The activity of MAO A and B in rat renal cells and tubules

The present study reports on the presence of type A and B monoamine oxidase (MAO) activity and their sensitivity to selective MAO-A and MAO-B inhibition by Ro 41-1049 and lazabemide, respectively, in homogenates of isolated rat renal tubules. Non-linear analysis of the saturation curve of H-5-hydroxytryptamine (3H-5-HT ) deamination revealed a Km of 351+/-71 microM (n=4) and a Vmax of 25+/-2 nmol mg protein(-1) h(-1). Deamination of 14C-beta-phenylethylamine (14C-beta-PEA) was also a saturable process yielding Km values of 58+/-12 microM and Vmax values of 24+/-2 nmol mg protein(-1) h(-1). Ro 41-1049 produced a concentration-dependent inhibition of 3H-5-HT deamination with a Ki of 24 nM. Deamination of 14C-beta-PEA was found to be reduced by lazabemide in a concentration-dependent manner with a Ki value of 17 nM. The effect of these selective MAO inhibitors on dopamine fate and DOPAC formation in isolated tubular epithelial cells was also studied. In these studies a clear inhibition of DOPAC formation was observed with Ro 41-1049 (250 nM), while 250 nM lazabemide was found not to increase the accumulation of newly-formed DA in those tubular epithelial cells loaded with 50 microM L-DOPA. In conclusion, the results presented here confirm the presence of both MAO-A and MAO-B activity in renal tubular epithelial cells, that MAO-A is the predominant enzyme involved in the deamination of the natriuretic hormone dopamine and that the deamination of newly-formed dopamine is a time-dependent process which occurs early after the decarboxylation of L-DOPA.     

Identification and characterization of human cytochrome P450 isoforms interacting with pimozide

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.     

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

Interactions between transport of triiodothyronine and tryptophan in JAR cells

We studied the effect of a number of amino acids on uptake of L-triiodothyronine (T3) in the human choriocarcinoma cell line, JAR. Tryptophan inhibited saturable T3 uptake by about 57% without any significant effect on the non-saturable uptake. Michaelis constant (Km) for T3 uptake was 1.06 +/- 0.15 microM (n = 15) with the corresponding maximum velocity (Vmax) of 24.2 +/- 3.1 pmol/min/mg cellular protein. For tryptophan uptake the Km was 1.31 +/- 0.26 microM (n = 7) and Vmax was 166.4 +/- 35.7 pmol/min/mg protein. The kinetic parameters for both uptake processes were similar to those reported in normal placenta. Uptake of T3 was inhibited by tryptophan but not phenylalanine, but tryptophan uptake was inhibited both by T3 and phenylalanine. Inhibition of T3 uptake by tryptophan was dose dependent, with an inhibition constant (Ki) of 2.9 +/- 0.5 mM. Similarly, tryptophan uptake was inhibited by T3 and phenylalanine in a dose dependent way with Ki values of 4.9 +/- 0.5 microM and 15.6 +/- 4.8 microM respectively. Km for T3 uptake was significantly increased to 1.86 +/- 0.42 microM (n = 4) in the presence of 3 mM unlabelled tryptophan and, similarly, Km for tryptophan uptake was significantly increased to 9.91 +/- 2.57 microM (n = 3) in the presence of 5 microM unlabelled T3. Efflux of T3 was progressively inhibited by increasing concentrations of both ligands, i.e. was saturable. We conclude that there is mutual competitive inhibition between uptake systems for T3 and tryptophan in JAR cells, but the kinetic parameters of cross-inhibition of uptake by the substrates suggest that the carriers are distinct. T3 may be transported in JAR cells by at least two transport systems with differing substrate specificities. We also demonstrated the presence of a saturable membrane carrier mediating the efflux of T3 from the cells which was subject to trans-inhibition by T3 and tryptophan.     

Kinetic and allosteric cooperativity in L-adenosine transport in chromaffin cells. A mnemonical transporter

In cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue, the transport of D-[3H]adenosine followed Michaelis-Menten saturation kinetics, with Km values of 1.5 +/- 0.3 microM and 1.9 +/- 0.2 microM, respectively. The transport of the isomer, L-[3H]adenosine, showed sigmoidal kinetics in both preparations. In plasma membrane vesicles the S0.5 was 2.5 +/- 0.2 microM with a Hill coefficient of 2.8 and the Vmax value of 0.26 +/- 0.01 pmol s-1 (mg of protein)-1. In cultured chromaffin cells the kinetic parameters for L-[3H]adenosine were S0.5 = 6.2 +/- 0.2 microM and a Vmax 19.7 +/- 0.5 pmol/min per 10(6) cells, with a pronounced positive cooperativity. The Hill coefficient was 4.9. The transport of the L-isomer in cultured cells followed Michaelis-Menten kinetics at the lowest concentrations employed, below 2 microM. On the basis of these results, we propose a kinetic model whereby the adenosine transporter functions mnemonically.     

