Forskolin-induced cAMP Formation Research Articles

In vitro exposure to ambient fine and ultrafine particles alters dopamine uptake and release, and D2 receptor affinity and signaling

The exposure to environmental pollutants, such as fine and ultrafine particles (FP and UFP), has been associated with increased risk for Parkinson’s disease, depression and schizophrenia, disorders related to altered dopaminergic transmission. The striatum, a neuronal nucleus with extensive dopaminergic afferents, is a target site for particle toxicity, which results in oxidative stress, inflammation, astrocyte activation and modifications in dopamine content and D2 receptor (D2R) density. In this study we assessed the in vitro effect of the exposure to FP and UFP on dopaminergic transmission, by evaluating [3H]-dopamine uptake and release by rat striatal isolated nerve terminals (synaptosomes), as well as modifications in the affinity and signaling of native and cloned D2Rs. FP and UFP collected from the air of Mexico City inhibited [3H]-dopamine uptake and increased depolarization-evoked [3H]-dopamine release in striatal synaptosomes. FP and UFP also enhanced D2R affinity for dopamine in membranes from either rat striatum or CHO-K1 cells transfected with the long isoform of the human D2R (hD2LR)2LR). In CHO-K1-hD2L In CHO-K1-hD2LR cells or striatal slices, FP and UFP increased the potency of dopamine or the D2R agonist quinpirole, respectively, to inhibit forskolin-induced cAMP formation. The effects were concentration-dependent, with UFP being more potent than FP. These results indicate that FP and UFP directly affect dopaminergic transmission.

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H3 receptor (hH3R445 and hH3R365) on [35S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells, respectively, with similar dissociation constants (Kd, 0.86 nM and 0.81 nM). The mRNA of the hH3R365 isoform was 40.9 ± 7.9% of the hH3R445 isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [35S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH3R445 cells ([35S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH3R365 cells; cAMP accumulation, −74.0 ± 4.9% versus −43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [3 H]-dopamine release evoked by 100 mM K+ (−18.9 ± 3.0% and −20.5 ± 3.3%, for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH3R445 cells 0.639 ± 0.049, SH-SY5Y-hH3R365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH3R445 and hH3R365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

Inhibition of RhoA-dependent pathway and contraction by endogenous hydrogen sulfide in rabbit gastric smooth muscle cells.

Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, increase cyclic nucleotide levels and inhibit muscle contraction via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase (MLCP). H2S produced as an endogenous signaling molecule synthesized mainly from l-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) regulates muscle contraction. The aim of this study was to analyze the expression of CSE and H2S function in the regulation of MLCP activity, 20-kDa regulatory light chain of myosin II (MLC20) phosphorylation, and contraction in isolated gastric smooth muscle cells. Both mRNA expression and protein expression of CSE, but not CBS, were detected in smooth muscle cells of rabbit, human, and mouse stomach. l-cysteine, an activator of CSE, and NaHS, a donor of H2S, inhibited carbachol-induced Rho kinase and PKC activity, Rho kinase-sensitive phosphorylation of MYPT1, PKC-sensitive phosphorylation of CPI-17, and MLC20 phosphorylation and sustained muscle contraction. The inhibitory effects of l-cysteine, but not NaHS, were blocked upon suppression of CSE expression by siRNA or inhibition of its activity by dl-propargylglycine (PPG) suggesting that the effect of l-cysteine is mediated via activation of CSE. Glibenclamide, an inhibitor of KATP channels, had no effect on the inhibition of contraction by H2S. Both l-cysteine and NaHS had no effect on basal cAMP and cGMP levels but augmented forskolin-induced cAMP and SNP-induced cGMP formation. We conclude that both endogenous and exogenous H2S inhibit muscle contraction, and the mechanism involves inhibition of Rho kinase and PKC activities and stimulation of MLCP activity leading to MLC20 dephosphorylation and inhibition of muscle contraction.

Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12‐receptor

The G-protein-coupled P2Y12 -receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12 -receptor antagonist. The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12 -receptors. Recombinant wild-type or mutant human P2Y12 -receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [(3) H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay. The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12 -receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A(5.43) -mutant P2Y12 -receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP. The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12 -receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys194(5.43) is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12 -receptor.

