The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells.

Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson's disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson's disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson's disease, providing novel opportunities for therapeutic intervention.

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-ß hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-ß hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

The end of a myth: cloning and characterization of the ovine melatonin MT(2) receptor.

BACKGROUND AND PURPOSE: For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT(1) from other mammal species). Here we report the cloning of another melatonin receptor, MT(2), from sheep. EXPERIMENTAL APPROACH: Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT(2) mRNA sequence, we have cloned and characterized MT(2) receptors from sheep retina. KEY RESULTS: The ovine MT(2) receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT(2) receptor stably expressed in CHO-K1 cells showed high-affinity 2[(125)I]-iodomelatonin binding (K(D)= 0.04 nM). The rank order of inhibition of 2[(125)I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT(2) receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT(2) receptors. Functionally, the ovine MT(2) receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT(2) mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT(1) and MT(2) mRNA was not parallel in the three tissues. CONCLUSION AND IMPLICATIONS: Co-expression of MT(1) and MT(2) receptors in all analysed sheep brain tissues suggests that MT(2) receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT(1) receptor action.

Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake".

Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

Use-dependent inhibition of hHCN4 by ivabradine and relationship with reduction in pacemaker activity.

BACKGROUND AND PURPOSE: Ivabradine, a specific and use-dependent I(f) inhibitor, exerts anti-ischaemic activity purely by reducing heart rate. The aim of this work was to characterize its effect on the predominant HCN channel isoform expressed in human sino-atrial nodes (hSAN), to determine its kinetics in HCN channels from multicellular preparations and rate-dependency of its action. EXPERIMENTAL APPROACH: RT-PCR analysis of the four HCN channel isoforms was carried out on RNAs from hSAN. Patch-clamp and intracellular recordings were obtained from CHO cells stably expressing hHCN4 and isolated SAN, respectively. Beating rate of rat isolated atria was followed using a transducer. KEY RESULTS: hHCN4 mRNAs were predominant in hSAN. Ivabradine induced a time-dependent inhibition of hHCN4 with an IC(50) of 0.5 microM. In rabbit SAN, ivabradine progressively reduced the frequency of action potentials: by 10% after 3 h at 0.1 microM, by 14% after 2 h at 0.3 microM and by 17% after 1.5 h at 1 microM. After 3h, ivabradine reduced the beating rate of rat right atria with an IC(30) of 0.2 microM. The onset of action of ivabradine was use-dependent rather than time-dependent with slower effects than caesium, an extracellular I (f) blocker. Ivabradine 3 microM decreased the frequency of action potentials in SAN from guinea-pig, rabbit and pig by 33%, 21% and 15% at 40 min, respectively. CONCLUSIONS AND IMPLICATIONS: The use-dependent inhibition of hHCN4 current by ivabradine probably contributes to its slow developing effect in isolated SAN and right atria and to its increased effectiveness in species with rapid SAN activity.

The use of IRES-based bicistronic vectors allows the stable expression of recombinant G-protein coupled receptors such as NPY5 and histamine 4.

Stable expression of G protein coupled receptors in cell lines is a crucial tool for the characterization of the molecular pharmacology of receptors and the screening for new antagonists. However, in some instances, many difficulties have been encountered to obtain stable cell lines expressing functional receptors. Here, we addressed the question of vector optimization to establish cell lines expressing the human neuropeptide Y receptor 5 (NPY5-R) or histamine receptor 4 (HH4R). We have compared bicistronic vectors containing viral or cellular internal ribosome entry sites (IRES), co-expressing the receptor and the neomycine resistance gene from a single mRNA, to a bigenic vector containing two distinct promoters upstream each different genes. This study is the first one to validate the use of three cellular IRESs for long-term transgene expression. Our results demonstrate for both NPY5-R and HH4R that the bicistronic vectors with EMCV, VEGF, FGF1A or FGF2 IRES provide clones expressing functional receptors with yields between 25% and 100%. In contrast, the bigenic vector provided no functional clones, related to a low expression of NPY5R mRNA. The cell lines expressing active receptor were stable after more than 50 passages. These data indicate that IRES-based bicistronic vectors are particularly appropriate to establish cell clones expressing active G-coupled protein receptors with a high yield. In the case of NPY5, it was a new way to produce such a stable cell line. Furthermore, the characteristics-presented herein-of this receptor pharmacological property are perfectly in line with those reported in the literature.

