Fluorescein-methotrexate transport in dogfish shark (Squalus acanthias) choroid plexus

2006 ◽  
Vol 291 (2) ◽  
pp. R464-R472 ◽  
Author(s):  
Carsten H. Baehr ◽  
Gert Fricker ◽  
David S. Miller
Keyword(s):  
Protein Kinase ◽  
Choroid Plexus ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Squalus Acanthias ◽  
Cellular Accumulation ◽  
Vascular Space ◽  
Dogfish Shark ◽  
Tissue Compartments

The vertebrate choroid plexus removes potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We used confocal microscopy and quantitative image analysis to characterize the mechanisms driving transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact lateral choroid plexus from dogfish shark, Squalus acanthias, an evolutionarily ancient vertebrate. With 2 μM FL-MTX in the bath, steady-state fluorescence in the subepithelium/vascular space exceeded bath levels by 5- to 10-fold, and fluorescence in the epithelial cells was slightly below bath levels. FL-MTX accumulation in both tissue compartments was reduced by NaCN, Na removal, and ouabain, but not by a 10-fold increase in medium K. Certain organic anions, e.g., probenecid, MTX, and taurocholate, reduced FL-MTX accumulation in both tissue compartments; p-aminohippurate and estrone sulfate reduced subepithelial/vascular accumulation, but not cellular accumulation. At low concentrations, digoxin, leukotriene C4, and MK-571 reduced fluorescence in the subepithelium/vascular space while increasing cellular fluorescence, indicating preferential inhibition of efflux over uptake. In the presence of 10 μM digoxin (reduced efflux, enhanced cellular accumulation), cellular FL-MTX accumulation was specific, concentrative, and Na dependent. Thus transepithelial FL-MTX transport involved the following two carrier-mediated steps: electroneutral, Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane. Finally, FL-MTX accumulation in both tissue compartments was reduced by phorbol ester and increased by forskolin, indicating antagonistic modulation by protein kinase C and protein kinase A.

scholarly journals Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus

2008 ◽  
Vol 295 (4) ◽  
pp. R1311-R1319 ◽  
Author(s):  
Valeska Reichel ◽  
David S. Miller ◽  
Gert Fricker
Keyword(s):  
Steady State ◽  
Choroid Plexus ◽  
Driving Forces ◽  
Free Acid ◽  
Organic Anion ◽  
Squalus Acanthias ◽  
Vascular Space ◽  
Dogfish Shark ◽  
Texas Red ◽  
Pkc Activation

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark ( Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na+-replacement, but was not affected by a 10-fold increase in buffer K+. In shark CP, Na+-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K+ medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.

Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy

2004 ◽  
Vol 287 (3) ◽  
pp. F562-F569 ◽  
Author(s):  
Christopher M. Breen ◽  
Destiny B. Sykes ◽  
Carsten Baehr ◽  
Gert Fricker ◽  
David S. Miller
Keyword(s):  
Confocal Microscopy ◽  
Choroid Plexus ◽  
Apical Membrane ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Cellular Accumulation ◽  
Estrone Sulfate ◽  
Steady State Fluorescence ◽  
Toxic Metabolites ◽  
Quantitative Image

One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 μM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.

Confocal imaging of organic anion transport in intact rat choroid plexus

2002 ◽  
Vol 282 (5) ◽  
pp. F877-F885 ◽  
Author(s):  
Christopher M. Breen ◽  
Destiny B. Sykes ◽  
Gert Fricker ◽  
David S. Miller
Keyword(s):  
Blood Vessel ◽  
Choroid Plexus ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Anion Transport ◽  
Confocal Imaging ◽  
Organic Anion Transport ◽  
Indirect Coupling ◽  
Quantitative Image

We used confocal microscopy and quantitative image analysis to follow the movement of the fluorescent organic anion fluorescein (FL) from bath to cell and cell to blood vessel in intact rat lateral choroid plexus. FL accumulation in epithelial cells and underlying vessels was rapid, concentrative, and reduced by other organic anions. At steady state, cell fluorescence exceeded bath fluorescence by a factor of 3–5, and vessel fluorescence exceeded cell fluorescence by a factor of ∼2. In cells, FL distributed between diffuse and punctate compartments. Cell and vessel accumulation of FL decreased when metabolism was inhibited by KCN, when bath Na+ was reduced from 130 to 26 mM, and when the Na+ gradient was collapsed with ouabain. Cell and vessel accumulation increased by >50% when 1–10 μM glutarate was added to the bath. Finally, transport of FL and carboxyfluorescein (generated intracellularly from carboxyfluorescein diacetate) from cell to blood vessel was greatly diminished when medium K+ concentration ([K+]) was increased 10-fold. These results 1) validate a new approach to the study of choroid plexus function, and 2) indicate a two-step mechanism for transepithelial organic anion transport: indirect coupling of uptake to Na+ at the apical membrane and electrical potential-driven efflux at the basolateral membrane.

