scholarly journals Vasoconstrictor agonists activate G-protein-dependent receptor-operated calcium channels in pig aortic microsomes

1992 ◽  
Vol 282 (1) ◽  
pp. 81-84 ◽  
Author(s):  
L M Blayney ◽  
P W Gapper ◽  
A C Newby
Keyword(s):  
Steady State ◽  
Angiotensin Ii ◽  
Calcium Channels ◽  
G Protein ◽  
G Proteins ◽  
Intact Cells ◽  
Vasoconstrictor Agents ◽  
Receptor Sites ◽  
Alpha Beta ◽  
Ca2 Channels

Receptor-operated Ca2+ channels were characterized by their ability to decrease steady-state ATP-dependent Ca2+ accumulation into pig aortic microsomes. The vasoconstrictor agents noradrenaline, angiotensin II and adenosine 5′-[alpha beta-methylene]triphosphate (pp[CH2]pA) all decreased Ca2+ accumulation only when sonicated into vesicles (to allow access to receptor sites) and in the presence of guanosine 5′-[beta gamma-imido]triphosphate to activate transducing G-proteins. The effect of noradrenaline was inhibited by the alpha 2 antagonist yohimbine, but not by the alpha 1 antagonist prazosin. The effect of none of the agonists was reversed by diltiazem. SK&F 96365 (an inhibitor of receptor-mediated Ca2+ influx into intact cells) reversed the effect of noradrenaline, but not that of pp[CH2]pA, which suggests that at least two receptor-operated channels may be present in this preparation.

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

scholarly journals G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

1998 ◽  
Vol 332 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Hisashi KAI ◽  
R. Wayne ALEXANDER ◽  
Masuko USHIO-FUKAI ◽  
P. Reid LYONS ◽  
Marjorie AKERS ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Binding ◽  
G Protein ◽  
G Proteins ◽  
Synthetic Peptides ◽  
Terminal Region ◽  
Functional Coupling ◽  
Intracellular Loop ◽  
Binding Domains ◽  
Gtpase Activation

The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR–G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125–137), the N-terminal region of the third intracellular loop (217–227) and the juxtamembranous region of the C-terminal tail (304–316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217–227 and 304–316) are predicted to form amphiphilic α-helices. Only the peptide representing residues 217–227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

scholarly journals Histamine and a guanine nucleotide increase calcium permeability in pig aortic microsomal fractions

1990 ◽  
Vol 267 (1) ◽  
pp. 105-109 ◽  
Author(s):  
L M Blayney ◽  
A C Newby
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Receptor Sites ◽  
H2 Antagonist ◽  
Dependent Inhibition ◽  
Calcium Permeability ◽  
H1 Antagonist ◽  
Stimulation Of

ATP-dependent Ca2+ accumulation was measured in pig aortic microsomal fractions containing plasmalemma and endoplasmic reticulum. In vesicles sonicated with histamine, to allow access to internally located receptor sites, guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG), added to activate externally located guanine-nucleotide-transducing proteins, caused a concentration-dependent decrease in steady-state Ca2+ accumulation that was reversed by guanosine 5′-[beta-thio]diphosphate. In the presence of p[NH]ppG, sonication with histamine produced a concentration-dependent inhibition of Ca2+ accumulation that could be antagonized by the H1 antagonist mepyramine, but not by the H2 antagonist cimetidine. The inhibition of steady-state Ca2+ accumulation could have resulted from an inhibition of ATP-dependent Ca2+ uptake or a stimulation of Ca2+ release. We observed, however, that p[NH]ppG plus histamine stimulated, rather than inhibited, Ca2(+)-ATPase activity. We concluded that p[NH]ppG and histamine acted together to increase Ca2+ permeability. In support of this, p[NH]ppG accelerated efflux of Ca2+ from passively loaded vesicles sonicated with, but not without, histamine. The effect of p[NH]ppG was unlikely to be due to Ins(1,4,5)P3 (and hence release from endoplasmic-reticulum vesicles), since addition of Ins(1,4,5)P3 to vesicles sonicated with histamine did not alter steady-state Ca2+ accumulation. Our results therefore suggest that histamine and p[NH]ppG increased the permeability of the plasmalemma vesicles and may thus model the process of receptor-mediated Ca2+ entry into intact cells.

scholarly journals Hormonal regulation of Gi2 α-subunit phosphorylation in intact hepatocytes

1990 ◽  
Vol 268 (2) ◽  
pp. 449-457 ◽  
Author(s):  
M Bushfield ◽  
G J Murphy ◽  
B E Lavan ◽  
P J Parker ◽  
V J Hruby ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Kinase ◽  
G Protein ◽  
Hormonal Regulation ◽  
Dose Effect ◽  
Alpha Subunit ◽  
Intact Cells ◽  
Α Subunit ◽  
Guanine Nucleotide Binding ◽  
Dose Dependent

