Effect of Rodent Hepatocarcinogenic Peroxisome Proliferators on Fatty Acyl-CoA Oxidase, DNA Synthesis, and Apoptosis in Cultured Human and Rat Hepatocytes

The effects of the rodent hepatocarcinogens clofibric acid and ciprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP–biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-β (TGFβ)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGFβ-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGFβ-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.     

Comparative Effects of Fibrates on Drug Metabolizing Enzymes in Human Hepatocytes

No HeadingPurpose. The induction potential of different fibric acid derivatives on human drug metabolizing enzymes was evaluated to help assess the role of enzyme induction on pharmacokinetic drug interactions.Methods. Effects of gemfibrozil, fenofibric acid, and clofibric acid on expression levels of cytochromes P450 (CYPs) 3A4 and 2C8 and UDP-glucuronyltransferase (UGT) 1A1 were evaluated in primary human hepatocyte cultures. The potential for these fibrates to activate human pregnane X receptor (PXR) also was studied in a cell-based PXR reporter gene assay.Results. All three fibrates caused increases in mRNA levels of CYP3A4 (2- to 5-fold), CYP2C8 (2- to 6-fold), and UGT1A1 (2- to 3-fold). On average, the effects on CYP3A4 were less than (30% of rifampin), while those on CYP2C8 and UGT1A1 were comparable to or slightly higher than (up to 200% of rifampin) the corresponding effects observed with rifampin (10 M). Consistent with the mRNA results, all fibrates caused moderate (<2- to 3-fold) increases in CYP3A4 activity (measured by testosterone 6 hydroxylase), as compared to about a 10-fold increase by rifampin. Significant increases (3- to 6-fold) in amodiaquine N-deethylase (a functional probe for CYP2C8 activity) also were observed with clofibric acid, fenofibric acid, and rifampin, in agreement with the mRNA finding. However, in contrast to the mRNA induction, marked decreases (>60%) in CYP2C8 activity were obtained with gemfibrozil treatment. Consistent with this finding, co-incubation of amodiaquine with gemfibrozil, but not with fenofibric acid, clofibric acid, or rifampin, in human liver microsomes or hepatocytes resulted in significantly decreased amodiaquine N-deethylase activity (IC₅₀ = 80 M for gemfibrozil, >500 M for fenofibric or clofibric acid, and >50 M for rifampin). Similar to rifampin, all three fibrates caused a modest change in the glucuronidation of chrysin, a nonspecific substrate of UGTs. No significant activation on human pregnane X receptor (PXR) was observed with the three fibrates in a PXR reporter gene assay.Conclusions. In human hepatocytes, both fenofibric acid and clofibric acid are inducers of CYP3A4 and CYP2C8. Gemfibrozil is also an inducer of CYP3A4, but acts as both an inducer and an inhibitor of CYP2C8. In this system, all fibrates are weak inducers of UGT1A1. The enzyme inducing effects of fibrates appear to be mediated via a mechanism(s) other than PXR activation. These results suggest that fibrates may have potential to cause various pharmacokinetic drug interactions via their differential effects on enzyme induction and/or inhibition.     

Fast uptake kinetics in vitro of 51Cr (VI) by red blood cells of man and rat

Fast transport kinetics of ⁵¹Cr (VI) into red blood cells (RBCs) in vitro were studied. No significant species differences were found between RBCs of man and rat. The uptake of ⁵¹Cr (VI) by RBCs in whole blood was composed of two different first order processes of different velocities (apparent t_1/2 of 22.7 s and 10.4 min for man and 6.9 s and 10.1 min for rat, respectively). However, even after longer time periods a fixed portion of approximately 15% of the administered dose remained in the plasma and did not penetrate into RBCs Over the entire concentration range studied (10 M–50 mM), the fast initial uptake followed Michaelis-Menten kinetics. The maximal capacity of this Cr(VI) transport into RBCs of man and rat was 3.1×10⁸ CrO₄ ^2– ions × cell^–1 × min^–1 and 2.5×10⁸ CrO₄ ^–2 ions × cell^–1 × min^–1, respectively. It is likely that Cr(VI) is transported into RBCs via a physiological anion carrier (band-3-protein).     

