Effects of antifolates on the binding of 5-fluoro-2'-deoxyuridine monophosphate to thymidylate synthase

Folate based inhibitors of thymidylate synthase (TS) might facilitate binding of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) to TS similar to the natural reduced folate 5,10-methylenetetrahydrofolate (CH(2)-H(4)-folate). We studied the lipophilic, non-polyglutamatable antifolates Nolatrexed (NTX) and AG331 and antifolates, that can have a polyglutamate side chain like the natural folate CH(2)-H(4)-folate; GW1843U89, Raltitrexed (RTX) and Multi-targetted antifolate (MTA) and pentaglutamates (RTX-Glu(5) and MTA-Glu(5)). The capacity of these compounds to facilitate the binding of [(3)H]FdUMP to Lactobacillus casei TS and an ammoniumsulphate precipitate of human TS was investigated. Only NTX, RTX-Glu(5) and MTA-Glu(5) facilitated FdUMP binding to L. casei TS and their dissociation constant K(d) (0.2-0.7 microM) was low compared to CH(2)-H(4)-folate (2.0 microM). The small lipophilic molecule NTX was favorable to the larger AG331. Polyglutamylation, as indicated by the difference in effect of RTX vs. RTX-Glu(5) and MTA vs. MTA-Glu(5), seems to be important for a classical antifolate to facilitate binding of FdUMP to bacterial TS. Effects of antifolates on FdUMP binding to human TS were different. At a low concentration (0.05 microM) NTX, RTX-Glu(5) and MTA-Glu(5) facilitated 3-5 times higher binding of [(3)H]FdUMP to TS than CH(2)-H(4)-folate. At higher concentrations (0.3-5 microM) of NTX, RTX-Glu(5) and MTA-Glu(5) the FdUMP binding decreased. The complex remained stable in the absence of (anti)folate for at least 24hr. The K(d) values of the antifolates for human TS varied from 19 to 387 nM, while the K(d) of CH(2)-H(4)-folate for human TS was 351 nM. The Hill coefficients, which indicated the type of cooperativity of the antifolates in the binding of FdUMP to TS were positive (0.58-0.99) at low concentrations (<0.3 microM) and negative (-0.35 to -0.81) at concentrations >0.3 microM except for GW1843U89, which only showed negative cooperativity (-1.70). It was shown with [(14)C]NTX that when the binding of FdUMP decreased at high NTX concentrations, the binding of NTX to TS still increased. This also held for the natural substrate dUMP. The negative cooperativity of the antifolates was clearly concentration dependent. The difference between human and L. casei TS in the FdUMP binding assays with antifolates can possibly be explained by interaction of the two subunits of human TS, which was absent in L. casei TS. The binding of antifolates to one of the two subunits induced a conformational change of the other subunit. This change no longer allowed the binding of FdUMP or dUMP at the active site. In conclusion this study showed that antifolates enhanced the binding of FdUMP to TS, especially at low antifolate concentrations, that are also clinically achievable, e.g. in human plasma.     

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.     

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.     

Carbonyl reduction of the potential cytostatic drugs benfluron and 3,9-dimethoxybenfluron in human in vitro

