Binding properties of 3-[125I]iodophencyclidine, a new radioligand for N-methyl-D-aspartate-gated ionic channels.

The binding properties of the 125I-labeled phencyclidine derivative N-[1-(3-[125I]iodophenyl)cyclohexyl]piperidine (3-[125I]iodo-PCP), a new ligand of the N-methyl-D-aspartate (NMDA)-gated ionic channel, were investigated. Association and dissociation kinetic curves of 3-[125I]iodo-PCP with rat brain homogenates were well described by two components. About 32% of the binding was of fast association and fast dissociation, and the remaining binding was of slow association and slow dissociation. Saturation curves of 3-[125I]iodo-PCP also were well described using two binding sites: one of a high affinity (KDH = 15.8 +/- 2.3 nM) and the other of a low affinity (KDL = 250 +/- 40 nM). 3-Iodo-PCP inhibited the binding of 3-[125I]iodo-PCP with inhibition curves that were well fitted by a two-site model. The binding constants (KiH, BmaxH; KiL, BmaxL) so obtained were close to those obtained in saturation experiments. Ligands of NMDA-gated ionic channels also inhibited the binding of 3-[125I]iodo-PCP with two constants, KiH and KiL. There was a very good correlation (r = 0.987) between the affinities of these ligands to bind to NMDA-gated ionic channels and their potencies to inhibit the binding of 3-[125I]iodo-PCP with a high affinity. Moreover, the regional distribution of the high-affinity binding of 3-[125I]-iodo-PCP paralleled that of tritiated N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). In contrast to that of [3H] TCP, the binding of 3-[125I]iodo-PCP to well-washed rat brain membranes was fast and insensitive to glutamate and glycine.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved synthesis of [1-14C]acyl-sphingosine-galactose-3-sulfate (sulfatide) for diagnosis of metachromatic leukodystrophy: usefulness of radioscanning.

We report an improved method for the radiolabelling of [1-14C 00acyl-sphingosine-galactose-3-sulfate (sulfatide), requiring preparation of lysosulfatide (sulfogalactosyl-sphingosine) by alkaline hydrolysis of sulfatide and reacylation of the sphingosine amino group with a [1-14C]stearoyl chloride. We found that the yield of labeled sulfatide could be considerably increased using stringent chromatographic conditions for the preparation of lysosulfatide and strict anhydrous conditions for the formation of the acylchloride and its coupling to lysosulfatide. Radioscanning was used at different steps to check the purity of the labeled compounds. Radioscanning was also used to determine the formation of cerebroside when measuring cerebroside sulfate sulfatase activity and sulfatide metabolism in intact fibroblasts in controls and patients with metachromatic leukodystrophy. It could demonstrate and measure with accuracy the cerebroside sulfate storage characteristic of the disease.

Distribution of tritium labeled 12(S) hydroxy-eicosatetraenoic acid (12-HETE) in the rat.

The in vivo metabolism of 12-(S)-Hydroxy-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets, has been investigated in the rat. Fifty microcuries of 5,6-[3H]-12-HETE (50 Ci/mmol) were injected to anesthetized rats and the radioactivity was followed in plasma. At the end of the experiment, various organs of the animal were removed and the radioactivity attached to them was determined. The label of the plasma plateaued to approximately one third of the initial radioactivity ten minutes after the injection. Among the various organs tested (brain, heart, intestine, kidney, liver, lungs, spleen, testis/uterus) the kidney was far the most active to accumulate 12-HETE and/or its labeled metabolites, and no radioactivity could be detected in urine during the course of the experiment. The analysis of lipid extracts from the various tissues revealed that 12-HETE was not accumulating in its unesterified form but was likely bound to phospholipids. We conclude that, although the label providing from the initial 12-HETE did not completely disappear from plasma, circulating 12-HETE cannot be considered as a circulating marker of cell activation.

Comparative biological activities of the four synthetic (5,6)-dihete isomers.