Molecular and pharmacological characterization of zebrafish ‘contractile’ and ‘inhibitory’ prostanoid receptors

Prostanoids comprising prostaglandins (PGs) and thromboxanes (TXs) have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been established. Here, we demonstrate that there exist at least five ‘contractile’ (Ca2+-mobilizing) and one ‘inhibitory’ (Gi-coupled) prostanoid receptors in zebrafish; five ‘contractile’ receptors consisting of two PGE2 receptors (EP1a and EP1b), two PGF2α receptors (FP1 and FP2), and one TXA2 receptor TP, and one ‘inhibitory’ receptor, the PGE2 receptor EP3. [3H]PGE2 specifically bound to the membranes of cells expressing zebrafish EP1a, EP1b and EP3 with a Kd of 4.8, 1.8 and 13.6nM, respectively, and [3H]PGF2α specifically bound to the membranes of cells expressing zebrafish FP1 and FP2, with a Kd of 6.5 and 1.6nM, respectively. U-46619, a stable agonist for human and mouse TP receptors, significantly increased the specific binding of [35S]GTPγS to membranes expressing the zebrafish TP receptor. Upon agonist stimulation, all six receptors showed an increase in intracellular Ca2+ levels, although the increase was very weak in EP1b, and pertussis toxin abolished only the EP3-mediated response. Zebrafish EP3 receptor also suppressed forskolin-induced cAMP formation in a pertussis toxin-sensitive manner. In association with the low structural conservation with mammalian receptors, most agonists and antagonists specific for mammalian EP1, EP3 and TP failed to work on each corresponding zebrafish receptor. This work provides further insights into the diverse prostanoid actions mediated by their receptors in zebrafish.

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N 6-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A3 receptor (hA3R) ligands with affinities in the high nanomolar range (K i values of 159 and 649 nM, respectively). These values were comparable to the observed K i value of adenosine on hA3R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A3R. In a functional assay in Chinese hamster ovary cells transfected with hA3R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A3R antagonist VUF5574. Both IPA and reference A3R agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A3R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A3R-independent mechanism, as was previously reported for other A3R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A3R.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-011-9244-9) contains supplementary material, which is available to authorized users.

Novel sterically hindered cannabinoid CB 1 receptor ligands

In the present study, 11 novel N-(3,3-diphenyl)propyl-2,2-diphenylacetamide derivatives ( 4a– d and 9a– g) and six triphenylacetamides ( 10a– c and 11a– c) were synthesized and tested as ligands of cannabinoid CB 1 and CB 2 receptors. All compounds exhibited affinity for CB 1 and CB 2 receptors. Four compounds ( 4b, 9a, 9b, and 11a) showed selectivity for CB 1 versus CB 2 receptors, although only the N-(3,3-diphenyl)propyl-2,2-diphenylacetamide ( 4b) can be considered a potent CB 1 ligand ( K i = 58 nM). It was 140-fold selective over CB 2 receptors ( K i = 7800 nM) and behaved as an inverse agonist by stimulating forskolin-induced cAMP formation in mouse N18TG2 neuroblastoma cells. This compound is the first of a novel class of tetraphenyl CB 1 ligands that, in view of its easy synthesis and high affinity for CB 1 receptors and despite its sterical hindrance, will be useful for the design of new blockers of this therapeutically exploitable receptor type.

Functional Characterization of Three G Protein-coupled Receptors for Pigment Dispersing Factors in Caenorhabditis elegans

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.

Effect of GABAA receptor activation on UT-coupled signaling pathways in rat cortical astrocytes

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.

Analysis of Promoter Regions Regulating Basal and Interleukin-4-Inducible Expression of the Human CB1 Receptor Gene in T Lymphocytes

The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) -3086 to -2490 and nts -1950 to -1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts -648 and -559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt -2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R(+)-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells.

Chemokine Fractalkine/CX3CL1 Negatively Modulates Active Glutamatergic Synapses in Rat Hippocampal Neurons

We examined the effects of the chemokine fractalkine (CX3CL1) on EPSCs evoked by electrical stimulation of Schaffer collaterals in patch-clamped CA1 pyramidal neurons from rat hippocampal slices. Acute application of CX3CL1 caused a sustained reduction of EPSC amplitude, with partial recovery after washout. CX3CL1-induced EPSC depression is postsynaptic in nature, because paired-pulse ratio was maintained, amplitude distribution of spontaneous excitatory postsynaptic currents shifted to lower values, and whole-cell current responses to AMPA were reversibly inhibited. EPSC depression by CX3CL1 is mediated by CX3CL1 receptor (CX3CR1), because CX3CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons from CX3CR1 knock-out mice. CX3CL1-induced depression of both EPSC and AMPA current was not observed in the absence of afferent fiber stimulation or AMPA receptor activation, respectively, indicating the requirement of sustained receptor activity for its development. Findings obtained from hippocampal slices, cultured hippocampal neurons, and transfected human embryonic kidney cells indicate that a Ca2+-, cAMP-, and phosphatase-dependent process is likely to modulate CX3CL1 effects because of the following: (1) CX3CL1-induced depression was antagonized by intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors, and pertussis toxin (PTX); (2) CX3CL1 inhibited forskolin-induced cAMP formation sensitive to PTX; and (3) CX3CL1 inhibited forskolin-induced Ser845 GluR1 phosphorylation, which was sensitive to PTX and dependent on Ca2+ and phosphatase activity. Together, these findings indicate that CX3CL1 negatively modulates AMPA receptor function at active glutamatergic synapses through cell-signaling pathways by influencing the balance between kinase and phosphatase activity.

