Alterations of adenylyl cyclase-linked G proteins in rat liver during aging

1996 ◽  
Vol 270 (1) ◽  
pp. E126-E132 ◽  
Author(s):  
A. T. Eakes ◽  
T. K. Hymer ◽  
M. J. Rosenthal ◽  
J. Moss ◽  
M. S. Katz
Keyword(s):  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
G Proteins ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Adp Ribosylation ◽  
Old Rats ◽  
Gs Alpha

beta-Adrenergic stimulation of adenylyl cyclase in rat liver increases during aging. We examined whether this increase is related to alterations in the stimulatory and inhibitory G proteins (Gs and Gi) linked to adenylyl cyclase. Levels of immunoreactive alpha- and beta-subunits of Ga and Gi in liver plasma membranes from 6-, 12-, 18-, and 24-mo-old rats were unchanged with age, as was pertussis toxin-catalyzed [32P]ADP ribosylation of Gi alpha. Cholera toxin-catalyzed [32P]ADP ribosylation of Ga alpha and Gs bioactivity, assessed as reconstitution of adenylyl cyclase activity in S49 cyc- cell membranes, increased two- to threefold between 6 and 12-18 mo, and declined by 24 mo. Recombinant ADP ribosylation factor (ARF) enhanced cholera toxin labeling of Gs alpha at all ages, yet abolished the increase in toxin labeling at 12-18 mo. Auto-ADP ribosylation of the cholera toxin A1 peptide also increased transiently with age. Alteration of Gs alpha, as reflected by increased cholera toxin labeling and Gs bioactivity, may be involved in the regulation of beta-adrenergic-responsive adenylyl cyclase in rat liver during aging. Moreover, changes in endogenous ARF levels could contribute to age differences in cholera toxin labeling of Gs alpha.

scholarly journals Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase

1980 ◽  
Vol 186 (3) ◽  
pp. 749-754 ◽  
Author(s):  
C A Doberska ◽  
A J S MacPherson ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Activation Process ◽  
Liver Plasma Membrane ◽  
Adp Ribosylation ◽  
A Subunit

1. Cholera toxin was shown to require the presence of GTP to activate rat liver plasma-membrane adenylate cyclase. ATP did not affect the activation process. 2. Cholera toxin catalysed the incorporation of 32P from NAD labelled in the alpha-phosphate group of the ADP moiety into a rat liver plasma-membrane protein with a subunit mol.wt. of 42 500. This is taken to demonstrate ADP-ribosylation. The ADP-ribosylation of this protein also required GTP and was unaffected by ATP. 3. Nicotinamide inhibited both the activation of adenylate cyclase by cholera toxin and the ADP-ribosylation of the protein of 42 500 subunit mol wt. Neither the activation nor the ADP-ribosylation could be reversed by treatment with nicotinamide in the presence of cholera toxin.

scholarly journals Epoxyeicosatrienoic acid stimulates ADP-ribosylation of a 52 kDa protein in rat liver cytosol

1992 ◽  
Vol 281 (1) ◽  
pp. 185-190 ◽  
Author(s):  
K Seki ◽  
A Hirai ◽  
M Noda ◽  
Y Tamura ◽  
I Kato ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Rat Liver ◽  
Epoxyeicosatrienoic Acid ◽  
Liver Cytosol ◽  
Adp Ribosylation ◽  
A Subunit ◽  
Gs Alpha ◽  
Adp Ribosyltransferase ◽  
Rat Liver Cytosol

In rat liver cytosol, rapid ADP-ribosylation of a 52 kDa protein by endogenous ADP-ribosyltransferase(s) was observed. This ADP-ribosylation was stimulated dose-dependently by 14,15-epoxyeicosatrienoic acid (14,15-EET), one of the metabolites of arachidonic acid by NADPH-dependent cytochrome P-450 mono-oxygenase. This stimulatory effect required the presence of GTP or its non-hydrolysable analogues, guanosine 5′-[beta gamma-imido]triphosphate or guanosine 5′-[gamma-thio]triphosphate. Of four regioisomeric EETs, 14,15-EET was the most potent. No stimulatory effect was observed with addition of 14,15-dihydroxyeicosatrienoic acid, a stable metabolite of 14,15-EET. The 52 kDa protein was not ADP-ribosylated by cholera toxin A subunit and pertussis toxin, and was not recognized by anti-Gs alpha and anti-Gi alpha antibodies. However, the 52 kDa protein could be photoaffinity-labelled with 8-azidoguanosine 5′-[alpha-32P]triphosphate. These results suggest that the 52 kDa protein is neither Gs nor Gi, though it may have a GTP-binding site. These results contribute to the understanding of the role of mono-oxygenase metabolites of arachidonic acid in intracellular signal transduction.

