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PubMed2006

Update on drug-eluting stents for prevention of restenosis.

Despite the success of coronary stent implantations in the last decade, in-stent restenosis due to neointimal hyperplasia remains a problem to overcome. Neointimal hyperplasia is a vascular response to stent injury and mainly consists of proliferation of smooth muscle cells and deposition of extracellular matrix. Recently, local drug delivery has been advocated as a potential strategy to prevent in-stent restenosis. Unprecedented results have been obtained in early clinical studies on sirolimus-eluting and paclitaxel-eluting stents. Trials using various pharmaceutical coatings on different coronary stents are ongoing. More types of drug-eluting stents are expected on the market in the near future. Meanwhile, the evaluation of drug-eluting stents is entering the second phase in which the safety and efficacy in more complex lesion subsets and different clinical presentations are being investigated. Results including cost-benefit analyses are expected to have a tremendous impact on the practice of interventional cardiology in the next decade.

Animals↗
PubMed2005

A radioligand binding assay for antitubulin activity in tumor cells.

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.

Benzamides↗
PubMed2006

Miniaturization and validation of a sensitive multiparametric cell-based assay for the concomitant detection of microtubule-destabilizing and microtubule-stabilizing agents.

The authors describe a cell-based assay for anti-microtubule compounds suitable for automation. This assay allows the identification, in a single screening campaign, of both microtubule-destabilizing and microtubule-stabilizing agents. Its rationale is based on the substrate properties of the tubulin-modifying enzymes involved in the tubulin tyrosination cycle. This cycle involves the removal of the C-terminal tyrosine of the tubulin alpha-subunit by an ill-defined tubulin carboxypeptidase and its readdition by tubulin tyrosine ligase. Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin, whereas non-dynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can easily be detected with double-immunofluorescence staining using antibodies specific to tyrosinated and detyrosinated tubulin. The authors have scaled this assay to the 96-well plate format and adapted its process for an automated handling, including a readout using a microplate reader. They describe the different steps of this adaptation. This assay was validated using known compounds. This new cell-based assay represents an alternative to both global cytotoxicity assays and in vitro tubulin assembly assays commonly used for the detection of microtubule poisons.

Cell Count↗
PubMed2006

Aggresome induction by proteasome inhibitor bortezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells.

Histone deacetylase (HDAC) inhibitors have shown cytotoxicity as single agents in preclinical studies for multiple myeloma (MM) cells. LBH589 is a novel hydroxamic acid derivative that at low nanomolar concentrations induces apoptosis in MM cells resistant to conventional therapies via caspase activation and poly-(ADP-ribose) polymerase (PARP) cleavage. Significant synergistic cytotoxicity was observed with LBH589 in combination with bortezomib against MM cells that were sensitive and resistant to dexamethasone (Dex), as well as primary patient MM cells. LBH589 at low nanomolar concentrations also induced alpha-tubulin hyperacetylation. Aggresome formation was observed in the presence of bortezomib, and the combination of LBH589 plus bortezomib induced the formation of abnormal bundles of hyeracetylated alpha-tubulin but with diminished aggresome size and apoptotic nuclei. These data confirm the potential clinical benefit of combining HDAC inhibitors with proteasome inhibitors, and provide insight into the mechanisms of synergistic anti-MM activity of bortezomib in combination with LBH589.

Boronic Acids↗
PubMed2006

Integrated molecular profiling of SOD2 expression in multiple myeloma.

Reactive oxygen species are known to be involved in several cellular processes, including cell signaling. SOD2 is a key enzyme in the conversion of reactive oxygen species and has been implicated in a host of disease states, including cancer. Using an integrated, whole-cell approach encompassing epigenetics, genomics, and proteomics, we have defined the role of SOD2 in multiple myeloma. We show that the SOD2 promoter is methylated in several cell lines and there is a correlative decrease in expression. Furthermore, myeloma patient samples have decreased SOD2 expression compared with healthy donors. Overexpression of SOD2 results in decreased proliferation and altered sensitivity to 2-methoxyestradiol-induced DNA damage and apoptosis. Genomic profiling revealed regulation of 65 genes, including genes involved in tumorigenesis, and proteomic analysis identified activation of the JAK/STAT pathway. Analysis of nearly 400 activated transcription factors identified 31 transcription factors with altered DNA binding activity, including XBP1, NFAT, forkhead, and GAS binding sites. Integration of data from our gestalt molecular analysis has defined a role for SOD2 in cellular proliferation, JAK/STAT signaling, and regulation of several transcription factors.

