Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Probenecid”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,387 records · Page 77Linked to original sources
PubMed2006

Multidrug resistance related protein (ABCC1) and its role on nitrite production by the murine macrophage cell line RAW 264.7.

Multidrug resistance related protein 1 (MRP1/ABCC1) is an ABC transporter protein related to the extrusion of reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH-conjugates, as well as leukotriene C(4) and cyclopentane prostaglandins. Inhibition of ABCC1 activity impairs lymphocyte activation. The present work studied ABCC1 expression and activity on a murine macrophage cell line, RAW 267.4 and the effects of ABCC1 classical inhibitors, as well as GSH metabolism modulators, on LPS induced activation. Approximately, 75% of resting cells were positive for ABCC1 and the classical ABCC1 reversors (indomethacin, 0.1-2mM; probenecid, 0.1-10mM and MK571, 0.01-1mM) were able to enhance intracellular CFDA accumulation in a concentration-dependent manner, suggesting ABCC1 inhibition. After LPS (100ng/ml) activation 50% of the population was positive for ABCC1, and this protein was still active. In LPS-activated cells, ABCC1 activity was also impaired by BSO (1mM), an inhibitor of GSH synthesis. Conversely, GSH (5mM) reversed the BSO effect. ABCC1 inhibition by indomethacin, probenecid or MK571 decreased LPS induced nitrite production in a concentration-dependent manner, the same result was observed with BSO and again GSH reversed its effect. The ABCC1 reversors were also able to inhibit iNOS expression. In conclusion, LPS modulated the expression and activity of ABCC1 transporters in RAW macrophages and inhibitors of these transporters were capable of inhibiting nitrite production suggesting a role for ABCC1 transporters in the inflammatory process.

Acetylcysteine↗
PubMed2005

Characterization of prostaglandin E1 transport by rat renal cortical slices.

The purpose of this study was to examine prostaglandin E1 (PGE1) transport in rat kidney. [3H]PGE1 administered intravenously was accumulated most abundantly in the renal cortex. Infusion of probenecid and bromcresol green (BCG) decreased [3H]PGE1 accumulation in the renal cortex after injection of [3H]PGE1. To further investigate PGE1 transport in the kidney, [3H]PGE1 uptake by renal cortical slices was examined. Probenecid and BCG inhibited [3H]PGE1 uptake by the slices. Unlabeled PGE1 decreased [3H]PGE1 uptake by renal cortical slices in a concentration-dependent manner. The inhibitory effect of various dicarboxylates with different carbon atoms on [3H]PGE1 uptake was maximal at 6 carbon atoms. Preloading cortical slices with glutarate significantly increased [3H]PGE1 uptake. [3H]PGE1 uptake was inhibited by various eicosanoids and compounds with other structures (p-aminohippurate, benzylpenicillin, estrone-3-sulfate, etc.). These findings suggest that PGE1 uptake by renal cortical slices may be mediated by the members of the organic anion transporter family.

Alprostadil↗
PubMed2005

Mrp2/Abcc2 transport activity is stimulated by protein kinase Calpha in a baculo virus co-expression system.

Cholestatic and choleretic effect are well known for protein kinase C activator and inhibitor, respectively. However, post-translational regulation, especially the effect of phosphorylation status of the biliary transporters on their intrinsic transport activity has not been fully understood. In this study, effect of phosphorylation on the transport activity of Mrp2, a biliary organic anion transporter, was examined in membrane vesicles isolated from Sf9 cells co-expressing excess amount of protein kinase Calpha (PKCalpha). Mrp2-mediated transport activity was enhanced to three-fold by co-expressing PKCalpha. At the same time, phosphorylation of Mrp2 was also detected. The Km and Vmax values for the transport of [3H]estradiol-17beta-D-glucuronide exhibited a 1.5-fold decrease and a 1.9-fold increase, respectively. Probenecid (100 microM) and benzylpenicillin (1 mM), both are activator of Mrp2, did not stimulated the transport activity of phosphorylated Mrp2. On the other hand, transport activity was further stimulated by Estron-3-sulfate and taurocholic acid. Similar mechanism that occurred in the presence of probenecid and benzylpenicillin, but different from that occurred in the presence of Estron-3-sulfate and taurocholic acid seems to be involved in the stimulation. Considering the discrepancy between the previous in vivo inhibitory effect of PKC activators and our in vitro stimulatory effect of PKCalpha on Mrp2 transport activity, direct modulation of Mrp2-transport activity may be minor if any under in vivo condition.

