Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A + C + 0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A + C + 0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation, thermal or oxidative stress.

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.

Hepatomegaly in transgenic mice expressing an oncogenic form of beta-catenin.

Inappropriate activation of the Wnt/beta-catenin signaling, resulting mainly from activating mutations of the beta-catenin gene, has been implicated recently in the development of hepatocellular carcinoma (HCC). We have generated transgenic mice expressing an oncogenic form of beta-catenin in their hepatocytes to analyze the effect of deregulated beta-catenin signaling on liver homeostasis. These mice rapidly developed hepatomegaly soon after birth, with livers three to four times heavier than those of nontransgenic littermates. The liver cell hyperplasia resulted from increased cell proliferation without any compensatory apoptosis. Although the genes encoding c-myc and cyclin D1 are potential targets of the beta-catenin signaling pathway, neither of them was overexpressed in the hyperplastic livers of beta-catenin transgenic mice. Thus, the key target genes of the beta-catenin signaling pathway in the liver remain to be identified.

Treatment with 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin.

BACKGROUND: We previously reported that skin closure is improved by photoirradiation of the wound margins with an 815-nm diode laser system. OBJECTIVES: To determine whether the beneficial effects of laser treatment involve the overexpression of the inducible 72-kDa heat shock protein, Hsp70. METHODS: Expression of Hsp70 was investigated by immunocytochemistry in normal hairless rat dorsal skin and compared with its expression after laser photoirradiation. RESULTS: Constitutive expression of Hsp70 was mainly confined to the upper epidermal layer. Laser irradiation further increased epidermal expression of Hsp70 while inducing de novo synthesis of the protein in dermal structures, particularly around blood vessels, hair follicles and sebaceous glands. Laser-induced expression of Hsp70 was still present 7 days after photoirradiation. CONCLUSIONS: Laser-induced expression of Hsp70 might contribute to improved tissue regeneration and wound healing.

Laser assisted skin closure (LASC) by using a 815-nm diode-laser system accelerates and improves wound healing.

BACKGROUND AND OBJECTIVE: This study aimed to evaluate a 815-nm diode-laser system to assist wound closure to accelerate and improve healing process. STUDY DESIGN/MATERIALS AND METHODS: A total of 25 male hairless rats (mutant OFA Sprague-Dawley rats, IFFA-CREDO, L'Arbresle, France) with four dorsal skin incisions were used for the study. For each wound, the good apposition of the edges was obtained with buried absorbable suture. In the laser group, the laser beam was applied spot by spot through a transparent adhesive dressing along two incisions with the following parameters: 1.5 W; 3 seconds; spot diameter, 2 mm; fluence, 145 J/cm(2). Both control wounds were closed with conventional suture techniques. The duration of the closure procedure was noted for each group. Clinical examination, histologic study, and measurement of tensile strength were performed at 3, 7, 15, and 21 days after surgery. Determination of activation of heat shock protein 70 (Hsp70) through immunocytochemistry was performed at days 1 and 7. RESULTS: LASC was 4 times faster to process than conventional suture: 1 minute 49 +/- 20.6 seconds vs. 7 minutes 26 +/- 62.2 seconds. In the laser group, healing was accelerated resulting in a more indiscernible scar than in the control groups. Histologic aspect was better with earlier continuous epidermis and dermis and a thinner resulting scar. Tensile strength was 30 to 58% greater than in control groups at 7 and 15 days (P < 0.001). Expression of Hsp70 was markedly induced in skin structures examined after laser exposure. CONCLUSIONS: This study shows the ability of the 815-nm diode-laser system to assist wound closure leading to an acceleration and an improvement of wound healing with indiscernible resulting scar. The mechanisms of this phenomenon are still unclear but further investigations are in progress to attempt to explain them.

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells.

