Metabolism of 5-hydroxytryptophan-14c after intracisternal injection with and without the influence of drugs in the rat brain

After intracisternal injection of 5-hydroxytryptophan-¹⁴C (5-HTP-¹⁴C), the disappearance of 5-HTP-¹⁴C, 5-hydroxytryptamine-¹⁴C (5-HT-¹⁴C) and 5-hydroxyindoleaceticacid-¹⁴C (5-HIAA-¹⁴C) in the rat brain, as a function of time, was measured. By this method, the half-life of 5-HTP-¹⁴C was 3.5 hrs. Indirectly, the synthesis rate of 5-HT-¹⁴C was calculated to be 1.75 nMol/g/hr. The effect of pargyline, a MAO inhibitor, and Ro 4-1284, a depletor, on the distribution of these metabolites was studied. If imipramine is given i.p. before the injection of 5-HTP-¹⁴C, an increase in 5-HIAA-¹⁴C occurred, while the application of imipramine after 5-HTP-¹⁴C resulted in a decrease in 5-HIAA-¹⁴C. The discussion considers whether results could be explained by 5-HTP-¹⁴C following the serotonin and the transamination pathway, which leads to 5-HIAA-¹⁴C without 5-HT-¹⁴C as an intermediate.This work is a part of a Thesis for the Ph.D. of P.B.     

Effects of edrophonium on end-plate currents in frog skeletal muscle

The effects of edrophonium on end-plate currents measured at frog neuromuscular junctions were assessed using the two microelectrode voltage clamp technique. Low concentrations of edrophonium (10^?5 M and 2.5 × 10^?5 M) increased the peak amplitude and the kinetic parameters of end-plate currents measured in curare-Ringer and in 5 mM Ca²⁺–10 mM Mg²⁺-Ringer solutions. At the highest concentration tested (6 × 10^?5 M), edrophonium decreased the peak amplitude and maximum rate of rise but had little effect on the time to peak. Time to half decay was the only parameter studied which continued to increase as edrophonium concentration was increased from 10^?5 M to 6 × 10^?5 M. Normalized values of the parameters for both 10^?5 M and 2.5 × 10^?5 M edrophonium revealed no significant difference between end plates bathed in curare-Ringer and those bathed in 5 mM Ca²⁺–10 mM Mg²⁺-Ringer.It is suggested that the low concentration of edrophonium inhibit the postjunctional acetylcholinesterase thereby causing an increase in transmitter concentration in the end-plate region, but that the highest concentration (6 × 10^?5 M) has a direct desensitizing action as well as the anticholinesterase action on the end-plate membrane. This desensitizing action is present although no depolarization of the end-plate membrane is observable.     

Competitive inhibition of Asp1-beta-amide-Val5-angiotensin II by Sar1-Ile5-Ile8-angiotensin II in cat isolated cardiac muscle and coronary vessels

Interaction of Asp¹-β-amide-Val⁵-angiotensin II with the 8-substituted analogue of Asp¹-Ile⁵-angiotensin II, Sar¹-Ile⁵-Ile⁸-angiotensin II, has been examined on the isolated perfused heart and isolated papillary muscle of the cat. The constrictor effect of angiotensin in the coronary vessels as well as its positive inotropic effect in the heart muscle have been shown to be competitively inhibited by Sar¹-Ile⁵-Ile⁸-angiotensin II. The antagonistic potency and the duration of the antagonistic effect of the analogue in both coronary vessels and myocardium have been evaluated separately. The analogue has a higher antagonistic potency in coronary vessels than in heart muscle. The duration of the antagonistic effect of Sar¹-Ile⁵-Ile⁸-angiotensin II was found to be longer in the heart muscle than in coronary vesesels. The possible mechanism of the antagonistic effect of Sar¹-Ile⁵-Ile⁸-angiotensin II against Asp¹-β-amide-Va1⁵-angiotensin II is discussed.     