Hepatocytes contain the Gi2 and Gi3 forms of the ‘Gi-family’ of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity (‘Gi’-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a ‘Gi-like’ G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

scholarly journals Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

2018 ◽  
Vol 115 (19) ◽  
pp. 5016-5021 ◽  
Author(s):  
Olga S. Ilyaskina ◽  
Horst Lemoine ◽  
Moritz Bünemann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Coupling Efficiency ◽  
Dissociation Kinetics ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Subtype Selectivity ◽  
Intracellular Signals

G-protein–coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR–G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein–M3–R interactions but rather the affinities of Gq and Go proteins to M3–Rs, their GPCR–G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

Modulation of Ca(2+)-channel currents by protein kinase C in adult rat sympathetic neurons

1994 ◽  
Vol 72 (4) ◽  
pp. 1549-1560 ◽  
Author(s):  
Y. Zhu ◽  
S. R. Ikeda
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
G Protein ◽  
G Proteins ◽  
Sympathetic Neurons ◽  
Adult Rat ◽  
Kinase C ◽  
Channel Inhibition ◽  
Channel Currents ◽  
Ca2 Channels

1. Modulation of Ca(2+)-channel currents by phorbol-12-myristate-13-acetate (PMA) was investigated in acutely dissociated adult rat superior cervical ganglion neurons using the whole cell variant of the patch-clamp technique. 2. PMA (500 nM) increased the current amplitudes, accelerated the inactivation of step currents, retarded the deactivation of tail currents, and shifted the tail current activation to more negative potentials. 3. The effects of PMA were concentration and voltage dependent and mediated through activation of protein kinase C (PKC). PMA also increased Ca2+ currents recorded with the perforated patch technique. 4. PMA affected the N-type Ca2+ channels and an omega-conotoxin GVIA-resistant current component. Ca2+ currents affected by PMA were not sensitive to omega-agatoxin IVA or nimodipine. 5. PMA not only attenuated Ca(2+)-channel inhibition induced by alpha 2-adrenoceptor agonist, which modulates Ca2+ channels via a pertussis toxin (PTX)-sensitive pathway, but also attenuated current inhibition by vasoactive intestinal polypeptide, which modulates Ca2+ channels via a PTX-insensitive but cholera toxin-sensitive pathway. 6. PMA reversed Ca(2+)-channel inhibition induced by tonic activation of G-protein in the absence of neurotransmitter (even in neurons pretreated with PTX) or induced by activation of G-proteins with guanosine 5'-O-(3-thiotriphosphate) (GTP)-gamma-S. 7. Inhibition of phosphatase by okadaic acid or substitution of Ba2+ for Ca2+ in the external solutions accelerated the PMA effect. 8. Our results suggest that activation of PKC antagonizes G-protein mediated inhibition of Ca2+ channels by shifting Ca2+ channels from the “reluctant” state to the “willing” state. The G-proteins and, more likely, the N-type Ca2+ channels may be the target of PKC phosphorylation. Protein phosphatases may be involved in counteracting the PKC phosphorylation in rat sympathetic neurons.

scholarly journals Angiotensin II potentiates adrenocorticotrophic hormone-induced cAMP formation in bovine adrenal glomerulosa cells through a capacitative calcium influx

1998 ◽  
Vol 330 (1) ◽  
pp. 21-27 ◽  
Author(s):  
M. Muriel BURNAY ◽  
B. Michel VALLOTTON ◽  
M. Alessandro CAPPONI ◽  
F. Michel ROSSIER
Keyword(s):  
Angiotensin Ii ◽  
Calcium Channels ◽  
Adenylyl Cyclase ◽  
Adrenocorticotrophic Hormone ◽  
Camp Production ◽  
Glomerulosa Cells ◽  
Camp Formation ◽  
Voltage Operated Calcium Channels ◽  
Ca2 Channels ◽  
Stimulation Of

