Interaction of central nervous system drugs with synaptosomal transport processes.

Interaction of a wide range of central nervous system (CNS) drugs with the transport of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) into synaptosomes from cerebral cortex was investigated. By determining the effect on Na,K-ATPase, the interaction of these drugs with the synaptic transport of Na ⁺ and K⁺ was also assessed. Although of different numerical value, the order of potency of the drugs in inhibiting the uptakes of both biogenic amines was comparable. Furthermore, similar inhibition constants were obtained for the low affinity uptake of 5-HT and the uptake of NE. Desipramine and amphetamine strongly inhibited both uptake processes. The narcotic drugs displayed a wide range of inhibitory potency. An exceptionally strong, but not stereospecific, effect was obtained with levorphanol, which inhibited 5-HT uptake with a K_i of 0.7 μM. While methadone and pentazocine had inhibition constants between 20 and 50 μM, morphine, codeine and naloxone were markedly weak inhibitors with K_i, values extending into the mM range. The uptake of both 5-HT and NE was strongly inhibited by quinacrine. Among the drugs investigated, only methadone showed substantial inhibition of synaptosomal Na,K-ATPase. The reversible inhibition of the enzyme by the drug was diminished at concentrations of Na ⁺ and K ⁺ reflecting synaptic discharge. The drug partially bound to the ouabain site of Na,K-ATPase and also inhibited the Mg-activated component. The feasibility and experimental conditions were established for using a modified crude mitochondrial fraction as synaptosomal preparation to study transport processes in general and the uptake of biogenic amines in particular.     

Affinity of Drugs for Cytochrome P-450 Determined by Inhibition of p-Nitrophenetole O-Deethylation by Rat Liver Microsomes

Abstract: The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent K_m for oxygen was 0.07 μM. Furthermore, cytochrome b₅ seemed to be involved in the formation of p-nitrophenol. The effect on p-nitrophenol formation of drugs known to be involved in drug interaction in clinical practice was studied. There was a competitive inhibition by phenytoin (inhibitor constant, K_i, 30 μM), disulfiram (K_i, 2 μM) and chloramphenicol (K_i, 20 μM), whereas a mixed-type inhibition by isoniazid was observed (K_is, 1,3 mM and K_ii, 10,6 mM).     

Uptake of benzylpenicillin,cefpiramide and cefazolin by freshly prepared rat hepatocytes: Evidence for a carrier-mediated transport system

The kinetics and mechanism of the hepatic uptake of β-lactam antibiotics were studied by using freshly prepared rat hepatocytes. The initial uptake rates of benzylpenicillin and cefpiramide represented both saturable and nonsaturable transport processes, whereas that of cefazolin showed an apparently nonsaturable uptake process within the concentration range below 4mM. The apparent nonsaturable uptake rate constants for benzylpenicillin, cefpiramide and cefazolin were 0.580, 0.047 and 0.289 nmoles/min/mg protein/mM respectively. The apparent values of K_t and V_max describing the saturable transport were 0.473 ± 0.158 mM and 2.02 ± 0.48 nmoles/min/mg protein for benzylpenicillin and 0.847 ± 0.254 mM and 0.70 ± 0.18 nmoles/min/mg protein for cefpiramide respectively. The Arrhenius plot of benzylpenicillin uptake of 200 μM presented a single straight line in the range of 22°–37° with an activation energy of 16.8 kcal/mole. An energy requirement was also demonstrated for benzylpenicillin uptake as metabolic inhibitors (antimycin A, NaCN, rotenone and 2,4-dinitrophenol) significantly reduced the initial uptake rate of benzylpenicillin (P < 0.05). Uptake of benzylpenicillin (200 μM) was not inhibited by ouabain (1 mM). Benzylpenicillin uptake was inhibited competitively by phenoxymethylpenicillin, cefpiramide and cefazolin with the inhibition constants, K_i of 0.680, 0.583 and 11.7 mM respectively. Benzylpenicillin also inhibited competitively the uptake of cefpiramide with a K_i of 0.655 mM. From these results it was considered that a carrier-mediated uptake system participates in the hepatic uptake of at least four of the β-lactam antibiotics examined in this study.     