Hydroxypropyl-β-Cyclodextrin Increases the Aqueous Solubility and Stability of Pilocarpine Prodrugs

Purpose. The effects of 2-hydroxypropyl--cyclodextrin (HP--CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. Methods. The solubility of prodrugs was studied by phase-solubility method (0–72.5 mM HP--CD). The stability of one of the prodrugs was investigated as a function of temperature (40°C–70°C) and HP--CD concentration (0–72.5 mM). The apparent rate constants (k ₁, k ₂) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K _1:1, K _l:2) were calculated by the curve-fitting method. Results. The phase-solubility diagrams were classified as A_p-type and the apparent stability constants (K _l:l, K _l:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143–815 M^–l and 29–825 M^–1, respectively. The stability of prodrug increased as a function of HP--CD concentration over the studied temperature range. The shelf-life (t _90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP--CD solution increased 5.1-fold and 6.1-fold at 25°C and 4°C, respectively. Conclusions. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the l:2-complex than in the l:l-complex and the relative amounts of complex species were found to be dependent on CD concentration.     

In vivo studies of mucosal-serosal transfer in rat jejunum

Summary In anesthetized rats, the appearance rates of a series of labeled substances in jejunal venous blood ( _B) and serosal bath ( _S) were measured in vivo (intestinal blood flow rate 1.5 ml min^–1g^–1) after intraluminal administration of 0.5 ml buffer solution (initial concentration 1 mmol/l or 1 GBq/l tritiated water) into a closed jejunal segment (length 4–5cm). Between 32% (erythritol) and 93% (salicylic acid) of the administered activity (unchanged substance and possible metabolites) appeared in the intestinal venous blood within 60 min. The fraction recovered from the serosal bath after 15 (60) min was 11 (6) % for tritiated water, 7 (4) % for aniline, 3 (7)% for aminopyrine, 5 (4) % for butanol, 3 (3) % for benzyl alcohol, 2 (4) % for benzylamine, 1–2% for benzoic acid, theophyline, methyl--d-glucopyranoside,l-lysine, antipyrine, and urea, and less than 1% forl-phenylalanine,d-galactose, erythritol, and salicylic acid. During single pass perfusion of a jejunal segment (length 3–4 cm) the fraction of serosal transfer _S/( _B+ _S) was 19% for tritiated water, 4.9% for antipyrine, 0.5% for benzoic acid, and 0.08% for salicylic acid.Distension of the intestinal wall by administration of 1 ml buffer solution instead of 0.5 ml increased the appearance rate of benzoic acid and antipyrine in intestinal venous blood by a factor of 2 and serosal transfer by a factor of approximately 3. Reduction of blood flow rate from 1.26 to 0.91 ml min^–1g^–1 decreased the fraction of antipyrine recovered in the intestinal venous blood within 60 min from 84% to 72% and increased the fraction transferred into the serosal bath from 2.3% to 9.1%. A high mucosal-serosal transfer in rat small intestine with intact blood supply can be expected for substances with high epithelial permeability and low binding to plasma proteins.     

Motor effects following application of putative excitatory amino acid antagonists to the region of the mesencephalic dopamine cell bodies in the rat