Benfluron (B, [5-(2-N-oxo-2-N',N"-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene]) is a potential benzo[c]fluorene antineoplastic agent with high activity against a broad spectrum of experimental tumors in vitro and in vivo. The structure of B has been modified to repress its rapid deactivation through carbonyl reduction on C7. 3,9-Dimethoxybenfluron (D, [3,9-dimethoxy-5-(2-N-oxo-2-N',N"-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene]) is one of the B derivatives developed. The present paper was designed to compare the C7 carbonyl reduction of B and D in microsomes, cytosol and hepatocytes from human liver. Two purified human enzymes, microsomal 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1) and cytosolic carbonyl reductase, were tested if they are responsible for B and D carbonyl reduction in the respective fractions. Indeed, carbonyl reduction of D in comparison to that of B was 4 and 6-10 times less extensive in human liver microsomes and cytosol, respectively. Moreover, about 10-20 times higher amounts of dihydro B than dihydro D were detected in primary culture of human hepatocytes. 11beta-HSD 1 was shown to be able to reduce B and D. For this enzyme, about 10 times higher rates of carbonyl reduction were observed for B than for D. Likewise, CR participates in B and D carbonyl reduction, although smaller amounts of both reduced metabolites were detected. In summary, carbonyl reduction of D was significantly less extensive than that of B in all in vitro experiments. This lower rate of D inactivation was especially pronounced in hepatocytes which represent a close to in vivo situation. Our results clearly demonstrate that dimethoxy substitution protects the carbonyl group of the benzo[c]fluorene moiety against the deactivation by microsomal and cytosolic reductases. Detailed knowledge on the participating enzymes may serve as a basis for the co-application of specific inhibitors in chemotherapy to further improve the pharmacokinetics of benzo[c]fluorene derivatives.     

Cellular transformation of the investigational new anticancer drug NB1011, a phosphoramidate of 5-(2-bromovinyl)-2'-deoxyuridine,results in modification of cellular proteins not DNA

NB1011 [E-5-(2-bromovinyl)-2'-deoxyuridine-5'-(L-methylalaninyl)-phenylphosphoramidate], a phosphoramidate prodrug of E-5-(2-bromovinyl)-2'-deoxyuridine-5'-monophosphate (BVdUMP), is an investigational new anticancer drug. NB1011 targets thymidylate synthase (TS), which catalyzes the transformation of BVdUMP into cytotoxic reaction products. Due to the elevated levels of TS expression in tumor cells compared to normal cells, these cytotoxic products are preferentially generated inside tumor cells, and, as expected, NB1011 is more toxic to cells with higher levels of TS expression. Therefore, NB1011 therapy should kill tumor cells without severely damaging normal cells. Radiolabeled NB1011 was used to determine the intracellular fate of NB1011 reaction products and, possibly, the mechanism of action of this investigational new drug. We found significant incorporation of the radiolabel into cellular macromolecules. In contrast to our expectations that NB1011 product(s) would be incorporated into DNA, we discovered that cellular proteins were the labeled macromolecular fraction. Herein, we report that the intracellular transformation of NB1011 involves formation of the corresponding monophosphate, TS-dependent transformation into highly reactive intermediates, and subsequent incorporation into cellular proteins. TS itself appears to escape irreversible inactivation. Our data suggest that protein modification not DNA incorporation accounts for the therapeutic effect of NB1011. The proposed mechanism is rather unexpected for a nucleotide analogue and could lead to the discovery of new cellular protein targets for future drug design.     

Deconjugation of N-glucuronide conjugated metabolites with hydrazine hydrate--biomarkers for exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been investigated in rabbit liver S9. Two phase I metabolites, N(2)-OH-PhIP and 4'-OH-PhIP were identified based on UV and mass spectra and co-elution with reference standards. Fortification of the incubation with UDGPA resulted in a complete glucuronidation of PhIP and N(2)-OH-PhIP, while 4'-OH-PhIP was only partly glucuronidated. Also, the PhIP metabolite 5-OH-PhIP was completely glucuronidated by rabbit liver S9, while 5-OH-PhIP was a poor substrate for CYP mediated hydroxylation. The glucuronic acid conjugates of PhIP metabolites were unsusceptible to treatment with beta-glucuronidase indicating that these are N-glucuronides. Treatment of the conjugates with hydrazine hydrate, however, resulted in complete hydrolysis of the glucuronic acid conjugates as well as in reduction to the parent amine of metabolites hydroxylated in the exocyclic amino group. Urine was collected from a male volunteer following consumption of fried chicken. Treatment of the urine with beta-glucuronidase/sulfatase resulted in release of 4'-OH-PhIP, while treatment with hydrazine hydrate in addition resulted in release of substantial amounts of PhIP and 5-OH-PhIP. The data show that hydrazine hydrate can hydrolyse N-glucuronides of metabolites of PhIP, glucuronides that are unsusceptible to enzymatic hydrolysis. In addition the data indicate that humans metabolise a large fraction of ingested PhIP to genotoxic metabolites. The chemical hydrolysis of glucuronide conjugates of PhIP metabolites with hydrazine hydrate observed in this study may also be a useful approach in the development of biomarkers for exposure and effect of other xenobiotics.     

Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance

Temozolomide (TMZ) is a newly approved alkylating agent for the treatment of malignant gliomas. To investigate resistance mechanisms in a multidrug therapeutic approach, a TMZ-resistant human glioma cell line, SF188/TR, was established by stepwise exposure of human SF188 parental cells to TMZ for approximately 6 months. SF188/TR showed 6-fold resistance to TMZ and cross-resistance to a broad spectrum of other anticancer agents that included 3-5-fold resistance to melphalan (MEL), gemcitabine (GEM), paclitaxel (PAC), methotrexate (MTX), and doxorubicin (DOX), and 1.6-2-fold resistance to cisplatin (CDDP) and topotecan (TPT). Alkylguanine alkyltransferase (AGT) activity was increased significantly in the resistant cell line compared with the parental cell line (P<0.05), whereas no significant differences occurred in the cellular uptake of TMZ and PAC between resistant and parental cells. Depletion of AGT by O(6)-benzylguanine significantly increased the cytotoxicity of TMZ in both the sensitive and resistant cell lines, but did not influence the cytotoxicity of the other drugs tested. Treatment with TMZ caused SF188 cells to accumulate in S phase, whereas SF188/TR cells were unaffected. Expression of Bcl-2 family members in SF188/TR cells compared with SF188 cells indicated that the pro-apoptotic proteins (i.e. Bad, Bax, Bcl-X(S)) were reduced 2-4-fold in the resistant cell line, whereas the anti-apoptotic proteins Bcl-2 and Bcl-X(L) were expressed at similar levels in both cell lines. In conclusion, the mechanism of resistance of SF188/TR cells to TMZ involved increased activity of AGT, a primary resistance mechanism, whereas the broad cross-resistance pattern to other anticancer drugs was due to a common secondary resistance mechanism related to alterations in the relative expression of the pro-apoptotic and anti-apoptotic proteins.     

Biomonitoring of urinary metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) following human consumption of cooked chicken

Human risk assessment of exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through the diet may be improved by conducting biomonitoring studies comparing metabolism in humans and rodents. Eleven volunteers ingested a meal of cooked chicken containing 4 -OH-PhIP and PhIP in amounts of 0.6 and 0.8mug/kg, respectively and urine was collected for the next 16h. The large number of PhIP metabolites was by treatment of the urine samples with hydrazine hydrate and hydrolytic enzymes reduced to three substances, 4'-OH-PhIP, PhIP and 5-OH-PhIP of which the first is a biomarker for detoxification and the last a biomarker for activation. The eleven volunteers eliminated large amounts of 4'-OH-PhIP in the urine. The majority of which could be accounted for by the presence of 4'-OH-PhIP in the fried chicken, showing that PhIP only to a small extent (11%) was metabolised to 4'-OH-PhIP. A larger fraction of the PhIP exposure, 38%, was recovered as PhIP and the largest fraction (51%) was recovered as 5-OH-PhIP suggesting that PhIP in humans to a large extent is metabolised to reactive substances. In rats, less than 1% of the dose of PhIP was eliminated as 5-OH-PhIP, suggesting that human cancer risk from exposure to PhIP is considerable higher than risk estimations based on extrapolation from rodent bioassays.     

Differential allosteric modulation by amiloride analogues of agonist and antagonist binding at A(1) and A(3) adenosine receptors

The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A(2A) adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A(1) and A(3) receptors, as well as agonist binding at A(1), A(2A), and A(3) receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([3H]PSB-11), from A(1) and A(3) receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A(1) receptors than at A(3) receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A(3) receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A(1), A(2A), and A(3) adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A(1) agonist [3H]N(6)-[(R)-phenylisopropyl]adenosine ([3H]R-PIA) from A(1) receptors or the A(2A) agonist [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) from A(2A) receptors. The dissociation rate of the A(3) agonist radioligand [125I]N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]I-AB-MECA) from A(3) receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A(3) receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A(3) receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A(3) receptors.     