Neurochemical and electrophysiological evidence for the existence of a functional gamma-hydroxybutyrate system in NCB-20 neurons

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into gamma-hydroxybutyrate. This activity (1.14+/-0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [3H]gamma-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes (Kd=250+/-44.4nM, Bmax=180+/-16.2fmol/mg protein) that apparently represents specific gamma-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na+-dependent uptake of [3H]gamma-hydroxybutyrate was expressed in NCB-20 cells with a Km of 35+21.1 microM and a Vmax of 80+/-14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K+-evoked release of [3H]gamma-hydroxybutyrate occurred. This release was Ca2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [3H]gamma-hydroxybutyrate binding sites, which could be due to spontaneous gamma-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca2+ conductances which were partially inhibited by 50 microM gamma-hydroxybutyrate. This gamma-hydroxybutyrate-induced effect was blocked by the gamma-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA(B) antagonist CGP-55845. These results demonstrate the presence of an active gamma-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release gamma-hydroxybutyrate. These cells express specific gamma-hydroxybutyrate receptors which modulate Ca2+ currents independently of GABA(B) receptors.     

Inhibitory actions of emodin on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acetyltransferase (NAT) activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori, a gram-negative rod bacteria collected from peptic ulcer patients. The NAT activity was determined using a acetyl CoA recycling assay and HPLC. Cytosols or suspensions of H. pylori with and without selected concentrations of emodin co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there were decreased NAT activity associated with increased emodin in H. pylori cytosols. As 400 microns of emodin can obviously inhibit NAT activity both in vitro and in vivo (inhibition rate 90% and 93% for 2-aminofluorene and p-aminobenzoic acid in vitro, and 90% and 92%, respectively, for both substrate in vivo). For in vitro examination, the apparent values of Km and Vmax were 3.12 +/- 0.38 mM and 15.20 +/- 3.16 nmol/min/mg protein for 2-aminofluorene, and 0.56 +/- 0.12 mM and 0.74 +/- 0.09 nmol/min mg protein for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 2.40 +/- 0.32 mM and 10.62 +/- 0.04 nmol/min/mg protein for 2-aminofluorene, and 0.23 +/- 0.02 mM and 0.62 +/- 0.08 nmol/min/mg protein for p-aminobenzoic acid. For in vivo examination, the apparent Km and Vmax were 0.82 +/- 0.18 mM and 0.92 +/- 0.21 nmol/min/10 x 10(10) colony forming units (CFU) for 2-aminofluorene, and 0.78 +/- 0.14 mM and 0.52 +/- 0.06 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 0.50 +/- 0.08 mM and 0.62 +/- 0.22 nmol/min/ 10 x 10(10) (CFU) for 2-aminofluorene, and 0.52 +/- 0.21 mM and 0.26 +/- 0.04 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. This report is the first finding of emodin inhibition of arylamine N-acetyltransferase activity in a strain of H. pylori.     

High concentration of L-arginine suppresses nitric oxide synthase activity and produces reactive oxygen species in NB9 human neuroblastoma cells

Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6) cells to 1310.0 +/- 5 pmol/10(6) cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 +/- 0.2 pmol/10(6) cells (mean +/- SD of three independent analyses). This increase in intracellular L-Arg levels caused a decrease in NOS activity by approximately 71%. Flow cytometric analysis showed that reactive oxygen species (ROS) are produced in NB9 exposed to 2 mM L-Arg. The production of ROS was abolished by a NOS inhibitor, NG-nitro-L arginine-methylester. Production of ROS was also observed when NB9 were treated with L-Cit for 48 hr. To investigate the effect of L-Cit on the activity of NOS, a kinetic study on nNOS was conducted using cellular extracts from NB9. The apparent Km value of nNOS for L-Arg was 8.4 microM, with a Vmax value of 8.2 pmol/min/mg protein. L-Cit competitively inhibited NOS activity, as indicated by an apparent Ki value of 65 nM. These results suggest that L-Cit formed by nNOS in L-Arg-loaded neuronal cells inhibits NOS activity and nNOS in these L-Arg-loaded cells functions as a NADPH oxidase to produce ROS, which may cause neurotoxicity in argininemia.     