BACKGROUND: We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes. METHODS: and results Here, we have analyzed the ability of histidylated polylysine to transfer reporter or CFTR genes into immortalized cystic fibrosis airway surface epithelial cells (sigmaCFTE29o- cells) and airway gland serous cells (CF-KM4 cells) which are both important targets for cystic fibrosis gene therapy. The luciferase reporter gene expression measured after gene transfer with histidylated polylysine into both cell lines was quite high and similar to that obtained with commercially available vectors. In addition, the level of expression was not dependent on the presence of a membrane disrupting agent such as chloroquine. Histidylated complexes were present in slightly acidic non-lysosomal cellular compartments as shown by a cytological approach using biotinylated plasmid, lysosome-specific antibodies and confocal microscopy. Histidylated complexes appeared to be of small size when prepared at low ionic strength and formed aggregates upon increasing the ionic strength. However, aggregate formation was prevented by the addition of 10% fetal bovine serum. Gene transfer efficiency varied with the size of the complexes and decreased when small particles were used. CONCLUSIONS: These results suggest that histidylated polylysine may be an efficient non-viral vector for gene transfer into cystic fibrosis airway surface epithelial cells and airway gland serous cells.

Maturational changes of endothelial vasoactive factors and pulmonary vascular tone at birth.

The aim of this study was to determine which endothelial factors were involved in the decrease of pulmonary vascular resistance at birth, and how they changed with maturation. Response of intrapulmonary artery rings precontracted with prostaglandin F2alpha were studied from piglets aged <2 h, 2-3 day, 10 day and adult pigs for pharmacological responses to acetylcholine (ACh) and cromakalim (CMK) in the presence and the absence of the nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NA), the adenosine triphosphate sensitive potassium (K(ATP)) channel blocker, glibenclamide and the endothelin (ET)-A receptor antagonist, BQ123. In situ hybridization and immunochemistry studies were performed in lung tissues of the same animals in order to determine the expression of NOS and ET. There was a small contractile effect of ACh in the newborn. Relaxation to ACh, which was blocked by L-NA and reduced by glibenclamide, only appeared from the age of 3 days. The significantly greater relaxation to CMK in rings without endothelium (p<0.05) was abolished by BQ123 in the newborn, and then disappeared by 2 days of age. Glibenclamide had a greater inhibitory effect on relaxation induced by CMK at 10 days than in the newborn and 2 days old piglets. NOS expression was low in pulmonary arteries of the newborn and increased by 2 days of age whereas the converse was seen with ET expression. It is concluded that: 1) relaxant response to acetylcholine was absent at birth and appeared at 2 days; 2) the reduced relaxant response to cromakalin in rings with endothelium at birth could be blocked by BQ123; and 3) the expression of endothelin decreased whereas the expression of nitric oxide synthase increased from birth to 2 days of age.

Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors.

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.

Differential role of nitric oxide pathway and heat shock protein in preconditioning and lipopolysaccharide-induced brain ischemic tolerance.

The purposes of this study were to investigate the role of nitric oxide (NO), nitric oxide synthase (NOS), and 70 kDa heat shock protein in brain ischemic tolerance induced by ischemic preconditioning and lipopolysaccharide. Focal cerebral ischemia was induced in rats by intraluminal middle cerebral artery occlusion. Infarct volume was significantly reduced (1) in rats subjected to 3 min ischemia 72 h prior to 60 min ischemia; (2) in rats administered lipopolysaccharide (0.5 mg/kg; i.p.) 72 h prior to 60 min ischemia compared with controls. The beneficial effect of ischemic preconditioning was unchanged despite prior administration of nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. Conversely, the protective effect of lipopolysaccharide was nullified by L-NAME. Using immunohistochemical techniques, we observed that (1) ischemic preconditioning but not lipopolysaccharide induces the expression of 70 kDa heat shock protein in cerebral cortex and (2) lipopolysaccharide induces early increased expression of endothelial NOS in cerebral blood vessels. The results suggest that (1) endothelium-derived NO plays a role of a trigger in the brain tolerance induced by lipopolysaccharide, and (2) 70 kDa heat shock protein is involved in the protection afforded by ischemic preconditioning but not by lipopolysaccharide.