Summary The excitatory amino acid antagonists 2-amino-5-phosphonovalerate and -D-glutamylglycine have been applied focally to the ventral tegmental area and both the pars compacta and pars reticulata of the substantia nigra of the rat. The injections were performed under halothane anaesthesia so that behavioural effects could be observed 5 min afterwards. Bilateral application of either antagonist to the ventral tegmental area and the pars compacta of the substantia nigra induced enhanced locomotor activity in an open field. This effect was blocked by pretreatment of the animals with a low dose of the dopamine receptor antagonist fluphenazine. Bilateral application of either antagonist to the pars reticulata of the substantia nigra produced sedation and a reduction in locomotor activity. Unilateral injection of either of the excitatory amino acid antagonists into the pars reticulata or pars compacta of the substantia nigra both resulted in contraversive circling behaviour The effect of intranigral (both pars compacta and reticulata) 2-APV and -DGG was accompanied by a significant increase in concentrations of both 3,4-dihydroxyphenylacetic acid (to 158–160% of control following injection into pars compacta, and 134–146% of control injected into pars reticulata) and homovanillic acid (to 161–166% of control following injection into pars compacta, and 186–210% of control injected into pars reticulata) in the ipsilateral, striatum. Pretreatment of these animals with fluphenazine (0.3 mg/kg) antagonized this circling behaviour.These results indicate that antagonism, of excitatory amino acid receptors in the region of the midbrain of the rat leads to specific behavioural effects, which may in part be mediated through the ascending dopaminergic projections.Abbreviations used 2-APV 2-amino-5-phosphonovaleric acid - -DGG -D-glutamylglycine - SN substantia nigra - VTA ventral tegmental area - HVA homovanillic acid - DOPAC 3,4-dihydroxyphenylacetic acid A preliminary communication of this work was presented to the British Pharmacological Society in Bradford, April 1981     

Evidence that histamine H3 receptors are involved in the control of gastric acid secretion in the conscious cat

Summary In an attempt to assess the role of histamine H₃ receptors in the control of gastric acid secretion, the effects of the selective histamine H3 receptor agonist, (R)-methylhistamine and antagonist, thioperamide were evaluated in the conscious gastric fistula cat under basal conditions and against different stimuli. (R)-methylhistamine (0.05–0.2 mol/kg/h) was ineffective against spontaneous and dimaprit-induced acid secretion; it also did not reduce significantly pentagastr-ininduced acid output, but caused a dose-dependent (0.05–0.1 mol/kg/h) and significant inhibition of the acid response to 2-deoxy-d-glucose. Thioperamide (0.02–0.04 mol/kg/h) did not modify spontaneous acid secretion, whereas it evoked a significant enhancement of the acid response to submaximal doses (50 mg/kg i. v.) of 2-deoxy-d-glucose. Thioperamide completely reversed the inhibitory effect of (R)-methylhistamine against 2-deoxy-d-glucose-induced secretion, while leaving unaffected the inhibition induced by somatostatin. These data suggest that histamine H3 receptors may be involved in the control of acid secretion stimulated by indirectly acting secretagogues. Send offprint requests to G. Bertaccini at the above address     

Cost-effectiveness of periconceptional supplementation of folic acid

Background: Supplementation of folic acid prior to and in the beginning of pregnancy may prevent neural tube defects (NTDs) in newborns – such as spina bifida – and possibly other congenital malformations.Objective: To estimate cost effectiveness of periconceptional supplementation of folic acid using pharmacoeconomic model calculation.Method: Probabilities for NTDs, risk reductions through periconceptional supplementation of folic acid and lifetime costs of care for children with spina bifida were estimated using Dutch registrations and international literature.Main outcome measure: Cost effectiveness was expressed in net costs per discounted lifeyear gained. Cost effectiveness was calculated in the baseline and in sensitivity analysis.Results: Estimated cost effectiveness of periconceptional supplementation of folic acid amounts to NLG 3900(D1800) in the base case. In sensitivity analysis cost effectiveness mostly remains below NLG 10.000(D4500).Conclusion: Periconceptional supplementation of folic acid shows a favorable cost effectiveness. From pharmacoeconomic point of view this justifies further stimulation of folicacid supplementation prior to pregnancy. This can be done through targeted education by healthcare workers, such as pharmacists.     