The role of the matrix metalloproteinases during in vitro vessel formation.

Matrix metalloproteinases (MMPs) constitute a large family of extracellular matrix degrading proteases implicated in a number of physiological and pathological processes, including angiogenesis. However, the relative importance of the individual MMPs in vessel formation is poorly understood. Using the three-dimensional rat aortic model, the role of the MMPs in angiogenesis in vitro was investigated both by the use of synthetic MMP inhibitors, and by a study of the expression of nine MMPs and three of their endogenous inhibitors (the TIMPs) during vessel formation. Inhibition of microvessel growth in this model by the MMP inhibitor Marimastat demonstrated the requirement of the MMPs for angiogenesis in both collagen and fibrin matrices (half-maximal inhibition at 5 and 80 nM, respectively). The profile of MMP expression was seen to be modified by both matrix composition and exogenous growth factors. For example, whilst the gelatinase MMP-2 and stromelysin MMP-3 were present at high levels in fibrin culture, the stromelysin MMP-11 and membrane-type-1-MMP were more highly expressed during vessel formation in collagen. The angiogenic basic fibroblast growth factor (bFGF) upregulated the expression of the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10 and MMP-11) and the interstitial collagenase MMP-13, whereas vascular endothelial growth factor (VEGF) led to a marked increase in expression of MMP-2 only. Together, the environment-dependent upregulation in expression of a number of MMPs during angiogenesis, and the total inhibition of vessel growth observed at nanomolar concentrations of synthetic MMP inhibitors, suggests a major collective role of these enzymes in angiogenesis, and provides a basis for further development of MMP inhibitors for anti-angiogenic therapy.

Development of new assays and improved procedures for the purification of recombinant human chymase.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

Regulation of murine airway responsiveness by endothelial nitric oxide synthase.

Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(-/-)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(-/-) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-L-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine. eNOS(-/-) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-L-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity.

Structure and expression of the human histamine H4-receptor gene.

We report the characterization by genomics-based approach of the human H4-receptor gene structure. The H4-receptor gene have been mapped by radiation hybrid experiments (Gene Bridge 4) on chromosome 18q11.2, between the AFMBB11WH5 and CHLC.GATA85D10 markers. The H4-receptor gene spans more than 21 kbp and contains three exons separated by two large introns (>7 kbp). RT-PCR analysis showed that the H4-receptor gene encoded a 3.7 kb mRNA which did not seem to be alternatively spliced within its coding region. The H4-receptor transcripts were found to be highly expressed in peripheral tissues implicated in inflammatory responses such as leukocytes, spleen, lung, and liver. In addition, low expression level of the H4-receptor mRNA was also detected in several human brain regions. Analysis of the 5'-flanking region of the H4-receptor gene did not reveal the existence of canonical TATA or CAAT-box. However, several putative regulatory elements mediating TNFalpha or IL-6-stimulated transcriptional activation were detected. The uteroglobin promoter binding factor, known to mediate anti-inflammatory response of uteroglobin, in the lung, was also found in this region. Thus, the description of the H4-receptor gene promoter region will facilitate the elucidation of its transcriptional control by factors secreted during inflammatory responses.

Genomic organization and characterization of splice variants of the human histamine H3 receptor.

