scholarly journals Quantification of signalling components and amplification in the β-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes

1995 ◽  
Vol 311 (1) ◽  
pp. 75-80 ◽  
Author(s):  
S R Post ◽  
R Hilal-Dandan ◽  
K Urasawa ◽  
L L Brunton ◽  
P A Insel
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Cardiac Myocytes ◽  
Signal Amplification ◽  
Adrenergic Receptor ◽  
Ventricular Myocytes ◽  
Binding Assays ◽  
Beta Adrenergic ◽  
Adult Rat ◽  
Gs Alpha

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.

scholarly journals Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein α-subunits

1995 ◽  
Vol 307 (3) ◽  
pp. 831-841 ◽  
Author(s):  
S W Bahouth
Keyword(s):  
Thyroid Hormone ◽  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Half Life ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Rat Ventricular Myocytes ◽  
Gs Alpha ◽  
Neonatal Rat Ventricular Myocytes

Thyroid hormone exerts profound effects on the activity of the hormone-sensitive adenylate cyclase system in the heart. Distinct guanine nucleotide-binding regulatory proteins (G-proteins) mediate stimulatory and inhibitory influences on adenylate cyclase activity. To examine whether the effects of thyroid hormone on adenylate cyclase involve specific changes in G-protein subunit expression, the influence of tri-iodothyronine (T3) on the biosynthesis and activity of G-proteins in neonatal rat ventricular myocytes was determined. In myocytes challenged with T3 for 5 days, Gs alpha levels increased by 4 +/- 0.5-fold, whereas Gi2 alpha levels declined by more than 80%. T3 down-regulated Gi2 alpha mRNA by 60% within 3 days, but had no effect on Gs alpha mRNA. The basis for the decline in Gi2 alpha mRNA was the T3-mediated suppression of Gi2 alpha gene transcription by 80 +/- 9% within 4 h. The decline in Gi2 alpha mRNA in response to T3 produced a 2-fold decrease in relative rate of synthesis of Gi2 alpha but not in its half-life (46 +/- 7 h). Gs alpha synthesis was not altered by T3, but the half-life of Gs alpha increased from 50 +/- 6 h in control cells to 72 +/- 8 h in T3-treated cells. In addition, T3 provoked the translocation of Gs alpha from the cytoplasmic to the membranous compartment. Membranous Gs alpha increased from 30 +/- 6% to 61 +/- 7% of total cellular Gs alpha, whereas cytoplasmic Gs alpha declined from 68 +/- 6% to 33 +/- 8% within 1 day of exposure to T3. T3-mediated up-regulation of Gs alpha enhanced the activation of myocardial adenylate cyclase by the stimulatory pathway whereas the down-regulation of Gi2 alpha attenuated the deactivation of myocardial adenylate cyclase by the inhibitory pathway.

Expression of G proteins in rat cardiac myocytes: effect of KCl depolarization

1990 ◽  
Vol 259 (2) ◽  
pp. H432-H441 ◽  
Author(s):  
K. A. Foster ◽  
P. J. McDermott ◽  
J. D. Robishaw
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cardiac Myocytes ◽  
Protein Composition ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Adult Rat ◽  
Rat Hearts ◽  
Gs Alpha ◽  
Rat Cardiac Myocytes

As a first step in understanding the functioning of hormonal signaling pathways in the heart, the G protein composition of neonatal and adult rat hearts was determined by immunoblotting analysis. Neonatal rat cardiac myocytes and nonmuscle heart cells contained Gs alpha 52, Gi alpha 1,3, and Gi alpha 2 with little or no Gs alpha 45 or Go alpha 39. Interestingly, Go alpha 39 accumulated fourfold in cardiac myocytes between days 1 and 6 of culture. Atrial membranes from adult rat hearts had approximately equal amounts of the 45- and 52-kDa forms of Gs alpha, whereas adult ventricles had predominantly Gs alpha 45. In addition, adult atria contained relatively more Gi alpha 1,3, Gi alpha 2, and Go alpha 39 than adult ventricle. Moreover, the increase in Go alpha 39 observed in cardiac myocytes cultured for 6 days could be prevented by culturing cells in medium containing 50 mM KCl. The observed differences in G protein expression between cell types and between contracting and KCl-depolarized cardiac myocytes may provide a system to investigate the function of various G proteins and to study regulation of their expression.

scholarly journals G-protein-mediated activation of turkey erythrocyte phospholipase C by β-adrenergic and P2y-purinergic receptors

1992 ◽  
Vol 284 (3) ◽  
pp. 917-922 ◽  
Author(s):  
C Vaziri ◽  
C P Downes
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
Adrenergic Receptor ◽  
Purinergic Receptor ◽  
Single Species ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic

Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5′-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5′-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.

scholarly journals The turkey erythrocyte β-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein α subunits

1994 ◽  
Vol 304 (2) ◽  
pp. 359-364 ◽  
Author(s):  
S R James ◽  
C Vaziri ◽  
T R Walker ◽  
G Milligan ◽  
C P Downes
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Adenosine Monophosphate ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic

By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response.

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