Integrity of extracellular loop 1 of the human cannabinoid receptor 1 is critical for high-affinity binding of the ligand CP 55,940 but not SR 141716A

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (-)-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.     

Induction of interleukin 8 release from the HMC-1 mast cell line: synergy between stem cell factor and activators of the adenosine A(2b) receptor

The HMC-1 mast cell line has both adenosine A(3) and A(2b) receptors on its surface, but only agonists of the A(2b) receptor are effective at releasing interleukin 8. Object of this study was to look for co-factors for adenosine A(2b) receptor activation. There was a powerful and statistically significant synergy for release of IL-8, both at the mRNA level (measured after 4 hr) and protein level (measured after 24 hr), between adenosine A(2b) receptor agonists and stem cell factor (SCF). Suitable concentrations for showing synergy were 100 ng/mL SCF and 3 microM 5'-N-ethylcarboxamidoadenosine (NECA). At these concentrations, the IL-8 released into the culture medium after SCF and NECA together was typically 3-5-fold greater in amount than the sum of the amounts of IL-8 released after exposure to the same concentrations of NECA and SCF separately. Since mast cells may be exposed to both adenosine and stem cell factor in the diseased lung, the synergy observed in this model system may have implications for asthma.     

The direct stimulation of Gi proteins by neuropeptide Y (NPY) in the rat left ventricle

Neuropeptide Y (NPY) is a neuropeptide with high distribution in the cardiovascular system of mammals, where it modulates heart and vessel contractility. In the rat heart, the presence of at least three different NPY receptor subtypes has been hypothesised. Notwithstanding this, receptor activation might not be the only mechanism responsible for the complex cardiac effects of the peptide. In this study, we investigated the effect of NPY on the GTPase activity of G-proteins in the rat left ventricle as a possible alternative mechanism of action for the peptide in the rat heart. Our results show that NPY, but also the neuropeptide fragment (18-36) (NPY (18-36)), stimulated the basal, spontaneous GTPase activity of ventricle membranes only when it was measured under the condition of an absence of Mg2+. This stimulation was resistant to BIBP3226 a non-peptidergic antagonist at Y1 receptors, but it was significantly reduced in membranes treated with selective antibodies against the Gialpha subunits. NPYs effect was concentration-dependent with a maximum of activity at 10nM. At this concentration, NPY (and NPY 18-36) was able to inhibit forskolin (FSK)-induced cyclic adenosine-5'-monophosphate (cAMP) elevation in rat left ventricle slices. Our results assess that NPY in the rat heart is able to activate the GTPase activity of Gi proteins, in a receptor-independent way.     

Inhibition of leptin release by atrial natriuretic peptide (ANP) in human adipocytes

The addition of atrial natriuretic peptide (ANP) to isolated human adipocytes in primary culture from very obese individuals resulted in an inhibition of leptin release after a 24- or 48-hr incubation. There was also an inhibition of leptin release by isoproterenol (ISO) that was partially reversed by insulin, whereas the inhibition due to ANP was unaffected. Similar results were seen with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89), which is a cell-permeable inhibitor of protein kinase A. H-89 markedly reduced the effects of ISO on both lipolysis and leptin release without affecting the stimulation of lipolysis or the inhibition of leptin release due to ANP. Inhibition of endogenous nitric oxide formation using N(omega)-nitro-L-arginine resulted in a 20% increase in leptin release over 48 hr, which suggests that the nitric oxide/cyclic GMP pathway might play a small role in the regulation of endogenous leptin release. Similarly, the addition of the nitric oxide donor (Z)-1-[2-aminoethyl)-N-(2-aminoethyl)diazen-1-ium-1,2-diolate (DETA NONOate) at 0.1 or 1 microM to explants of human adipose tissue enhanced lipolysis by 29%. Our data demonstrate that the lipolytic effect of ANP is probably secondary to stimulation of cyclic GMP accumulation in human adipocytes, and this is accompanied by an inhibition of leptin release.     