In the present paper we report the genomic organization of the human histamine H3-receptor gene, which consists of four exons spanning 5.5 kb on chromosome 20. Using PCR, six alternative splice variants of the H3 receptor were cloned from human thalamus. These variants were found to be coexpressed in human brain, but their relative distribution varied in a region-specific manner. These isoforms displayed either a deletion in the putative second transmembrane domain (TM), H3(DeltaTM2, 431aa) or a variable deletion in the third intracellular loop (i3), H3(Deltai3, 415aa), H3(Deltai3, 365aa), H3(Deltai3, 329aa) and H3(DeltaTM5+Deltai3, 326aa). In order to determine the biological role of the H3 receptor variants compared with the 'original' H3(445aa) receptor, three isoforms, namely H3(445aa), H3(DeltaTM2, 431aa) and H3(Deltai3, 365aa), were expressed in CHO cells and their pharmacological properties were investigated. Binding studies showed that H3(DeltaTM2, 431aa) transiently expressed in CHO cells was unable to bind [125I]iodoproxyfan, whereas both the H3(445aa) and H3(Deltai3, 365aa) receptors displayed a high affinity for [125I]iodoproxyfan [K(d)=28+/-5 pM (n=4) and 8+/-1 pM (n=5) respectively]. In addition, H3(Deltai3, 365aa) possessed the same pharmacological profile as the H3(445aa) receptor. However, in CHO cells expressing H3(Deltai3, 365aa), H3 agonists did not inhibit forskolin-induced cAMP production, stimulate [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) binding or stimulate intracellular Ca(2+) mobilization. Therefore the 80-amino-acid sequence located at the C-terminal portion of i3 plays an essential role in H3 agonist-mediated signal transduction. The existence of multiple H3 isoforms with different signal transduction capabilities suggests that H3-mediated biological functions might be tightly regulated through alternative splicing mechanisms.

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

Endothelin-1 pathway in human alveolar epithelial cell line A549 and human umbilical vein endothelial cells.

AIM: This study was designed to characterize the endothelin pathway in an immortalized human adenocarcinoma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC). METHODS: The release of ET-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin isoforms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-1a, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody. RESULTS: Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in both cell lines phosphoramidon produced a concentration-dependent inhibition of ET-1 release and an enhanced accumulation of big-ET-1. Both HUVEC and A549 cells express the mRNAs for ppET-1, ET-A, and ET-B receptor subtypes and ECE-1 (isoforms ECE-1b, c and/or d). In addition, in HUVEC the mRNAs for ppET-2 and for the isoform ECE-1a were also detected. In A549 cells, ECE-1 had a preferential subcellular localization in the membrane fraction but was not detected in the cytosol. CONCLUSION: Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involved remains to be determined. A549 might be used as a screening assay for drug discovery such as for inhibitors of endothelin-1 release.

Agonist and antagonist actions of yohimbine as compared to fluparoxan at alpha(2)-adrenergic receptors (AR)s, serotonin (5-HT)(1A), 5-HT(1B), 5-HT(1D) and dopamine D(2) and D(3) receptors. Significance for the modulation of frontocortical monoaminergic transmission and depressive states.