scholarly journals Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C

2000 ◽  
Vol 350 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Philip A. HELLIWELL ◽  
Michael RICHARDSON ◽  
Julie AFFLECK ◽  
George L. KELLETT
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Sugar Absorption ◽  
Kinase C

Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC βII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3–4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.

scholarly journals Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1205-1215 ◽  
Author(s):  
Rozmin T K Janoo ◽  
Lori A Neely ◽  
Burkhard R Braun ◽  
Simon K Whitehall ◽  
Charles S Hoffman
Keyword(s):  
Protein Kinase ◽  
Fold Increase ◽  
Transcriptional Regulators ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Lacz Expression ◽  
Protein Subunit ◽  
Plasmid Library ◽  
Binding Factor ◽  
Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

scholarly journals Protein kinase C-independent stimulation of activator protein-1 and c-Jun N-terminal kinase activity in human endometrial cancer cells by the LHRH agonist triptorelin

2001 ◽  
pp. 651-658 ◽  
Author(s):  
C Grundker ◽  
L Schlotawa ◽  
V Viereck ◽  
G Emons
Keyword(s):  
Cell Proliferation ◽  
Endometrial Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Fold Increase ◽  
Lhrh Agonist ◽  
Activator Protein 1 ◽  
Kinase C ◽  
Lhrh Receptor

OBJECTIVE: The expression of luteinizing hormone-releasing hormone (LHRH) and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumours, including cancers of the endometrium. The signalling pathway through which LHRH acts in endometrial cancer is distinct from that in pituitary gonadotrophs. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors via activation of a phosphotyrosine phosphatase, resulting in down-regulation of cancer cell proliferation. In addition, LHRH activates nucleus factor kappaB (NFkappaB) and protects the cancer cells from apoptosis. This study was conducted to investigate additional signalling mechanisms of the LHRH receptor cooperating with NFkappaB in endometrial cancer cells. DESIGN: The LHRH agonist triptorelin-induced activator protein-1 (AP-1) activation was analysed using a pAP-1-SEAP reporter gene assay. Expression of c-jun mRNA was quantified using quantitative reverse transcription (RT)-PCR. c-Jun N-terminal kinase (JNK) activity was measured by quantification of phosphorylated c-Jun protein. RESULTS: Treatment of Ishikawa and Hec-1A human endometrial cancer cells with 100 nM triptorelin resulted in a 3.1-fold and 3.5-fold activation of AP-1 respectively (P<0.05). If the cells had been made quiescent, treatment with triptorelin (100 nM) resulted in a 41.7-fold and 48.6-fold increase of AP-1 activation respectively (P<0.001). This effect was completely blocked by simultaneous treatment with pertussis toxin (PTX). A 17.6-fold and 17.3-fold increase of c-jun mRNA expression respectively (P<0.001) was obtained after 20 min of stimulation with triptorelin (100 nM). Treatment with 1 nM triptorelin resulted in a 12.5-fold or an 11.9-fold increase, and treatment with 10 pM triptorelin resulted in a 6.5-fold or a 5.2-fold increase of maximal c-jun mRNA expression respectively (P<0.001). Maximal c-Jun phosphorylation (68.5-fold and 60.2-fold, respectively, P<0.001) was obtained after 90 min incubation with triptorelin (100 nM). CONCLUSIONS: These results suggest that the LHRH agonist triptorelin stimulates the activity of AP-1 in human endometrial cancer cells mediated through PTX-sensitive G-protein alphai. In addition, triptorelin activates JNK, known to activate AP-1. In earlier investigations we have shown that triptorelin does not activate phospholipase and protein kinase C (PKC) in endometrial cancer cells. In addition, it has been demonstrated that triptorelin inhibits growth factor-induced mitogen activated protein kinase (MAPK, ERK) activity. Thus triptorelin-induced activation of the JNK/AP-1 pathway in endometrial cancer cells is independent of the known AP-1 activators, PKC or MAPK (ERK).

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

2000 ◽  
Vol 78 (6) ◽  
pp. 715-723 ◽  
Author(s):  
John P Williams ◽  
Margaret A McKenna ◽  
Allyn M Thames III ◽  
Jay M McDonald
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Dependent Manner ◽  
Osteoclast Activity ◽  
Kinase C ◽  
Protein Kinase Cα

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1–0.3 µM). In contrast, tamoxifen inhibited osteoclast activity ~60% with an IC50 of 1.5 µM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase Cα recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase Cα, β, δ, ε, and ζ were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 ± 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.Key words: osteoclast, calmodulin, tamoxifen, osteoporosis, protein kinase C.

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