In vitro and in vivo pharmacology of synthetic olivetol‐ or resorcinol‐derived cannabinoid receptor ligands

We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined. CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

Respiratory Syncytial Virus Infection Reduces β2-Adrenergic Responses in Human Airway Smooth Muscle

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract illness in infants, the effect of RSV on human airway smooth muscle (HASM) has not been studied. We hypothesized that RSV has direct effects on cAMP formation and beta(2)-adrenergic receptor (ADRB2) density and that ADRB2 haplotype influences this response. A recombinant green-fluorescent protein (rg) expressing RSV was used to determine whether RSV could infect cultured HASM. Influence of RSV infection on beta(2)-adrenergic responsiveness was determined by measuring differences in isoproterenol (ISO)-induced cyclic AMP (cAMP) formation, ADRB2 density, and G(i) expression in HASM cells challenged with RSV, with ultraviolet-inactivated RSV, and with mock infection. The rgRSV efficiently infected cultured HASM cells. ISO-induced cAMP formation was significantly reduced in cells infected with RSV, compared with mock-infected and ultraviolet-inactivated RSV, in a time- and concentration-dependent manner. Forskolin-induced cAMP formation and Gi expression were not altered in cells infected with RSV, suggesting that the influence of RSV on beta(2)-adrenergic relaxation was upstream of cAMP formation. ADRB2 density was reduced in cells infected with RSV, compared with mock infection, and the Arg16Gln27 ADRB2 haplotype was associated with decreased ISO-induced cAMP formation (P < 0.05) and with decreased ADRB2 density at baseline (P < 0.05). The implications of these results are that limitations of beta(2)-agonists in the treatment of any airway obstruction associated with RSV infection may be related to direct effects of RSV on HASM, and ADRB2 genotype may predict beta(2)-adrenergic responses.

Compared pharmacology of human histamine H3 and H4 receptors: structure–activity relationships of histamine derivatives

Various histamine derivatives were investigated at the human H3 receptor (H3R) and H4 receptor (H4R) stably expressed in human embryonic kidney (HEK)-293 cells using [125I]iodoproxyfan and [3H]histamine binding, respectively. In Tris buffer, [3H]histamine binding to membranes of HEK(hH4R) cells was monophasic (K(D) of 3.8+/-0.8 nM). In phosphate buffer, the Hill coefficient was decreased (n(H) = 0.5+/-0.1) and a large fraction of the binding was converted into a low-affinity component (K(D) = 67+/-27 nM). The inhibition of [3H]histamine binding by two agonists, a protean agonist and five antagonists/inverse agonists confirms that the potency of many H3R ligands is retained or only slightly reduced at the H4R. Histamine derivatives substituted with methyl groups in alpha, beta or N(alpha) position of the side chain retained a nanomolar potency at the H3R, but their affinity was dramatically decreased at the H4R. With relative potencies to histamine of 282 and 0.13% at the H3R and H4R, respectively, (+/-)-alpha,beta-dimethylhistamine is a potent and selective H3R agonist. Chiral alpha-branched analogues exhibited a marked stereoselectivity at the H3R and H4R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H4R (67%) was similar to that reported at H2 receptors but, owing to its high affinity at the H4R (Ki = 7.0+/-1.2 nM) and very low potency at H1- and H3-receptors, it can be considered as a potent and selective H4R agonist. On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H4R (approximately -30%) was much lower than at the H3R (approximately -80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (approximately +25%). In conclusion, although the pharmacological profiles of the human H3R and H4R overlap, the structure-activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds.