[Role of nitric oxide synthase III and guanosine 3':5'- cyclic monophosphate in the protection exerted by nitric oxide on hepatic ischemia-reperfusion injury]. / Rôle de la monoxyde d'azote synthase-III et du guanosine 3':5'-monophosphate cyclique dans la protection exercée par le monoxyde d'azote lors d'une ischémie-reperfusion hépatique.

Nitric oxide (NO) exerts cytoprotective effects against hepatic ischemia-reperfusion damage. This study was designed to evaluate which isoform of NO synthase (NOS) is implicated in the generation of cytoprotective NO and to investigate whether NO effects are mediated by cyclic GMP (cGMP). After partial ischemia for 45 min, liver damage was estimated by the release into plasma of cytolytic enzymes. Ischemia-reperfusion induced marked increases in plasma creatine kinase and lactate dehydrogenase after 1 h of reperfusion and of aminotransferases after 6 h of reperfusion. The pretreatment of ischemic rats with 8-bromo-cGMP (16 mg/kg i.v. 30 min before ischemia) or with L-arginine (the endogenous precursor of NO, 100 mg/kg i.v.) significantly diminished the ischemia-reperfusion-induced release of all these enzymes. This demonstrates that cGMP possesses hepatoprotective properties. By immunohistochemistry, we observed, after 6 h of reperfusion, an increase in endothelial NOS-III immunoreactivity, particularly in the small arteries and sinusoids. This NOS-III accumulation in endothelial cells could protect the liver against ischemia-reperfusion by the local generation of NO probably via cGMP.

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes.

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.

Coexpression of dopamine D1 and D3 receptors in islands of Calleja and shell of nucleus accumbens of the rat: opposite and synergistic functional interactions.

Using double in situ hybridization, we found extensive coexpression of dopamine D1 and D3 receptor (D1R and D3R) mRNAs in neurons of the island of Calleja major (ICjM) and ventromedial shell of nucleus accumbens (ShV), respectively. Thus, at least 79 and 63% of D3R mRNA-expressing neurons in ICjM and ShV also expressed the D1R mRNA. Coexpression of D1R and D3R mRNAs was found to occur in substance P (SP) mRNA-expressing neurons in both areas, suggesting SP mRNA as a marker of the activity of coexpressing neurons. Administration of SKF 38393, a D1R receptor agonist, increased c-fos mRNA in ICjM, whereas administration of quinpirole, a D2R/D3R agonist, decreased it; SCH 23390, a D1 R antagonist and nafadotride, a preferential D3R antagonist, given alone, had effects opposite to those of the corresponding agonists. These data indicate that basal c-fos expression in ICjM is maintained by endogenous dopamine acting tonically upon two receptor subtypes subserving opposite effects on the same cell. However, in ShV, whereas SKF 38393 also increased c-fos mRNA, quinpirole had no effect, a difference presumably reflecting the lower fraction of neurons coexpressing D1R and D3R in this area. In contrast, in ShV from reserpine-treated rats, SKF 38393 increased SP mRNA and quinpirole potentiated this effect. These contrasting interactions of D1R- and D3R-mediated signalling events, i.e. in either opposite or synergistic directions, most likely occurring at the single cell level, may serve to increase the dopamine response threshold of the target cells in ICjM and to maintain a strong tonic activity of ShV neurons.

Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes.

