Kurt A. Krobert

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.

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.

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.

PDE3, but not PDE4, reduces β1‐ and β2‐adrenoceptor‐mediated inotropic and lusitropic effects in failing ventricle from metoprolol‐treated patients

PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3‐selective inhibitor cilostamide (0.3–1 μM) or PDE4 inhibitor rolipram (1–10 μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium.

Effects of treatment with a 5-HT4 receptor antagonist in heart failure

Positive inotropic responses (PIR) to 5‐hydroxytryptamine (5‐HT) are induced in the left ventricle (LV) in rats with congestive heart failure (CHF); this is associated with upregulation of the Gs‐coupled 5‐HT4 receptor. We investigated whether chronic 5‐HT4 receptor blockade improved cardiac function in CHF rats.

NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors

Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M2 muscarinic acetylcholine receptor (M2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly Giβγ-mediated, adenosine-induced activation of Gi protein–regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K+ current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca2+ concentrations in human embryonic kidney 293 (HEK-293) cells expressing M1, M2, or M3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M2 and M3 mAChR crystal structures confirmed this interpretation.

Diastolic dysfunction in alveolar hypoxia: a role for interleukin-18-mediated increase in protein phosphatase 2A

AIMS Chronic obstructive pulmonary disease with alveolar hypoxia is associated with diastolic dysfunction in the right and left ventricle (LV). LV diastolic dysfunction is not caused by increased afterload, and we recently showed that reduced phosphorylation of phospholamban at serine (Ser) 16 may explain the reduced relaxation of the myocardium. Here, we study the mechanisms leading to the hypoxia-induced reduction in phosphorylation of phospholamban at Ser16. METHODS AND RESULTS In C57Bl/6j mice exposed to 10% oxygen, signalling molecules were measured in cardiac tissue, sarcoplasmic reticulum (SR)-enriched membrane preparations, and serum. Cardiomyocytes isolated from neonatal mice were exposed to interleukin (IL)-18 for 24 h. The beta-adrenergic pathway in the myocardium was not altered by alveolar hypoxia, as assessed by measurements of beta-adrenergic receptor levels, adenylyl cyclase activity, and subunits of cyclic AMP-dependent protein kinase. However, alveolar hypoxia led to a significantly higher amount (124%) and activity (234%) of protein phosphatase (PP) 2A in SR-enriched membrane preparations from LV compared with control. Serum levels of an array of cytokines were assayed, and a pronounced increase in IL-18 was observed. In isolated cardiomyocytes, treatment with IL-18 increased the amount and activity of PP2A, and reduced phosphorylation of phospholamban at Ser16 to 54% of control. CONCLUSION Our results indicate that the diastolic dysfunction observed in alveolar hypoxia might be caused by increased circulating IL-18, thereby inducing an increase in PP2A and a reduction in phosphorylation of phospholamban at Ser16.

Unaltered Agonist Potency upon Inducible 5-HT 7(a) but not 5-HT 4(b) Receptor Expression Indicates Agonist-Independent Association of 5-HT 7(a) Receptor and G s

We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (approximately 400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-Gs stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.

Hormonal regulation of beta(2)-adrenergic receptor level in prostate cancer

Androgen deprivation is the only effective systemic therapy available for patients with prostatic carcinoma, but is associated with a gradual transition to a hormone‐refractory prostate cancer (HRCAP) in which ligand‐independent activation of the androgen receptor has been implicated. The β2‐adrenergic receptor (β2‐AR) is a well‐known activator of the androgen receptor.

Activation of Adenylyl Cyclase by Endogenous Gs-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT7 Receptor Expression

Human 5-hydroxytryptamine7 (5-HT7) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with Gi/o and Gq/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT7 receptor expression was sufficient to limit signaling of endogenously expressed Gs-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT4(b) and 5-HT7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous Gs-coupled β-adrenergic (βAR) and prostanoid EP (EPR) receptors. βAR- and EPR-stimulated AC activity was attenuated by 5-HT7 receptor expression in both membrane preparations and intact HEK293 cells. βAR- and EPR-stimulated AC activity was unaffected by expression of the Gs-coupled 5-HT4 receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) βAR- and EPR-stimulated AC activity was not restored to control values when Gαs was overexpressed; and 2) β1AR and β2AR activation of Gαs was unaffected by the expression of 5-HT7 receptors. In addition, overexpression of AC isoforms was unable to rescue βAR- and EPR-stimulated AC activity. Therefore, 5-HT7 receptors probably limit access and/or impede activation of AC by βAR and EP receptors. Although the 5-HT7 receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.