Effects of clofibric and beclobric acid in rat and monkey hepatocyte primary culture: influence on peroxisomal and mitochondrial β-oxidation and the activity of catalase,glutathione S-transferase and glutathione peroxidase

The effect of hypolipidaemic compounds on peroxisomal fatty acid β-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial β-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial β-oxidation activities in intact cells. Overall β-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate β-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal β-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate β-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells. When a comparison between both species is made for the ratio of hydrogen peroxide production over catalase activity, it can be concluded that this peroxide will have much smaller possibilities to escape from the peroxisomal compartment in monkey hepatocytes. These findings suggest that species differences in these enzyme activities can contribute to differences in susceptibility for peroxisome proliferator-induced carcinogenicity between rodents and primates. Received: 3 January 1994/Accepted: 11 April 1994     

The mycotoxin ochratoxin A is a substrate for phenylalanine hydroxylase in isolated rat hepatocytes and in vivo

Ochratoxin A (OTA), is a mycotoxin contaminating food and feed stuffs, consisting of a chlorinated dihydroisocoumarin linked through a 7-carboxyl group tol-phenylalanine by an amide bond. When OTA (0.12–1.4 mM) is incubated with freshly isolated rat hepatocytes, it inhibits both the hydroxylation of phenylalanine (0.05 mM) to tyrosine, catalyzed by phenylalanine hydroxylase and the subsequent metabolism of tyrosine as measured by homogentisate oxidation. The IC₅₀ of OTA for phenylalanine hydroxylation is 0.43 mM. OT, (0.5–1.0 mM), the dihydroisocoumarin moiety of OTA, does not inhibit phenylalanine hydroxylase activity under these conditions. During incubations of hepatocytes with uniformly labelled [³H]-OTA and unlabelled phenylalanine, tyrosine-ochratoxin A is formed (up to 6% of the total mycotoxin added), indicating that ochratoxin can act as a substrate for phenylalanine hydroxylase. In vivo tyrosine-OTA is also found in liver of poisoned animals.     

Multidrug Resistance—Associated Protein 1 Functions as an Efflux Pump of Xenobiotics in the Skin

Purpose Recent research has identified gene expression of several types of xenobiotic transporters in the skin. The aim of this study was to investigate whether multidrug resistance–associated protein 1 (MRP1) functions in the skin.Methods The distribution of [¹⁴C]grepafloxacin in vivo and the transport of 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N,N-tetraacetic acid (fluo 3) were examined in the skin of Mrp1 knockout mice [FVB/Mrp1(–/–)] and normal mice [FVB/Mrp1(+/+)].Results The tissue-to-plasma concentration ratio of [¹⁴C]grepafloxacin was higher in the skin of FVB/Mrp1(–/–) mice than that of FVB/Mrp1(+/+) mice. In skin slices of hairless mouse incubated with fluo 3 pentaacetoxymethyl ester, the accumulation of fluo 3 was significantly increased in the presence of probenecid (2 mM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 M) in a time-dependent manner but did not change in the presence of tetraethylammonium (2 mM). In FVB/Mrp1(–/–) mouse skin, the accumulation of fluo 3 increased time-dependently, while no increase was observed in FVB/Mrp1(+/+) mouse skin.Conclusions These findings suggest that Mrp1 is involved in the efflux of [¹⁴C]grepafloxacin and fluo 3 in the skin, possibly acting as part of a barrier system against xenobiotic compounds.     

Hydrophobic membrane interaction of etidocaine,bupivacaine and 2-chloroprocaine

Summary It has been suggested that local anesthetics may block sodium conductance through nervous membranes also by hydrophobic interaction, e.g., by expanding the membrane. Decreased anisotropy (fluidization) and depressed phase transition temperatures have been shown by relatively high local anesthetic concentrations. We studied the dose dependence of the effect of three clinically used local anesthetics, with different lipid solubility, on lipid fluidity parameters of four different model membranes. With stearic acid spin labels in dipalmitoyl lecithin vesicles etidocaine (1–5 mM) had the clearest fluidizing effect followed by bupivacaine (5 mM); 2-chloroprocaine was without effect on lipid fluidity. In synaptic plasma membranes a fluidizing effect near the hydrophilic part of the lipid bilayer was similar with etidocaine and bupivacaine (5–10 mM); 2-chloroprocaine had no effect. Bupivacaine at 125 and 250 M had a small ordering effect, which was not seen at a more hydrophobic site of the membrane. Etidocaine had the strongest fluidizing effect at the latter site of the synaptic plasma membranes. In erythrocyte ghost membranes, probed by stearic acid spin labels near the hydrophilic end, none of local anesthetics affected fluidity at 24° C, while at 37° C etidocaine (1–5 mM) and bupivacaine (5 mM) had a fluidizing effect. Dimyristoyl lecithin vesicles were probed by cis-and trans-parinaric acid. Etidocaine and bupivacaine (5–10 mM) clearly depressed the phase transition temperature evaluated from fluorescence intensity scans. The effect was most marked with bupivacaine (1–10 mM) when dis-parinaric acid was used. While isolated mammalian nerves are blocked by local anesthetic concentrations below 100 M, this study shows that the clinically used local anesthetics increase fluidity and depress phase transition temperature only at 10–100 times higher concentrations at physiological pH. This kind of hydrophobic membrane interaction may not be important for the nerve blocking effect.     