Simulataneous and independent versus antagonistic inhibition of muscle carbonic anhydrase (CA III) by acetazolamide and cyanate

The inhibition by cyanate and acetazolamide of pig muscle carbonic anhydrase III (CA III) CO₂ hydratase activity was studied in order to explore mechanistic features possibly unique to the muscle isoenzyme. The turnover number for CO₂ hydration was found to be 6000 sec^?1 with a K_m of 83 mM for CO₂. Cyanate inhibition (K_i, 3 μM) and acetazolamide inhibition (K_i, 44 μM) were both found to be noncompetitive with respect to CO₂. Significantly, acetazolamide and cyanate displayed non-exclusive binding to pig muscle carbonic anhydrase. The similarity of mode and degree of inhibition of muscle carbonic anhydrase by cyanate as compared with the inhibition of the erythrocyte isoenzymes suggests the existence of a similar metal environment. However, the observation that cyanate and acetazolamide bind simultaneously to CA III and the comparatively large K_i for acetazolamide per se appear to be more compatible with a different mode of coordination of the zinc with the sulfonamide, thus supporting a five-coordinant zinc in the catalytic mechanism of CO₂ hydration for CA III.     

Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-propranolol

The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contamination with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site (K_d) = 265 μM, B_max = 65 $\text{nmoles}\text{mg}$ protein) was detected by equilibrium dialysis. Monovalent metal ions exhibited 10–100-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K_i values found for divalent metal ions was similar to the K_d value for calcium. La⁺³ produced a potent non-competitive inhibition. A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack β-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with K_i values ranging from 40–900 μM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experimental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation (K_i = 795 μM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to dl-propranolol (k_i = 115 μM and 108 μM, respectively).     

Thiopental inhibition of γ-aminobutyrate transaminase in rat brain synaptosomes

The kinetic parameters for thiopental inhibition of γ-aminobutyrate: 2-oxoglutarate transaminase solubilized from rat brain synaptosomes were studied, V_max is 0.76 μmole/hr/mg of protein. K_m for GABA (γ-aminobutyric acid) is 16.0 mM and K_m for 2-OG (2-oxoglutarate: α-ketoglutarate) is 0.35 mM. Thiopental inhibition is noncompetitive and K_i 1.1 mM. is similar when either GABA or 2-OG is the limiting substrate. The implication of these findings in clinical anesthesia is discussed.     

Cytotoxicities of the l and d isomers of phenylalanine mustard in L1210 cells

The cytotoxicity of d-phenylalanine mustard (medphalan) to murine L1210 leukemia cells in culture was reduced by both the d and l isomers of leucine. l-Leucine only partially protected L1210 cells from medphalan cytotoxicity at drug concentration's above 10 μM, suggesting that medphalan uptake occured via both an amino acid carrier and an as yet undetermined agency, possibly passive diffusion. At equitoxic concentrations of l-phenylalanine mustard (melphalan) and medphalan, l-leucine reduced medphalan cytotoxicity by only one-sixth that obtained with melphalan. Analysis of melphalan and medphalan inhibition of the initial rate of l-leucine transport indicated a melphalan K_i of 0.085 mM, a value one-seventh that of melphalan (K_i, 0.635 mM).     

Protective effects of verapamil and diltiazem against inorganic phosphate induced impairment of oxidative phosphorylation of isolated heart mitochondria

