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Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.

ABSTRACT: We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

SUBMITTER: Post SR

PROVIDER: S-EPMC1136121 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Project description:AimsCardiomyocyte β2-adrenergic receptor (β2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors' subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), β2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon β2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown.Methods and resultsRat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), β2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local β2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the β2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell β2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve β2AR-mediated signal compartmentation or reduce cAMP diffusion.ConclusionAlthough changes in T-tubule structure and β2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of β2AR-mediated cAMP is temporarily reversed. Complete disorganization of β2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.

Project description:BackgroundIn Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling.ResultsIn contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2'H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1- cells showed relatively thin stalks, aca2- showed delayed secondary sorogen formation and aca3- formed less aggregation centers. The aca1-aca2- and aca1-aca3- mutants combined individual defects, while aca2-aca3- and aca1-aca3-aca2- additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2-aca3- and aca1-aca3-aca2- by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis aca-/ACG cells produced a pulse of cAMP upon stimulation with 2'H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone.ConclusionsThe three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1-aca3-aca2- mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour.

Project description:The ?1-adrenergic signaling system plays an important role in the functioning of cardiac cells. Experimental data shows that the activation of this system produces inotropy, lusitropy, and chronotropy in the heart, such as increased magnitude and relaxation rates of [Ca(2+)]i transients and contraction force, and increased heart rhythm. However, excessive stimulation of ?1-adrenergic receptors leads to heart dysfunction and heart failure. In this paper, a comprehensive, experimentally based mathematical model of the ?1-adrenergic signaling system for mouse ventricular myocytes is developed, which includes major subcellular functional compartments (caveolae, extracaveolae, and cytosol). The model describes biochemical reactions that occur during stimulation of ?1-adrenoceptors, changes in ionic currents, and modifications of Ca(2+) handling system. Simulations describe the dynamics of major signaling molecules, such as cyclic AMP and protein kinase A, in different subcellular compartments; the effects of inhibition of phosphodiesterases on cAMP production; kinetics and magnitudes of phosphorylation of ion channels, transporters, and Ca(2+) handling proteins; modifications of action potential shape and duration; magnitudes and relaxation rates of [Ca(2+)]i transients; changes in intracellular and transmembrane Ca(2+) fluxes; and [Na(+)]i fluxes and dynamics. The model elucidates complex interactions of ionic currents upon activation of ?1-adrenoceptors at different stimulation frequencies, which ultimately lead to a relatively modest increase in action potential duration and significant increase in [Ca(2+)]i transients. In particular, the model includes two subpopulations of the L-type Ca(2+) channels, in caveolae and extracaveolae compartments, and their effects on the action potential and [Ca(2+)]i transients are investigated. The presented model can be used by researchers for the interpretation of experimental data and for the developments of mathematical models for other species or for pathological conditions.

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Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.

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