2-Methoxyestradiol↗
PubMed2006

Long-term clinical effects of interferon gamma-1b and colchicine in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a deadly disease with no effective treatment. The purpose of this randomised prospective multicentric study was to characterise the clinical effects of interferon gamma (IFN-gamma) 1b administered subcutaneously thrice weekly versus colchicine for 2 yrs. This study had no pre-specified end-points. Fifty consecutive IPF patients were randomised. Patients with mild-to-moderate IPF were eligible for the study if they had histologically proven IPF, or, in the absence of surgical biopsy, fulfilled the European Respiratory Society/American Thoracic Society criteria. In the intent-to-treat population, five out of 32 (15.6%) IFN-gamma-1b patients and seven out of 18 (38.8%) colchicine patients died after a median follow-up period of 25 months Patients treated with IFN-gamma 1b showed a better outcome after 2 yrs of therapy, and fewer symptoms, as assessed using the St George's Respiratory Questionnaire, after 12 months of therapy. Also, the IFN-gamma-1b group exhibited a higher forced vital capacity (percentage of the predicted value) after 24 months of treatment. No significant differences were detected in resting arterial oxygen tension, total lung capacity (% pred), transfer factor of the lung for carbon monoxide (% pred) and high-resolution computed tomographic scoring between the two treatment groups. These data suggest that long-term treatment with interferon gamma 1b may improve survival and outcome in patients with mild-to-moderate idiopathic pulmonary fibrosis. Further studies are needed to verify these results.

Adult↗
PubMed2006

Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: a cytometric study.

BACKGROUND: Despite the great advances in the understanding of programmed cell death, little attention has been paid to the sequence of the events that characterise it. In particular, the course of apoptotic events induced by microtubule-interfering agents such as taxanes is poorly understood. In order to increase such knowledge, we studied a number of independent biochemical and cytological modifications using cytometric methods in a bladder cancer cell line treated with the second generation taxane, docetaxel. RESULTS: Within a few hours, drug treatment had induced mitochondrial membrane transition, cell shrinkage and a decrease in granularity. Cell cycle was almost completely blocked in G2/M phase within 24 hours. The hypodiploid peak started to become prominent 48 hours after the treatment. At the same time, the appearance of a DNA ladder demonstrated caspase-dependent chromatin fragmentation. Concurrently, specific cell surface modifications took place, involving at first glycoprotein syalilation and later phospholipid asymmetry. DNA fragmentation was subsequently detected by TUNEL assay. Over time, cell membranes became permeable to propidium iodide. A very similar time-course of apoptotic events was found after treatment of a myelomonocytic cell line with the same drug. CONCLUSION: After discussing some characteristics of the methods employed and their limitations, a succession of apoptotic events over time is suggested, in which the collapse of mitochondrial transmembrane potential (Deltapsim) is the earliest sign of apoptosis.

Antineoplastic Agents↗
PubMed2006

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs. RESULTS: Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20-22 h p.i.), but the logarithmic infection phase (days 2-3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-gamma (IFN-gamma), as well as the more-potent experimental antiviral agent AK-2. CONCLUSION: The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.

Animals↗
PubMed2007

Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress.

BACKGROUND: The VP2 outer capsid protein Bluetongue Virus (BTV) is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown. RESULTS: In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of amino acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells. CONCLUSION: The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i) the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii) Deletion of amino acids 65-114 or mutation of amino acids 70-72 to DVD abrogates vimentin association. iii) Finally, disruption of vimentin structures results in an increase in cell associated BTV and a reduction in the amount of released virus from infected cells.

Acrylamide↗
PubMed2004

Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils.