ATP-Binding Cassette Transporters↗
PubMed2006

Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson's disease.

Parkinson's disease is associated with a progressive loss of substantia nigra pars compacta dopaminergic neurons. The cellular and molecular mechanisms underlying Parkinson's disease neurodegeneration have not been fully determined. Clinical investigations and subacute in vivo studies using the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have generated some observations suggesting that apoptosis is involved in neurodegeneration; however, this view remains equivocal. In this study, the substantia nigra pars compacta neurodegenerative process was examined in the chronic mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson's disease treated with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) and probenecid (250 mg/kg) over five weeks. One day after chronic treatment, numerous terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were detected specifically in the substantia nigra pars compacta displaying shrunken volume, chromatin condensation, and DNA fragmentation. The number of apoptotic cells declined over time. No terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were found in untreated or probenecid-treated control animals. Cytomorphometric analysis of substantia nigra pars compacta nuclear loci revealed eccentric nucleoli dislocation and vesicular degranulation in all of the apoptotic neurons for the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells phenotypically showed neuronal origin (NeuN-positive) with a loss of tyrosine hydroxylase immunoreactivity. While the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were not co-localized with astroglial (GFAP-positive) cells, some apoptotic cells were clearly associated with the activated microglial (macrophage antigen complex-1 and isolectin B(4)-positive) cells suggesting an active process of dead cell removal. In the one-day and seven-day post-treated mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease, marked depression of tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and striatum was observed, which was correlated with significant reductions of striatal dopamine content and uptake. These results suggest that initial neuronal apoptosis and morphological changes are involved, at least in part, in the chronic neurodegeneration of mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Inactivation of atrial natriuretic factor-stimulated cyclic guanosine 3',5'-monophosphate (cGMP) in UMR-106 osteoblast-like cells.

Previous studies have suggested a role of cyclic guanosine 3', 5'-monophosphate (cGMP) in the differentiation and proliferation of osteoblasts. We studied the effect of ANF (atrial natriuretic factor) on intracellular cGMP accumulation, cGMP efflux, and cGMP-phosphodiesterase (PDE) activity in UMR-106 osteoblast-like cells. ANF rapidly increased both intracellular cGMP and cGMP efflux. ANF-stimulated intracellular cGMP peaked at 2 min in the absence and at 10 min in the presence of 0.25 mM 3-isobutyl-1-methylxanthine. Probenecid, an antagonist of anion transport, blocked the efflux of cGMP (IC(50) = 0.1 mM), ruling out simple diffusion as a mechanism of the efflux. cGMP-PDE activity was increased threefold in crude homogenates from ANF-treated cells (IC(50) = 23 nM). ANF-evoked stimulation of cGMP-PDE activity was reached simultaneously with the peak in intracellular cGMP. Separation of the PDEs by Q-Sepharose chromatography revealed three cGMP-hydrolyzing peaks. The first peak was sensitive to the PDE5 (cGMP-specific PDE) isoenzyme-selective inhibitor zaprinast (IC(50) = 0.45 microM). The second peak was stimulated fourfold by the addition of calcium/calmodulin, indicating the presence of PDE1. The third peak was sensitive to the PDE2 (cGMP-stimulated PDE) isoenzyme-selective inhibitor 9-[2-hydroxy-3-nonyl]adenine (EHNA) (IC(50) = 3 microM), and was activated by over 300% in the presence of 4 microM cGMP. Our results show that ANF-stimulated cGMP is released from UMR-106 cells by a probenecid-sensitive mechanism. ANF also stimulates cGMP hydrolysis by activating cGMP-PDE activity. Three distinct cGMP-hydrolyzing PDEs, namely PDE5, PDE1, and PDE2, are present in the studied cells.

Animals↗
PubMed2000

Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes.

