Modulation of P-glycoprotein activity by estramustine is limited by binding to plasma proteins

BACKGROUND: Estramustine previously has been shown to interact with P-glycoprotein and to restore intracellular accumulation of vinblastine and paclitaxel in cells overexpressing this drug transporter. However, the ability of estramustine to potentiate the cytotoxicities of several drugs was less than that expected. To resolve this apparent discordance, the authors examined the effects of serum on the actions of estramustine. METHODS: The cytotoxicities of anticancer drugs with or without estramustine or verapamil toward MCF-7 breast carcinoma cells and a P-glycoprotein-overexpressing subline MCF-7/ADR were determined using the sulforhodamine-binding assay. The extent of intracellular accumulation of [3H]vinblastine and [3H]paclitaxel was determined for each using standard methods, and the binding of radiolabeled drugs to plasma proteins was characterized by equilibrium dialysis. RESULTS: Without serum, the sensitivities of MCF-7/ADR cells to several P-glycoprotein-transported drugs were increased by estramustine and verapamil. Conversely, when the cells were treated with a 10% serum, the cytotoxicities of these drugs were increased by verapamil, but not by estramustine. Without serum, intracellular accumulation of [3H]vinblastine and [3H]paclitaxel by MCF-7/ADR cells was increased markedly by verapamil and estramustine; however, serum suppressed the effects of estramustine much more strongly than those of verapamil. Equilibrium dialysis experiments demonstrated that [3H]estramustine binds to plasma proteins, predominantly albumin, whereas [3H]paclitaxel binds to albumin and alpha 1-acid-glycoprotein, and [3H]vinblastine binds predominantly to alpha 1-acid-glycoprotein. CONCLUSION: Although estramustine can bind to P-glycoprotein, its effectiveness as a reversing agent in vivo likely is limited by binding to plasma proteins.     

Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment

Rats were treated with daily injections of SDZ-PSC 833 (PSC) to study the interaction of this potent modulator of multidrug resistance (MDR) with P-glycoprotein (P-gp) expressed in normal tissues. After 2 days of treatment, the level of P-gp expression, detected by Western blot analysis, was not modified in renal brush border membranes (BBMs) and brain capillaries. However, the amount of P-gp detected with the photoaffinity probe [125I]-arylazidoprazosin (IAAP) was decreased in both tissues, suggesting that the drug binding properties of P-gp were altered by PSC treatment. This effect was further characterized by treating rats with PSC for 10 days. Following these treatments, the amount of immunodetected P-gp was increased in renal BBMs and brain capillaries. However, no increase in P-gp expression was observed in photolabeling experiments, suggesting that induced P-gp was not functional. In vitro experiments performed with renal BBMs showed that the inhibition of P-gp photolabeling by cyclosporin A (CsA), verapamil and vinblastine could be reversed by performing washing steps to remove these drugs before incubating the samples with IAAP. However, the inhibition mediated by PSC was less reversible since photolabeling of P-gp remained inhibited following the washing steps. Pre-incubation of intact CHRC5 cells with PSC, CsA and verapamil also inhibited P-gp photolabeling and increased rhodamine 123 accumulation. For PSC pre-treated samples, these effects were not completely reversed following washing, but were abolished for CsA and Ver pre-treated samples. Our results suggest that PSC could block P-gp function by a different mechanism from that of CsA and verapamil, involving modification of the drug binding sites.     

Characterization of P-glycoprotein mediated transport of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor, across MDR1-MDCK and Caco-2 cell monolayers