The effects of verapamil and diltiazem on oxidative phosphorylation of isolated rabbit heart mitochondria were related to the experimental conditions employed. In an assay medium containing 250 mM sucrose, 1 mM pyruvate and 5 mM potassium phosphate buffer (pH 7) at 37° (sucrose medium), only a high concentration of verapamil (200–800 μM) or diltiazem (400–600 μM) affected mitochondria. State 4 respiration was stimulated, state 3 respiration was inhibited, and the ADP: O ratio was decreased by these drugs in sucrose medium. These effects resulted in a depression of the respiratory control index (RCI) and oxidative phosphorylation rate (OPR). On the other hand, in an assay medium containing 150 mM KCl, 1 mM pyruvate and 2 mM potassium phosphate buffer (pH 7) at 37° (KCl medium), the high rate of state 3 respiration and the normal value of the ADP: O ratio were not influenced significantly by diltiazem (400–800 μM) or verapamil (200–400 μM). These data indicate that neither verapamil nor diltiazem has an effect on the normal, functioning, isolated mitochondria in KCl medium. Elevation of inorganic phosphate (P₁) from 2 to 5 mM in the KCl medium induced a swelling of the mitochondria, inhibition of state 3 respiration, and a decrease in the ADP: O ratio, RCI and OPR. Under these conditions, a low concentration of verapamil (25–200 μM) or diltiazem (50–800 μM) inhibited the swelling effect of P_i and at the same time prevented the P_i-induced decrease in state 3 respiration, and the ADP: O ratio, RCI and OPR. In a medium containing 150 mM KCl, 1 mM pyruvate, 2 mM ADP and 10 μM palmitoyl-CoA, the addition of 5 mM P_i induced swelling of mitochondria and a decreased rate of state 3 respiration. Under these conditions, even a low concentration of verapamil (6–200 μM) or diltiazem (25–400 μM) inhibited swelling and prevented the inhibition of state 3 respiration. It is concluded that low concentrations of verapamil and diltiazem had no effect on unswollen heart mitochondria. An increase in the free P_i concentration induced swelling of mitochondria and resulted in an inhibition of oxidative phosphorylation, provided that the extramitochondrial potassium concentration was as high as that normally found in the cytosol. Under these conditions, a low concentration of verapamil and diltiazem was able to affect the mitochondrial membranes so as to prevent P_i-induced swelling and the related inhibition of oxidative phosphorylation.     

Inhibition of choline uptake in syncytial microvillus membrane vesicles of human term placenta: Specificity and nature of interaction

The potency and nature of the inhibitory effect of various cationic drugs on the transport of choline across the placental syncytial microvillus membrane was investigated. Tetraethylammonium, a model substrate for organic cation transport, was a poor inhibitor. Enlarging the degree of alkylation of the quaternary ammonium increased the inhibitory effect, in proportion with increasing lipophilicity. Log concentration vs % control uptake curves showed marked differences in inhibitory potency for the different cationic drugs. Hemicholinium-3 inhibited mediated choline uptake in the micromolar range, whereas atropine and mepiperphenidol were less potent. The H₂-receptor antagonists cimetidine, ranitidine, and famotidine inhibited choline uptake in the millimolar ranges. Dixon analysis revealed a competitive nature of inhibition for hemicholinium-3 and atropine (K_i = 40 μM and 1.2 mM, respectively). Cimetidine interacted noncompetitively (K_i = 3.4 mM). Since relatively high concentrations were needed to reach half maximal inhibition, impairment of fetal choline supply due to maternal drug use during pregnancy is not to be expected.     

Intestinal metabolism of tyramine by both forms of monoamine oxidase in the rat

The two forms of monoamine oxidase (MAO) in rat intestine and brain homogenates were found to have different K_m and V_max values towards tyramine. The K_m values for the A-form of the enzyme towards this substrate were around 120 μM in both cases, whereas the values for the B-form were about 240 μM. As a consequence, the ratio of activities (MAO-A: MAO-B) towards tyramine are dependent upon the substrate concentration. The MAO-A-selective inhibitors, toloxatone and cimoxatone, were found to be competitive inhibitors of the oxidation of tyramine by the A-form of this enzyme in the rat intestine, with K_i values of 3.4 μM and 3.7 nM respectively. The significance of these results in relation to the “cheese effect”, a pressor response to tyramine after monoamine oxidase inhibition, are discussed.     