Combined use of NMR,distance geometry and MD calculations for the conformational analysis of opioid peptides of the type [D(L)-Cys2, D(L)-Cys5]enkephalin

The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr¹-Cys²-Gly³ Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H-Tyr¹-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H-Tyr¹-D-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H-Tyr^l-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, which contains solely L-Cys (LCLCE), there is no single structure compatible with the NMR data. © Munksgaard 1996.     

177Lu‐5‐Fluorouracil a potential theranostic radiopharmaceutical: radiosynthesis,quality control,biodistribution, and scintigraphy

The aim of this study is to develop ¹⁷⁷Lu‐5‐Flourouracil as a potential cancer therapeutic radiopharmaceutical. 5‐Flourouracil (5‐FU) is widely accepted as an anticancer drug of broad spectrum fame. The labeling of 5‐FU was carried out at different set of experimental conditions using high specific activity of ¹⁷⁷LuCl₃. The optimum conditions for maximum radiochemical yield was set: 5‐FU (5 mg), ¹⁷⁷LuCl₃ (185 MBq), diethylenetriaminepentaacetic acid (10 µg), reaction volume (2 mL), pH (5.5), temperature (80°C), and reaction time (20 min). The radiochemical labeling was assessed with Whatman No. 2 paper, instant thin layer chromatographic, and radio‐HPLC, which revealed >94% labeling results with sufficient stability up to 6 h. Serum stability study also showed ¹⁷⁷Lu‐5‐FU promising stability. Biodistribution study in normal rats and rabbits showed liver, stomach, kidney, and heart as area of increased tracer accumulation just after injection, which decreased to 1.4%, 0.4%, 0.2%, and 0.39% ID/g, respectively, after 72 h. Glomerular filtration rate and cytotoxicity study results of ¹⁷⁷Lu‐5‐FU showed it had no adverse effect on renal function and nontoxic to blood cells. The promising characteristics of ¹⁷⁷Lu‐5‐FU, that is, clever elimination from kidney and nontoxic nature toward blood cells make it the radiopharmaceutical for further testing in patients for therapeutic purposes.     

Effects of 5-(N,N-diethylamino)-n-pentyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-5) on cardiovascular systems

The effects of 5-(N,N-diethylamino)-pentyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-5) were studied pharmacologically on smooth muscle, skeletal muscle, blood vessel and cardiac preparations. In all cases, TMB-5 inhibited muscle contractions induced by muscle stimulants such as acetylcholine, norepinephrine, KCl and BaCl₂, indicating that the muscle inhibition induced by TMB-5 is unrelated to specific receptors. TMB-5 was found to be most potent in inhibiting skeletal muscles (at 10^?6 –10^?5 M level) and least effective inhibiting smooth muscles (at 10^?4 –10^?3 M level). The potency of vascular inhibition was in between these two levels (at 10^?4 M level). The ability of TMB-5 to raise the threshold of cardiac arrhythmias was quite good at 7 × 10^?7 –7 × 10^?6 M. It is concluded that TMB-5 could be a good antiarrhythmic agent with some skeletal muscle relaxation action.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets

Antidepressant agents with properties to inhibit 5-hydroxytryptamine (5-HT, serotonin) uptake in brain tissue and platelets bind with high affinities to neuronal and platelet membranes. [³H]Imipramine, [³H]paroxetine and [³H]citalopram label specific binding sites related to the 5-HT transporter. [³H]Paroxetine and [³H]citalopram appear to be better ligands than [³H]imipramine. The former label a homogenous population of binding sites, whereas the displaceable binding of [³H]imipramine is heterogenous. Recent observations in several laboratories, which have taken the heterogeneity of [³H]imipramine binding into account, indicate that the binding of antidepressants to the 5-HT transporter probably occurs to the same site that binds 5-HT for transport and not to a separate site as previously suggested. Additional bonds to subsites in close vicinity to the 5-HT recognition site may contribute to the binding. No convincing evidence has been presented of the existence of an endogenous ligand other than 5-HT itself that binds to the [³H]imipramine binding site. Recent studies also suggest that repeated treatment of rats with antidepressant agents does not produce any alterations of the binding of [³H]imipramine or [³H]paroxetine to membranes of cerebral cortex. It is also doubtful whether the density of the 5-HT uptake site in platelets measured with these ligands is decreased in affective disorders as first reported.     