Organic anion secretion by human hepatocytes was characterized using primary liver parenchymal cell cultures and the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF). Probenecid, a well-known common blocker of the membrane transport process for anions, was shown to increase CF accumulation in primary human hepatocytes by inhibiting cellular CF efflux in a dose-dependent manner, thereby establishing the presence of an efflux system for organic anions in cultured hepatocytes. Outwardly directed transport of CF from hepatocytes was found to be temperature-dependent; it was not altered by changes in the ionic composition of the incubation medium used in efflux experiments. In addition to probenecid, various structurally and functionally unrelated xenobiotics such as glibenclamide, rifampicin, vinblastine, MK-571, indomethacin, and cyclosporin A were shown to inhibit secretion of CF by primary human hepatocytes, thus suggesting that organic anion excretion by human liver may be impaired by various drugs. Northern blot and Western blot analyses of the expression of multidrug resistance proteins (MRP), such as MRP1 and MRP2, which are known to mediate cellular outwardly directed transport of organic anions indicated that MRP2 was present at substantial levels in cultured human hepatocytes as well as in their in vivo counterparts, whereas MRP1 expression was only barely detectable. These results therefore suggest that MRP2, unlike MRP1, may contribute to the organic anion efflux system displayed by primary human hepatocytes and inhibited by a wide range of xenobiotics.

Anion Transport Proteins↗
PubMed2003

Transport of 5-aminolevulinic acid between blood and brain.

Little is known about the movement of 5-aminolevulinic acid (delta-aminolevulinic acid; ALA) between blood and brain. This is despite the fact that increases in brain ALA may be involved in generating the neuropsychiatric symptoms in porphyrias and that systemic administration of ALA is currently being used to delineate the borders of malignant gliomas. The current study examines the mechanisms involved in the movement of [(14)C]ALA across the blood-brain and blood-CSF barriers in the rat. In the adult rat, the influx rate constant (K(i)) for [(14)C]ALA movement into brain was low ( approximately 0.2 microl/g per min), was unaffected by increasing plasma concentrations of non-radioactive ALA or probenecid (an organic anion transport inhibitor) and, therefore, appears to be a diffusional process. The K(i) for [(14)C]ALA was 3-fold less than that for [(14)C]mannitol, a molecule of similar size. This difference appears to result from a lower lipid solubility rather than saturable [(14)C]ALA transport from brain to blood. The K(i) for [(14)C]ALA for uptake into the neonatal brain was 7-fold higher than in the adult. However, again, this was unaffected by increasing plasma ALA concentrations suggesting a diffusional process. In contrast, at the blood-CSF barrier, there was evidence of carrier-mediated [(14)C]ALA transport from blood to choroid plexus and blood to CSF. Both processes were inhibited by administration of non-radioactive ALA and probenecid. However, experiments in choroid plexus epithelial cell primary cultures indicated that transport in these cells was polarized with [(14)C]ALA uptake from the apical (CSF) side being about 7-fold greater than uptake from the basolateral (blood) side. In total, these results suggest that the brain is normally fairly well protected from changes in plasma ALA concentration by the very low blood-brain barrier permeability of this compound and by a saturable efflux mechanism present at the choroid plexus.

Aminolevulinic Acid↗
PubMed2002

Novel quinolinone-phosphonic acid AMPA antagonists devoid of nephrotoxicity.

We reported previously the synthesis and structure-activity relationships (SAR) in a series of 2-(1H)-oxoquinolines bearing different acidic functions in the 3-position. Exploiting these SAR, we were able to identify 6,7-dichloro-2-(1H)-oxoquinoline-3-phosphonic acid compound 3 (S 17625) as a potent, in vivo active AMPA antagonist. Unfortunately, during the course of the development, nephrotoxicity was manifest at therapeutically effective doses. Considering that some similitude exists between S 17625 and probenecid, a compound known to protect against the nephrotoxicity and/or slow the clearance of different drugs, we decided to synthesise some new analogues of S 17625 incorporating some of the salient features of probenecid. Replacement of the chlorine in position 6 by a sulfonylamine led to very potent AMPA antagonists endowed with good in vivo activity and lacking nephrotoxicity potential. Amongst the compounds evaluated, derivatives 7a and 7s appear to be the most promising and are currently evaluated in therapeutically relevant stroke models.