PURPOSE: Here we characterized the transport properties of morpholine-urea-phenylalanine-homophenylalanine-vinylsulfone-phenyl (K02), a newly developed peptidomimetic cysteine protease inhibitor, across monolayers of P-gp-expressed MDRI transfected MDCK cells (MDR1-MDCK) and Caco-2 cells. METHODS: MDR1-MDCK, MDCK and Caco-2 cells, grown to confluence on Transwell insert membranes, were used to investigate transcellular transport of [14C]-K02. RESULTS: The basolateral to apical (B-A) flux of 10 microM [14C]-K02 across MDR1-MDCK cells was markedly greater than its apical to basolateral (A-B) flux (ratio = 39). This specific B-A transport was temperature dependent and saturable, with an apparent Michaelis-Menten constant and maximum velocity of 69.1 +/- 19.5 microM and 148.9 +/- 16.3 pmol/min/cm2, respectively. This B-A flux was significantly inhibited by cyclosporine (IC50 = 17.1 +/- 0.7 microM), vinblastine (IC50 = 75.9 +/- 13.0 microM) and verapamil (IC50 = 236 +/- 63 microM). In Caco-2 cell monolayers, the B-A flux was reduced about 50% compared to that in MDR1-MDCK and the A-B flux was increased about 8-fold. The apparent Michaelis-Menten constant and maximum velocity values for the B-A transport were 71.8 +/- 45.9 microM and 35.3 +/- 9.0 pmol/min/ cm2. This B-A flux was also significantly inhibited by P-gp substrates/ inhibitors. Western blots showed that the P-gp expression in MDR1-MDCK cells was about 10-fold that in Caco-2 cells. CONCLUSIONS: K02 is transported by P-gp in both MDR1-MDCK and Caco-2 cells, and the in vitro interactions between K02 and various P-gp substrates may provide strategies to overcome the bioavailability barrier by intestinal P-gp.     

Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein

Saquinavir, a peptidomimetic HIV protease inhibitor, has been shown to be effective in reducing patient viral load and reducing mortality. In this report we investigated whether saquinavir is a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), which may reduce the effective intracellular concentration of the drug. G185 cells, which highly express P-gp, are resistant to saquinavir-mediated cytotoxicity, and co-administration of cyclosporine reversed this resistance. Saquinavir and saquinavir mesylate inhibited basolateral to apical transport of the fluorescent dye rhodamine 123 in a polarized epithelial transport assay, a result that suggests competition of these drugs for the P-gp transporter. Finally, we measured specific, directional transport of saquinavir and saquinavir mesylate in an epithelial monolayer model. Transport in the basolateral to apical direction was 3-fold greater than apical to basolateral flux for both saquinavir and saquinavir mesylate and was blocked by co-incubation with the established P-gp reversal agents cyclosporine and verapamil. These data provide evidence that saquinavir is a substrate for the P-gp transporter and suggest that this protein may affect intracellular accumulation of the drug and contribute to its poor oral bioavailability.     

Enhanced separation of antidepressant drugs using a polymerized nonionic surfactant as a transient capillary coating

PURPOSE: To investigate the role of the P-glycoprotein (P-gp) drug efflux pump in the intracellular disposition of colchicine and vinblastine. METHODS: Uptake and efflux kinetics were studied in vitro in human lymphocytes and in HL-60 cells with or without the P-gp modulator, verapamil. RESULTS: In human lymphocytes, colchicine was slowly taken up (uptake half-life was 18.9+/-1.1 hr.) and verapamil increased colchicine uptake by 37%, whereas it did not modify colchicine efflux from cells. In HL-60 cells, colchicine uptake was non-linear and slower than that of vinblastine, the colchicine uptake half-life (11.1+/-0.5 hr.) being 25-fold longer than that of vinblastine at 25 nM. Verapamil did not significantly modify colchicine uptake half-life, but increased its intracellular accumulation by 23% and that of vinblastine by 81%. Immuno-flow cytometry showed that P-gp expression in HL-60 cells increased significantly from 24 hr. following colchicine or vinblastine exposure. The significant increase in colchicine uptake induced by verapamil at 24 hr. was correlated with this enhanced P-gp expression. The drug efflux half-life was 11.5-fold higher for colchicine (23+/-0.9 hr) than vinblastine, indicating a much slower elimination of colchicine from cells that could be related to its longer dissociation half-life from the tubulin receptor. Verapamil treatment did not modulate either colchicine or vinblastine efflux kinetics, suggesting that the intracellular drugs are not available to the transmembrane P-gp binding sites. CONCLUSIONS: P-gp may not be the main reason for the slowness of colchicine uptake. It may be more efficient at controlling entry of colchicine and vinblastine through the plasma membrane than at mediating their efflux from HL-60 cells.     