Hypoxia initiates pulmonary vasoconstriction (HPV) by inhibiting one or more voltage-gated potassium channels (Kv) in the pulmonary artery smooth muscle cells (PASMCs) of resistance arteries. The resulting membrane depolarization increases opening of voltage-gated calcium channels, raising cytosolic Ca2+ and initiating HPV. There are presently nine families of Kv channels known and pharmacological inhibitors lack the specificity to distinguish those involved in control of resting membrane potential (Em) or HPV. However, the Kv channels involved in Em and HPV have characteristic electrophysiological and pharmacological properties which suggest their molecular identity. They are slowly inactivating, delayed rectifier currents, inhibited by 4-aminopyridine (4-AP) but insensitive to charybdotoxin. Candidate Kv channels with these traits (Kv1.5 and Kv2.1) were studied. Antibodies were used to immunolocalize and functionally characterize the contribution of Kv1. 5 and Kv2.1 to PASMC electrophysiology and vascular tone. Immunoblotting confirmed the presence of Kv1.1, 1.2, 1.3, 1.5, 1.6, and 2.1, but not Kv1.4, in PASMCs. Intracellular administration of anti-Kv2.1 inhibited whole cell K+ current (IK) and depolarized Em. Anti-Kv2.1 also elevated resting tension and diminished 4-AP-induced vasoconstriction in membrane-permeabilized pulmonary artery rings. Anti-Kv1.5 inhibited IK and selectively reduced the rise in [Ca2+]i and constriction caused by hypoxia and 4-AP. However, anti-Kv1.5 neither caused depolarization nor elevated basal pulmonary artery tone. This study demonstrates that antibodies can be used to dissect the whole cell K+ currents in mammalian cells. We conclude that Kv2. 1 is an important determinant of resting Em in PASMCs from resistance arteries. Both Kv2.1 and Kv1.5 contribute to the initiation of HPV.

Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain.

Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [(125)I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance. high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [(125)I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [(125)I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

The guinea pig histamine H2 receptor: gene cloning, tissue expression and chromosomal localization of its human counterpart.

The guinea pig is the prototypic animal species for the histamine H2 receptor. Using a strategy based upon nucleotide sequence homology and starting from the sequence of the rat histamine H2 receptor (Ruat et al., Biochem. Biophys. Res. Commun. 1991, 179: 1470-78), we have cloned an intronless highly homologous DNA very likely encoding the guinea pig H2 receptor. The encoded 359 amino acid protein displays 83 to 86% identity with the rat-, human- or dog-H2 receptors. Northern blot analysis identified a single transcript of 4.6 kb in peripheral tissues and brain areas in which the presence of the H2 receptor had been revealed previously by either photoaffinity labeling or binding studies. In brain, the distribution of transcripts, established by either Northern blots or in situ hybridization studies, was consistent with the localization of the H2-receptor. In addition, using Southern analysis of a chromosome mapping panel constructed from human x hamster hybridomas, we assigned the H2 receptor gene to human chromosome 5.

Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin.

We have identified 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probe for the recently cloned dopamine D3 receptor and used it to assess the presence of this receptor and establish its distribution and properties in brain. In transfected Chinese hamster ovary (CHO) cells, it binds to D3 receptors with subnanomolar affinity, whereas its affinity is approximately 100-, 1000-, and 10,000-fold lower at D2, D4, and D1 receptors, respectively. Specific [3H]7-OH-DPAT binding sites, with a Kd of 0.8 nM and a pharmacology similar to those at reference D3 receptors of CHO cells, were identified in rat brain. D3 receptors differ from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas--e.g., paleostriatum and archicerebellum--as evidenced by membrane binding are autoradiography studies. Native D3 receptors in brain are characterized by an unusually high nanomolar affinity for dopamine and a low modulatory influence of guanyl nucleotides on agonist binding. These various features suggest that D3 receptors are involved in a peculiar mode of neurotransmission in a restricted subpopulation of dopamine neurons.

Localization and function of the D3 dopamine receptor.

