Claude Gros

Dopamine D3 Receptors Expressed by All Mesencephalic Dopamine Neurons

A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D3 receptor (D3R). Using transfected cells expressing recombinant D3R, but not D2 receptor, this antibody labeled 45–80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [125I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D3R binding, revealed using [125I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino] tetralin ([125I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D3R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D3R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D3R binding in both AccSh and SN/VTA. D3R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D3autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D3R-deficient mice.

Effects of histamine H3-receptor ligands on various biochemical indices of histaminergic neuron activity in rat brain

The interaction of the potent histamine H3-receptor ligands i.e. (R)alpha-methylhistamine, an agonist, and thioperamide, an antagonist, with the three classes of cerebral histamine receptors was studied in vitro and in vivo. The histamine-induced stimulation of 3,5-cyclic AMP accumulation in slices of guinea-pig hippocampus was not modified by thioperamide (up to 0.1 mM) and (R)alpha-methylhistamine stimulated cyclic AMP accumulation only at millimolar concentrations. Hence, both (R)alpha-methylhistamine and thioperamide were at least 100,000-fold more potent at H3- than at H1- or H2-receptors in brain. In vivo, the turnover of histamine in rat cerebral cortex, as determined from its depletion elicited by alpha-fluoromethylhistidine in a synaptosomal fraction was not modified by mepyramine and zolantidine but was markedly enhanced by thioperamide at a low dose (ED50 = 2 mg/kg). Thioperamide also elicited a long-lasting decrease in synaptosomal histamine and increase in radioimmunoassayable N tau-methylhistamine. In contrast, (R)alpha-methylhistamine markedly reduced cortical [3H]histamine synthesis (ED50 = 5 mg/kg). This long-lasting action was accompanied by an increase in synaptosomal histamine and a decrease in N tau-methylhistamine levels. These changes were compared with those in plasma drug levels. Hence the two H3-receptor ligands appear to modify the activity of cerebral histamine neurons markedly and in a long-lasting and opposite manner.

Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: A member of a super family of zinc-metallohydrolases

Using a polyclonal antibody, a partial cDNA clone for rat aminopeptidase M was identified in a lambda gt11 library from rat kidney. A synthetic oligonucleotide probe derived from the sequence of the insert was used to screen a randomly primed lambda gt10 library. This allowed the identification of several overlapping clones encoding the full sequence of the enzyme. The reading frame, 2898 base pairs in length, encodes a 966 amino acid polypeptide. A highly hydrophobic segment, 24 amino acids in length, located close to the aminoterminus, is proposed to serve as the membrane-spanning domain for this membrane-bound enzyme. The sequence includes nine potential N-linked glycosylation sites and one potential sulfation site. In addition, the rat aminopeptidase M sequence contains an eight amino acid consensus sequence believed to serve as the zinc binding domain in a family of zinc-metallohydrolases. Rat aminopeptidase M shows 77% similarity with the recently cloned human enzyme, as well as weaker but significant similarity with aminopeptidase N from E. coli (18%) and with human leukotriene A4 hydrolase (21%).

Aminopeptidase N mediates the utilization of the GSH precursor CysGly by cultured neurons.

Neurons in culture rely on the supply of exogenous cysteine for their glutathione synthesis. After application of cysteine to neuron‐rich primary cultures, the glutathione content was doubled after a 4‐hr incubation. The dipeptide cysteinylglycine (CysGly) was able to substitute for cysteine as exogenous glutathione precursor. In kidneys, the ectopeptidase aminopeptidase N (ApN) has been reported to hydrolyze CysGly. Expression of mRNA of ApN in rat brain and cultured rat neurons was demonstrated by reverse transcriptase polymerase chain reaction and sequencing of the cDNA fragment obtained. In addition, the presence of ApN protein in cultured neurons was demonstrated by its immunocytochemical localization. In the presence of an activity‐inhibiting antiserum against ApN the utilization of CysGly as neuronal glutathione precursor was completely prevented, whereas that of cysteine plus glycine was not affected. The data presented demonstrates that cultured rat neurons express ApN and that this ectopeptidase participates in the utilization of CysGly as precursor for neuronal glutathione.

Major localization of aminopeptidase M in rat brain microvessels

The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 3.4.11.2) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.

