Benzo(a)pyrene metabolites: efficient and rapid separation by high-pressure liquid chromatography.

High-pressure liquid chromatography can separate eight metabolites of benzo[a] pyrene formed by rat liver microsomes. This method offers major advantages over previous techniques used for the separation of oxygenated polycyclic aromatic hydrocarbons.

Identification of differential melanocortin 4 receptor agonist profiles at natively expressed receptors in rat cortical astrocytes and recombinantly expressed receptors in human embryonic kidney cells.

Using cAMP accumulation as a functional readout, we pharmacologically characterized the response of native melanocortin receptors in cultured rat astrocytes, and found this response to be mediated by the melanocortin 4 receptor (MC4R). Melancortin agonists stimulate cAMP in a concentration-dependent manner in both astrocytes and human embryonic kidney cells recombinantly expressing rat MC4R (HEK-rMC4R), however, the relative potency and intrinsic activity of both small molecule and peptide agonists are reduced in the native system. As such, the small molecules THIQ, NBI-702 and MB243 display 43, 30 and 18% of the maximal response elicited by alpha-MSH in astrocytes. Likewise, the peptides MTII and ACTH display 55 and 72% of the maximal response elicited by alpha-MSH in these cells. In contrast, all of these compounds elicit full agonist responses with similar intrinsic activity to alpha-MSH in HEK-rMC4R cells. MC4R mRNA was detected in astrocytes, however radioligand binding experiments failed to detect measurable MC4R in astrocyte membranes, in contrast to membranes from HEK-rMC4R cells that display a binding site density of 18.1+/-1.5 fmol/mg. We propose that the divergent observations in functional activity between the cell types reflect differences in receptor expression and that caution should be exercised when interpreting agonist activity in over-expression systems for the purposes of drug discovery.

Comparison of epoxide and free-radical mechanisms for activation of benzo[a]pyrene by Sprague-Dawley rat liver microsomes.

Coincubation of [6-3H]benzo[a]pyrene ([6-3H]BP) and [14C]BP with SD rat liver microsomes produced metabolic profiles that showed that the C-6 of BP was not affected by formation of 4,5-dihydro-4,5-dihydroxy-BP, 7,8-dihydro-7,8-dihydroxy-BP, and 9,10-dihydro-9,10-dihydroxy-BP nor the 3- and 9-phenols of BP. Complete retention of tritium at C-6, except in the three quinones, confirmed the radical-cation model for formation of the 6-oxo-radical followed by oxidation to quinones. Epoxide formation at the carcinogenically active regions of BP appeared to biochemically isolate from 6-position activation and suggested that the microsomal epoxide pathway is unrelated to the radicalcation scheme. These molar ratios derived from double-label experiments reinforced the current literature that indicates the epoxide mechanism as the major pathway toward carcinogenic forms of BP.

Effect of trichloropropene oxide on the ability of polyaromatic hydrocarbons and their "K-region" oxides to initiate skin tumors in mice and to bind to DNA in vitro.

The potent epoxide hydrase inhibitor, 1,1,1-trichloro-2,3-propene oxide (TCPO), enhanced the tumor-initiating ability of benzo[alpha]pyrene (BP) and 3-methylcholanthrene (MCA) but had no effect on 9,10-dimethyl-1,2-anthracene (DMBA) initiation in a two-stage system of tumorigenesis in female Charles River CD-1 mice. The tumor-initiating ability of dibenz[alpha,h]-anthracene (DBA) was decreased by prior topical treatment with 10 mumoles of TCPO. The tumor latency period of BP and MCA was decreased by TCPO but had no effect on DMBA or DBA. Topical treatment with 10 mumoles of TCPO did not initiate tumors in a two-stage system in mouse skin nor did it cause any histopathologic changes in the skin. The "K-region" epoxides of BP, DMBA, and MCA were weak tumor initiators when compared to the parent compounds. TCPO only slightly increased or had no effect on the tumor-initiating activity of the above epoxides. Pretreatment with Croton oil 18 hours prior to initiation with BP-4,5-epoxide also slightly enhanced the tumorigenic response in mouse skin. DBA-5,6-epoxide, when tested as a complete carcinogen at high doses (1 mg daily/10 days), was found to be a weak carcinogen but with activity comparable to that of DBA. TCPO only slightly increased the in vitro epidermally mediated covalent binding of the above parent polycyclic hydrocarbons to DNA.