Anandamide hydrolysis by human cells in culture and brain

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet, AnNH inactivation has not been studied in humans. Here, a human brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 +/- 0.2 microM and 800 +/- 75 pmol.min-1.mg of protein-1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 degreesC, respectively, and the activation energy of the reaction was 43.5 +/- 4.5 kJ.mol-1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Ki values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 +/- 0.9 microM) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 +/- 0.02 microM and 30 +/- 3 pmol.min-1.mg of protein-1 (CHP100 cells) and 0.13 +/- 0.01 microM and 140 +/- 15 pmol.min-1.mg of protein-1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.     

Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation

AIMS: Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. METHODS: Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. RESULTS: Km and Vmax values determined in human liver microsomes were lower for the demethylation (61 +/- 21 microM, 159 +/- 42 pmol min(-1) mg protein(-1) mean +/- s.d.; n = 4), than for the N-oxidation of CLZ (308 +/- 1.5 microM, 456 +/- 167 pmol min(-1) mg protein(-1); n = 3). Formation of DCLZ was inhibited by fluvoxamine (53 +/- 28% at 10 microM), triacetyloleandomycin (33 +/- 15% at 10 microM), and ketoconazole (51 +/- 28% at 2 microM) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34 +/- 16% at 10 microM) and ketoconazole (51 +/- 13% at 2 microM), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. Km and Vmax values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. CONCLUSIONS: It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).     

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.     

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT)

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).     

Organization and transcription of the gene encoding potato UDP-glucose pyrophosphorylase

Fenspiride inhibits the calcium signal evoked by the inflammatory peptide formyl-Met-Leu-Phe (fMLP) in peritoneal macrophages, but at concentrations (approximately 1 mM) far above the therapeutic range (approximately 1 microM). Here, in rat alveolar macrophages, high fenspiride concentrations (1 mM) were required to inhibit the calcium signals evoked by the calcium agonist Bay K8644 or by ionomycin. Moreover, fenspiride (1 mM) was a poor inhibitor of the cell membrane depolarization induced by gramicidine D. By contrast, fenspiride blocked Na+-H+ antiport activation by (i) fMLP with an IC50 = 3.1 +/- 1.9 nM and (ii) PMA (phorbol 12-myristate 13-acetate) with an IC50 = 9.2 +/- 3.1 nM. Finally, protein kinase C (PKC) activity of macrophage homogenate was not significantly modified by 10 or 100 microM fenspiride (at 100 microM: 2.57 +/- 1.60 vs. 2.80 +/- 1.71 pmol/10(6) cells/min). In conclusion, fenspiride inhibits fMLP- and PMA-induced pH signals in rat alveolar macrophages, probably by acting distally on the PKC transduction signal. This pH antagonistic action may be relevant for the antiinflammatory mechanism of fenspiride and requires further investigation.     

Guanine nucleotide-binding inhibitory protein-mediated inhibition of adenylyl cyclase is enhanced in spontaneously hypertensive rat preglomerular arteriolar smooth muscle cells

The purpose of our study was to determine whether Gi-mediated control over adenylyl cyclase in preglomerular arteriolar smooth muscle cells (PGASMC) is enhanced in the spontaneously hypertensive rat (SHR). PGASMC were cultured from preglomerular microvessels isolated from adult SHR (14-15 wk of age) and age-matched WKY rats. Confluent monolayers of cells in third passage were used for the experiments. cAMP released into the media (30 min) as well as cellular levels of cAMP were measured in the presence of a phosphodiesterase inhibitor, 1-isobutyl-3-methyl-xanthine (IBMX; 100 microM) and expressed as pmol/mg protein. Total (released + cellular) cAMP was significantly lower in SHR (14.19 +/- 2.30 pmol/mg protein) as compared with WKY (28.3 +/- 3.04 pmol/mg protein). Correspondingly, the released (4.6 +/- 0.4 pmol/mg protein) as well as cellular (9.78 +/- 2.18 pmol/mg protein) cAMP levels were also significantly lower in SHR when compared with WKY (8.85 +/- 1.26 and 18.86 +/- 2.0 pmol/mg protein, respectively). The steady-state levels of none of the Gi alpha subunits, namely Gi alpha 1, Gi alpha 2 and Gi alpha 3, were higher in the SHR PGASMC. Pertussis toxin treatment (PTX; 100 ng/ml; 24 hr) caused complete ADP-ribosylation of Gi alpha subunits in both WKY and SHR PGASMC. The same treatment of PTX also produced a significant increase in total cAMP in SHR, but not in WKY, such that the total cAMP levels after PTX treatment were not significantly different between the two strains. Interestingly, PTX significantly increased the released (20.26 +/- 0.90 pmol/mg protein) but not the cellular (13.63 +/- 1.63 pmol/mg protein) cAMP in SHR. Forskolin (1 microM) induced similar increases in total cAMP and isoproterenol (1 microM) caused greater increases in total cAMP in SHR cells compared with WKY cells. These data strongly suggest that in SHR PGASMC total adenylyl cyclase activity is not altered. Furthermore, steady-state expressions of Gi alpha-1, Gi alpha-2 and Gi alpha-3 are not increased whereas Gi-mediated inhibition of adenylyl cyclase is augmented in SHR PGASMC.     