Analysis of Variance↗
PubMed2002

Elimination of phenolsulfonphthalein from the cerebrospinal fluid via capillaries in central nervous system in cats by active transport.

It was recently proposed that organic anions, such as cerebral acidic metabolites and phenolsulfonphthalein (PSP), are eliminated from cerebrospinal fluid (CSF) by diffusion into the central nervous system (CNS) and further by active transport into capillaries. To test this hypothesis, PSP was injected into cisternal CSF and its distribution into various parts of the CNS was measured 1 and 3 h later in control cats and those pretreated with probenecid, which blocks active transport of organic anions into capillaries. PSP in tissue shows an intensive pink color when exposed to 1 N NaOH. Planimetric analysis of color pictures of coronal CNS slices showed that at the first hour, diffusion and distribution of PSP into the CNS in both groups of animals was similar, while at the third hour, a great reduction of PSP distribution in the CNS in control and only a slight reduction in probenecid pretreated cats was observed. The results support the hypothesis that active transport across the capillary wall in the CNS is the main avenue for elimination of cerebral acidic metabolites from both CSF and CNS and in such a way that central homeostasis is maintained.

Animals↗
PubMedDate not supplied

Nucleobase- and p-glycoprotein-mediated transport of AG337 in a Caco-2 cell culture model.

This study aimed to determine the intestinal transport characteristics of AG337. Caco-2 cell monolayers were used for directional transport of AG337. The results indicated that the rate of basolateral (BL) to apical (AP) or secretory transport of AG337 was always higher than the rate of AP to BL or absorptive transport. The BL-AP/AP-BL ratio increased from 7.6 to 17.2 as the concentration increased from 10 to 75 microM and stabilized afterward. The following results suggest that the p-glycoprotein is involved in the AP efflux of AG337. (1) Verapamil completely abolished the directional transport. (2) The apparent activation energy for secretory transport was high (33 kcal/mol). (3) Secretory transport was inhibited by verapamil (63%) and dipyridamole (35%), but not by probenecid. (4) AP to BL transport was enhanced by verapamil (232%) and dipyridamole (41%), but was not altered by probenecid or an AP pH change. An additional carrier mechanism may be involved in the AP uptake of AG337 because (1) absorptive permeabilities decreased as the concentration increased even though secretory permeabilities remained the same, (2) the absorptive transport rate was 37% lower in a medium containing verapamil and dipyridamole than in a medium containing only verapamil, and (3) the absorptive transport rate was also lower in a medium containing verapamil and a nucleobase mixture (consisting of adenine, hypoxanthine, and xanthine at 100 microM each) than in a medium containing verapamil only. Apical transport of AG337 in the Caco-2 cells is mediated, at least in part, by p-glycoprotein (efflux) and a nucleobase transporter that transports (uptake) nucleic bases.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

A human lymphocyte based ex vivo assay to study the effect of drugs on P-glycoprotein (P-gp) function.

PURPOSE: The effect of drugs on P-glycoprotein (P-gp) is normally studied in transfected or overexpressing cell lines derived from tumor cells or animal tissue. We wanted to develop an assay using normal healthy human tissue to study and characterize the drug-transporter interaction. METHODS: Lymphocytes were isolated from healthy human blood. The effect of inhibitors of P-gp (cyclosporine, tacrolimus, verapamil, quinidine, vinblastine) and of other transporters (indomethacin, probenecid, sulfinpyrazone) on intracellular accumulation of rhodamine 123 was evaluated by flow cytometry. RESULTS: The efflux of rhodamine 123 was inhibited by P-gp inhibitors in a saturable, concentration-dependent manner. The potency of inhibition of P-gp was cyclosporine > tacrolimus > quinidine > verapamil > vinblastine. Vinblastine inhibited P-gp at lower concentrations, whereas at high concentrations, there was an activation of rhodamine 123 efflux from lymphocytes. The multidrug resistance associated protein (MRP) inhibitors, sulfinpyrazone and probenecid, did not have any significant effect on intracellular accumulation of rhodamine 123, but indomethacin caused a concentration-dependent increase in retention of rhodamine 123, indicating the involvement of other uncharacterized transporters. CONCLUSIONS: Lymphocytes can serve as a model tissue for studying modulation of P-gp activity by drugs. Both inhibitors and inducers of P-gp activity can be evaluated.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Study on brain interstitial fluid distribution and blood-brain barrier transport of baclofen in rats by microdialysis.

PURPOSE: This study was performed to examine the distribution in the brain interstitial fluid (ISF) and the blood-brain barrier (BBB) transport of baclofen in rats by a microdialysis technique. METHODS: Following an i.v. bolus administration and/or the constant i.v. infusion of baclofen to the microdialysis cannula-bearing anesthetized rats, the concentrations of baclofen in the hippocampal ISF, whole brain tissue, cerebrospinal fluid (CSF), and plasma were determined by high-performance liquid chromatography (HPLC). Data were kinetically analyzed to estimate the transport parameters, i.e., the influx clearance (CLin) from plasma to brain and the efflux rate constant (keff) from brain to plasma, and the steady-state volume of distribution in the brain (Vd). RESULTS: The concentrations of baclofen in ISF, whole brain tissue, and CSF at the pseudo-steady state were almost 30-fold lower than the plasma unbound concentration, suggesting the restricted distribution of baclofen in the brain. The estimated values of CLin and keff were 0.00157 +/- 0.00076 ml/min/g of brain and 0.0872 +/- 0.0252 min-1, respectively. The efflux clearance (CLout) calculated by multiplying keff by Vd (0.816 +/- 0.559 ml/g of brain) was 0.0712 +/- 0.0529 ml/min/g of brain, and it was significantly 40-fold greater than the CLin value and fully greater than the convective flow in ISF. Furthermore, no significant concentration gradient was observed between ISF and CSF. These results suggest that the CLout value mainly reflects the efflux clearance through the BBB. Additionally, the hippocampal ISF/plasma concentration ratio of baclofen was markedly increased by both systemic administration of probenecid and its direct instillation into ISF. CONCLUSIONS: The restricted distribution of baclofen in the brain ISF may be ascribed to the efficient efflux from the brain through the BBB which is regulated possibly by a probenecid-sensitive organic anion transport system.

Animals↗
PubMed2004

Determination of carrier-mediated transport of 2',3'-dideoxypurine nucleosides in the rat ileum using a bidirectional perfusion technique.

PURPOSE: Previous attempts to ascertain the role of uptake and efflux transporters in the oral absorption of anti-HIV dideoxypurine nucleosides have been inconclusive. A novel in situ intestinal perfusion technique with complete mesenteric arterial/venous cannulation was used to examine the asymmetry of ileal dideoxynucleoside permeability under near in vivo conditions. METHODS: Intestinal perfusions were performed in the rat ileum, with cannulation of the artery and vein immediately entering and leaving the segment. Urea and mannitol were used as passive permeability markers, and the directional transport of 2',3'-dideoxyinosine (ddI), 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), and 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) were examined. RESULTS: Urea and mannitol exhibited symmetric permeability (PLtoB = PBtoL), whereas F-ddI and ddI showed significantly higher permeability in blood-to-lumen transport (PBtoL > PLtoB). PBtoL for F-ddA exceeded PLtoB, but the difference did not reach significance at p < 0.05. PBtoL for ddI was demonstrated to be saturable with increasing ddI concentrations, but PLtoB was independent of ddI concentration. PBtoL for ddI was shown to be dependent on sodium concentration and inhibited by probenecid. CONCLUSIONS: Symmetric transport was demonstrated for urea and mannitol as expected for these passive permeability markers. F-ddI and ddI were shown to be preferentially transported from blood to lumen. The basolateral to luminal transport of ddI is saturable, inhibited by probenecid, and sodium ion dependent. These results are consistent with carrier-mediated uptake on the basolateral membrane.

Animals↗
PubMed1989

Contraluminal organic anion and cation transport in the proximal renal tubule: V. Interaction with sulfamoyl- and phenoxy diuretics, and with beta-lactam antibiotics.

In order to study the interaction of sulfamoyl- and phenoxy diuretics as well as of beta-lactam antibiotics with the contraluminal anion and cation transport systems the inhibitory potency of these substances against the influx of 3H-para-aminohippurate, 14C-succinate, 35S-sulfate and 3H-N1-methylnicotinamide into cortical tubular cells have been determined. 1.) 2-, 3- and 4-sulfamoylbenzoate inhibit contraluminal PAH influx. N-dipropyl substitution to yield probenecid or ring-substitution to yield furosemide and piretanide augment the inhibitory potency. However, hydrochlorothiazide and acetazolamide exert only a moderate inhibitory potency. Succinate transport was inhibited by furosemide only. Sulfate transport was inhibited by furosemide and 3-sulfamoyl-4-phenoxybenzoate as well as by probenecid, piretanide, hydrochlorothiazide and acetazolamide. 2.) Phenoxyacetate, -propionate, and -butyrate exert increasing inhibition against PAH transport. The weed-killers 2,4-dichloro-, and 2,4,5-trichlorophenoxyacetate (2,4 D and 2,4,5 T) had a similar inhibitory potency, while ethacrynic acid showed a lower and the uricosuric tienilic acid a higher inhibitory potency. None of the compounds of this group interact with contraluminal succinate transport, and only the multiring-substituted compounds 2,4 D, 2,4,5 T, ethacrynic and tienilic acid interact slightly with the sulfate transporter. 3.) The monocarboxylic penicillins benzylpenicillin and phenoxymethylpenicillin as well as the dicarboxylic ticarcillin interact with the contraluminal PAH transport. The aminopenicillin ampicillin had a lower, and apalcillin a higher inhibitory potency than monocarboxylic penicillin. Benzylpenicillin showed small inhibition against succinate transport and ticarcillin against sulfate transport. 4.) The monocarboxylic cephalosporine, 6315 S Shionogi, and the aminocephalosporines, cephalexin and cefadroxil, showed an app. Ki.PAH as the comparable penicillins. The zwitterions cephaloridine and cefpirome did not interact with the PAH transporter, but with the organic cation (NMN) transporter. Amongst the amino-thiazol-containing compounds cefotaxime, ceftriaxone, and cefodizime, increasing interaction with the PAH transporter was seen dependent of a second ionizable anionic group. Compounds with two ionizable anionic groups (cefsulodin, ceftriaxone, cefodizime) exert also a small inhibitory potency against sulfate transport. None of the cephalosporins interacted with the dicarboxylate transporter. The interaction pattern of the tested compounds is in accordance with the specificity requirements for the contraluminal transporters depending on electrical charge and hydrophobicity.

Animals↗
PubMed2003

Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment.

(1) This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). (2) Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. (3) Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. (4) Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. (5) The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. (6) This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). (7) Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. (8) Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid.

Animals↗
PubMed2003

Hydroxyurea transport across the blood-brain and blood-cerebrospinal fluid barriers of the guinea-pig.

Hydroxyurea is used in the treatment of HIV infection in combination with nucleoside analogues, 2'3'-didehydro-3'deoxythymidine (D4T), 2'3'-dideoxyinosine or abacavir. It is distributed into human CSF and is transported from the CSF to sub-ependymal brain sites, but its movement into the brain directly from the blood has not been studied. This study addressed this by a brain perfusion technique in anaesthetized guinea-pigs. The carotid arteries were perfused with an artificial plasma containing [14C]hydroxyurea (1.6 microm) and a vascular marker, [3H]mannitol (4.6 nm). Brain uptake of [14C]hydroxyurea (8.0 +/- 0.9%) was greater than [3H]mannitol (2.4 +/- 0.2%; 20-min perfusion, n = 8). CSF uptake of [14C]hydroxyurea (5.6 +/- 1.5%) was also greater than [3H]mannitol (0.9 +/- 0.3%; n = 4). Brain uptake of [14C]hydroxyurea was increased by 200 microm hydroxyurea, 90 microm D4T, 350 microm probenecid, 25 microm digoxin, but not by 120 microm hydroxyurea, 16.5-50 microm D4T, 90 microm 2'3'-dideoxyinosine or 90 microm abacavir. [14C]Hydroxyurea distribution to the CSF, choroid plexus and pituitary gland remained unaffected by all these drugs. The metabolic half-life of hydroxyurea was > 15 h in brain and plasma. Results indicate that intact hydroxyurea can cross the brain barriers, but is removed from the brain by probenecid- and digoxin-sensitive transport mechanisms at the blood-brain barrier, which are also affected by D4T. These sensitivities implicate an organic anion transporter (probably organic anion transporting polypeptide 2) and possibly p-glycoprotein in the brain distribution of hydroxyurea and D4T.

Animals↗
PubMed2000

Developmental changes in multispecific organic anion transporter 1 expression in the rat kidney.

BACKGROUND: The cDNA of the multispecific organic anion transporter 1 (OAT1) responsible for the tubular secretion of organic anions was recently isolated. In the current study, we investigated the developmental changes in OAT1 expression in the rat kidney. METHODS: Ontogenic expression of rat OAT1 was investigated by Northern blot, in situ hybridization, Western blot, and immunohistochemical analysis. In addition, para-aminohippurate (PAH) accumulation was measured using fetal, neonatal, and adult rat kidney slices. RESULTS: In Northern blot analysis, OAT1 was detected as early as on embryonic day 18 in the fetal kidney. The expression level of OAT1 mRNA increased remarkably just after birth (postnatal day 0). In situ hybridization revealed OAT1 expression on embryonic day 19. In both the fetal and neonatal kidneys, OAT1 mRNA was localized in a relatively deep region in the cortex. Western blot analysis detected OAT1 protein on embryonic day 20, and the expression level increased after birth. Immunohistochemical analysis did not reveal OAT1 staining in the fetal kidneys. A faint signal of OAT1 protein was detected on postnatal day 0; thereafter, the expression level increased. In the functional study using kidney slices, low but definite probenecid-sensitive PAH accumulation was noted in fetal rat kidney on embryonic day 20. After birth, probenecid-sensitive PAH uptake was increased. CONCLUSIONS: The present study consistently demonstrates the remarkable increase of OAT1 expression after birth, and the immature excretory capacity of the proximal tubules of the neonatal kidney can be attributed, at least in part, to the low expression level of OAT1.

Aging↗
PubMed2002

Transport of anionic drugs across the basolateral membrane of proximal S2 segments of the rabbit kidney. Inverse relationship between the affinity to the p-aminohippurate transport system and the transport rate.

The renal p-aminohippurate (PAH) transport system, which resides in the S2 segments of proximal tubules, is a main tubular secretory system for drugs. Previous studies have shown that the hydrophobicity of drugs is positively correlated with their affinity for the basolateral PAH transporter. Affinity was deduced from inhibition of tubular 3H-PAH uptake. However, up to now, no studies are available which tested the relationship between hydrophobicity and transport rate of drugs. Therefore, in the present study, the basolateral transport rates of several drugs (probenecid, CAS 57-66-9, furosemide, CAS 54-31-9, bumetanide, CAS 28395-03-1, mefruside, CAS 7195-27-9) which are substrates of the PAH transporter and which differ markedly regarding their hydrophobic properties (log P values ranging from 1-3) were determined on non-perfused proximal S2 segments microdissected from rabbit kidneys without the use of enzymatic solutions. To measure the transport rate advantage of the finding was taken that the PAH transporter in the basolateral membrane of proximal S2 segments acts as a countertransporter which exchanges PAH with dicarboxylates (glutarate or alpha-keto-glutarate). Hence, the stimulatory effect of a drug on the 14C-glutarate efflux rate of 14C-glutarate preloaded S2 segments is a measure of its translocation across the basolateral cell membrane. The results revealed that with increasing hydrophobicity (mefruside < furosemide < bumetanide < probenecid) the stimulating effect of the drugs on 14C-glutarate efflux decreased indicating that the compounds are more slowly translocated although their potency to inhibit 3H-PAH uptake increased. Thus, an inverse relationship between hydrophobicity and hence affinity of the drugs for the PAH transporter and transport rate could be established.

Algorithms↗