Role of tissue transglutaminase in GTP depletion-induced apoptosis of insulin-secreting (HIT-T15) cells

The role of tissue transglutaminase (tTG), a calcium-dependent and GTP-modulated enzyme, in apoptotic death induced by GTP depletion in islet beta-cells was investigated. GTP depletion and apoptosis were induced by mycophenolic acid (MPA) in insulin-secreting HIT-T15 cells. MPA treatment increased in situ tTG activity (but not protein levels) in a dose- and time-dependent manner in parallel with the induction of apoptosis. MPA-induced increases of both tTG activity and apoptosis were entirely blocked by co-provision of guanosine but not adenosine. MPA-enhanced tTG activity could be substantially reduced by co-exposure to monodansylcadaverine or putrescine (tTG inhibitors), and largely blocked by lowering free Ca(2+) concentrations in the culture medium. However, MPA-induced cell death was either not changed or was only slightly reduced under these conditions. By contrast, a pan-caspase inhibitor (Z-VAD-FMK) entirely prevented apoptosis induced by MPA, but did not block the enhanced tTG activity, indicating that GTP depletion can induce apoptosis and activate tTG either independently or as part of a cascade of events involving caspases. Importantly, the morphological changes accompanying apoptosis could be markedly prevented by tTG inhibitors. These findings suggest that the effect of the marked increase in tTG activity in GTP depletion-induced apoptosis of insulin-secreting cells may be restricted to some terminal morphological changes.     

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5)

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.     

Novel effect of carvedilol on Ca2+ movement in renal tubular cells

The effect of carvedilol on intracellular free Ca(2+) levels ([Ca(2+)](i)) has not been explored previously. This study was aimed to examine the effect of carvedilol on Ca(2+) handling in renal tubular cells. Madin-Darby canine kidney cells were used as a model for renal tubular cells and fura-2 was used as a fluorescent Ca(2+) probe. Carvedilol increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 5 microM. Extracellular Ca(2+) removal partly inhibited the [Ca(2+)](i) signals. Carvedilol-induced Ca(2+) influx was verified by measuring Mn(2+)-induced quench of fura-2 fluorescence. Carvedilol-induced store Ca(2+) release was reduced by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) but not with 5 microM ryanodine or 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Carvedilol (30 microM)-induced Ca(2+) release was not affected by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-l)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; 2 microM), but was potentiated by increasing cAMP levels or inhibiting protein kinase C. The carvedilol-induced Ca(2+) mobilization was not significantly sequestered by the endoplasmic reticulum or mitochondria. This study shows that carvedilol increased [Ca(2+)](i) in renal tubular cells by causing Ca(2+) release from the endoplasmic reticulum and other unknown stores in an inositol-1,4,5-trisphosphate-independent manner, and by inducing Ca(2+) influx. The Ca(2+) release was modulated by cAMP and protein kinase C.     

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.     

The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A(1), A(2A), A(2B) and A(3). Previous studies have identified the involvement of A(2) receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A(2A) versus A(2B) receptors have not been determined in human primary monocytes. Nor has the role of A(1) and A(3) been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the A(2A) receptor agonist, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-alpha production, with IC(50)s of 58.4nM (32.7-104.5nM, 95% confidence interval) and 49.2nM (22.7-105.9nM, 95% confidence interval), respectively. The selective A(2A) receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 (pK(B) estimates were 8.7+/-0.1 and 8.9+/-0.1, respectively), while the selective A(2B) antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) nor the A(3) receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A(2A) receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-alpha production from LPS-stimulated monocytes.     

Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: comparison with human and rat receptor

Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48 h. Radioligand diprenorphine and [D-AIa(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-stimulated GTP gamma S binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTP gamma S binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor.