Pregnancy and estradiol decrease GTPase activity in the guinea pig uterine artery

2001 ◽  
Vol 281 (5) ◽  
pp. H2168-H2175 ◽  
Author(s):  
Irina A. Buhimschi ◽  
Gentzon Hall ◽  
Loren P. Thompson ◽  
Carl P. Weiner
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Uterine Artery ◽  
Mesenteric Arteries ◽  
Gtpase Activity ◽  
G Protein Coupling ◽  
High Affinity ◽  
Specific Manner ◽  
Organ Specific ◽  
17Β Estradiol

The mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner are poorly understood. We propose that it is consequential to estrogen-mediated alterations in G protein-mediated signal transduction. Aortas and uterine (UAs) and mesenteric arteries (MAs) were obtained from late-pregnant, nonpregnant, or ovariectomized guinea pigs chronically treated with 17β-estradiol. High-affinity GTPase activity was assayed enzymatically. The cGMP generated in response to the endothelium-dependent agonist ACh was measured in UAs incubated with or without cholera toxin (CTX, which inhibits Gsα). Pregnancy significantly decreased UA but not aorta or MA GTPase activity. 17β-Estradiol decreased UA GTPase activity compared with untreated ovariectomized animals. ACh increased cGMP in pregnant but not nonpregnant UAs. Pretreatment of nonpregnant UAs with CTX increased ACh-induced cGMP levels similar to pregnancy. Thus pregnancy and estradiol decrease the GTPase activity of a CTX-sensitive G protein in UAs, increasing receptor-dependent cGMP release. This alteration in receptor-mediated G protein coupling in UAs may contribute to the characteristic cardiovascular adaptation to pregnancy.

scholarly journals Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1α when constrained within an α2A-adrenoceptor-Gi1α fusion protein

1997 ◽  
Vol 325 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Alan WISE ◽  
I. Craig CARR ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Pertussis Toxin ◽  
Gtpase Activity ◽  
Α Subunit ◽  
Fusion Construct ◽  
High Affinity ◽  
C Terminus ◽  
Adrenoceptor Agonist ◽  
Stimulation Of

A fusion protein was generated between the porcine α2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351 → Gly) variant of the α subunit of Gi1α by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the α2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min-1 for Gi1α when the α2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37±0.07 μM. Co-expression of excess β1γ2 along with the α2A-adrenoceptor-Gi1α fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1±0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with βγ complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.

scholarly journals Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates

1988 ◽  
Vol 249 (3) ◽  
pp. 653-659 ◽  
Author(s):  
F R McKenzie ◽  
E C H Kelly ◽  
C G Unson ◽  
A M Spiegel ◽  
G Milligan
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
High Affinity ◽  
C Terminus ◽  
Inhibitory G Protein ◽  
Guanine Nucleotide Binding

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.

scholarly journals δ-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2

1990 ◽  
Vol 267 (2) ◽  
pp. 391-398 ◽  
Author(s):  
F R McKenzie ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Opioid Receptor ◽  
Protein S ◽  
Guanine Nucleotide ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
High Affinity ◽  
Guanine Nucleotide Binding

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.

scholarly journals Characterization of CCKA receptor affinity states and Ca2+ signal transduction in vagal nodose ganglia

2002 ◽  
Vol 282 (6) ◽  
pp. G1002-G1008 ◽  
Author(s):  
Tim O. Lankisch ◽  
Yasuhiro Tsunoda ◽  
Yuanxu Lu ◽  
Chung Owyang
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Ganglion Cells ◽  
Protein S ◽  
Calcium Signal ◽  
High Affinity ◽  
Nodose Ganglia ◽  
Afferent Fibers ◽  
Voltage Dependent ◽  
Stimulation Of

CCKA receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCKA receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca2+ transient followed by a sustained Ca2+plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca2+ oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCKAreceptors, primarily elicited Ca2+ oscillations (29% of cells responded). CCK-OPE inhibited the Ca2+ transient induced by 1 nM CCK-8 but not by carbachol and high K+. This result suggests the presence of high- and low-affinity states of CCKA receptors on nodose ganglia. We further demonstrated that nicardipine (10 μM) but not ω-conotoxins GVIA and MVIIC (10–100 nM) abolished Ca2+ signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca2+ channel and not an N- or Q-type Ca2+ channel is coupled to CCKA receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca2+ transient and inhibited CCK-8-evoked Ca2+ signaling, indicative of G protein(s) involvement in CCKA receptor activity. The Gq protein antagonist Gp antagonist-2A (10 μM) also abolished the action of CCK-8. This study indicates that CCKA receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCKAreceptors elicits Ca2+ oscillations, whereas stimulation of low-affinity CCKA receptors evokes a sustained Ca2+ plateau. These Ca2+-signaling modes are mediated through the L-type Ca2+ channel and involve the participation of Gq protein.

scholarly journals Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and α2C10 adrenoceptor-transfected Rat 1 fibroblasts

1994 ◽  
Vol 298 (2) ◽  
pp. 493-497 ◽  
Author(s):  
C Carr ◽  
M Grassie ◽  
G Milligan
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Lysophosphatidic Acid ◽  
Gtpase Activity ◽  
Response Curves ◽  
Protein Activation ◽  
Adp Ribosylation ◽  
High Affinity ◽  
Stimulation Of

Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 microM LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a Gi-like G-protein. LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor. The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells. LPA was also able to promote cholera toxin-catalysed [32P]ADP-ribosylation of Gi. This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin. LPA-stimulated cholera toxin-catalysed [32P]ADP-ribosylation of Gi in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304. Dose-response curves for LPA in the two assays of G-protein activation were coincident. The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of Gi.

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