adenocarcinoma cell line.
(28/2012)
The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma. (+info)
Canalicular membrane transport is primarily responsible for the difference in hepatobiliary excretion of triethylmethylammonium and tributylmethylammonium in rats.
(29/2012)
Two structurally similar quaternary ammonium compounds, triethylmethylammonium (TEMA, M(r) 116) and tributylmethylammonium (TBuMA, M(r) 200) were used as model compounds to identify the unit process of hepatobiliary excretion that is responsible for markedly different biliary excretion of organic cations (OCs). Cumulative biliary excretion (in percentage of dose; i.v., 12 micromol/kg) was 0.17 for TEMA and 34.5 for TBuMA. In vivo uptake clearance into the liver was 0.686 +/- 0.020 ml/min for TEMA and 0.421 +/- 0.028 ml/min for TBuMA. When the uptake clearance was examined in an isolated hepatocyte system, comparable clearance between TEMA and TBuMA was obtained, consistent with the in vivo result. These observations suggest that uptake into the liver is not the major determinant for the difference in biliary excretion of the OCs. Coadministration of colchicine, an inhibitor of microtubule formation, had no effect on biliary excretion of the model compounds, and the primary site of subcellular distribution of the OCs appears to be the cytosol, suggesting that intracellular movement does not play a major role in the markedly different biliary excretion of the OCs. In contrast, in vivo excretion clearance across the canalicular membrane for TBuMA was 180-fold greater than that for TEMA, and in vitro efflux clearance of TBuMA was smaller than that of TEMA (p <.01), indicative of involvement of these processes in the markedly different biliary excretion of the OCs. Therefore, these data indicate that canalicular transport is primarily responsible for the markedly different biliary excretion of TEMA and TBuMA. (+info)
Protein kinase C-mediated regulation of the renal Na(+)/dicarboxylate cotransporter, NaDC-1.
(30/2012)
The Na(+)/dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the tubular filtrate. Although long-term regulation of this transporter by chronic metabolic acidosis and K(+) deficiency is well documented, there is no information on acute regulation of NaDC-1. In the present study, the transport of succinate in Xenopus oocytes expressing NaDC-1 was inhibited up to 95% by two activators of protein kinase C, phorbol 12-myristate, 13-acetate (PMA) and sn-1, 2-dioctanoylglycerol (DOG). Activation of protein kinase A had no effect on NaDC-1 activity. The inhibition of NaDC-1 transport by PMA was dose-dependent, and could be prevented by incubation of the oocytes with staurosporine. Mutations of the two consensus protein kinase C phosphorylation sites in NaDC-1 did not affect inhibition by PMA. The inhibitory effects of PMA were partially prevented by cytochalasin D, which disrupts microfilaments and endocytosis. PMA treatment was also associated with a decrease of approximately 30% in the amount of NaDC-1 protein found on the plasma membrane. We conclude that the inhibition of NaDC-1 transport activity by PMA occurs by a combination of endocytosis and inhibition of transport activity. (+info)
Superimposed primary chancre in a patient with Adamantiades-Behcet's disease.
(31/2012)
Adamantiades-Behcet's disease was diagnosed in a 42 year old Turkish patient with recurrent oral aphthae, genital ulcerations, papules, and sterile pustules, histologically presenting as cutaneous vasculitis, and intermittent arthritis with joint effusion particularly of the knees. Six months after initial improvement under treatment with colchicine 2 mg/day, a solitary genital ulcer with enlarged inguinal lymph nodes appeared and persisted for 7 weeks despite the continuation of colchicine treatment and the introduction of clindamycin 2 mg/day intravenously. The unusual persistence of the ulcer and the failure of clindamycin therapy led to further differential diagnostic considerations and the identification of primary syphilis. The genital lesion healed 4 weeks after initiation of treatment with tetracycline 2 mg/day by mouth for 15 days. (+info)
Rapid downregulation of rat renal Na/P(i) cotransporter in response to parathyroid hormone involves microtubule rearrangement.
(32/2012)
Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/P(i) cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/P(i) cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs. (+info)