Enkephalinase (EC 3.4.24.11) inhibitors: Protection of endogenous ANF against inactivation and potential therapeutic applications

Atrial natriuretic factor (ANF) is a cardiac hormone exerting potent cardiovascular and renal effects but its poor intestinal absorption and rapid inactivation have prevented so far its therapeutic utilisation. However inhibition of endogenous ANF metabolism progressively emerges as a novel therapeutic approach in cardiovascular and renal disorders. The critical role played by enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) in ANF inactivation was deduced from the effects of inhibitors. These compounds not only protect partially exogenous ANF from hydrolysis by some tissue preparations in vitro but also, in vivo, they increase the half-life of the exogenous hormone in plasma and, even more markedly, its recovery in intact form in kidney, a major target organ. In addition, enkephalinase inhibitors increase by two- to three-fold the circulating level of endogenous ANF, even when the latter is already markedly elevated, such as in patients with chronic heart failure. Finally, enkephalinase inhibitors induce a series of ANF-like responses such as natriuresis, diuresis or increase in cGMP excretion which are attributable to the hormone. These pharmacological observations, as well as preliminary clinical trials, suggest that enkephalinase inhibitors may represent a novel class of therapeutic agents with potential applications in congestive heart failure, essential hypertension and various sodium-retaining states.

Selective participation of both “enkephalinase” and aminopeptidase activities in the metabolism of endogenous enkephalins

Abstract Striatal slices metabolise 3 H-(Met 5 ) enkephalin via two main pathways : the “enkephalinase” pathway inhibited by thiorphan and an aminopeptidase pathway inhibited by bestatin but only slightly affected by puromycin. In addition in contrast to other inhibitors thiorphan and bestatin allow to completely prevent the extensive hydrolysis of endogenous (Met 5 ) enkephalin occuring when it is released and before it reaches the incubation medium. Finally the compounds additively delay the response to those nociceptive stimuli which seem to be accompanied by a release of endogenous opioids.

Antihypertensive Effects of Fasidotril, a Dual Inhibitor of Neprilysin and Angiotensin-Converting Enzyme, in Rats and Humans

The aim of this study was to assess the antihypertensive activity of fasidotril, a dual inhibitor of neprilysin (NEP) and angiotensin I-converting enzyme (ACE), in various models of hypertension in rats (spontaneously hypertensive rats [SHR]; renovascular Goldblatt 2-kidney, 1-clip rats; and deoxycorticosterone acetate [DOCA]-salt hypertensive rats) and in patients with mild-to-moderate essential hypertension. Fasidotril treatment (100 mg/kg PO twice daily for 3 weeks) resulted in a progressive and sustained decrease in systolic blood pressure (-20 to -30 mm Hg) in SHR and Goldblatt rats compared with vehicle-treated rats and prevented the progressive rise in blood pressure in DOCA-salt hypertensive rats. After a 4-week placebo run-in period, 57 patients with essential hypertension were included in a randomized double-blind, placebo-controlled, parallel-group study and received orally either fasidotril (100 mg twice daily) or placebo for 6 weeks. Blood pressure was measured during the 6 hours after the first intake and then at trough (12 hours after the last intake) on days 7, 28, and 42. The first dose of fasidotril had no significant effect on blood pressure. After 42 days, compared with placebo, fasidotril lowered supine systolic and diastolic blood pressures by 7.4/5.4 mm Hg and standing blood pressure by 7.6/6.8 mm Hg. Fasidotril, a dual NEP/ACE inhibitor, was an effective oral antihypertensive agent during chronic treatment in high-renin renovascular rats, normal-renin SHR, and low-renin DOCA-salt hypertensive rats and in patients with essential hypertension.

[125I]Iodobolpyramine, a highly sensitive probe for histamine H1-receptors in guinea-pig brain.

[125I]Iodobolpyramine is a novel 125I-ligand for histamine H1-receptors, synthesised using the 125I-Bolton Hunter reagent (2000 Ci/mmol) for acylation of an aminopentyl analogue of mepyramine. Its specific binding varied linearly with the concentration of guinea-pig cerebellar membranes and represented about 80% of the total. Selective interaction with H1-receptors was demonstrated by estimation of Ki values of known agonists and antagonists and confirmed by the low affinity of histamine H2- and H3-receptor antagonists and of non-histaminergic agents. At 25 degrees C, [125I]iodobolpyramine exhibited a slow association rate (180-240 min to reach equilibrium) and a slow dissociation rate (t1/2 = 201 min). Kinetic and saturation data yielded KD values of 0.05 and 0.15 nM, respectively, indicating that it is among the most potent H1-receptor antagonists known. The sensitivity for detecting H1-receptors in guinea-pig cerebellum using [125I]iodobolpyramine was increased 50-fold relative to use of [3H]mepyramine. Well-contrasted autoradiograms of guinea-pig brain, obtained after a short exposure time, confirmed previous H1-receptor localisation established with [3H]mepyramine and revealed new localisations, e.g. in cerebral cortex and nucleus accumbens.

Two splice variants of the hypoxia-inducible factor HIF-1α as potential dimerization partners of ARNT2 in neurons

The hypoxia‐inducible factor (HIF‐1α), a basic helix‐loop‐helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF‐1α and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF‐1α is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single‐minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF‐1α in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF‐1α/ARNT1 complex, does not recognize the HIF‐1‐binding site of the hypoxia‐induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF‐1α and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF‐1α, ARNT1 and ARNT2, downregulation of either HIF‐1α or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.