Metabolism of benzo(a)pyrene by variant mouse hepatoma cells.

Four mouse hepatoma cell lines, a parent (Hepa-1c1c7) and three variants (MUL12, BPrc1, and TAOc1BPrc1) which had been derived from Hepa-1c1c7 by the fluorescence-activated cell sorter, were incubated with benzo(a)pyrene, and the metabolites were analyzed by high-pressure liquid chromatography. Among these four cell lines, Hepa-1c1c7 and MUL12 metabolized benzo(a)pyrene the most quickly and to the greatest extent, and BPrc1 had the weakest metabolic activity for this substrate. TAOc1BPrc1 had intermediate benzo(a)pyrene-metabolizing activity, depending on cell density and incubation time. At low cell density, the active variant TAOc1Bprc1 resembled the weakly active Bprc1 in accumulating a low amount of ethyl acetate-soluble metabolites in the medium while, at high cell density, TAOc1Bprc1 resembled the parent clone Hepa-1c1c7 and the highly active variant MUL12. At short incubation times, TAOc1Bprc1 also had low conjugating activity while, at longer incubation times, the conjugating activity approached that of Hepa-1c1c7 and MUL12. At low cell density, Bprc1 was able to produce phenols, but this variant did not seem to have this ability at high cell density. When the substrate concentration was 4 microM and the incubation time was 24 h, beta-glucuronidase treatment of water-soluble metabolites released about 5.3 times more pmol of quinones compared with phenols. But when the substrate concentration was 25 nM, beta-glucuronidase released about 2.0 times as many phenols compared with quinones. The parent and the two more actively metabolizing variants showed differences in the peak times of accumulation of 9,10-diol and 7,8-diol of benzo(a)pyrene, which may have implications for binding to DNA and nuclear proteins. It was concluded that BPrc1 has basal but not easily inducible aryl hydrocarbon hydroxylase activity, whereas Hepa-1c1c7, MUL12, and TAOc1Bprc1 have basal and inducible aryl hydrocarbon hydroxylase activity. These results show that variants of a single parent cell line can exhibit significant differences in the rate and extent of metabolism of benzo(a)pyrene.

High-pressure liquid chromatographic separation of 10 benzo(a)pyrene phenols and the identification of 1-phenol and 7-phenol as new metabolites.

The separation of ten isomeric benzo(a)pyrene phenols has been accomplished by the use of high-pressure liquid chromatography utilizing a newly developed recycling technique and new column and solvent systems. Using this new system and comparing the metabolites obtained with authentic standards, we have isolated 1-hydroxybenzo(a)pyrene and 7-hydroxybenzo(a)pyrene and identified them as metabolites formed by rat liver microsomes. In previously reported chromatography systems, the new metabolites migrated with another metabolite, 3-hydroxybenzo(a)pyrene.

Two-dimensional gel electrophoretic analysis of cytoplasmic proteins from Friend erythroleukemia cells chemically induced to undergo terminal erythroid differentiation.