The neutrophil cytoplasmic protein S100A8/A9 (along with S100A8 and S100A9) is chemotactic and stimulates neutrophil adhesion by activating the beta2-integrin CD11b/CD18. It is also essential to neutrophil migration in vivo in response to monosodium urate monohydrate (MSUM) crystals, the principal etiologic agent of gout. S100A8/A9 is present in the synovial fluid of patients with gout and arthritis and is secreted by activated monocytes; however, its mechanism of release by neutrophils remains unknown. The aim of this study was to identify the mechanism of stimulation of the release of S100A8/A9 by MSUM-activated neutrophils. Here, we show that S100A8/A9 is released by neutrophils stimulated with MSUM crystals and that this release could be enhanced by preincubating neutrophils with granulocyte macrophage-colony stimulating factor. Antibodies directed against CD11b and CD16 blocked the release induced by MSUM crystals, suggesting that Fc receptor for immunoglobulin G (FcgammaR)IIIB (CD16) and CD11b/CD18 were involved in the stimulation by MSUM crystals. Neutrophil preincubation with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine and the Syk tyrosine kinase inhibitor trans-3,3',4,5'-tetrahydrozystilbene significantly reduced the release of S100A8/A9, suggesting that the Src tyrosine kinase family and Syk were involved. In addition, wortmannin reduced neutrophil release of S100A8/A9, indicating a potential involvement of phosphatidylinolitol-3 kinase in this release. Preincubation of neutrophils with the tubulin depolymerization promoters nocodazole and vincristine reduced MSUM-induced release, suggesting a tubulin-associated pathway of release. These results indicate that S100A8/A9 is released by MSUM crystal-stimulated neutrophils following activation of CD11b, CD16, Src kinases, Syk, and tubulin polymerization.

Calgranulin A↗
PubMed1999

Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death.

PURPOSE: To analyze the available data concerning mechanisms of action of and mechanisms of resistance to the antitubulin agents, vinca alkaloids and taxanes, and more recently described compounds. DESIGN: We conducted a review of the literature on classic and recent antitubulin agents, focusing particularly on the relationships between antitubulin agents and their intracellular target, the soluble tubulin/microtubule complex. RESULTS AND CONCLUSION: Although it is widely accepted that antitubulin agents block cell division by inhibition of the mitotic spindle, the mechanism of action of antitubulin agents on microtubules remains to be determined. The classic approach is that vinca alkaloids depolymerize microtubules, thereby increasing the soluble tubulin pool, whereas taxanes stabilize microtubules and increase the microtubular mass. More recent data suggest that both classes of agents have a similar mechanism of action, involving the inhibition of microtubule dynamics. These data suggest that vinca alkaloids and taxanes may act synergistically as antitumor agents and may be administered as combination chemotherapy in the clinic. However, enhanced myeloid and neurologic toxicity, as well as a strong dependence on the sequence of administration, presently exclude these combinations outside the context of clinical trials. Although the multidrug resistance phenotype mediated by Pgp appears to be an important mechanism of resistance to these agents, alterations of microtubule structure resulting in altered microtubule dynamics and/or altered binding of antitubulin agents may constitute a significant mechanism of drug resistance.

Amino Acid Sequence↗
PubMed2006

Peripheral neuropathy induced by microtubule-stabilizing agents.

Microtubule-stabilizing agents (MTSAs), including the taxanes and epothilones, are effective chemotherapeutic agents for the treatment of many cancers. Neuropathy is a major adverse effect of MTSA-based chemotherapy, with severe peripheral neuropathy (grade 3 or 4) occurring in as many as 30% of patients treated with a MTSA. MTSA-induced neuropathy usually resolves gradually after cessation of the treatment. The most reliable method to accurately assess MTSA-induced neuropathy is by clinical evaluation, although additional techniques are being developed and evaluated. Among MTSA-induced neuropathy, the most extensively studied is that induced by taxanes; such a neuropathy usually presents as sensory neuropathy and is more common with paclitaxel than docetaxel. The incidence of MTSA-induced neuropathy seems to depend on the MTSA dose per treatment cycle, the schedule of treatment, and the duration of the infusion. Although there have been several small clinical trials with neuroprotective agents, early recognition and supportive care are the best approaches for prevention and management of MTSA-induced neuropathy. In the future, research should focus on elucidating the mechanism of MTSA-induced neuropathy, developing reliable in vivo and in vitro preclinical models to study MTSA-induced neuropathy, developing a more reliable grading system for MTSA-induced neuropathy, developing more reliable methods for evaluating MTSA-induced neuropathy, and evaluating the efficacy of potential neuroprotective agents in clinical trials.

