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

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 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

Possible mechanisms mediating the receptor-independent role of G_i in regulating basal AC activity.

<p>(A) βγ-sink hypothesis: In the absence of PTX (top panel), spontaneously active G_i dominates, inhibiting basal AC activity and maintaining low cAMP levels which are incapable of eliciting an inotropic response even after inhibition of PDE3 and 4 (inhibition marked by X). After PTX treatment (bottom panel), G_i is not only inactivated (through ADP-ribosylation, marked by X) removing its spontaneous intrinsic inhibition upon AC, but also sequesters a large proportion of the shared Gβγ pool. This indirectly increases the proportion of receptor-independent spontaneously active G_s, leading to increased basal AC activity and cAMP that is normally readily degraded by PDE3 and 4. However, inhibition of PDE3 and 4 (marked by X) allows for translation of this cAMP increase into an inotropic response (see Fig. 4A). The net result is a shift from predominantly spontaneous G_i activity towards G_s activity, increasing basal AC activity and promoting activation of AC and positive inotropic effects of all inotropic agents working through increased cAMP signalling. (B) NDPK-hypothesis: In the absence of PTX (top panel), NDPK-activation of G_i dominates due to excess of G_i protein levels over G_s, resulting in low cAMP production readily degraded by PDE3 and 4. In the presence of PTX (bottom panel), G_i is inactivated by permanent ADP-ribosylation (marked by X). Thus, NDPK B could predominantly activate G_s, leading to both increased basal contractile force and cAMP accumulation that becomes revealed after PDE3 and 4 inhibition (marked by X).</p