The proerythroblastoid Friend erythroleukemia cell (FELC) line, clone TR 19-9, was treated with 4 mM hexamethylene bisacetamide (HMBA) over a 6-day period. Greater than 94% of the FELC reacted positively to the benzidine assay for hemoglobin by Day 4 of treatment. Protein accumulation during the final 4 days of treatment (from Days 2 to 6) was monitored by labeling for 24-h periods with a 14C-labeled amino acid mixture. At the end of each radiolabeling time point, cells were harvested and cytoplasmic proteins were isolated and subjected to two-dimensional gel electrophoresis in triplicate. Short-term fluorographic exposures were made in the linear X-ray film response range to monitor those polypeptides which were most rapidly accumulated. Fluorographs were digitized for computer image analysis and gel data comparison rationales were used to combine the polypeptides contained on the replicate fluorographs into a single cytoplasmic polypeptide profile or Master Image for each of the two experimental conditions, control and HMBA-treated FELC. These two images were merged into a single Master Composite Image containing a total of 211 polypeptides so that those polypeptides common to both and/or unique to each of the experimental conditions could be viewed graphically in the same plane. A total of 98 polypeptides in HMBA-treated FELC were shown to have large accumulation rate differences from the control FELC;32 of these polypeptides were present in the HMBA Master Image which were not detected in the Control Master Image and 66 polypeptides were present in the Control Master Image but not detected in the HMBA Master Image. Five polypeptides, found in both Master Images, were shown to vary quantitatively in the HMBA-treated FELC from the corresponding polypeptides in the control. These quantitative data measurements on the rates of accumulation of various common polypeptides offer a mode for simultaneously monitoring the kinetics of induction and repression of many gene products throughout an experimental time course.

Differences in benzo(a)pyrene metabolism between rodent liver microsomes and embryonic cells.

Differences in benzo(a)pyrene metabolite pattern have been shown by rodent liver microsomes (Sprague-Dawley) and rodent embryo cells from Syrian hamsters and NIH Swiss mice. Rodent liver induced by methylcholanthrene shows marked quantitative variation between species. Additional pattern changes were found in mouse and hamster embryo secondary cultures with a reduction of the K-region metabolites and a marked increase in 9-hydroxybenzo(a)-pyrene. These results are indicative of a region-specific attack on the carcinogen by the cell monooxygenases which is distinct from the liver attack of microsomal enzymes on benzo(a)pyrene. These results suggest that activation and detoxification of benzo(a)pyrene may be species and tissue variable, and susceptibility and resistence to malignant transformation may be predicted on induction of a fortuitous combination of intermediate metabolic steps.

Metabolism and macromolecular binding of carcinogenic and noncarcinogenic metabolites of benzo(a)pyrene by hamster embryo cells.

The metabolism and macromolecular binding of four metabolites of benzo(a)pyrene in hamster embryo fibroblasts has been studied. Two noncarcinogenic phenolic derivatives, 3-hydroxybenz(a)pyrene and 9-hydroxybenzo(a)pyrene, are rapidly metabolized, primarily to their respective glucuronic acid conjugates and other H2O-soluble conjugates (78.4 to 80.8% of total radioactivity). Water-soluble conjugates were also formed from the carcinogenic phenol, 2-hydroxybenzo(a)pyrene, and from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, but in lower amounts (36.8 to 43.8% of total radioactivity. With each of the compounds, from 10 to 20% of the radioactivity was converted to ethyl acetate-soluble metabolites. The amount of unmetabolized 2-hydroxybenzo(a)pyrene recovered intracellularly was 20-fold higher than that recovered in incubations with the other phenols. Covalent binding to nuclear macromolecules was monitored after isopyknic separation. Binding of the three phenols tested was similar and was lower than the binding of benzo(a)pyrene to nuclear DNA, RNA, and protein. In contrast to the results with the monohydroxybenzo(a)pyrenes, high levels of covalent binding were observed with 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene; binding to DNA was 8-fold higher (315 pmol bound per mg DNA) than binding of benzo(a)pyrene to DNA.

Kinetic analysis of the metabolism of benzo(a)pyrene to phenols, dihydrodiols, and quinones by high-pressure chromatography compared to analysis by aryl hydrocarbon hydroxylase assay, and the effect of enzyme induction.