(5,6)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acids [5,6)-DiHETEs) were synthesized and separated into four pure diastereoisomers. They were tested for comparative binding affinities to leukotriene receptors (LTC4, LTD4, LTB4) in guinea pig lung membranes. Only (5S,6R)-DiHETE was recognized by the LTD4 receptor, the other receptors interacted with neither of the four isomers. (5S,6R)-DiHETE also contracted ileum in vitro and this effect was inhibited by the LTD4 receptor antagonists ICI 198,615 and SKF104,353. These data suggest that the bioproduct (5S,6R)-DiHETE generated by enzymatic conversion of LTA4 could have some LTD4-like activity when produced in large concentrations.

Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus.

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

In vivo formation of beta-oxidized metabolites of leukotriene E4 in the rat.

Intraperitoneal administration of [3H]-leukotriene E4 in the rat resulted in the appearance of radiolabel in urine and feces. Separation of polar urinary metabolites and chromatographic comparison of synthetic metabolites indicated the in vivo formation of omega-oxidized metabolites of LTE4 with sequential beta-oxidation. Furthermore, the metabolite identified as 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl-LTE4 substantiates the biochemical pathway of beta-oxidation in vivo involving the 2,4-dienoyl CoA reductase as an integral step. These results substantiate beta-oxidation of sulfidopeptide leukotrienes in vivo and these metabolites account for some of the major urinary metabolites of this class of lipid mediator.

Synthesis and contractile activity of new acetylenic and allenic analogues of leukotrienes C4 and D4: importance of the Z-11,12 double bond.

The (5S,6R) isomers of new acetylenic and allenic analogues of leukotrienes C4 and D4 were synthesized for comparative pharmacological studies on intestinal smooth muscle preparations. These new analogues are poor spasmogenic agonists, the replacement of the 11,12-ene with a relatively more stable triple bond causing an important reduction in intrinsic activity. They did not show any significant antagonist activity. Unexpectedly, these results prove that the 11,12 portion in the triene structure of the lipophilic chain is critical for an agonist activity.

125I-Bolton-Hunter-8-methoxy-2-[N-propyl-N-propylamino]tetralin as a new selective radioligand of 5-HT1A sites in the rat brain. In vitro binding and autoradiographic studies.

In vitro binding assays with 125I-[8-methoxy-2-[N-propyl-N-(3'-iodo-4'-hydroxyphenyl)-propionamido -N'- propylamino] tetralin] (125I-BH-8-MeO-N-PAT), a 125I-labeled derivative of the potent serotonin (5-HT) agonist 8-hydroxy-2-[di-n-propylamino]tetralin [( 3H]-8-OH-DPAT), showed that this compound recognized specific sites with nanomolar affinity for 5-HT and 5-HT1A ligands such as spiroxatrine, ipsapirone, buspirone and gepirone in rat hippocampal membranes. Comparison of the binding characteristics of 125I-BH-8-MeO-N-PAT with those of [3H]-8-OH-DPAT revealed striking similarities: at the hippocampal level, both binding sites exhibited nanomolar affinity for their respective ligands and the same Bmax; their pharmacological profiles defined by the inhibition of each bound ligand by a series of 26 serotonin, dopamine- or norepinephrine-related agonists and antagonists were identical; and their regional distributions examined by membrane binding assays and autoradiography of labeled brain sections were highly correlated. These observations indicate that 125I-BH-8-MeO-N-PAT is the first 125I-reversible ligand for the selective labeling of 5-HT1A sites in the rat central nervous system.

[125I-BH-8-MeO-N-PAT, a new ligand for the study of 5-HT1A receptors in the central nervous system]. / 125I-BH-8-MeO-N-PAT, un nouveau ligand pour l'étude des récepteurs 5-HT1A dans le système nerveux central.

Specific radioactive ligands are needed for studying the pharmacological properties and the regional distribution of the different classes of 5-HT1 receptors within the central nervous system. We describe here the synthesis and some characteristics of the first iodinated specific ligand of 5-HT1A receptors. Like its parent compound, the agonist 8-hydroxy-2-(di-n-propylamino)tetralin or 8-OH-DPAT, [125I]-BH-8-MeO-N-PAT, exhibits a high affinity and excellent selectivity for 5-HT1A sites. Its high specific radioactivity makes this ligand a useful tool for studying 5-HT1A receptors in membranes and sections of the rat brain.