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Cytosolic calcium concentrations (Ca(i)) of barley aleurone protoplasts after stimulation with the plant hormone abscisic acid (ABA) were measured by using the calcium-sensitive fluorescent dye Indo-1. The measuredd basal Ca(i) is about 200 nM. Stimulation with ABA induces a strong dose-dependent decrease in Ca(i) to a minimal value of about 50 nM. This decrease occurs within 5 s. The Ca2+ antagonists La3+ and Cd2+ inhibit the ABA-induced Ca(i) decrease in a dose-dependent manner, while the Ca2+ channel blockers verapamil and nifedipine give no inhibition. The induction of Ca(i) decrease by ABA is consistent with activation of the plasma membrane Ca2+-ATPase by ABA. The possible role of this ABA-induced Ca(i) decrease in ABA signal transduction and in counteracting the effects of gibberellic acid are discussed. Chemicals/CAS: Histocompatibility Antigens Class IIChemicals/CAS: Abscisic Acid, 21293-29-8; Ca(2+)-Transporting ATPase, EC 3.6.1.38; Calcium, 7440-70-2; Fluorescent Dyes; indo-1, 96314-96-4; Indoles; Nifedipine, 21829-25-4; Verapamil, 52-53-9

Purpose: previous studies have shown that the rat small intestinal cell line IEC-18 provides a size-selective barrier for paracellularly transported hydrophilic macromolecules. In order to determine the utility of IEC-18 cells as an in vitro model to screen the passive paracellular and transcellular components of the intestinal transport of nutrients and drugs, we have now examined the transport of GlySar (H+-coupled di/tripeptide carrier), O-methyl-D-glucose (glucose carrier), vincristine and rhodamine 123 (P-glycoprotein), and calcein and DNPSG (MRPs) and the bidirectional transport of paracellularly transported compounds. Transport of these compounds across the filter grown IEC-18 cells was compared with transport across the human colon carcinoma Caco-2 cells. Results: in IEC-18 cells, transepithelial transport of GlySar and methylglucose was as fast as the transport of mannitol, which is transported passively via the paracellular route. Whereas in Caco-2 cells, mannitol transport was much slower than the transport of GlySar and methylglucose. In contrast to Caco-2 cells, no H+-coupled transport of GlySar could be measured in IEC-18 cells. P-Glycoprotein-mediated transport was characterised in Caco-2 cells by an enhanced transport of vincristine and rhodamine 123 in the basolateral to apical direction and by the inhibition of this transport by verapamil. In IEC-18 cells, permeability of vincristine and rhodamine 123 was similar in both directions and verapamil had no effect on the transport of these compounds. Both IEC-18 and Caco-2 cells efflux the organic anions calcein and DNPSG to the apical and basolateral compartments, and this efflux could be inhibited by probenecid. Conclusions: in conclusion, no carrier-mediated transport of GlySar, methylglucose, vincristine and rhodamine 123 could be determined in IEC-18 cells in contrast to Caco-2 cells. However, both IEC-18 and Caco-2 cells showed MRP-mediated eflux system(s) in the apical and basolateral membrane. Monolayers of IEC-18 cells appear to be more suitable than monolayers of Caco-2 cells as an in vitro system to screen the passive component of the intestinal transport in a deconvoluted screening regimen, where passive transport is represented by the IEC-18 monolayer permeability and active transport is represented by monolayers of cells expressing the transport proteins heterologously. © 2002 Elsevier Science B.V. All rights reserved.