Albumin-Bound Paclitaxel↗
PubMed2001

Potential transition state phosphoramidate inhibitors of beta-tubulin as antifilarial agents.

Transition state phosphoramidate inhibitors of beta-tubulin were designed as potential antifilarial agents. The reaction of 2-aminobenzimidazole with diisopropyl phosphite and carbon tetrachloride at a low temperature gave the unexpected 1-diisopropoxyphosphoryl-2-aminobenzimidazole, which on heating gave the novel benzimidazole derivative, 2-(diisopropoxyphosphoryl)aminobenzimidazole. Both products were fully characterized and the synthetic procedure to both compounds was optimized. The procedure was used to prepare the related 5-benzoyl-2-(diisopropoxyphosphoryl)aminobenzimidazole and 5-benzoyl-2-(diethoxyphosphoryl)aminobenzimidazole (1d). In a preliminary trial against Brugia pahangi compound 1d was found to have no antifilarial activity. This lack of activity may be attributed to its extreme insolubility and thus low bioavailability. The synthesis of analogous, more soluble, phosphorothioate-substituted benzimidazoles using the same methods may yield compounds with greater antifilarial activity.

Amides↗
PubMed2006

Cytotoxic activity and cytostatic mechanism of novel 2-arylbenzo[b]furans.

The aims of this study were to screen cytotoxic compounds from 14 newly-synthesized 2-arylbenzo[b]furans and explore their mechanisms of action. Cytotoxicity was determined by the MTT method. Cell-cycle distribution was detected by flow cytometry. Wright-Giemsa staining was performed to demonstrate the morphological features of cells in mitotic phase. Polymerization of tubulin was detected by tubulin assembly assay, and the cellular microtubule network was observed by immunocytochemical study. Among the 14 compounds screened, 4-formyl-2-(4-hydroxy-3-methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo[b]furan (ERJT-12) showed significant cytotoxicity. Our results demonstrated that ERJT-12 exhibited anti-cancer activity in a variety of tumour cell lines with an IC50 value (concentration resulting in 50% inhibition of cell growth) of 5.75 approximately 17.29 microM. Cell cycle analysis showed a concentration-dependent accumulation of tumour cells in G2/M phase after treatment with ERJT-12. Further investigation indicated that ERJT-12 blocked the cell cycle in M phase, with separation and dispersion of chromosomes. ERJT-12 inhibited tubulin polymerization in-vitro. Changes of the cellular microtubule network caused by ERJT-12 were also detected, which were similar to the changes caused by colchicine. These results suggested that the anti-cancer activity of ERJT-12 is worth further investigation.

Antineoplastic Agents↗
PubMed2005

Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1.

Human enhancer of filamentation 1 (HEF1), a multifunctional docking protein of the Cas family, participates in integrin and growth factor signaling pathways that regulate global cellular processes including growth, motility and apoptosis. HEF1 consists of two isoforms, p105 and p115, the larger molecular weight form resulting from Ser/Thr phosphorylation of p105HEF1. The molecular mechanisms that regulate the interconversion of the two HEF1 species as well as the function of HEF1 Ser/Thr phosphorylation are unknown. Our study reveals that cell adhesion and detachment regulate the interconversion of the two HEF1 isoforms. Experiments using various inhibitors of cytoskeletal organization indicated that disruption of actin microfilaments but not intermediate filaments or microtubules resulted in a complete conversion of p115HEF1 to p105HEF1. The conversion of p115HEF1 to p105HEF1 was prevented by inhibition of protein phosphatase 2A (PP2A), suggesting that cytoskeletal regulation of PP2A activity controlled the dephosphorylation of p115HEF1. Degradation of endogenous HEF1 was dependent on proteasomes with the p115 species of HEF1 being preferentially targeted for turnover. Dephosphorylation of HEF1 by suspending cells or disrupting actin filaments protected HEF1 from degradation. These results suggest that the adhesion-dependent actin organization regulates proteasomal turnover of HEF1 through the activity of PP2A.