Opposing effects on blood pressure following the activation of metabotropic and ionotropic glutamate receptors in raphe obscurus in the anaesthetized rat

The microinjection of l-glutamate (1–6 nmol/rat) and N-methyl-d-aspartate (NMDA 1–10 nmol/rat), ionotropic glutamate receptor (iGluR) agonists, into the nucleus raphe obscurus caused a concentration-dependent increase of arterial blood pressure. In contrast, (±)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD, 14–42 nmol/rat), a metabotropic glutamate receptor (mGluRs) agonist, caused a concentration-dependent decrease in blood pressure. Pretreatment with D, L-2-amino-phosphono valeric acid (2-APV, 5 nmol/rat) a selective NMDA iGluR antagonist, and (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, b] cyclohepten-5,10-imine hydrogen maleate (MK801, 0.9 nmol/rat), a noncompetitive NMDA iGluR antagonist, blocked both the glutamate and NMDA pressor responses, while pretreatment with (+)--methyl-4-carboxyphenylglycine (MCPG, 0.05 nmol/rat), a mGluR₁ antagonist, increased the glutamate-induced pressor effects and blocked the fall in blood pressure induced by t-ACPD. 6-Cyano-7-nitroquinoxaline-2,3dione-(CNQX, 0.4 nmol/rat) a non-NMDA iGluR antagonist, did not affected the glutamate-induced hypertension. These observations indicate opposing roles for ionotropic and metabotropic receptors in the glutamate-induced blood pressure changes elicited from the nucleus raphe obscurus. Moreover, we suggest that the glutamate-induced hypertension may be due to the activation of NMDA ionotropic receptor subtypes and the metabotropic receptors may influence this activaction through a reduction of excitability at level of synapses.     

Improved Corneal Pilocarpine Permeability with O,O′-(l,4-Xylylene) Bispilocarpic Acid Ester Double Prodrugs

0,0-(l,4-Xylylene) bispilocarpic acid esters are pilocarpine pro-drugs containing two pilocarpic acid monoesters linked with one pro-moiety. Each mole of prodrug forms two pilocarpine moles in the presence of esterases. Corneal uptake and permeability of various bispilocarpic acid diesters were investigated in vitro using isolated albino rabbit corneas. The permeability coefficient of pilocarpine was 2.8 × 10 ^–6 cm/sec, whereas for bispilocarpic acid diesters, despite their large molecular weights (between 638 and 722), permeability coefficients were 6.5–20.2 × 10 ^–6 cm/sec. Only pilocarpine, and no intact prodrug, was observed at the endothelial side. Corneal uptake was increased with increasing lipophilicity, but a parabolic relationship between the logarithm of the apparent partition coefficient (1-octanol–pH 7.4 phosphate buffer) (log PC) and the corneal permeability was noticed. Corneal permeability and the rate of enzymatic hydrolysis of the compounds correlated well. The corneal permeability of pilocarpine given as lipophilic bispilocarpic acid diester (log PC 3) prodrugs seems to be controlled by the formation of pilocarpine in the corneal epithelium rather than by the absorption of prodrugs into the epithelium or their epithelium–stroma transport rate.     