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.     

3H]Alnespirone: a novel specific radioligand of 5-HT1A receptors in the rat brain

Determination of the optimal assay conditions for the specific binding of a tritiated derivative of the novel potential anxiolytic drug alnespirone (S-20499, (+)-4-[N-(5-methoxy-chroman-3-yl)-N-propylamino]butyl-8-azaspiro-( 4,5)-decane-7,9-dione) allowed the demonstration that this radioligand bound with a high affinity (Kd = 0.36 nM) to a homogeneous class of sites in rat hippocampal membranes. The pharmacological properties of [3H]alnespirone specific binding sites matched exactly (r = 0.95) those of 5-HT1A receptors identified with [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) as radioligand. Furthermore, membrane binding experiments and autoradiographic labeling of tissue sections showed that the regional distribution of [3H]alnespirone specific binding sites in the rat brain and spinal cord superimposed over that of 5-HT1A receptors specifically labeled by [3H]8-OH-DPAT. However, the differential sensitivity of [3H]alnespirone and [3H]8-OH-DPAT specific binding to various physicochemical effectors (temperature, pH, Mn2+, N-ethyl-maleimide) supports the idea that these two agonist radioligands did not recognize 5-HT1A receptors exactly in the same way. These differences probably account for the reported inability of alnespirone, in contrast to 8-OH-DPAT, to induce some 5-HT1A receptor-mediated behavioural effects in rats.     

3H]-BIBP3226 and [3H]-NPY binding to intact SK-N-MC cells and CHO cells expressing the human Y1 receptor

We have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80% of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged.     

Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.     

Microsatellite instability is correlated with lymph node-positive breast cancer

1. P-glycoprotein, a 170-180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein. 2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay. 3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity. 4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833. 5. The binding of 8-azido-[alpha-32P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein. 6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.     

High affinity [3H]imipramine and [3H]paroxetine binding sites in suicide brains

Specific binding of [3H]imipramine and [3H]paroxetine was simultaneously examined in human brains (frontal cortex, temporal cortex, cingulate cortex, hypothalamus, hippocampus and amygdala) from 11 controls and 11 depressed suicide victims. A single saturable high affinity site was obtained for both radioligands. Age was not related to significant changes in [3H]imipramine and [3H]paroxetine binding parameters, which indicates the stability of the brain serotonergic system with increasing age. A major finding of the present study concerns the existence of a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in hippocampus from depressed suicides as compared with the control group, without changes in the binding affinity (Kd). In contrast, when [3H]paroxetine was used as radioligand, no changes in either Bmax or Kd were detected in any of the brain regions studied. These findings suggest that [3H]imipramine may be a better marker than [3H]paroxetine when alterations in the presynaptic serotonergic uptake site are to be detected.     

Binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binding

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.     

Studies of human MDR1-MDR2 chimeras demonstrate the functional exchangeability of a major transmembrane segment of the multidrug transporter and phosphatidylcholine flippase

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 and MDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).     

3H]paroxetine binding is altered in the hippocampus but not the frontal cortex or caudate nucleus from subjects with schizophrenia

[3H]Paroxetine binding to particulate membrane from tissue, obtained at autopsy, from the hippocampus, frontal cortex, and caudate nucleus from subjects who had or had not had schizophrenia was measured. The density of [3H]paroxetine binding to membranes from subjects who had or had not had schizophrenia did not differ. Similarly, the affinity of [3H]paroxetine binding in the frontal cortex and caudate nucleus was not different. By contrast, the affinity of [3H]paroxetine binding to hippocampal membrane from subjects who had schizophrenia was significantly lower than the affinity of binding for the nonschizophrenic subjects (0.40 +/- 0.06 vs. 0.26 +/- 0.02; p < 0.05). As [3H]paroxetine binds to the serotonin transporter, these data suggest that the serotonin transporter is altered in the hippocampus in subjects with schizophrenia.     