Actin Cytoskeleton↗
PubMed1980

Effect of antitubulins on spontaneous and chemotactic migration of neutrophils under agarose.

The effect of colchicine, Nocodazole or vinblastine on the movement of mouse bone marrow neutrophils was examined by the method of migration under agarose. Migration was estimated by counts of the numbers of cells that left the wells and moved under the gel. Micromolar concentrations of the agents increased spontaneous migration 2- to 4-fold, while lumicolchicine was ineffective. The increase may be due to the disassembly of the microtubular skeleton, making the cells more fluid, or to some other effect of the drugs. Since other agents, such as serum, low osmolality and low pH also enhance migration, these were tried in combination with a pretreatment of the cells with colchicine. Serum and colchicine acted synergistically, low osmolality and colchicine were additive. Possible, the enhancement of neutrophil migration by serum or lowered osmolality does not require intact microtubules. At low pH, colchicine had very little stimulatory effect, perhaps indicating that the microtubules are more colchicine-resistant at low pH. Although antibular drugs showed such marked effects on spontaneous migration, the chemotactic attraction of the neutrophils towards activated serum was unaffected. This indicates that the sensory and control mechanisms of the cells are resistant to antibulins.

Animals↗
PubMed1982

Effect of tubulin-binding agents on the infiltration of tumour cells into primary hepatocyte cultures.

The influence of tubulin-binding agents on the infiltration of murine MB6A lymphosarcoma and TA3 mammary carcinoma cells into primary rat hepatocyte cultures was studied. Colchicine, nocodazole and vinblastine reduced the number of infiltrating lymphosarcoma cells, probably by interfering with the adhesion of these cells to the exposed hepatocyte surface. However, the subsequent infiltration of cells that did adhere was not affected or even slightly stimulated. The reduced adhesion appears to be due to an effect on both the MB6A cells and the hepatocytes. In contrasts, adhesion of TA3 cells was not reduced and infiltration was markedly enhanced by these agents, due to an effect on the TA3 cells but probably not on the hepatocytes. These observations support previously described morphological evidence for differences between the infiltration mechanisms of the two tumour cell types. It is concluded that the system within the hepatocytes involved in adhesiveness of the exposed surface to MB6A cells is distinct from that mediating other types of adhesion. The tendency of TA3 cells to invaginate hepatocytes may be due to disturbances in tubulin-dependent processes.

Animals↗
PubMed2006

Microtubule-dependent relocation of branchial V-H+-ATPase to the basolateral membrane in the Pacific spiny dogfish (Squalus acanthias): a role in base secretion.

We have previously shown that continuous intravenous infusion of NaHCO3 for 24 h ( approximately 1000 micromol kg(-1) h(-1)) results in the relocation of V-H+-ATPase from the cytoplasm to the basolateral membrane in the gills of the Pacific dogfish. To further investigate this putative base-secretive process we performed similar experiments with the addition of colchicine, an inhibitor of cytoskeleton-dependent cellular trafficking processes. Blood pH and plasma total CO2 were significantly higher in the colchicines-treated, HCO3- -infused fish compared with fish infused with HCO3- alone. The effect of colchicine was highest after 24 h of infusion (8.33+/-0.06 vs 8.02+/-0.03 pH units, 15.72+/-3.29 vs 6.74+/-1.34 mmol CO2 l(-1), N=5). Immunohistochemistry and western blotting confirmed that colchicine blocked the transit of V-H+-ATPase to the basolateral membrane. Furthermore, western blotting analyses from whole gill and cell membrane samples suggest that the short-term (6 h) response to alkaline stress consists of relocation of V-H+-ATPases already present in the cell to the basolateral membrane, while in the longer term (24 h) there is both relocation of preexistent enzyme and upregulation in the synthesis of new units. Our results strongly suggest that cellular relocation of V-H+-ATPase is necessary for enhanced HCO3- secretion across the gills of the Pacific dogfish.

Acid-Base Equilibrium↗