High-pressure liquid chromatography was used to analyze the rate of benzo(a)pyrene metabolite formation by liver microsomes from control and 3-methylcholanthrene-treated rats. The relative amounts of each metabolite formed were determined with several concentrations of microsomal mixed-function oxidases. The specific activity, i.e., amount formed per mg protein per min, was found to be constant for the formation of 3-hydroxybenzo(a)pyrene and 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene. The specific activity for the formation of 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene was higher at high microsomal enzyme concentration. The formation of 9-hydroxybenzo(a)pyrene, however, did not increase with greater amounts of microsomes. The data indicate that 9-hydroxybenzo(a)pyrene is a nonenzymatic product derived from a reactive intermediate, probably benzo(a)pyrene-9,10-oxide. The relatively constant specific activity for the formation of 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene with several enzyme concentrations suggests that the K-region epoxide, benzo(a)pyrene-4,5-oxide, is the most stable of the benzo(a)pyrene epoxide intermediates. The relative percentages of each metabolite fraction found are as follows: 3-hydroxybenzo(a)pyrene, 36; 9-hydroxybenzo(a)pyrene, 3 to 13; 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, 15 to 25; 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, 8; 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 12 to 14; benzo(a)pyrene quinones, 14 to 17. Induction of the enzyme system by 3-methylcholanthrene increases the amount of each metabolite formed to a different extent. The amount of 9,10-dihydro-9,10-dihydroxy- and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene formed are markedly increased relative to the increase in the other metabolites. Thus the induction of the enzyme may specifically alter pathways of metabolism relevant to carcinogenesis. This study also makes a detailed comparison between the results obtained by high-pressure liquid chromatography analysis and the standard aryl hydrocarbon hydroxylase assay and further develops the chromatographic analysis of benzo(a)pyrene metabolites.

In vitro metabolism of benzo(a)pyrene by human liver microsomes and lymphocytes.

The metabolism of benzo(a)pyrene by human liver microsomes and human lymphocytes has been analyzed by high-pressure liquid chromatography. Human liver forms seven known metabolites and at least five additional unidentified metabolites that migrate as distinct peaks. Lymphocytes incubated with benzo(a)pyrene for 30 min do not form dihydrodiols. Lymphocytes incubated for 24 hr with benzo(a)pyrene form all of the metabolites produced by liver including dihydrodiols as well as additional metabolites. The ratios of phenols formed by liver and lymphocytes are different, and preparations from humans form a different profile of metabolites than that formed by rat liver.

p53-dependent p21 induction following gamma-irradiation without concomitant p53 induction in a human peripheral neuroepithelioma cell line.

We previously generated cell hybrids between a derivative of the E6-containing HeLa cell line and a p53 null peripheral neuroepithelioma (PNET) cell line. Although p53 protein from the hybrids was genotypically wild type, it did not demonstrate wild-type behavior. Therefore, in the present study, we introduced wild-type p53 into the PNET parent to investigate whether p53 retained wild-type function within this cell line. Although the p53 null PNET parent lacked detectable p21 protein, introduction of wild-type p53 resulted in a detectable expression of p21 protein in all clones tested, suggestive of wild-type p53 function. In addition, p53 expression was necessary for induction of p21 in response to irradiation, and, furthermore, we show this induction to occur at the transcriptional level. Although introduction of wild-type p53 seems to be responsible for p21 induction, the overall protein levels of p53 were not induced. The involvement of p53 in up-regulating p21 is further substantiated by the observation that p21 up-regulation was dependent on the introduction of the wild-type protein. Our results suggest that wild-type p53 is capable of up-regulating p21 in response to DNA damage in the absence of p53 induction.

Carcinogenicity and mutagenicity of benz(a)anthracene diols and diol-epoxides.

Benz(a)anthracene (BA) and its five possible trans-dihydrodiols were evaluated for determination of their skin tumor-initiating activity and their mutagenic activity in Chinese hamster V79 cells. In addition, the skin tumor-initiating abilities of five diol-epoxides of BA were tested. Results showed (+/-)-trans-3,4-dihydroxy-3,4-dihydrobenz(a)anthracene (BA 3,4-dihydrodiol) to be approximately 10 times more mutagenic than was BA and about 20 times more mutagenic than were the other possible dihydrodiols in the V79 cells cocultivated with irradiated hamster embryo cells. As a skin tumor initiator, BA 3,4-dihydrodiol was approximately 5 times more active than BA, whereas the other BA dihydrodiols were all less active tumor initiators. (+/-)-trans-3alpha,4beta-Dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenz(a)anthracene was found to be approximately 20% more active as a tumor initiator than was BA 3,4-dihydrodiol, whereas the other diol-epoxides of BA were less active than BA itself. The results suggest that the bay-region diol-epoxide of BA may be the ultimate carcinogen and mutagenic form of BA.