K+-evoked [3H]-5-HT release from rat frontal cortex slices: the effect of 5-HT agonists and antagonists

The pharmacological characteristics of a pre-junctional 5-HT autoreceptor have been studied by following the Ca²⁺-dependent, K⁺-evoked release of [³H]-5-HT from preloaded rat frontal cortex slices. Added 5-HT, in the presence of the 5-HT uptake inhibitor chlorimipramine. caused a dose related inhibition of the K²⁺-evoked release of [³H]-5-HT in this system as did the 5-HT analogues 5-methoxytryptamine, N-methyltryptamine, 5-methoxy-NN'-dimethyltryptainine, N-methyl 5-hydroxytryptamine and tryptamine.The inhibitory effect of 1 μM 5-HT on the K⁺evoked release of [³H]-5-HT was reversed in a doserelated manner by the 5-HT antagonist drug, methiothepin (pA₁₀ value 6.7). At a concentration of 1 μM, the 5-HT antagonists drugs cinanserin and mianserin produced a small but significant reversal of the 5-HT induced inhibition of K⁺-evoked [³H]-5-HT release, but methysergide, metergoline and cyproheptadine were completely without effect at this concentration. The results are interpreted as evidence for a pre-junctional autoreceptor for 5-HT in the frontal cortex of the rat with a different pharmacological specificity for 5-HT antagonists from previously studied 5-HT receptors.     

Selective ligands for opioid receptors: β-Cyclopropylalanyl containing analogs of enkephalin

The synthesis and resolution of the amino acid β-cyclopropylalanine (Cpr) and its incorporation into four enkephalin analogs is reported. The analogs prepared were: Tyr - l - Cpr - Gly - Phe - Pen (des - COOH - Nle = n - pentylamide = Pen) (l -Cpr²-Pen⁵-ENK), Tyr-d -Cpr-Gly-Phe-Pen (d -Cpr²-Pen⁵-ENK), l -Cpr-Tyr-d -Ala-Gly-Phe-Pen (l -Cpr⁰-d -Ala²-Pen⁵-ENK) and d -Cpr-Tyr-d -Ala-Gly-Phe-Pen (d -Cpr⁰-d -Ala²-Pen⁵-ENK). Each was tested for its ability to inhibit the field stimulated guinea pig ileum (GPI) and rat vas deferens (RVD) and the results compared to the effect d -Ala²-d -Leu⁵-enkephalin (DADLE) has on the same preparations. The results show that at concentrations up to 10^-5 m all four analogs, as well as DADLE, are full agonists on the GPI preparation. The concentrations necessary to produce a 50% inhibition of the twitch response were, DADLE, 3.5 °× 10^-8 m ; l - Cpr⁰-d -Ala²-Pen⁵-ENK, 6.0 × 10^-8 m ; d -Cpr²-Pen⁵-ENK, 1.1 × 10^-7 m ; l -Cpr²-Pen⁵-ENK, 1.2 × 10^-6 m and d -Cpr⁰-d -Ala²-Pen⁵-ENK, > 10^-5 m . On RVD a different result was observed with only DADLE (1.3 × 10^-6 m ) and l -Cpr⁰-Pen⁵-enkephalin (1.8 × 10^-6 m ) showing full agonist activity. d -Cpr²-Pen⁵-ENK was a partial agonist (29 · 5% inhibition of the twitch at 10^-5 m ) while d -Cpr⁰-d -Ala²-Pen⁵-ENK and l -Cpr²-Pen⁵-ENK did not inhibit the twitch at concentrations up to 10^-5 m . These compounds which were inactive or of low potency on each preparation were also tested as antagonists. Only d -Cpr²-Pen⁵-ENK was an antagonist (pA₂ = 6.09) versus DADLE on RVD while d -Cpr⁰-d -Ala²-Pen⁵-ENK was inactive as an antagonist on both GPI and RVD. d -Cpr²-Pen⁵-ENK, therefore, represents the first enkephalin analog to be categorized as a mixed agonist-antagonist.     

