Finn Olav Levy

The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects

Using membranes from stably or transiently transfected HEK293 cells cultured in 5‐HT‐free medium and expressing the recombinant human 5‐HT7 receptor splice variants (h5‐HT7(a), h5‐HT7(b) and h5‐HT7(d)), we compared their abilities to constitutively activate adenylyl cyclase (AC). All h5‐HT7 splice variants elevated basal and forskolin‐stimulated AC. The basal AC activity was reduced by the 5‐HT7 antagonist methiothepin and this effect was blocked by mesulergine (neutral 5‐HT7 antagonist) indicating that the inhibitory effect of methiothepin is inverse agonism at the 5‐HT7 receptor. Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin‐stimulated AC was significantly higher for the h5‐HT7(b) splice variant compared to the h5‐HT7(a) and h5‐HT7(d) splice variants but only in stable cell lines. All eight 5‐HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration‐dependent manner. No differences in inverse agonist potencies (pIC50) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pKb) determined by antagonism of 5‐HT‐stimulated AC activity (methiothepin>metergoline>mesulergineclozapinespiperoneritanserin>methysergide>ketanserin). The efficacy of inverse agonism was not receptor level dependent and varied for several 5‐HT antagonists between membrane preparations of transiently and stably transfected cells. It is concluded that the h5‐HT7 splice variants display similar constitutive activity and inverse agonist properties.

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity.

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA α-isoforms RIα, RIIα, and Cα and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3′,5′-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3′,5′-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.

Activation of the CAMP signaling pathway increases apoptosis in human B‐precursor cells and is associated with downregulation of Mcl‐1 expression

During B‐ and T‐cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B‐precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10+ cells in a dose‐dependent manner (19%–94% with 0–1,000 μM forskolin after 48 hours incubation, IC50 = 150 μM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8‐chlorophenylthio‐cAMP (8‐CPT‐cAMP). Specific involvement of cAMP‐dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp‐isomer of 8‐bromo‐adenosine‐ 3′, 5′‐ monophosphorothioate (Rp‐8‐Br‐cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp‐8‐Br‐cAMPS. Furthermore, we investigated the expression of Bcl‐2 family proteins. We found that treatment of the cells with forskolin or 8‐CPT‐cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl‐1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl‐2, Bcl‐xl, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8‐CPT‐cAMP (1.3‐fold, n = 6, P = 0.045) compared to B‐precursor cells, and a smaller decrease in Mcl‐1 levels (1.5‐fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B‐progenitor and mature B cells and suggest that cAMP‐increased apoptosis could be mediated, at least in part, by a decrease in Mcl‐1 levels. J. Cell. Physiol. 180:71–80, 1999.

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

BackgroundQuantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.ResultsThe abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.ConclusionsThe most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

Dual Serotonergic Regulation of Ventricular Contractile Force Through 5-HT2A and 5-HT4 Receptors Induced in the Acute Failing Heart

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of Gs-coupled 5-HT4 receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca2+ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT2A mRNA levels. Functionally, this receptor dominated over 5-HT4 receptors that were also induced. The 5-HT2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca2+-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca2+ transients were slightly decreased by 5-HT2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT4 receptors.

The serotonin 5-HT7 receptors: two decades of research

Abstract Like most neurotransmitters, serotonin possesses a simple structure. However, the pharmacological consequences are more complex and diverse. Serotonin is involved in numerous functions in the human body including the control of appetite, sleep, memory and learning, temperature regulation, mood, behavior, cardiovascular function, muscle contraction, endocrine regulation, and depression. Low levels of serotonin may be associated with several disorders, namely increase in aggressive and angry behaviors, clinical depression, Parkinson’s disease, obsessive–compulsive disorder, eating disorders, migraine, irritable bowel syndrome, tinnitus, and bipolar disease. These effects are mediated via different serotonin (5-HT) receptors. In this review, we will focus on the last discovered member of this serotonin receptor family, the 5-HT7 receptor. This receptor belongs to the G protein-coupled receptor superfamily and was cloned two decades ago. Later, different splice variants were described but no major functional differences have been described so far. All 5-HT7 receptor variants are coupled to Gαs proteins and stimulate cAMP formation. Recently, several interacting proteins have been reported, which can influence receptor signaling and trafficking.