Herein, we evaluate the interaction of the alpha(2)-AR antagonist, yohimbine, as compared to fluparoxan, at multiple monoaminergic receptors and examine their roles in the modulation of adrenergic, dopaminergic and serotonergic transmission in freely-moving rats. Yohimbine displays marked affinity at human (h)alpha(2A)-, halpha(2B)- and halpha(2C)-ARs, significant affinity for h5-HT(1A), h5-HT(1B), h5-HT(1D), and hD(2) receptors and weak affinity for hD(3) receptors. In [(35)S]GTPgammaS binding protocols, yohimbine exerts antagonist actions at halpha(2A)-AR, h5-HT(1B), h5-HT(1D), and hD(2) sites, yet partial agonist actions at h5-HT(1A) sites. In vivo, agonist actions of yohimbine at 5-HT(1A) sites are revealed by WAY100,635-reversible induction of hypothermia in the rat. In guinea pigs, antagonist actions of yohimbine at 5-HT(1B) receptors are revealed by blockade of hypothermia evoked by the 5-HT(1B) agonist, GR46,611. In distinction to yohimbine, fluparoxan shows only modest partial agonist actions at h5-HT(1A) sites versus marked antagonist actions at halpha(2)-ARs. While fluparoxan selectively enhances hippocampal noradrenaline (NAD) turnover, yohimbine also enhances striatal dopamine (DA) turnover and suppresses striatal turnover of 5-HT. Further, yohimbine decreases firing of serotonergic neurones in raphe nuclei, an action reversed by WAY100,635. Fluparoxan increases extracellular levels of DA and NAD, but not 5-HT, in frontal cortex. In analogy, yohimbine enhances FCX levels of DA and NAD, yet suppresses those of 5-HT, the latter effect being antagonized by WAY100,635. The induction by fluoxetine of FCX levels of 5-HT, DA, and NAD is potentiated by fluparoxan. Yohimbine likewise facilitates the influence of fluoxetine upon DA and NAD levels, but not those of 5-HT. In conclusion, the alpha(2)-AR antagonist properties of yohimbine increase DA and NAD levels both alone and in association with fluoxetine. However, in contrast to the selective alpha(2)-AR antagonist, fluparoxan, the 5-HT(1A) agonist actions of yohimbine suppress 5-HT levels alone and underlie its inability to augment the influence of fluoxetine upon 5-HT levels.

Truncated isoforms inhibit [3H]prazosin binding and cellular trafficking of native human alpha1A-adrenoceptors.

We have identified from human liver eight alpha(1A)-adrenoceptor (alpha(1A)-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these alpha(1A)-AR isoforms (alpha(1A-1)-AR, alpha(1A-2a)-AR and alpha(1A-3a)-AR) gave rise to receptors with seven transmembrane domains (7TMalpha(1A)-AR). The other five (alpha(1A-2b)-AR, alpha(1A-2c)-AR, alpha(1A-3c)-AR, alpha(1A-5)-AR and alpha(1A-6)-AR) led to truncated receptors lacking transmembrane domain VII (6TMalpha(1A)-AR). The 7TMalpha(1A)-AR isoforms transiently expressed in COS-7 cells bound [(3)H]prazosin with high affinity (K(d) 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca(2+) concentration, whereas the 6TMalpha(1A)-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged alpha(1A)-AR isoforms showed that the 7TMalpha(1A)-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMalpha(1A)-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated alpha(1A)-AR isoform inhibited [(3)H]prazosin binding and cell-surface trafficking of the co-expressed 'original' 7TMalpha(1A-1)-AR. However, there was no modification of either the [(3)H]prazosin-binding affinity or the pharmacological properties of alpha(1A-1)-AR. Immunoblotting experiments revealed that co-expression of the alpha(1A-1)-AR with 6TMalpha(1A)-AR isoforms did not impair alpha(1A-1)-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of alpha(1A)-AR.

In vitro down-regulation of antigen-induced IL-5 gene expression and protein production by cAMP-specific phosphodiesterase type 4 inhibitor.

The effects of cAMP-elevating agents on antigen-induced IL-5 (interleukin-5) messenger RNA expression and protein production were examined in vitro in an antigen-driven system of splenocytes from ovalbumin sensitized BALB/c mice. IL-5 production was inhibited by rolipram, a type 4 phosphodiesterase (PDE4) inhibitor, dose-dependently (maximally at 10(-5) M) and by dibutyryl-cAMP (db-cAMP) (3 x 10(-4) M), but not by the type 3 and type 5 PDE inhibitors milrinone and zaprinast (10(-5) M), respectively. Forskolin (10(-5) M), an adenylate cyclase activator, was noninhibitory alone but potentiated inhibition by rolipram. Inhibition was associated with a decrease in IL-5 mRNA expression. Cycloheximide 10(-6) M and actinomycin 2 micrograms/ml abolished IL-5 production and mRNA expression. We conclude that in splenocytes from sensitized mice, IL-5 production and mRNA expression depend on antigen stimulation. The time course of IL-5 protein production is closely related to IL-5 mRNA expression and depends on de novo protein synthesis. db-cAMP and a selective PDE4 inhibitor, alone or in combination with forskolin, are the only cAMP-elevating agents that dose-dependently inhibited antigen-induced IL-5 mRNA expression and protein production. These results are in agreement with in vivo inhibition by a selective PDE4 inhibitor of antigen-induced pulmonary eosinophil infiltration and IL-5 production in sensitized mice, and they suggest that PDE4 inhibitors have potential for treating respiratory allergy.