Angiotensin II (AngII) plays a crucial role in the control of aldosterone biosynthesis in adrenal glomerulosa cells through the stimulation of two distinct Ca2+ entry pathways: (1) opening of voltage-operated calcium channels, and (2) activation of a capacitative Ca2+ entry that is dependent on calcium release from intracellular pools. Adrenocorticotrophic hormone (ACTH), on the other hand, a major hormonal regulator of steroidogenesis, induces an increase in intracellular cAMP through the activation of a G-protein-coupled adenylyl cyclase. Recent studies have demonstrated that the rise in cAMP induced by ACTH can be potentiated by AngII in bovine glomerulosa cells. The aim of the present study was to investigate the mechanism of AngII action on ACTH-induced cAMP production. In primary cultures of bovine glomerulosa cells, we found that AngII (100 nM), which had no effect by itself on cAMP production, significantly potentiated maximal ACTH-induced cAMP formation in the presence of extracellular calcium (1.2 mM). In contrast, in the absence of extracellular calcium, AngII did not affect ACTH-induced cAMP production. These results suggest that calcium entry into the cell plays an important role in the activation of the cyclase by AngII. The inhibition of voltage-operated calcium channels by nicardipine, a dihydropyridine calcium antagonist blocking both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels, did not significantly affect the potentiating effect of AngII. Moreover, the cAMP response to ACTH was insensitive to activation of these Ca2+ channels induced by potassium ions and, even when cytosolic free-calcium concentration ([Ca2+]c) was kept elevated with the Ca2+ ionophore, ionomycin, no stimulation of adenylyl cyclase was observed at concentrations of [Ca2+]c up to 640 nM. In contrast, thapsigargin, an activator of capacitative Ca2+ influx, mimicked the potentiating effect of AngII on ACTH-induced cAMP formation. In agreement with the characteristics of cAMP modulation by Ca2+ in these cells, the presence of type III adenylyl cyclase was observed by immunodetection in bovine glomerulosa cell membranes. In conclusion, these data suggest a tight coupling between the capacitative Ca2+ influx induced upon stimulation by either AngII or thapsigargin and a calcium-sensitive isoform of adenylyl cyclase, probably type III, in bovine glomerulosa cells.

Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, Gi

1996 ◽  
Vol 271 (4) ◽  
pp. C1340-C1349 ◽  
Author(s):  
H. K. Lu ◽  
R. J. Fern ◽  
D. Luthin ◽  
J. Linden ◽  
L. P. Liu ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
Cell Pretreatment ◽  
Dependent Protein ◽  
Channel Currents ◽  
Ca2 Channels ◽  
Sustained Increase

Angiotensin II (ANG II) is the most potent and the most physiologically important stimulator of aldosterone synthesis and secretion from the adrenal zona glomerulosa. Because steroidogenesis by adrenal glomerulosa (AG) cells is mediated in part by Ca2+ influx through T- and L-type Ca2+ channels, we evaluated whether T-type Ca2+ channels are regulated by ANG II. We observe that ANG II enhances T-type Ca2+ current by shifting the voltage dependence of channel activation to more negative potentials. This shift is transduced by the ANG II type 1 receptor. The effect of the hormone is not mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) as it is not prevented by CaMKII(281-302), a peptide inhibitor of the catalytic region of the kinase. Rather, this shift is mediated by the activation of a G protein, Gi, because it is abolished by cell pretreatment with pertussis toxin and by cell dialysis with a monoclonal antibody generated against recombinant Gi alpha. This effect of ANG II on T-type Ca2+ channels should increase Ca2+ entry in AG cells at physiologically relevant voltages and result in a sustained increase in aldosterone secretion.

scholarly journals Heparin and other polyanions uncouple α1-adrenoceptors from G-proteins

1991 ◽  
Vol 280 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L L T Dasso ◽  
C W Taylor
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Alkaline Treatment ◽  
Receptor Activation ◽  
Maximal Effect ◽  
Intact Cells ◽  
High Affinity ◽  
Antagonist Binding ◽  
Coupling Mechanisms

Several polyanionic compounds antagonize the interaction between receptors and the G-proteins that regulate adenylate cyclase or K+ channels, possibly by binding to a basic stretch of the receptor that is proposed to mediate its interaction with the G-proteins. We have studied the effects of polyanions on the interaction between the liver alpha 1-adrenoceptor and the G-protein through which it stimulates polyphosphoinositide turnover. Heparin [concn. causing 50% of maximal effect (EC50) = 0.5 microM], Trypan Blue (EC50 7.1 microM) or suramin (EC50 2.1 microM) prevented formation of the high-affinity adrenaline-receptor-G-protein complex without affecting antagonist binding. After alkaline treatment of the membranes, previously reported to cause G-protein removal, binding of agonists was insensitive to both guanine nucleotides and heparin. We conclude that these polyanions uncouple the alpha 1-adrenoceptor from its G-protein, suggesting that similar coupling mechanisms may underlie receptor activation of the G-proteins that activate polyphosphoinositide hydrolysis and those that regulate adenylate cyclase. This action of heparin severely limits its utility as a selective antagonist of the Ins(1,4,5)P3 receptor in intact cells.

scholarly journals Angiotensin II Negatively Modulates L-type Calcium Channels through a Pertussis Toxin-sensitive G Protein in Adrenal Glomerulosa Cells

1999 ◽  
Vol 274 (28) ◽  
pp. 19943-19948 ◽  
Author(s):  
Andrés D. Maturana ◽  
Andrés J. Casal ◽  
Nicolas Demaurex ◽  
Michel B. Vallotton ◽  
Alessandro M. Capponi ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Calcium Channels ◽  
G Protein ◽  
Pertussis Toxin ◽  
Glomerulosa Cells
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