Binding of benzo(a)pyrene derivatives to specific proteins in nuclei of intact hamster embryo cells.

In hamster embryo cells incubated for 24 hr with 4 microM [3H]benzo(a)pyrene, a major portion of the nonextractable radioactivity in nuclear preparations copurifies with the protein fraction. When these proteins are analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis, significant variations in the labeling intensities of the various proteins are seen. Control experiments demonstrate that the labeling is due to covalent binding to protein. Histones H3 an H2A are heavily labeled while the other histones of the nucleosome core, H2B and H4, are devoid of radioactivity. Large amounts of label are associated with proteins with mobilities similar to the very lysine-rich histones H1. However, the results of differential extraction experiments suggest that the labeled proteins do not belong to either the H1 or the high-mobility-group class of chromosomal proteins. During 6 hr of inhibition of protein synthesis by cycloheximide, the metabolism of [3H]benzo(a)pyrene, as monitored by high-pressure liquid chromatography, remained normal. Patterns of labelling of nuclear proteins after 3 or 6 hr were identical in the presence and absence of cycloheximide. This finding strongly suggests that binding of benzo(a)pyrene derivatives to nuclear proteins occurs in situ.

Benzo(a)pyrene:DNA adduct formation in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line.

The benzo(a)pyrene (BaP):DNA adducts formed in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line were analyzed by chromatography and acid hydrolysis of the BaP:deoxyribonucleoside adducts to BaP:purine adducts and BaP:tetraols. Human mammary epithelial cell cultures and human mammary carcinoma T47D cells were exposed to [3H]BaP for 24 h, and the levels of binding were 81 and 182 pmol BaP/mg DNA in normal and T47D cultures, respectively. Analysis of BaP:deoxyribonucleoside adducts resolved by immobilized boronate chromatography and reversephase high-performance liquid chromatography demonstrated the presence of three BaP:deoxyribonucleoside adducts in both cells: M2, MS1, and MS2 in a ratio of 1.6:1:14. Two adducts (MS1 and MS2) bound to the immobilized boronate column indicating the presence of cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA. MS2 was identified as (+)-anti-BaPDE:deoxyguanosine (dGuo) for it cochromatographed with a [14C]-(+)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS2 cochromatographed with [14C]-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (+/-)-anti-BaPDE:tetraols. MS1 was identified as (-)-anti-BaPDE:dGuo for MS1 eluted in the same relative position as a (-)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS1 cochromatographed with [14C]-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (+/-)-anti-BaPDE:tetraols. Thus, both adducts that bound to the immobilized boronate column were formed from (+/-)-anti-BaPDE. One major adduct that did not contain cis-vicinal hydroxy groups, M2, was detected in both cell types. M2 was formed from (+/-)-7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydroBaP (syn-BaPDE) as M2 eluted in the same relative position as a syn-BaPDE:dGuo adduct marker and the tetraol hydrolysis products of M2 cochromatographed with tetraols formed from (+/-)-syn-BaPDE. The isolation of the individual BaP:DNA adducts followed by acid hydrolysis allowed the identification of the BaP:DNA adducts formed in human mammary cell cultures and demonstrated the presence of (-)-anti-BaPDE:dGuo. Thus, this work provides the first evidence, other than cochromatography, that (-)-anti-BaPDE is formed in cell systems and reacts with DNA in cells to form (-)-anti-BaPDE:dGuo.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of benzo(a)pyrene by human epithelial cells in vitro.