Spermine inhibits [3H]glycine binding at the NMDA receptors from plexiform layers of chick retina

Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [3H]glycine binding to membranes from synaptosomal fractions from the outer (P1) and the inner (P2) plexiform layers of 1-3 day-old chick retinas in a dose-dependent manner with an IC50 = 35 microM for the P1 fraction and 32 microM for the P2 fraction. Kinetic experiments and non-linear regression analysis of [3H]glycine-specific binding showed a Kd approximately 100-150 nM in both fractions, and a higher Bmax (4.11 +/- 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (Bmax = 2.76 +/- 0.25 pmol/mg protein). Strychnine-insensitive [3H]glycine binding was inhibited by 100 microM spermine, due to a reduction in Bmax (P1 = 0.84 +/- 0.16 pmol/mg protein; P2 = 0.81 +/- 0.16 pmol/mg protein) without affecting the Kd. Association and dissociation constants in the absence and presence of 50 microM spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina.     

The inducible and cytochrome P450-containing dehydroepiandrosterone 7alpha-hydroxylating enzyme system of Fusarium moniliforme

7Alpha-hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7alpha-position, with minor hydroxylation occurring at the 7beta-position. Constitutive 7alpha-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7alpha-hydroxylation via an increase in protein synthesis. DHEA 7alpha-hydroxylase was found to be mainly microsomal, and the best production yields of 7alpha-hydroxy-DHEA (28.5 +/- 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (KM=1.18 +/- 0.035 microM and Vmax=909 +/- 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7alpha-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7alpha-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7alpha-hydroxylase; (iii) this enzyme complex contains a cytochrome P450.     

Endogenous dopamine regulates phosphate reabsorption but not NaK-ATPase in spontaneously hypertensive rat kidneys

Dopamine's modulatory actions on signal transduction in the spontaneously hypertensive rat (SHR) proximal tubule are blunted; therefore, it was predicted that dopamine does not regulate phosphate (Pi) reabsorption in SHR. To test this hypothesis, dopamine production was inhibited with carbidopa (10 mg/kg ip) 18 h before and during clearance measurements of chronically denervated SHR and Wistar-Kyoto (WKY) rat kidneys. Dopamine excretion decreased 80% from SHR and 85% from WKY rats. Pi excretion decreased 60 to 67%. Plasma Pi and calcium, inulin clearance, and Na excretion did not change. Citrate excretion, which reflects proton secretion by proximal tubules, decreased 72% from WKY rats. Citrate excretion was significantly lower from SHR (5 +/- 10 pmol/min) than from WKY rats (73 +/- 11 pmol/min) and was not altered by carbidopa. Carbidopa, injected 18 and 1 h before kidneys were collected, increased NaK-ATPase in cortical basolateral membranes from WKY rats (27%) but not in membranes from SHR. After the incubation of renal cortical minceates for 15 min with L-DOPA (10(-5) M), there was no change in brush border membrane vesicle uptake of 32Pi, (3H)glucose, or (14C)citrate. Incubation with carbidopa (10(-4) M) increased 32Pi uptake by 11% (P < 0.001) and (3H)glucose uptake by 9% (P = 0.02). (14C)citrate uptake was not increased by carbidopa but was higher in SHR (977 +/- 2 pmol/10 s.mg) than in WKY rats (823 +/- 43 pmol/10 s.mg; P = 0.04). In summary, dopamine produced in WKY rat and SHR proximal tubules decreases Pi uptake by using a signaling process distinct from those that regulate NaK-ATPase and the antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-3,4-dihydroxyphenylalanine increases the quantal size of exocytotic dopamine release in vitro

The catecholamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of L-DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 mM potassium-containing media, L-DOPA (100 microM for 1 h) increased DA release by > 10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, L-DOPA (50 microM for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that L-DOPA (50 microM for 40-70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that L-DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.