Zur Oxidation von 2,5-Dihydroxyphenylethylamin-Derivaten

Oxidation of 2,5-Dihydroxyphenylethylamine Derivatives The syntheses of the free bases 1c , 1f and 11b , their hydrochlorides and the acetyl derivatives 12a and 12b are described. Treatment of the 2,5-dihydroxyphenylethylamine derivatives 1c and 1f with catalytical amounts of oxidants affords the indolines 5c and 5f . Compound 5c is characterised as hydrochloride, diacetate 13a and monoacetate 5g . Indoline 5f is synthesized as base and hydrochloride and transformed to the monoacetate 13b . The structures are analysed by MS, ¹H-NMR and ¹³C-NMR spectroscopy. The course of the reaction that leads to the indolines 5 is discussed.     

Riluzole activates TRPC5 channels independently of PLC activity

BACKGROUND AND PURPOSE

The transient receptor potential channel C5 (TRPC5) is a Ca²⁺-permeable cation channel, which is predominantly expressed in the brain. TRPC5 is activated in a PLC-dependent manner by, as yet, unidentified endogenous messengers. Recently, modulators of TRPC5, like Ca²⁺, pH and phospholipids, have been identified. However, the role of TRPC5 in vivo is only poorly understood. Novel specific modulators of TRPC5 might help to elucidate its function.

EXPERIMENTAL APPROACH

Novel modulators of TRPC5 were identified in a compound screening of approved drugs and natural compounds. The potency and selectivity of TRPC5-activating compounds were determined by fluorometric calcium imaging. The biophysical properties of channel activation by these compounds were analysed using electrophysiological measurements.

KEY RESULTS

Riluzole was identified as a novel activator of TRPC5 (EC₅₀ 9.2 ± 0.5 μM) and its mechanism of action was shown to be independent of G protein signalling and PLC activity. Riluzole-induced TRPC5 currents were potentiated by La³⁺ and, utilizing TRPC5 mutants that lack La³⁺ binding sites, it was confirmed that riluzole and La³⁺ activate TRPC5 by different mechanisms. Recordings of excised inside-out patches revealed a relatively direct effect of riluzole on TRPC5.

CONCLUSIONS AND IMPLICATIONS

Riluzole can activate TRPC5 heterologously expressed in HEK293 cells as well as those endogenously expressed in the U-87 glioblastoma cell line. Riluzole does not activate any other member of the TRPC family and could, therefore, despite its action on other ion channels, be a useful pharmacological tool for identifying TRPC5-specific currents in immortalized cell lines or in acutely isolated primary cells.     

Lipidosis-like alterations in cultured macrophages exposed to local anaesthetics

In this ultrastructural study, the simple model of cultured rat peritoneal macrophages was used to examine whether local anaesthetics can induce lipidosis-like alterations. Exposure (24 h) of macrophages to 1×10^–5 M dibucaine, or to 5×10^–5 M tetracaine, quinidine, and quinine, respectively, led to the occurrence of lamellated cytoplasmic inclusions in most cells. This is interpreted as indicating lipidosis. Type and degree of alterations were similar to those induced by the reference compound chlorphentermine (5×10^–5 M) for which lipidosis has previously been shown by biochemical methods. Tocainide (5×10^–5 M) caused weak alterations only; procaine (5×10^–5 M) was without effect. The differential potencies presently observed are paralleled by differential affinities of the local anaesthetics towards polar lipids as determined by other authors. The present results support the hypothesis that the lipidosis-inducing potency inherent to an amphiphilic cationic drug can be tentatively predicted on the basis of its affinity to polar lipids, although it may be obscured by secondary factors when the drug is administered to intact organisms. The present communication emphasizes the advantage of cell cultures over animal experiments (a) for studying the structure-response relationships underlying drug-induced lipidosis, and (b) to reliably ascertain that a given drug has only low lipidosis-inducing potency or none at all as found for tocainide and procaine, respectively.     