Opioid-induced regulation of gene expression in PC12 cells stably transfected with mu-opioid receptor

It has been postulated that opiates induce addictive behaviour via changes in gene expression. PC12 cells were stably transfected with the recombinant human mu-opioid receptor (MOR) to study opioid-induced gene expression. Expression was verified by binding assay, immunocytochemistry, and immunblotting experiments. Forskolin-induced cAMP formation was inhibited by [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO 1 μM), a specific MOR agonist. This effect was completely antagonized by naloxone. By using cDNA arrays, including approximately 1200 well-defined genes normally expressed in neural tissue, we monitored semi-quantitative changes in gene expression after 3 h short-term exposure to DAMGO. Incubation with DAMGO increased mRNA levels for 13 genes and down-regulated 13 other genes. Annexin V, RGS4 and CREB genes showed pronounced increase in expression after stimulation with DAMGO. Quantitative RT-PCR confirmed that DAMGO increased mRNA levels of Annexin V, an apoptosis-induced gene. We suggest that the PC12 cell transfected with the recombinant human MOR is a useful tool for identification of opioid-induced genes that may provide information on opiate effects of relevance for dependence.

L’apéline, un inhibiteur naturel de l’effet antidiurétique de la vasopressine

Apelin is a peptide that was recently isolated as the endogenous ligand for the human orphan APJ receptor, a G protein-coupled receptor which shares 31 % amino-acid sequence identity with the angiotensin type 1 receptor. Apelin naturally occurs in the brain and plasma as 13 (pE13F) and 17 amino-acid (K17F) fragments of a single pro-peptide precursor. In transfected CHO cells, K17F and pE13F bind with high affinity to the rat APJ receptor, promote receptor internalization, and inhibit forskolin-induced cAMP formation. In the same cells, pE13F activates MAP kinase and PI3 kinase pathways. Apelin and APJ receptors are both widely distributed in the brain but are particularly highly expressed in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Dual labeling studies demonstrate that within these two nuclei, apelin and its receptor are colocalized with vasopressin (AVP) in a subset of magnocellular neurons. In lactating rats, characterized by increases in both synthesis and release of AVP, central injection of apelin inhibits the phasic electrical activity of AVP neurons, reduces plasma AVP levels, and increases aqueous diuresis. Moreover, water deprivation, while increasing the activity of AVP neurons, reduces plasma apelin concentrations and induces an intra-neuronal pile up of the peptide, thereby decreasing the inhibitory effect of apelin on AVP release and preventing additional water loss at the kidney level. Taken together, these data demonstrate that apelin counteracts the effects of AVP in the maintenance of body fluid homeostasis. In addition, apelin and its receptor are present in the cardiovascular system, i.e. heart, kidney and vessels. Systemically administered apelin reduces arterial blood pressure, increases cardiac contractility and reduces cardiac loading. The development of non peptidic analogs of apelin may therefore offer new therapeutic avenues for the treatment of cardiovascular disorders.

Pharmacodynamic Characterization of ZP120 (Ac-RYYRWKKKKKKK-NH2), a Novel, Functionally Selective Nociceptin/Orphanin FQ Peptide Receptor Partial Agonist with Sodium-Potassium-Sparing Aquaretic Activity

In conscious rats, intravenous (i.v.) administration of the hexapeptide Ac-RYYRWK-NH(2), a partial agonist of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, produces a selective water diuresis without marked cardiovascular or behavioral effects. The present study examined the in vitro and in vivo pharmacodynamic profile of the novel and potentially metabolically stable NOP receptor ligand ZP120 (Ac-RYYRWKKKKKKK-NH(2)), which was created by conjugation of a structure-inducing probe (SIP) (i.e., K(6) sequence) to Ac-RYYRWK-NH(2). In cells transfected with human NOP receptors, both Ac-RYYRWK-NH(2) and ZP120 displaced [(3)H]N/OFQ (both peptides, pK(i) = 9.6), and similar to N/OFQ inhibited forskolin-induced cAMP formation (Ac-RYYRWK-NH(2), pEC(50) = 9.2; ZP120, 9.3; N/OFQ, 9.7). In the mouse vas deferens assay (MVD), Ac-RYYRWK-NH(2) and ZP120 behaved as partial agonists, inhibiting electrically induced contractions with similar pEC(50) values (9.0 and 8.6, respectively) but with submaximal efficacy compared with N/OFQ. In MVD, both peptides blocked the responses to N/OFQ, with ZP120 being approximately 50-fold more potent than Ac-RYYRWK-NH(2). In vivo, dose-response studies in rats showed that at doses (i.v. bolus or i.v. infusion) that produced a sodium-potassium-sparing aquaresis, ZP120 and Ac-RYYRWK-NH(2) elicited a mild vasodilatory response without reflex tachycardia. However, the renal responses to ZP120 were of greater magnitude and duration. Finally, each peptide blocked the bradycardia and hypotension to N/OFQ in conscious rats, but the effect of ZP120 was of much greater duration. Together, these findings demonstrate that ZP120 is a novel, functionally selective SIP-modified NOP receptor partial agonist with increased biological activity and sodium-potassium-sparing aquaretic activity, the actions of which may be useful in the management of hyponatremia/hypokalemia in water-retaining states.

Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases.

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol/L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on C(m) and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

B103 neuroblastoma cells predominantly express endothelin ET B receptor; effects of extracellular Ca 2+ influx on endothelin-1-induced mitogenesis

We sought to examine the effects of endothelin-1 on the intracellular free Ca 2+ concentration ([Ca 2+] i) and mitogenic response in the neuroblastoma cell line, B103 (B103 cells). The results obtained from an [ 125I] endothelin-1 binding assay demonstrated that B103 cells express the endothelin receptor. The B max and K d values for [ 125I]endothelin-1 binding were 70±36 fmol/mg protein and 52±13 pM, respectively. Endothelin-1 failed to stimulate cAMP formation, but it did inhibit forskolin-induced cAMP formation. Endothelin-1 also stimulated the accumulation of [ 3H]inositol phosphates. These results indicate that the endothelin receptor in B103 cells couples with G i and G q but not with G s. Monitoring of [Ca 2+] i showed that endothelin-1 evoked a transient increase in [Ca 2+] i; this remained even in the absence of extracellular Ca 2+. However, no sustained, endothelin-1-induced increase in [Ca 2+] i due to extracellular Ca 2+ influx was detected. The endothelin B receptor-selective antagonist, 2,6-Dimethylpiperidinecarbonyl-γ-Methyl-Leu- N in-[Methoxycarbonyl]- d-Trp- d-Nle (BQ 788), abolished the endothelin-1-induced increase in [Ca 2+] i, while the endothelin ET A receptor-selective antagonist, cyclo- d-Asp-Pro- d-Val-Leu- d-Trp (BQ 123), failed to inhibit it. These results indicate that B103 cells express endothelin ET B receptor or an endothelin ET B-like receptor predominantly and have no Ca 2+ channels activated by endothelin-1. Endothelin-1 activated mitogen-activated protein kinase in B103 cells. However, based on the data for 3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl tetrazolium bromide, [ 3H]thymidine incorporation, and apoptosis screening assays, endothelin-1 induces neither mitogenesis nor apoptosis. These results suggest that endothelin-1 has no role in the mitogenic response in B103 cells, and this is consistent with the notion that an endothelin-1-induced sustained increase in [Ca 2+] i plays a role in endothelin-1-induced cell proliferation.

Evidence for a G Protein‐Coupled γ‐Hydroxybutyric Acid Receptor

gamma-Hydroxybutyric acid (GHB) is a naturally occurring metabolite of GABA that has been postulated to exert ubiquitous neuropharmacological effects through GABA(B) receptor (GABA(B)R)-mediated mechanisms. The alternative hypothesis that GHB acts via a GHB-specific, G protein-coupled presynaptic receptor that is different from the GABA(B)R was tested. The effect of GHB on regional and subcellular brain adenylyl cyclase in adult and developing rats was determined and compared with that of the GABA(B)R agonist (-)-baclofen. Also, using guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding and low-K:(m) GTPase activity as markers the effects of GHB and (-)-baclofen on G protein activity in the brain were determined. Neither GHB nor baclofen had an effect on basal cyclic AMP (cAMP) levels. GHB significantly decreased forskolin-stimulated cAMP levels by 40-50% in cortex and hippocampus but not thalamus or cerebellum, whereas (-)-baclofen had an effect throughout the brain. The effect of GHB on adenylyl cyclase was observed in presynaptic and not postsynaptic subcellular tissue preparations, but the effect of baclofen was observed in both subcellular preparations. The GHB-induced alteration in forskolin-induced cAMP formation was blocked by a specific GHB antagonist but not a specific GABA(B)R antagonist. The (-)-baclofen-induced alteration in forskolin-induced cAMP formation was blocked by a specific GABA(B)R antagonist but not a specific GHB antagonist. The negative coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a developmental time point that is concordant with the developmental appearance of [(3)H]GHB binding in cerebral cortex, but the effects of (-)-baclofen were present by postnatal day 14. GHB and baclofen both stimulated [(35)S]GTPgammaS binding and low-K:(m) GTPase activity by 40-50%. The GHB-induced effect was blocked by GHB antagonists but not by GABA(B)R antagonists and was seen only in cortex and hippocampus. The (-)-baclofen-induced effect was blocked by GABA(B)R antagonists but not by GHB antagonists and was observed throughout the brain. These data support the hypothesis that GHB induces a G protein-mediated decrease in adenylyl cyclase via a GHB-specific G protein-coupled presynaptic receptor that is different from the GABA(B)R.