PURIFICATION AND PROPERTIES OF RAT SKIN ACID PHOSPHATASES

Four acid phosphatases (APases I-IV) were purified from Triton X-100 extracts of rat skin. APase I appeared as a high-molecular weight, most likely lysosomal lipo(glyco)-protein with a pI of 4.3. APase I, extractable as a Triton-protein complex with a mol.wt. of 2 10⁶, hydrolyzed α-naphthyl phosphate (α-NP; K_m= 0.18 mM) and p-nitrophenyl phosphate (PNP; K_m= 0.96 mM) at an almost equal rate at pH 5.0. Mo₇O₂₄^6-inhibited noncompetitively (K_i= 0.15 μM). APase II had a pI of 7.8–8.0 and a mol.wt. of 104000 ± 4000. APase II is likely lysosomal and at pH 5.0 it hydrolyzed most rapidly PNP (K_m= 0.057 mM), α-NP (K_m= 0.18 mM) and phenyl phosphate (PP; K_m= 0.41 mM). Mo₇O₂₄^6- (K_i=2.5 nM) inhibited competitively. APase III, also of lysosomal origin, showed a pI of 5.3–5.5 and a mol.wt. of 87000 ± 3000. This enzyme, at pH 5.0, hydrolyzed most rapidly α-NP (K_m= 0.16 mM), pyridoxal 5-phosphate (PLP; K_m= 0.41 mM), PNP (K_m= 0.16 mM) and PP (K_m= 0.16 mM). Mo₇O₂₄^6- inhibition (K_i= 0.75 μM) was of a mixed type. p-Chloromercuribenzoate (PCMB; K_i= 2.0 μM) inhibited noncompetitively. A pI of 5.6–6.2 and a mol.wt. of 14000 ± 400 was obtained for the cytoplasmic APase IV. This enzyme, at pH 5.0, hydrolyzed most rapidly PNP (K_m= 0.16 mM), PP (K_m= 0.81 mM) and riboflavin 5′-phosphate (RP; K_m= 0.20 mM). Mo₇O₂₄^6- inhibited competitively (K_i= 8.5 μM) but PCMB (K_i= 0.32 μM) showed a mixed type of inhibition. PLP may be a natural substrate of APase III and RP that of APases II and IV. The activity of APases II and III may depend on histidyl and SH-groups, that of APase I on histidyl groups, and SH-groups may be necessary for the activity of APase IV. All these APases seem to have an active tyrosyl and carboxyl residue. The pH dependence of the catalyses, inhibition characteristics and anatomic location showed APases I-IV to be true skin acid phosphatases.     

Probes of eukaryotic DNA-dependent RNA polymerase II-II: Covalent binding of two purine nucleoside dialdehydes to the initiation subsite

The catalytic center of wheat germ DNA-dependent RNA polymerase RNA polymerase II (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) as a model eukaryotic enzyme system was probed with two purine nucleoside dialdehydes, 6-methylthioinosinedicarboxaldehyde (MMPR-OP) and a derivative 6-[(acetylaminoethyl)-1-naphthylamine-5-sulfonyl]thioinosinedicarboxaldehyde (AMPR-OP). Both drugs gave noncompetitive inhibition with respect to [³H]UMP incorporations into RNA, and inhibitor bindings were reversed with initiation substrates. The K_i values for MMPR-OP and AMPR-OP were determined to be 0.64 mM and 1.0 μM respectively. The drugs were covalently bound to the catalytic center by NaBH₄ reduction. Both were found bound to the largest enzyme subunit, IIa. It is essential lysine in the initiation subsite of the catalytic center located on the IIa subunit.     

Biochemical pharmacology of acivicin in rat hepatoma cells

The antiglutamine agent acivicin, l-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, inhibited the growth of hepatoma 3924A cells in culture. After 7 days of incubation with the drug, an lc₅₀ of 1.4 μM was observed by determination of colony forming ability. A combination of cytidine (1 mM), deoxycytidine (10 μM) and guanosine (10 μM) completely protected the hepatoma cells against the cytotoxic action of acivicin, but each nucleoside by itself had no effect. Acivicin (0.1 mM) inhibited the incorporation of uridine and thymidine into macromolecules, but not that of leucine. Acivicin depressed the pools of CTP, GTP, dCTP, dGTP and dTTP to 46, 62, 40, 64 and 53%, respectively, but it increased UTP level to 152% of the values of untreated cancer cells. The activity of a highly purified CTP synthetase (EC 6.3.4.2) from rat liver and hepatoma 3924A was inhibited by acivicin. The inhibition was competitive with respect to l-glutamine, and the K_i values with liver and hepatoma enzymes, determined by Dixon and reciprocal plots, were 1.1 and 3.6 μM respectively. The hydroxy analog of acivicin was also a competitive inhibitor, but it was less effective than acivicin, with a K_i value of 1.8 mM for the hepatoma enzyme. Our observations on the impact of acivicin on the behavior of pools of ribonucleotides and deoxyribonucleotides and the competitive inhibition of purified CTP synthetase from hepatoma cells suggest that a major mechanism of action for this drug is the inhibition of CTP synthetase and GMP synthetase (EC 6.3.5.2).     

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 cells

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 μM [³H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (K_i = 2.5 μM), 4-biphenylacetic acid (K_i = 5.3 μM), and flurbiprofen (K_i = 5.2 μM), whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (K_i = 0.08 μM), ethacrynic acid (K_i = 0.52 μM), and 1-chloro-2,4-dinitrobenzene (K_i = 0.77 μM). Bilirubin ditaurate was a potent inhibitor of both transport components (K_i = 1.5 and 0.17 μM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 μM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave K_m values of 170 and 250 μM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.     

Influence of neuroleptics on the binding of met-enkephalin, morphine and dihydromorphine to synaptosome-enriched fractions of rat brain.

The influence of three neuroleptics on the binding of tritiated met-enkephalin to synaptosomeenriched fractions of rat brain was studied in the high-affinity binding region. All three drugs tested blocked the binding of met-enkephalin in a dose-dependent fashion with inhibition constants (k_i) of 11.6, 39.5 and 29.5 μM for haloperidol, chlorpromazine and prochlorperazine, respectively. Hill plots suggest that the inhibitory mechanism involves negative allosteric effects between neuroleptic molecules. The effects of these neuroleptics on the binding of tritiated morphine and dihydromorphine were also studied.     

Inhibitory effects of class I and IV antiarrhythmic drugs on the Na+-activated K+ channel current in guinea pig ventricular cells

Recently we have reported that class III antiarrhythmic drugs including amiodarone inhibit the Na⁺-activated K⁺ (K_Na) channels in isolated cardiac cells. In this study effects of antiarrhythmic drugs having class I and/or IV properties on the single K_Na channel current were examined in inside-out membrane patches of guinea pig ventricular cells by using patch clamp techniques. The K_Na channel current, which was activated by increasing [Na⁺]_i from 0 mM to 100 mM in the presence of 150 mM [K⁺]_o, showed a large slope conductance (212 pS) and inward-going rectification. Quinidine (100 μM), mexiletine (100 μM) and flecainide (10 μM) were selected as representative of class Ia, Ib and Ic drugs, respectively. These drugs at relatively high concentrations incompletely inhibited the K_Na channel by decreasing the open time (flickering block). The class IV drug verapamil inhibited the K_Na channel current mainly by decreasing the open probability although the IC₅₀ value of verapamil (3.36 μM) was higher than the therapeutic concentrations. Bepridil and SD-3212, antiarrhythmic drugs having both class I and IV properties, potently inhibited the K_Na channel current by decreasing the open probability. The IC₅₀ values of bepridil and SD-3212 for inhibiting the K_Na channel current was 0.51 μM and 0.53 μM, respectively, both of which are within the therapeutic range. Most antiarrhythmic drugs inhibit cardiac K_Na channels by different modes and at different concentrations. The K_Na channel blocking action of bepridil and SD-3212 may partly contribute to the prolongation of the action potential duration by these drugs at rapid stimulation rates. Received: 20 April 1998 / Accepted: 16 September 1998     

Diphenhydramine active uptake at the blood-brain barrier and its interaction with oxycodone in vitro and in vivo

Diphenhydramine (DPHM) and oxycodone are weak bases that are able to form cations. Both drugs show active uptake at the blood–brain barrier (BBB). There is thus a possibility for a pharmacokinetic interaction between them by competition for the same uptake transport system. The experiments of the present study were designed to study the transport of DPHM across the BBB and its interaction with oxycodone in vitro and in vivo. In vitro, the interaction between the drugs was studied using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13 cells). The in vivo relevance of the in vitro findings was studied in rats using brain and blood microdialysis. DPHM was actively transported across the BBB in vitro (TR-BBB13 cells). Oxycodone competitively inhibited DPHM uptake with a K_i value of 106 μM. DPHM also competitively inhibited oxycodone uptake with a K_i value of 34.7 μM. In rats, DPHM showed fivefold higher unbound concentration in brain interstitial fluid (ISF) than in blood, confirming a net active uptake. There was no significant interaction between DPHM and oxycodone in vivo. This accords with the results of the in vitro experiments because the unbound plasma concentrations that could be attained in vivo, without causing adverse effects, were far below the K_i values. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3912–3923, 2011     

Studies on rat kidney 15-hydroxy-prostaglandin dehydrogenase.