Novel Benzo[b]thiophene Derivatives as New Potential Antidepressants with Rapid Onset of Action

We report benzo[b]thiophene derivatives synthesized according to a dual strategy. 8j, 9c, and 9e with affinity values toward 5-HT(7)R and 5-HTT were selected to probe their antidepressant activity in vivo using the forced swimming text (FST). The results showed significant antidepressant activity after chronic treatment. 9c was effective in reducing the immobility time in FST even after acute treatment. These findings identify these compounds as a new class of antidepressants with a rapid onset of action.

Natriuretic peptides increase β1-adrenoceptor signalling in failing hearts through phosphodiesterase 3 inhibition

AIMS Whereas natriuretic peptides increase cGMP levels with beneficial cardiovascular effects through protein kinase G, we found an unexpected cardio-excitatory effect of C-type natriuretic peptide (CNP) through natriuretic peptide receptor B (NPR-B) stimulation in failing cardiac muscle and explored the mechanism. METHODS AND RESULTS Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban (PLB) and troponin I was measured by western blotting. Stimulation of NPR-B enhanced beta1-adrenoceptor (beta1-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced beta1-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased beta1-AR-stimulated protein kinase A activity, as demonstrated by increased PLB and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts. CONCLUSION An NPR-B-cGMP-PDE3 inhibitory pathway enhances beta(1)-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.

Expression of mRNA encoding G protein-coupled receptors involved in congestive heart failure--a quantitative RT-PCR study and the question of normalisation.

AbstractCongestive heart failure (CHF) induces changes in the neurohumoral system and gene expression in viable myocardium. Several of these genes encode G protein-coupled receptors (GPCRs) involved in mechanisms which compensate for impaired myocardial function. We used real-time quantitative RT-PCR (Q-RT-PCR) to investigate the expression of mRNA encoding 15 different GPCRs possibly involved in CHF, and the effect of normalisation to GAPDH mRNA (GAPDH) or 18S rRNA (18S). CHF was induced in rats by coronary artery ligation, with sham-operated controls (Sham). After 6 weeks, mRNA expression in viable left ventricular myocardium was determined using both 18S and GAPDH as the normalisation standard. An apparent 30% reduction in GAPDH mRNA levels vs. 18S in CHF compared to Sham, although not significant in itself, influenced the interpretation of regulation of other genes.Thus, levels of mRNA encoding receptors for angiotensin II (AT1), endothelin (ETA, ETB) and the muscarinic acetylcholine (mACh) receptor M1 increased significantly in CHF only when normalised to GAPDH. Levels of mRNA encoding the mACh receptors M3 and M4 and the serotonin receptors 5-HT2A and 5-HT4 increased, whereas α1D-adrenoceptor mRNA decreased in CHF irrespective of the normalisation standard. No significant change was detected for M2 and M5 mACh receptors or α1A-, α1B-, β1- or β2-adrenoceptors. Q-RT-PCR is a sensitive and powerful method to monitor changes in GPCR mRNA expression in CHF. However, the normalisation standard used is important for the interpretation of mRNA regulation.

Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6

No ligand has hitherto been designated for the Eph receptor tyrosine kinase family member, EphB6. Here, expression of an EphB6 ligand in the pro‐B leukemic cell line, Reh, is demonstrated by binding of soluble EphB6‐Fc fusion protein to the Reh cells. The ligand belongs to the subgroup of membrane spanning ligands, as suggested by the fact that phosphatidylinositol‐specific phospholipase C treatment did not abrogate binding of EphB6‐Fc. Two transmembrane Eph receptor ligands, ephrin‐B1 and ephrin‐B2, were identified in Reh cells. Analysis of EphB6‐Fc fusion protein binding to ephrin‐B1 or ephrin‐B2 transfected COS cells revealed a high‐affinity saturable binding between EphB6‐Fc and ephrin‐B2, but not with ephrin‐B1. In mice, EphB6 has previously been shown to be expressed in thymus. Here, we show expression of EphB6 in human thymus, as well as the expression of ephrin‐B2 in both human and mouse thymus. We conclude that ephrin‐B2 may be a physiological ligand for the EphB6 receptor.