Primary cell cultures derived from human skin epithelium metabolized benzo(a)pyrene to three classes of compounds: phenols, quinones, and dihydrodiols. The relative proportions of metabolites varied according to the skin donor but differed from the pattern of metabolites in rat liver microsome preparations. While appreciable amounts of 7,8- and 9,10-dihydrodiol; 1,6-, 3,6-, and 6,12-quinone; and 3- and 9-hydroxy derivatives were found in the medium, no 4,5 (K-region)-dihydrodiol or epoxide was detected. Reduced amounts of quinones were produced when the cultures were pretreated with hydrocortisone before exposure to the hydrocarbon. The cultures did not require a period of enzyme induction for efficient metabolism of the hydrocarbon. Cultures of fibroblasts derived from the same skin samples as the epithelial cells metabolized the hydrocarbon but to a much different extent. Preexposure of the epithelial cell cultures to mixtures of polycyclic hydrocarbons resulted in a decrease in the amounts of carcinogen metabolized to phenols and dihydrodiols. These findings suggest that the prevalence of carcinomatous disease in humans is due to the differential capacity of the epithelial cells to metabolize potential carcinogens to active forms, a capacity reduced in fibroblasts or other nonepithelial cells. This suggestion is supported by the observations that supposedly normal prostate cells also efficiently metabolize polycyclic hydrocarbons in a manner similar to that of epidermal cells. No evidence of neoplastic transformation was seen in cytological preparations of cells exfoliated into the medium.

HSP binding and mitochondrial localization of p53 protein in human HT1080 and mouse C3H10T1/2 cell lines.

In normal cells, the tumor suppressor actions of p53 protein are mediated by specific DNA binding and protein-protein interactions within the nucleus. Mutant p53 proteins, however, often assume an aberrant conformation devoid of tumor suppressor activity and newly capable of binding to the cognate or inducible HSP70. Recent reports from our laboratory and others show that additional unknown proteins may also complex with mutant p53. In this study, we characterize p53:HSP complexes and their subcellular location in the transformed cell lines, human HT1080 and murine C3H10T1/2, which both contain aberrant p53 conformers. Immunoprecipitation and SDS-PAGE of p53 from whole cell lysates revealed the additional presence of a broad 70 kDa band and a 90 kDa band in both lines, while p53 isolated from nuclear lysates was free from other proteins. 2D-PAGE was used to isolate and identify HSP members from cytoplasmic and nuclear lysates by immunoprecipitation, Western blotting and protein sequencing. Anti-p53 immune complexes from cytoplasmic lysates contained not only HSC70 but also GRP75, GRP78 and a weakly basic 90 kDa protein, which may be related to HSP90. The inducible form of HSP70 was not complexed to p53 protein, even though expressed in these cells. Analysis of anti-HSP70, anti-GRP75 and anti-HSP90 immune complexes suggests that HSP members exist as performed complexes in the cytoplasm, but not the nucleus. The presence of the mitochondrial and endoplasmic reticular chaperones, GRP75 and GRP78, in p53:HSP complexes suggested that p53 might be found in these cytoplasmic organelles which was confirmed in mitochondria by biochemical and immunoelectron microscopic evidence. These studies suggest that newly identified members of p53:HSP complexes represent components of a chaperone program which affects the subcellular distribution of p53 protein in these transformed lines.

Blockade of excitatory amino acid transporters in the rat hippocampus results in enhanced activation of group I and group III metabotropic glutamate receptors.