Electrical stimulation-evoked release of endogenous aspartate from rat medulla oblongata slices

Summary The effects of aminooxyacetic acid (AOAA), an aspartate aminotransferase (AAT) inhibitor, L-canaline, an ornithine aminotransferase inhibitor, and -acetylenic GABA and gabaculine, both y-aminobutyric acid transaminase (GABA-T) inhibitors, on the release of aspartate from slices of rat medulla oblongata and hippocampus were studied. The slices were superfused and electrically stimulated. There was ₁- Ca²⁺-dependent stimulus-evoked release of endogenous aspartate. AOAA (10^–4 and 10^–3 M) decreased the evoked release of aspartate in the medulla oblongata but not in the hippocampus. In addition, AOAA produced a decrease in the spontaneous efflux and tissue content of aspartate in the medulla oblongata. L-Canaline (5 × 10^–5 M), -acetylenic GABA (10^-4 M) and gabaculine (10^-5 M) did not affect the evoked release of aspartate in the medulla oblongata, while these agents produced ₁- decrease in spontaneous efflux and tissue content of aspartate. These findings suggest that AAT participates in the synthesis of transmitter aspartate in the medulla oblongata of the rat. It appears that there are the pools of transmitter aspartate and non-transmitter aspartate in the rat medulla oblongata Send offprint requests to T. Kubo at the above address     

X-Ray Structure and Crystal Lattice Interactions of the Taxol Side-Chain Methyl Ester

A colorless, parallelepiped crystal of methyl (2R,3S)-N-benzoyl-3-phenylisoserinate belonging to the space group P2_l with a = 5.414(4), b = 7.813(1), c = 17.802(7) , = 90.87(4)°, Z = 2, V = 752.9 ³, D _calc = 1.32 g cm^–3, and µ_calc = 1.02 cm^–1 was selected and the structure solved using direct methods. Refinement led to a final R = 0.079 for 819 [F _o 5(F_o)] reflections. Intermolecular hydrogen-bonding interactions are prevalent in the crystal lattice of this compound.     

Specific binding of 3H-adenosine to rat brain membranes

Summary The binding of ³H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of ³H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (K _d) were 1.7 M and 13.6 M and the maximal number of binding sites (B _max) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with ³H-adenosine binding. The phosphorylated adenosine compounds 5-AMP, cyclic AMP and ATP were most potent in displacing ³H-adenosine from its binding sites and the IC₅₀-values ranged from 11–25 M. N⁶-Phenylisopropyladenosine produced only partial inhibition (30%) of ³H-adenosine binding and no stereospecific difference between the (–)-and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the ³H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.     

Memory deficits induced byγ-l-glutamyl-l-aspartate andd-2-amino-5-phosphonovalerate in a Y-maze avoidance task: relationship to NMDA receptor antagonism

Post-training administration (ICV) of-l-glutamyl-l-aspartate (-lGLA) ord-2-amino-5-phosphonovalerate (d-AP5), a competitive NMDA antagonist, decreased retention of the temporal component but not the spatial discrimination component of a Y-maze active avoidance task. Inverted U-shaped dose-response curves were obtained for the ability of-lGLA andd-AP5 to decrease retention, with maximum effects occurring at doses of 2–20 nmol/mouse for-lGLA and 0.02 nmol/mouse ford-AP5.-lGLA andd-AP5 impaired the traction reflex only at doses (80 and 2 nmol/mouse, respectively) higher than those producing retention deficits. Convulsions induced by ICV administration of 1 nmol NMDA were antagonized by-lGLA andd-AP5 with ED₅₀ values of 46 (32–66) and 0.2 (0.16–0.25) nmol/mouse, respectively. The dose-effect curve of NMDA for producing convulsions was shifted to the right in a parallel manner and to the same extent by 80 nmol-lGLA and by 0.3 nmold-AP5. Taken together, these results are consistent with previous studies suggesting that the behavioral effects of-lGLA might be related to its NMDA receptor antagonist properties. The selectivity of the memory deficits induced by-lGLA andd-AP5 is in agreement with recent reports suggesting a role for NMDA receptors in the mechanisms underlying posttraining organization of memory traces.     