A reliable prediction of the oral bioavailability in humans is crucial and of high interest for pharmaceutical and food industry. The predictive value of currently used in silico methods, in vitro cell lines, ex vivo intestinal tissue and/or in vivo animal studies for human intestinal absorption, however, is often insufficient, especially when food-drug interactions are evaluated. Ideally, for this purpose healthy human intestinal tissue is used, but due to its limited availability there is a need for alternatives. The aim of this study was to evaluate the applicability of healthy porcine intestinal tissue mounted in a newly developed InTESTine™ system to predict human intestinal absorption of compounds with different chemical characteristics, and within biorelevant matrices. To that end, first, a representative set of compounds was chosen of which the apparent permeability (Papp) data in both Caco-2 cells and human intestinal tissue mounted in the Ussing chamber system, and absolute human oral bioavailability were reported. Thereafter, Papp values of the subset were determined in both porcine jejunal tissue and our own Caco-2 cells. In addition, the feasibility of this new approach to study regional differences (duodenum, jejunum, and ileum) in permeability of compounds and to study the effects of luminal factors on permeability was also investigated. For the latter, a comparison was made between the compatibility of porcine intestinal tissue, Caco-2 cells, and Caco-2 cells co-cultured with the mucin producing HT29-MTX cells with biorelevant samples as collected from an in vitro dynamic gastrointestinal model (TIM). The results demonstrated that for the paracellularly transported compounds atenolol, cimetidine, mannitol and ranitidine porcine Papp values are within 3-fold difference of human Papp values, whereas the Caco-2 Papp values are beyond 3-fold difference. Overall, the porcine intestinal tissue Papp values are more comparable to human Papp values (9 out of 12 are within 3-fold difference), compared to Caco-2 Papp values (4 out of 12 are within 3-fold difference). In addition, for the selected hydrophilic compounds a significant increase in the permeability was observed from duodenum to ileum. Finally, this study indicated that porcine jejunal tissue segments can be used with undiluted luminal samples to predict human intestinal permeability and the effect of biorelevant matrices on this. In conclusion, viable porcine intestinal tissue mounted in the InTESTine™ system can be applied as a reliable tool for the assessment of intestinal permeability in the absence and presence of biorelevant samples. This would enable an accessible opportunity for a reliable prediction of human intestinal absorption, and the effect of luminal compounds such as digested foods, early in drug development. © 2014 Elsevier B.V. All rights reserved. Chemicals/CAS: acebutolol, 34381-68-5, 37517-30-9; atenolol, 29122-68-7, 93379-54-5; candesartan, 139481-59-7; cetirizine, 83881-51-0, 83881-52-1; cimetidine, 51481-61-9, 70059-30-2; ciprofloxacin, 85721-33-1; diazepam, 439-14-5; digoxin, 20830-75-5, 57285-89-9; famotidine, 76824-35-6; hydrocortisone, 50-23-7; ibuprofen, 15687-27-1, 79261-49-7, 31121-93-4, 527688-20-6; indometacin, 53-86-1, 74252-25-8, 7681-54-1; mannitol, 69-65-8, 87-78-5; melagatran, 159776-70-2; metoprolol, 37350-58-6; oxprenolol, 22972-97-0, 6452-71-7, 6452-73-9; phenazone, 60-80-0; pindolol, 13523-86-9, 21870-06-4; propranolol, 13013-17-7, 318-98-9, 3506-09-0, 4199-09-1, 525-66-6; ranitidine, 66357-35-5, 66357-59-3; salazosulfapyridine, 599-79-1; salicylic acid, 63-36-5, 69-72-7; testosterone, 58-22-0; verapamil, 152-11-4, 52-53-9 Manufacturers: Gibco, United Kingdom; Sigma Aldrich, Netherlands; Newport, United States

Purpose. The purpose of this work was to study the relevant physicochemical properties for the absorption of steroids. Methods. Various physicochemical properties of steroids were calculated (molecular weight, ClogP, static polar surface area [PSA], etc.). Within this series of steroids, different pharmacological groups were defined. Based on the outcome of this survey, steroids were selected for the Caco-2 permeability study. The apparent permeability coefficients (Papp) were related to the calculated and measured physicochemical properties. Results. Between the defined groups of steroids, ClogP was the most discriminative descriptor. The steroids were well transported over the cell monolayers and the Papp was independent of the concentration and the transport direction. No relationship was found with the PSA; however, the Papp showed a weak inverse correlation with ClogP. Conclusions. The molecular descriptors and Papp values showed that all steroids are well transported. The small differences in the Papp values showed a weak inverse correlation with ClogP: the hydrophilic steroids (ClogP approximately 0-2) tend to diffuse faster over the cell monolayers compared with the more hydrophobic steroids (ClogP approximately 5). The relationship with ClogP suggests that partitioning of steroids between the biologic membrane and the surrounding aqueous phase is one of the main mechanisms for absorption.