The idea that excitatory amino acid transporters (EAATs) can control the activation of specific metabotropic glutamate receptors (mGluRs) was investigated in rat hippocampal slices. Using the accumulation of inositol phosphates as a measure of group I mGluR activity, we have shown that the broad spectrum, non-transportable EAAT blocker, TBOA, produces a significant shift to the left of agonist concentration-response curves. Moreover, this increase in potency did not occur if endogenous glutamate was enzymatically removed, suggesting a glutamate-dependent mechanism. This shift in potency was shown to be NMDA and group II mGlu receptor independent. Additionally, experiments with selective antagonists indicated that the group I receptor responsible for the stimulation of inositol phosphate production in this preparation is likely to be mGluR5. Inhibition of forskolin-stimulated cyclic AMP (cAMP) production was used as an index of group II/III mGluR activity. TBOA produced a rightward shift of the forskolin concentration-response curve. A group III, but not a group II, mGluR agonist also produced this effect, suggesting that the TBOA-mediated increase in glutamate activates a receptor, which appears to be a member of the group III mGluR subset. This was confirmed by the observation that an antagonist of group III mGluRs, prevented the TBOA-induced rightward shift in forskolin potency. These results provide evidence of a role for EAATs in the regulation of mGluR5 and group III mGluRs in the rat hippocampus, which may have therapeutic implications.

Cell type-specific differences in the coupling of recombinant mGlu1alpha receptors to endogenous G protein sub-populations.

In this study the effects of cell background on the coupling of the type 1alpha metabotropic glutamate (mGlu1alpha) receptor to different G protein sub-populations by recombinant expression of this receptor subtype in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells have been investigated. Receptor-G protein interactions were assessed using [(35)S]GTPgammaS binding and subsequent Galpha subunit-specific immunoprecipitation. In a CHO cell line (CHO-lac-mGlu1alpha), where mGlu1alpha receptor expression is under inducible control, stimulation of membranes with the mGlu receptor agonist quisqualate resulted in an increase in specific [(35)S]GTPgammaS binding to G(q/11)alpha only, whereas in a BHK cell line (BHK-mGlu1alpha) agonist stimulation increased [(35)S]GTPgammaS binding to G(q/11)alpha and also to pertussis toxin (PTx)-sensitive G(i/o) proteins (assessed using G(i1/2)alpha- and G(i3/o)alpha-specific antibodies). These data are consistent with our previous observations of dual, antagonistic G(q/11)/G(i/o) regulation of phospholipase C (PLC) in BHK-mGlu1alpha cells, whereas no evidence was found for a G(i/o) modulation of PLC activity in the CHO-lac-mGlu1alpha cell line. PTx pre-treatment of either cell line had no effect on either the magnitude or the concentration-dependency of agonist-stimulated [(35)S]GTPgammaS-G(q/11)alpha binding, excluding the possibility that receptor-G(i/o) uncoupling can unmask an increase in receptor-G(q/11) interaction. mGlu1alpha receptor expression per se had little effect on Galpha protein expression levels in either CHO or BHK cell lines, with the possible exception of a small, but consistent increase in G(o)alpha expression in BHK-mGlu1alpha cells compared to the vector-transfected control cell line (BHK-570). Semi-quantitative assessment of mGlu1alpha receptor immunoreactivity and [(3)H]quisqualate saturation binding analysis demonstrated a ca 10-fold higher mGlu1alpha receptor content in BHK cells. Whether the higher receptor expression level in BHK-mGlu1alpha cells underlies the additional G(i/o) coupling observed in this cell line, or additional factors contribute to the phenomenon are discussed.

The selective 5-HT1B receptor inverse agonist 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro- spiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289) potently blocks terminal 5-HT autoreceptor function both in vitro and in vivo.

5-HT1 receptors are members of the G-protein-coupled receptor superfamily and are negatively linked to adenylyl cyclase activity. The human 5-HT1B and 5-HT1D receptors (previously known as 5-HT1Dbeta and 5-HT1Dalpha, respectively), although encoded by two distinct genes, are structurally very similar. Pharmacologically, these two receptors have been differentiated using nonselective chemical tools such as ketanserin and ritanserin, but the absence of truly selective agents has meant that the precise function of the 5-HT1B and 5-HT1D receptors has not been defined. In this paper we describe how, using computational chemistry models as a guide, the nonselective 5-HT1B/5-HT1D receptor antagonist 4 was structurally modified to produce the selective 5-HT1B receptor inverse agonist 5, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6, 7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289). This compound is a potent antagonist of terminal 5-HT autoreceptor function both in vitro and in vivo.