Comparison of binding parameters of sigma 1 and sigma 2 binding sites in rat and guinea pig brain membranes: novel subtype-selective trishomocubanes

Comparisons of binding parameters of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) at sigma binding sites in guinea pig and rat brain membranes demonstrated that [3H](+)-pentazocine binds to a single high-affinity site, whereas [3H]DTG binds to two high-affinity sites in both species. The Kd values of the radioligands were similar in both types of membranes. However, the density of sigma 1 sites in guinea pig was significantly higher than that of rat. Novel trishomocubanes were tested for their affinities at sigma 1 and sigma 2 binding sites in guinea pig brain membranes using [3H](+)-pentazocine and [3H]DTG as the radioligands. N-(4-Phenylbutyl)-3-hydroxy-4- azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane (ANSTO-14) showed the highest affinity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity, as a result of increasing the alkyl chain between the cubane moiety and the aromatic ring. N-(3'-Fluorophenyl)methyl- 3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11]dodeca ne (ANSTO-19), displayed the highest affinity for sigma 2 sites (Ki = 19.6 nM) and 8-fold sigma 2/sigma 1 selectivity due to a fluoro substitution in the meta position of the aromatic ring. These represent structurally novel lead compounds, especially for the development of selective sigma 2 receptor ligands.     

Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes

Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.     

Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells

P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vinblastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min). MDCK-5'NU/- cells did not display functional expression of MDR. In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors. This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot. Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity. Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype. In conclusion: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.     

3H](+)-7-OH-DPAT and [3H]pramipexole binding in the striatum and nucleus accumbens of Sprague-Dawley and Fischer-344 rats

The binding of the D2-like agonists, (+)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin (7-OH-DPAT) and [3H]pramipexole (2-amino-4,5,6-tetrahydro-6-propylaminobenzthiazole; MIRAPEX) were determined in membranes from adult male Sprague-Dawley and Fischer-344 rats. Saturation analysis, which optimized binding to D3 receptors, revealed 3-6 fold differences in Bmax values between the two radioligands with no change in affinity. [3H](+)7-OH-DPAT labeled 41.4+/-4.1 to 61.8+/-3.0 fmol/mg protein in nucleus accumbens and striatal homogenates, yet [3H]pramipexole labeled only 7.0+/-1.2 to 18.9+/-5.3 fmol/mg protein. Regional differences with both radioligands were observed in Fischer-344 rats; the striatum exhibited a 52%-69% greater density of sites in comparison to the nucleus accumbens. These data suggest that D3 receptor density can vary significantly between animal strains depending on the radioligand used, and [3H]pramipexole identifies a different ratio of sites in the striatum and nucleus accumbens compared to [3H](+)7-OH-DPAT.     

3H]MDL 100,907 labels 5-HT2A serotonin receptors selectively in primate brain

The selective antagonist for the 5-HT2A serotonin receptor MDL 100,907, recently characterized autoradiographically in rat brain, has been characterized as a radioligand for the visualization of this receptor in human and monkey brain. In both species [3H]MDL 100,907 binding to brain sections was saturable, had sub-nanomolar affinity (Kd = 0.14-0.19 nM in human brain; Kd= 0.16-0.19 nM in monkey brain) and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL 100,907-labeled receptors: MDL 100,907 > spiperone > ketanserin > mesulergine). The autoradiographical signal obtained with [3H]MDL 100,907 was compared to the signal obtained with [3H]ketanserin, [3H]RP62203 and [3H]mesulergine in both species, and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization in monkey brain. At variance with the other radioligands, [3H]MDL 100,907 showed a single population of binding sites with extremely low levels of non-specific binding. As expected, mesulergine showed low affinity for [3H]MDL 100,907-labeled receptors and the autoradiographic pattern shown by [3H]mesulergine confirmed the lack of labeling of the 5-HT2A receptor by this radioligand in primate brain. The similarity of the distribution of [3H]MDL 100,907-labeled receptors and 5-HT2A mRNA in monkey brain, supports the selectivity of this radioligand for 5-HT2A receptors and suggests a somatodendritic localization of these receptors. The present results confirm [3H]MDL 100,907 as the radioligand of choice at present for the autoradiographic visualization of 5-HT2A receptors in mammalian brain including post-mortem human brain.