Neurotoxicity and pharmacology of Lathyrus sativus extracts of high- and low-toxicity strains

Neurolathyrism is characterized by spastic paraparesis of the legs. It is caused by overconsumption of grass pea (Lathyrus sativus L.; Leguminosae). We studied toxicity of extracts of L. sativus seeds from two different areas—Bangladesh and Canada—toward rat primary neuron/glia culture. Both extracts showed acute neurotoxicity within 24 h when the 75% ethanol extracts were added to the neuron/glia culture. Fractionation of the extracts showed that the water-soluble fraction accounted for ca. 75–84% of total toxicity in which 3-N-oxalyl-l-2,3-diaminopropanoic acid (l--ODAP) was present at the highest concentration. Toxicity of the water-soluble fraction obtained from Bangladeshi seeds was significantly higher than that obtained from Canada. Effects of these fractions were reversed almost completely by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX), an antagonist of AMPA-receptor. They were partially reversed by group I metabotropic glutamate receptor antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid, or (S)--methyl-4-carboxyphenyl-glycine [(S)-MCPG]. Nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME) strongly decreased the extracts toxicity. These data show that the neurotoxicity of grass pea seeds is attributable to l--ODAP, the toxicity of which is mediated by collective effects of l--ODAP on the AMPA-type receptor, metabotropic glutamate receptors, and NO production.     

Atorvastatin Transport in the Caco-2 Cell Model: Contributions of P-Glycoprotein and the Proton-Monocarboxylic Acid Co-Transporter

Purpose. The purpose of this study was to elucidate the mechanismsby which an HMG-CoA reductase inhibitor, atorvastatin (an organicacid with a pKa of 4.46), was transported in the secretory and absorptivedirections across Caco-2 cell monolayers. Methods. Caco-2 cells were grown on polycarbonate membrane insertsin 6-well Snapwell plates (Costar). The permeability of radiolabeledcompounds across Caco-2 cell monolayers was determined using aside-by-side diffusion apparatus (NaviCyte) and an automated liquidhandler (Hamilton Microlab 2200). The apical uptake of¹⁴C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 M) waspresent in the uptake media to block potential P-glycoprotein-mediatedatorvastatin efflux. Results. Polarized permeation of atorvastatin was observed with thebasolateral-to-apical (B-to-A) permeability being 7-fold greater thanthe A-to-B permeability (35.6 × 10^–6 and 4.9 × 10^–6 cm/s,respectively). The secretion of atorvastatin was a saturable process with anapparent K_m of 115 M. The B-to-A permeability of atorvastatin wassignificantly reduced by cyclosporin A (10 M), verapamil (100 M),and a P-glycoprotein specific monoclonal antibody, UIC2(10 g/ml)(43%, 25%, and 13%, respectively). Furthermore, both CsA andverapamil significantly increased the A-to-B permeability of atorvastatinby 60% however, UIC2 did not affect the A-to-B permeability ofatorvastatin. CsA uncompetitively inhibited the B-to-A flux ofatorvastatin with a K_i of 5 M. In addition, atorvastatin (100 M) significantlyinhibited the B-to-A permeability of vinblastine by 61%. The apicaluptake of atorvastatin increased 10.5-fold when the apical pH decreasedfrom pH 7.4 to pH 5.5 while the pH in the basolateral side wasfixed at pH 7.4. A proton ionophore, carbonylcyanidep-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatinuptake. In addition, atorvastatin uptake was significantly inhibited bybenzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%,14%, and 40%, respectively). Benzoic acid competitively inhibitedatorvastatin uptake with a K_i of 14 mM. Similarly, benzoic acid,nicotinic acid, and acetic acid significantly, inhibited the A-to-Bpermeability of atorvastatin by 71%, 21%, and 66%, respectively. Conclusion. This study demonstrated that atorvastatin was secretedacross the apical surface of Caco-2 cell monolayers viaP-glycoprotein-mediated efflux and transported across the apical membrane in theabsorptive direction via a H⁺-monocarboxylic acid cotransporter(MCT). In addition, this study provided the first evidence thatnegatively charged compounds, such as atorvastatin, can be a substrate forP-glycoprotein.