Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Background and purpose Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. Experimental approach We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. Key results PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Conclusions and implications Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents

The inotropic effect of the active metabolite of levosimendan, OR-1896, is mediated through inhibition of PDE3 in rat ventricular myocardium

Aims We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca2+ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors. Methods Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors. Results OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response. Conclusion The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896

Identification of essential residues for binding and activation in the human 5-HT7(a) receptor by molecular modeling and site-directed mutagenesis

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders.We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use

Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use

Hypothermia elongates the contraction-relaxation cycle in explanted human failing heart decreasing the time for ventricular filling during diastole

Targeted temperature management is part of the standardized treatment for patients in cardiac arrest. Hypothermia decreases cerebral oxygen consumption and induces bradycardia; thus, increasing the heart rate may be considered to maintain cardiac output. We hypothesized that increasing heart rate during hypothermia would impair diastolic function. Human left ventricular trabeculae obtained from explanted hearts of patients with terminal heart failure were stimulated at 0.5 Hz, and contraction-relaxation cycles were recorded. Maximal developed force (Fmax), maximal rate of development of force [(dF/dt)max], time to peak force (TPF), time to 80% relaxation (TR80), and relaxation time (RT = TR80 − TPF) were measured at 37, 33, 31, and 29°C. At these temperatures, stimulation frequency was increased from 0.5 to 1.0 and to 1.5 Hz. At 1.5 Hz, concentration-response curves for the β-adrenergic receptor (β-AR) agonist isoproterenol were performed. Fmax, TPF, and RT increased when temperature was lowered, whereas (dF/dt)max decreased. At all temperatures, increasing stimulation frequency increased Fmax and (dF/dt)max, whereas TPF and RT decreased. At 31 and 29°C, resting tension increased at 1.5 Hz, which was ameliorated by β-AR stimulation. At all temperatures, maximal β-AR stimulation increased Fmax, (dF/dt)max, and maximal systolic force, whereas resting tension decreased progressively with lowering temperature. β-AR stimulation reduced TPF and RT to the same extent at all temperatures, despite the more elongated contraction-relaxation cycle at lower temperatures. Diastolic dysfunction during hypothermia results from an elongation of the contraction-relaxation cycle, which decreases the time for ventricular filling. Hypothermic bradycardia protects the heart from diastolic dysfunction and increasing the heart rate during hypothermia should be avoided

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

BACKGROUND. 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 beta(2)-adrenergic receptor (beta(2)-AR) is a well-known activator of the androgen receptor. METHODS. Prostatic cell lines were analyzed using cDNA micro-array, real time RT-PCR, radioligand binding assay, cAMP measurements, transfection and thymidine incorporation assay. Clinical specimens were studied by immunohistochemistry and Affymetrix microarrays. RESULTS. Here, we show that beta(2)-AR was transiently down-regulated both at mRNA- and protein levels when hormone-sensitive prostate cancer cells, LNCaP, were cultured in steroid stripped medium (charcoal-stripped fetal calf serum) or when the cells were treated with the anti-androgen, bicalutamide (Casodex). The number of beta-adrenergic receptors was modestly up-regulated in androgen independent cell lines (LNCaP-C4, LNCaP-C4-2 and DU145) compared to LNCaP. Triiodothyronine (T3) increased the level of beta(2)-AR and the effect of T3 was inhibited by bicalutamide. Immunohistochemical staining of human prostate specimens showed high expression of beta(2)-AR in glandular, epithelial cells and increased expression in malignant cells compared to benign hyperplasia and normal tissue. Interestingly, beta(2)-AR mRNA was strongly down-regulated by androgen ablation therapy of prostate cancer patients. CONCLUSION. The level of beta(2)-AR was increased by T3 in prostatic adenocarcinoma cells and reduced in prostate cancer patients who had received androgen ablation therapy for 3 months

Effect of PTX upon (R,R)- and (R,S)-fenoterol-induced cAMP accumulation.

<p>(A) Representative autoradiogram showing ADP-ribosylated Gα_i protein levels in rat ventricle pre-treated with saline (control) or PTX. (B) Representative traces showing inotropic responses (mN) evoked by forskolin (FSK) and the subsequent effect of carbachol (Cch) and reversal of carbachol effects by atropine in left ventricular strips from rats pre-treated with saline (Control; top) or with PTX (bottom). Drug concentrations are given in -Log(M). (C) Concentration-response curves to (R,R)- and (R,S)-fenoterol-mediated cAMP accumulation in isolated ventricular cardiomyocytes in the presence of IBMX and the β₁AR antagonist CGP20712 (300 nM) in control or after PTX pre-treatment. Data are mean ± SEM. Basal cAMP accumulation was (in pmol cAMP/mg protein): (R,R-series) control: 18.5±2.8; PTX: 19.7±3.2; (R,S-series) control 11.9±2.5; PTX 16.7±2.0. RR: (R,R)-fenoterol; RS: (R,S)-fenoterol; *P<0.05, paired t-test.</p

Effect of PTX upon PDE3 and PDE4 inhibitor-induced cAMP accumulation and inotropic response.

<p>(A) Effect of simultaneous inhibition of PDE3 (cilostamide, 1 µM) and PDE4 (rolipram, 10 µM) to evoke an inotropic response in left ventricular strips in control (open bars) and after PTX pre-treatment (solid bars) in the presence of timolol (1 µM). Basal force was (in mN/mm²): control: 3.3±0.4; PTX: 3.1±0.3. (B) Effect of PTX, βAR inverse agonist timolol (1 µM), non-selective muscarinic inverse agonist atropine (1 µM) or non-selective adenosine receptor inverse agonist CGS 15943 (1 µM) upon PDE3 (cilostamide, 1 µM) and PDE4 (rolipram, 10 µM)-evoked cAMP accumulation. Basal cAMP accumulation (in pmol/mg protein): timolol control: 4.8±0.6 (n = 7). (C) Representative traces showing inotropic responses (mN) evoked by inactivation of PDE3 and 4 simultaneously (1 µM cilostamide and 10 µM rolipram) in left ventricular strips of saline-treated control (left) and after PTX pre-treatment (right) in the presence of 1 µM timolol and 0.1 µM prazosin. Isoproterenol (100 µM displacing timolol) was given to demonstrate that an inotropic effect could be elicited through βARs. Data are mean ± SEM. * P<0.05 PTX vs. control, unpaired t-test; ** P<0.05 vs. timolol, One-way ANOVA with Bonferroni adjustment for multiple comparisons.</p

Effect of PTX upon the forskolin-evoked cAMP accumulation and inotropic response.

<p>Concentration-response curves of forskolin-evoked cAMP accumulation in ventricular cardiomyocytes (A) and the inotropic response in ventricular strips (B) in the presence of the βAR blocker timolol (1 µM) in control or after PTX pre-treatment. Accumulation of cAMP was measured in the presence of IBMX, with basal cAMP accumulation (in pmol cAMP/mg protein): control: 38.8±5.3; PTX: 30.4±4.0. Basal force was (in mN/mm²): control: 4.05±0.56; PTX: 3.91±0.66. Data are mean ± SEM. *P<0.05, paired t-test (cAMP accumulation) or unpaired t-test (inotropic response).</p

Effect of PTX and PDE3 and 4 inhibition upon the inotropic response to (R,R)- and (R,S)-fenoterol stimulation.

<p>Maximal inotropic response (A) and concentration-response curves (B) to (R,R)- or (R,S)-fenoterol in left ventricular strips from saline-treated rats (control) or PTX-treated rats or strips from saline-treated rats given PDE3 (cilostamide, Cil, 1 µM) and PDE4 (rolipram, Rol, 10 µM) inhibitors. All experiments were conducted in the presence of the β₁AR blocker CGP20712 (300 nM). Inotropic responses are expressed as (dF/dt)_max as percent above basal. Basal force was (in mN/mm²) (R,R) control: 4.3±0.4; (R,S) control: 4.2±0.6; (R,R) PTX: 3.5±0.5; (R,S) PTX: 3.5±0.4; (R,R) with Cil/Rol: 3.6±0.3; (R,S) with Cil/Rol: 3.9±0.4. Data are mean ± SEM. RR: (R,R)-fenoterol; RS: (R,S)-fenoterol; *P<0.05 vs. control, One-way ANOVA with Bonferroni adjustment for multiple comparisons. **P<0.05 vs. (R,S) with Cil/Rol, One-way ANOVA with Bonferroni adjustment for multiple comparisons.</p