A novel dopamine receptor has been recently cloned, which differs from D1 and D2 receptors by its sequence, localization, pharmacology and possibly signalling system, hence its designation as D3 receptor. The D3 receptor cDNA was obtained by a combination of screenings of DNA libraries and PCR polymerase chain reaction experiments. It encodes a protein with a predicted structure consisting in 7 transmembrane domains indicating that it belongs to the G-protein coupled receptor family. Its global homology is 52% with the D2 receptor but 78% if only transmembrane domains are considered. Two shorter transcript variants, in addition to the full-length cDNA were detected by PCR in various rat brain regions. The shorter transcripts are generated by alternative splicing and encode two putative proteins respectively interrupted after the second transmembrane domain and lacking the second extracytoplasmic loop. After transfection of this latter isoform into cultured cells, no dopaminergic activity could be detected. These shorter splice variants may regulate the number of active D3 receptors. The human D3 receptor was also cloned using similar approaches with rat D3 receptor cDNA probes and was found highly homologous to the rat receptor, except in the third intracytoplasmic loop. The human D3 receptor gene was assigned to the chromosome 3 at q13.3 band. Visualisation of D3 receptor mRNA in rat brain by in situ hybridization indicated a predominant expression of the message in the ventral striatum and other "limbic" areas. There is no overlap in the distributions of D2 and D3 receptor mRNAs in discrete structures, suggesting that the two transcripts are expressed by different cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA.

The messenger RNA (mRNA) of the recently characterized D3 dopamine receptor was visualized on rat brain sections using in situ hybridization with a 32P-labeled ribonucleic acid probe corresponding to a major part of the third cytoplasmic loop, a domain in which D2 and D3 dopamine receptors display little homology. For the purpose of comparison, D2 receptor mRNA was also specifically visualized on adjacent sections. The areas that expressed D2 and/or D3 receptors were also compared with those previously detected using [125I]iodosulpride, a ligand that binds to both D2 and D3 receptors with a similar affinity. The localization of D3 receptor mRNa markedly differs from that of D2 receptor mRNA. Whereas D2 receptor mRNA is expressed in all major brain areas receiving dopaminergic projections, particularly in the whole striatal complex, D3 receptor mRNA is expressed in a more restricted manner. It is mainly detected in telencephalic areas receiving dopaminergic inputs from the A10 cell group, e.g. accumbens nucleus, islands of Calleja, bed nucleus of the stria terminalis and other limbic areas such as the hippocampus and the mammillary nuclei. D2 and D3 receptor mRNAs were also detected at the level of the substantia nigra, suggesting that these receptors function as both autoreceptor and postsynaptic receptors. In several dopaminergic projection areas, e.g. ventral straitum, septal or mammillary nuclei, the distributions of D2 and D3 receptor mRNAs appeared complementary without overlap. The distribution of [125I]iodosulpride binding sites generally overlapped that of D2 or D3 receptor mRNAs, the latter being most abundant in dopaminergic areas known to be associated with cognitive and emotional functions.

Reversible and irreversible labelling of H1- and H2 -receptors using novel [125I] probes.

We have recently designed the first 125I-labelled probes specific for the histamine H1 and H2 receptors. These reversible and irreversible antagonists are among the most potent H1 and H2 ligands and have enabled investigations into the biochemical and pharmacological properties of these two receptors. In various brain animal species, the ligand binding peptide of the H1 and H2 receptors, as determined by photoaffinity labeling, resides within 56-59 kDa peptides. In contrast, in guinea pig heart, the ligand binding domain of the H1 receptor is characterized by a higher molecular weight (68 kDa), suggesting the presence of an isoform of this protein, clearly differentiable by this biochemical property but not by its pharmacology. The reversible 125I-probes allowed us to extend the pharmacology of these receptors in several biological preparations and in human brain, and to establish their interaction with G-proteins. A detailed mapping of H1 and, for the first time, of H2 receptors, has been achieved in guinea pig brain, establishing their presence in almost all brain areas. These experiments show that there is no correlation between the density of H2 receptor and the activity of adenylate cyclase sensitive to histamine suggesting a molecular heterogeneity of this receptor.