The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich-culture and 14 different metabolites in microsomes. Ketoconazole did not affect the metabolism of ritonavir in sandwich-culture or microsomes and slightly inhibited the metabolism of saquinavir in sandwich-culture. This inhibition resulted in a different metabolite profile for saquinavir in microsomes. Ritonavir had a pronounced inhibiting effect on the metabolism of saquinavir and affected the hydroxylation of 6β-testosterone negatively. In the active transport studies, cyclosporin A and PSC833 enhanced the metabolism of ritonavir, suggesting that ritonavir is normally excreted into the bile canaliculi. Verapamil, showed no effect on the metabolism of ritonavir. The intrinsic clearance was estimated at 1.65 and 67.5 μl/min/1 × 106 cells and the hepatic metabolism clearance at 0.017 and 6.83 ml/min/SRW for ritonavir and saquinavir respectively. In conclusion, for saquinavir the metabolism rate and the amount of metabolites produced was higher than for ritonavir. Ritonavir had a strong inhibitory effect on the metabolism of saquinavir and seemed to be excreted into the bile. © 2008 Elsevier Ltd. All rights reserved.

Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer’s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. Chemicals/CAS: calcein, 1461-15-0; multidrug resistance protein, 149200-37-3, 208997-77-7

Pharmacological modulation of the in vivo induction of plasminogen activator inhibitor type-1 (PAI-1) synthesis was studied in rats using the induction of PAI-1 by endotoxin as a model system. Both the cyclooxygenase inhibitors acetylsalicylic acid and indomethacin enhanced PAI-1 induction. The combined cyclooxygenase-lipoxygenase inhibitor, BW755C, dose-dependently inhibited induction. Since five other lipoxygenase inhibitors, a phospholipase inhibitor, an inhibitor of leukotriene formation and dexamethasone had no effect on the endotoxin-induced increase in PAI-1 synthesis, the effect of BW755C could not be ascribed to its known pharmacological properties. In addition, induction of PAI was enhanced by isobutyl-methylxanthine, a phosphodiesterase inhibitor, but not, however, by other phosphodiesterase inhibitors, or by forskolin or N(G)-nitro-L-arginine, suggesting an effect of isobutyl-methylxanthine other than through cyclic nucleotides. Heparin and hirudin had no effect either. Overall, the data showed that the induction of PAI-1 synthesis by endotoxin in vivo can be up- and down-regulated pharmacologically, but the mechanisms involved remain elusive. Chemicals/CAS: 2 (3,4 dihydro 3,4 dioxo 1 naphthylamino)benzoic acid methyl ester, 114832-13-2; 3 [3 tert butylthio 1 (4 chlorobenzyl) 5 isopropyl 2 indolyl] 2,2 dimethylpropionic acid, 118414-82-7; 3 amino 1 (3 trifluoromethylphenyl) 2 pyrazoline, 66000-40-6; acetylsalicylic acid, 493-53-8, 50-78-2, 53663-74-4, 53664-49-6, 63781-77-1; cycloheximide, 642-81-9, 66-81-9; dexamethasone, 50-02-2; diltiazem, 33286-22-5, 42399-41-7; forskolin, 66575-29-9; ginkgolide B, 15291-77-7, 99796-69-7; heparin, 37187-54-5, 8057-48-5, 8065-01-8, 9005-48-5; hirudin, 8001-27-2; indometacin, 53-86-1, 74252-25-8, 7681-54-1; isobutylmethylxanthine, 28822-58-4; mepyramine maleate, 59-33-6; n [3 (3 phenoxyphenyl)allyl]acetohydroxamic acid, 106328-57-8; n(g) nitroarginine methyl ester, 50903-99-6; naloxone, 357-08-4, 465-65-6; phentolamine mesylate, 65-28-1; phentolamine, 50-60-2, 73-05-2; plasminogen activator inhibitor, 105844-41-5; propranolol, 13013-17-7, 318-98-9, 3506-09-0, 4199-09-1, 525-66-6; verapamil, 152-11-4, 52-53-9; 1-Methyl-3-isobutylxanthine, 28822-58-4; Aspirin, 50-78-2; BW-755C, 66000-40-6; Indomethacin, 53-86-1; Lipopolysaccharides; Plasminogen Activator Inhibitor 1; Tissue Plasminogen Activator, EC 3.4.21.68