Characterisation of cyclooxygenase 1 and 2 expression in mouse resident peritoneal macrophages in vitro; interactions of non steroidal anti-inflammatory drugs with COX2.

Resident peritoneal macrophages exposed to inflammatory stimuli (zymosan, lipopolysaccharide (LPS)) represent a widely used model for studying arachidonic acid metabolism and for screening of prostaglandin (PG) synthesis inhibitors. In the present study, cyclooxygenase 1 (COX1) was shown constitutively expressed in mouse adherent and non-adherent macrophages whereas expression of COX2 was observed only in adherent cells, even when cultured in minimal conditions (Ca-, Mg- and serum-free medium). The COX2 expression was amplified by arachidonic acid cascade stimulating agents (Ca, Mg, zymosan) and by LPS in a time-dependant manner; PGE2 by itself amplified LPS-induced COX2 expression. In well-defined experimental conditions of COX2 expression (LPS-stimulated adherent macrophages), we studied specific interactions of some representative anti-inflammatory drugs with COX2 enzymatic activity and expression. By contrast with dexamethasone, which reduced PGE2 release together with a strong reduction of COX2 expression (protein and mRNA), non steroidal anti-inflammatory drugs (NSAIDs) reduced PGE2 synthesis without any effect at the COX2 mRNA level. This reduction of PGE2 production by NSAIDs resulted from either an exclusive enzymatic inhibition (aspirin, NS398, 6-Methoxy naphtyl acetic acid) or an enzymatic inhibition associated with a slight decrease of COX2 protein level (indomethacin). For paracetamol and salicylic acid, two weak inhibitors of COX enzymatic activity, reduction of PGE2 synthesis appeared to be related to reduced level of COX2. These findings show that the macrophage can be used as a cellular model to study specifically COX1 and COX2. In this cell type, COX2 expression is dependent on adhesion, enhanced by stimulation of arachidonic acid metabolism, and auto amplified by PGE2. Furthermore, the results indicate that known NSAIDs differ in their interaction with cyclooxygenase, being able to inhibit either COX2 enzymatic activity, and/or COX2 expression. However, further studies are required to determine the mechanism and the role of COX2 expression during inflammation in vivo, and to define more precisely the best target for new potent and safe NSAIDs.

NK1 and NK2 receptors are similar in man and rabbit.

A series of 15-18 compounds that act on NK1 or NK2 receptors as agonists or antagonists have been tested in the monoreceptor systems of the rabbit vena cava (NK1) and the rabbit pulmonary artery (NK2) for biological activities and for their ability to displace [3H] [Sar9, Met(O2)11]SP or [125I] NKA respectively from NK1 or NK2 human binding sites obtained by transfection and functional expression of the cDNAs for these receptor subtypes in CHO-K1 cells. For the two tachykinin receptors studied, positive highly significant correlations have been shown between binding and biological assays. Slopes of correlations are linear and near unity (r = 0.918 and 0.938). For NK1 and NK2 receptors the pharmacology in human and rabbit tissues appears to be very similar. The assays of biological activity on rabbit tissues may be therefore used to complement binding studies on human transfected cells to identify new antagonists for human tachykinin receptors.

Different mRNAs code for dopa decarboxylase in tissues of neuronal and nonneuronal origin.

A cDNA clone for dopa decarboxylase (EC 4.1.1.28) has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5' end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Drosophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5' untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3' untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5' untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

Comparative and quantitative study of L-dopa decarboxylase mRNA in rat neuronal and non-neuronal tissues.

The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, showed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.