Metabolism in the rat of potassium nonan-5-sulphate,a symmetrical anionic surfactant

1. The metabolism of potassium nonan-5-[³⁵S]sulphate, a symmetrical secondary alkylsulphate ester, was investigated in the rat. Oral administration of the radiolabelled ester was followed by the elimination of the majority of radioactivity in the urine.

2. Potassium nonan-5-[³⁵S]sulphate is degraded in vivo to produce at least three radiolabelled sulphate esters.

3. The same metabolites were produced by isolated rat livers perfused with potassium nonan-5-[³⁵S]sulphate.

4. The three radioactive metabolites were identified by combined g.l.c.-mass spectroscopy as the unchanged parent ester, nonan-1-ol-5-sulphate and nonanoate-5 sulphate.

5. The nature of the latter two metabolites indicates that potassium nonan-5-sulphate is metabolized by ω-oxidation only and, moreover, the alkylsulphate ester is metabolized only at one end of the molecule.     

Characterisation of C5a receptor agonists from phage display libraries

C5a des-Arg⁷⁴ has a 10- to 100-fold lower receptor binding affinity than intact C5a and is only a partial agonist. We have used phage display selection from randomly mutated C5a des-Arg⁷⁴ libraries to isolate variant proteins that can activate C5a receptors with similar potency to C5a. Here we explore the interactions of three variants (V1-3) with C5aR mutated at residues involved in the differential response. The mutant Asp²⁸²Arg-C5aR is preferentially activated by C5a des-Arg⁷⁴, probably due to repulsion between Arg⁷⁴ of C5a and the substituent Arg²⁸². In accordance with this hypothesis, V2 (with a polar C-terminus which has no Arg residue) but not V1 (with a C-terminal Arg residue at position 73) could activate Asp²⁸²Arg-C5aR. V3, with a very hydrophobic C-terminus, was the most potent agonist at Asp²⁸²Arg-C5aR. Arg¹⁷⁵ is a potential counterion for the C-terminal carboxylate of C5a. C5aR mutated to either Ala or Asp at this position lost nearly all responsiveness to both C5a and C5a des-Arg⁷⁴, suggesting that mutation of Arg¹⁷⁵ caused a non-specific loss of receptor conformation and a loss of signalling capacity. However, V3 could still activate Arg¹⁷⁵Asp/Ala-C5aR with the same potency as wild-type C5aR, demonstrating that the mutant receptors retained high signalling capability and showed a specific loss of responsiveness. Thus C5a des-Arg⁷⁴ variants produced by phage display are potentially useful tools for the dissection of ligand-receptor interactions.     

Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells

5-HT_1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT_1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT_1B or 5-HT_1D receptors, it was shown that 5-HT-moduline prevents the binding of [³H]5-HT to 5-HT_1B as well as to 5-HT_1D receptors with similar biochemical characteristics: the IC₅₀ of the peptide was 1.2×10^–12 M for 5-HT_1B and 9×10^–13 M for 5-HT_1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [³H]5-HT on 5-HT_1B (–51.2±1%) as well as 5-HT_1D binding (–47.2±7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the “scrambled” peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT_1A or 5-ht₆ receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [³⁵S]GTPγS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC₅₀=1.2±0.7×10^–12 M for 5-HT_1B and 9.8±4.0×10^–12 M for 5-HT_1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT_1D as well as 5-HT_1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT_1B and 5-HT_1D receptors. Received: 26 March 1998 / Accepted: 22 May 1998     