Rat kidney 15-hydroxy-prostaglandin dehydrogenase (PGDH) was isolated, and its characteristics and the effects of various drugs upon it were examined. The enzyme was found in the cell cytosol; was labile when unfrozen; and most active at alkaline pH, at 41°, and with the E prostaglandins. Additionally, the enzyme was inhibited by furosemide (K_i = 0.019 mM), ethacrynic acid (K_i = 0.27 mM, phenylbutazone (K_i = 0.16 mM), acetylsalicylic acid (K_i = 3.8 mM), and potassium cyanide (K_i = 1.03 mM). Inhibition of PGDH may play a role in the mechanism of action of the diuretic and anti-inflammatory drugs. Little or no inhibition was seen with amobarbital, hydralazine, alpha-methyldopa, bethanidine and guanethidine. Amobarbital inhibits NADH oxidase (K_i = 0.5 mM), but does not inhibit PGDH. This drug, therefore, may be useful in permitting the use of the fluorometric assay for PGDH in preparations of PGDH contaminated by NADH oxidase.     

Reduction of proline transport in R3230AC mammary carcinomas by estrogens in vitro

The effects of estrogens on proline and leucine transport into enzymatically dissociated cells from the R3230AC mammary adenocarcinoma were studied. Estradiol-17β demonstrated a time- and dose-dependent reduction of proline transport; at 10^?6M, transport of proline was decreased by 50 percent. Kinetic analysis of these effects indicate that estradiol displayed characteristics of a non-competitive inhibitor, with a K_i of 1.79 μM. Other estrogens, and the anti-estrogen tamoxifen, gave somewhat higher estimated K_i values and could be ranked as inhibitors as follows: estradiol-17β > diethylstilbestrol > tamoxifen > estriol-estrone > estradiol-17α. No effects of these estrogens on leucine transport were observed, indicating their selectiveness for the A system. The synthetic glucocorticoid, dexamethasone, was comparable to diethylstilbestrol as an inhibitor of proline transport, but dexamethasone also decreased transport of leucine. Testosterone and progesterone were approximately comparable to estrone in their actions. It is proposed that these actions of estrogens represent one potential mechanism whereby pharmacological levels exert therapeutic benefit in the treatment of advanced breast cancer.     

Opiates inhibit paclitaxel uptake by P-glycoprotein in preparations of human placental inside-out vesicles

The use of either methadone or buprenorphine for treatment of the pregnant opiate-dependent patient improves maternal and neonatal outcome. However, patient outcomes are often complicated by neonatal abstinence syndrome (NAS). The incidence and severity of NAS should depend on opiate concentration in the fetal circulation. Efflux transporters expressed in human placental brush border membranes decrease fetal exposure to medications by their extrusion to the maternal circulation. Accordingly, the concentration of either methadone or buprenorphine in the fetal circulation is, in part, dependent on the activity of the efflux transporters. The objective of this study was to characterize the activity of P-gp and its interaction with opiates in the placental apical membrane. Therefore, brush border membrane vesicles were prepared from human placenta. The vesicles were oriented approximately 75% inside-out, exhibited saturable ATP-dependent uptake of P-gp substrate [³H]-paclitaxel with an apparent K_t of 66 ± 38 nM and V_max of 20 ± 3 pmol mg protein⁻¹ min⁻¹. Methadone, buprenorphine, and morphine inhibited paclitaxel transport with apparent K_i of 18, 44, and 90 μM, respectively. Our data indicate that a method has been established to determine the activity of the efflux transporter P-gp, expressed in placental brush border membranes, and the kinetics for the transfer of its prototypic substrate paclitaxel. Furthermore, the method was used to determine the effects of methadone, buprenorphine, and morphine on paclitaxel transfer by placental P-gp and revealed that they have higher affinity to the transporter than its classical inhibitor verapamil (K_i, 300 μM).