scholarly journals Evidence for G proteins in rat parotid plasma membranes and secretory granule membranes

1992 ◽  
Vol 285 (2) ◽  
pp. 441-449 ◽  
Author(s):  
E L Watson ◽  
D DiJulio ◽  
D Kauffman ◽  
J Iversen ◽  
M R Robinovitch ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Proteins ◽  
Secretory Granule ◽  
Membrane Fraction ◽  
Immunoblot Analysis ◽  
Plasma Membrane Fraction ◽  
Adp Ribosylation ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Gs Alpha

G proteins were identified in rat parotid plasma membrane-enriched fractions and in two populations of isolated secretory granule membrane fractions. Both [32P]ADP-ribosylation analysis with bacterial toxins and immunoblot analysis with crude and affinity-purified antisera specific for alpha subunits of G proteins were utilized. Pertussis toxin catalysed the ADP-ribosylation of a 41 kDa substrate in the plasma membrane fraction and both secretory granule membrane fractions. Cholera toxin catalysed the ADP-ribosylation of two substrates with molecular masses of 44 kDa and 48 kDa in the plasma membrane fraction but not in the secretory granule fractions. However, these substrates were detected in the secretory granule fractions when recombinant ADP-ribosylating factor was present in the assay medium. Immunoblot analysis of rat parotid membrane fractions using both affinity-purified and crude antisera revealed strong immunoreactivity of these membranes with anti-Gs alpha, -Gi alpha 1/alpha 2 and -Gi alpha 3 sera. In contrast Gs alpha was the major substrate found in both of the secretory granule fractions. Granule membrane fractions also reacted moderately with anti-Gi alpha 3 antiserum, and weakly with anti-Gi alpha 1/alpha 2 and -G(o) alpha sera. The results demonstrate that the parotid gland membranes express a number of G proteins. The presence of G proteins in secretory granule membranes suggests that they may play a direct role in regulating exocytosis in exocrine glands.

Mechanisms mediating responsiveness to beta-adrenergic stimulation after coronary reperfusion in conscious dogs

1994 ◽  
Vol 267 (4) ◽  
pp. H1578-H1588 ◽  
Author(s):  
K. Kiuchi ◽  
Y. T. Shen ◽  
S. F. Vatner ◽  
D. E. Vatner
Keyword(s):  
Adenylyl Cyclase ◽  
Coronary Artery Occlusion ◽  
Artery Occlusion ◽  
Ischemic Myocardium ◽  
Regional Myocardial Blood Flow ◽  
Wall Thickening ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
High Affinity ◽  
Total Beta

The goal of this study was to assess beta-adrenergic receptor (beta-AR) signaling mechanisms in mediating physiological responses to sympathomimetic amines after 45 min coronary artery occlusion followed by 45 min reperfusion. At this time, isoproterenol (Iso) infusion (0.1 microgram/kg-1.min-1, n = 5) increased percent wall thickening in previously ischemic subendocardium (Endo) more than in nonischemic Endo (12.6 +/- 1.2 vs. 7.2 +/- 0.6%, P < 0.05), whereas forskolin (25 nmol.kg-1.min-1, n = 6) elicited the opposite effect (3.6 +/- 0.6 vs. 10.4 +/- 2.8%, P < 0.05). During Iso and forskolin infusions increases in regional myocardial blood flow in the previously ischemic zone were similar to the nonischemic zone. In all groups, total beta-AR density was depressed in previously ischemic Endo compared with nonischemic Endo (65 +/- 7 vs. 82 +/- 8 fmol/mg, P < 0.05), but the fraction of beta-AR binding agonist with high affinity increased (82 +/- 4 vs. 49 +/- 1%, P < 0.05). The changes in beta-AR were associated with a decrease in Iso-stimulated adenylyl cyclase (22 +/- 8%), a decrease in guanosine 5'-triphosphate (GTP)-stimulatory protein (Gs) (23 +/- 6%), and a decrease in inhibitory G proteins (16 +/- 4%). However, regional Endo functional responsiveness to beta-AR stimulation was enhanced in reperfused myocardium in response to Iso but not to forskolin. Thus the mechanism of increased number of beta-AR binding agonist with high affinity in previously ischemic myocardium predominated over persistent downregulation of total beta-AR density and reductions in Gs and adenylyl cyclase activity and correlated best with the physiological response to beta-AR stimulation. These data may also suggest that Iso exerts an action distal to adenylyl cyclase in previously ischemic myocardium.

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