用Cu-PAN系统作EDTA对钙、钡、镁、铝、铬、锌、铁、铅、铋、汞、铜等化合物的测定

本文重新研究了EDTA絡合量法中用Cu-PAN系統滴定Ca⁺⁺,Ba⁺⁺,Mg⁺⁺,Al⁺⁺⁺,Cr⁺⁺⁺,Zn⁺⁺,Fe⁺⁺⁺,Pb⁺⁺⁺,Bi⁺⁺⁺,Hg⁺⁺,Cu⁺⁺等的pH、溫度、指示剂用量等条件,测定准确度为1%.建立了用Cu-PAN系統滴定鉻盐及鋇盐的方法,測定終点較用鉻黑T指示剂明显.測定了Cu-PAN絡合物的組成为1:1,稳定常数为:pH2,1.4×10⁶;pH4,1.9×10⁶;pH 6,1×10⁵;pH 8,7.5×10⁴;pH 10,2.1×10⁵.根据这些結論,改进了Cu-PAN系統的滴定方法。     

The Action of Thioridazine and Promazine on Biological Membranes: Relationship between ATPase Inhibition and Membrane Stabilization

The effects of promazine and thioridazine on hypotonic haemolysis of human erythrocytes are compared with their effect on Na⁺-K⁺-ATPase of washed human erythrocyte ghosts. Promazine (5 × 10^-5 - 5 × 10^-4 M) and thioridazine (10^-5 - 10^-4 M) stabilize erythrocytes against hypotonic haemolysis, but have lytic effects at higher concentrations. Both drugs inhibit Na⁺-K⁺-ATPase of erythrocyte membranes. This inhibition is slight at drug concentrations which have a membrane-stabilizing action, but is complete and irreversible at lytic concentrations of the drugs. Promazine and thioridazine also inhibit Na⁺-K⁺-ATPase in a membrane fraction prepared from rat hearts. The relative order of potency of the two drugs in this respect does not reflect their relative potency as general cardiac depressants. It is concluded that Na⁺-K⁺-ATPase is not a primary target for the action of promazine and thioridazine in the rat heart. It is suggested that inhibition of Na⁺-K⁺-ATPase by these agents is secondary to more general alterations of the physical properties of the cell membrane.     

Inhibition of the Uptake of 3H-Dopamine and 14C-5-Hydroxytryptamine in Mouse Striatum Slices

Abstract: The inhibitory activities of a number of compounds on the simultaneous uptake of ³H-dopamine (³H-DA) and ¹⁴C-5-hydroxytryptamine (¹⁴C-5-HT) in the same slices of the mouse striatum were examined. Experiments with inactive DA and 5-HT demonstrated that the uptake of the two amines occurs at different sites. In vitro (+)-amphetamine was 2.7 times more potent than the (-)-isomer whereas in vivo this difference was about 6 times. (+)-Amphetamine was also significantly more active than the (-)-enantiomer on the ³H-DA uptake in striatal slices from the rat. The methylester group in cocaine is essential for the inhibitory activity of cocaine, which had similar potencies on the two uptake mechanisms. Benztropine was much more active on the ³H-DA uptake than on the ¹⁴C-5-HT uptake. In vivo the amphetamine derivatives including prolintane and methylphenidate were the most active compounds on the ³H-DA uptake, but the 4-substituted amphetamines were more active on the ¹⁴C-5-HT uptake. Among other compounds benztropine was as active as (+)-amphetamine on the ³H-DA uptake. None of the compounds tested seem to be useful as model compounds for the selective inhibition of the DA uptake, since they have other pharmacological effects at lower doses.     

Deciphering μ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells

BACKGROUND AND PURPOSE

The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala²-MePhe⁴-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr³⁷⁰) and serine 375 (Ser³⁷⁵), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser³⁷⁵ without causing rapid receptor internalization.

EXPERIMENTAL APPROACH

Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells.

KEY RESULTS

We found that DAMGO-driven phosphorylation of Thr³⁷⁰ and Ser³⁷⁵ was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser³⁷⁵ phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser³⁷⁵ phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr³⁷⁰ and Ser³⁷⁵ dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling.

CONCLUSIONS AND IMPLICATIONS

Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser³⁷⁵, which is not sufficient to drive significant receptor internalization.