Search PubMed⌕ Search

Biomedical subjects

G Milligan

Publications and source records attributed to G Milligan.

At least 199 records · Page 11Linked to original sources
PubMed1992

Effects of dexamethasone on G protein levels and adenylyl cyclase activity in rat vascular smooth muscle cells.

Dexamethasone administration in vitro has been shown to increase adenylyl cyclase activity in vascular smooth muscle cells (VSMC) from renal arteries and in non-vascular cell lines. To investigate whether G proteins are involved in this response, cultured VSMC from mesenteric arteries of Sprague-Dawley rats were incubated in the presence and absence of 10 nM dexamethasone for 24 and 48 h. Basal and stimulated adenylyl cyclase activities were increased by approximately 50% after treatment with dexamethasone. The changes were neither specifically associated with ligands which stimulate adenylyl cyclase catalytic unit via Gs (isoproterenol and prostaglandin E1) nor with guanylylimidodiphosphate (0.1 nM), which inhibits the catalytic unit via Gi. This suggests that dexamethasone enhances adenylyl cyclase activity through changes at the level of the catalytic unit, rather than through the G proteins which modulate its activity. No differences were seen in immunoblotting studies of the levels of Gi alpha 2, Gs alpha, Gi alpha 3 and beta subunits. Similarly, dexamethasone had no effect on the expression of mRNA for Gi alpha 2 and Gs alpha. The results indicate that glucocorticoid-induced increases of adenylyl cyclase activity are due to changes at the level of the adenylyl cyclase catalytic unit rather than alteration of the levels or turnover of Gs alpha, Gi alpha 2, Gi alpha 3 and beta subunits in the membranes of VSMC.

Adenylyl Cyclases↗
PubMed1991

Widespread distribution of Gq alpha/G11 alpha detected immunologically by an antipeptide antiserum directed against the predicted C-terminal decapeptide.

Antisera were raised to a synthetic peptide which represents the predicted C-terminal decapeptide of the alpha subunit of the G-proteins Gq and G11. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS-PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gs alpha.

Adenosine Diphosphate Ribose↗
PubMed1991

Alterations in G-protein expression and the hormonal regulation of adenylate cyclase in the adipocytes of obese (fa/fa) Zucker rats.

Attenuated maximal activations by forskolin, Mn+. NaF or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) were noted for adenylate cyclase activity in adipocytes from obese (fa/fa) Zucker rats compared with their lean (Fa/Fa) littermates. GTP[S] achieved half-maximal activation of adenylate cyclase at some 10-fold lower concentrations in membranes from lean animals compared with those from obese. Levels of the 42 and 45 kDa forms of Gs were some 40-50% lower in membranes from obese animals, and levels of Gi-1 and Gi-3 were some 62-65% lower. No differences in levels of Gi-2 alpha-subunits or G-protein beta-subunits were observed. Gi function, as assessed by inhibiting forskolin-stimulated adenylate cyclase, achieved by prostaglandin E1, nicotinate and phenylisopropyladenosine, was similar in membranes from both lean and obese animals. Levels of beta-adrenoceptors were some 50% lower in membranes from obese animals. It is suggested that the attenuated activation of adenylate cyclase by stimulatory ligands in membranes from obese animals may be caused by decreases in both Gs and receptors, and that this may contribute to the attenuated lipolytic response seen in adipocytes from such animals.

Adenylyl Cyclases↗
PubMed1991

Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1991

Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma x glioma hybrid cells is due to a toxin-induced decrease in opioid receptor levels.

Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and 'Gi'. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.

Adenylate Cyclase Toxin↗
PubMed1991

Abundance of the alpha-subunits of Gi1, Gi2 and Go in synaptosomal membranes from several regions of the rat brain is increased in hypothyroidism.

1. Rats (4 weeks old) were made hypothyroid by treatment with propylthiouracil together with a low-iodine diet for a further period of 4 weeks. Synaptosomal membranes were obtained from six anatomical regions of the brain. 2. The abundances in these membranes of the G-protein alpha-subunits Gi1 alpha, Gi2 alpha and Go alpha were measured by quantitative immunoblotting. 3. Hypothyroidism significantly increased the abundances of all three G-protein subunits in membranes from the cerebral cortex and the striatum. In the medulla oblongata and the hippocampus the abundances of Gi2 alpha and Go alpha were increased significantly. By contrast, in the cerebellum only Go alpha was increased, and in the hypothalamus only Gi2 alpha was increased. 4. It is suggested that this up-regulation of G-protein abundances may modify signalling pathways and may contribute to the functional changes that are observed in the central nervous system in hypothyroidism.

Animals↗
PubMed1991

Altered G-protein expression and adenylate cyclase activity in platelets of non-insulin-dependent diabetic (NIDDM) male subjects.

Adenylate cyclase activity and levels of guanine nucleotide regulatory proteins (G-proteins) were compared in platelets from normal and non-insulin-dependent diabetic (NIDDM) male subjects. Whilst no differences were noted in basal and NaF-stimulated adenylate cyclase activities the degree of stimulation achieved by both forskolin and prostaglandin, E1 was lower by some 34 and 52% respectively, in platelet membranes from diabetic subjects compared with those from normal control subjects. Altered alpha 1-adrenoceptor-mediated inhibition of prostaglandin E1-stimulated adenylate cyclase activity was evident; it being some 34% lower in platelet membranes from diabetic subjects compared to controls. Analysis of G-protein alpha-subunits, using specific anti-peptide antisera, showed that platelets from all subjects exhibited the Gi-2 and Gi-3, but not the Gi-1 forms of the inhibitory G-protein 'Gi' and all expressed the 42 kDa species of alpha-subunit of the stimulatory G-protein Gs. Whilst platelets of diabetic subjects had levels of Gs which were comparable to those found in control subjects their levels of Gi-2 and Gi-3 were some 49 and 75%, respectively, of those found in platelets from control subjects. It is suggested that changes in adenylate cyclase functioning and G-protein expression may contribute to altered platelet functioning in non-insulin-dependent diabetic subjects.

Adenylyl Cyclases↗
PubMed1991

Characterization of G proteins in rat myometrium. A differential modulation of Gi2 alpha and Gi3 alpha during gestation.

Myometrial membranes, obtained from estrogen-dominated (day 0) rat uteri, were immunoblotted with antiserum (SG1), which recognizes the alpha subunits of both Gi1 and Gi2, with antiserum (LE2) specific for Gi2 alpha, and with I3B antiserum, specific for Gi3 alpha. The data revealed the absence of detectable levels of Gi1 alpha and the simultaneous presence of Gi2 alpha and Gi3 alpha as Gi subunits in rat myometrium. The expression of Gi proteins during gestation (days 0, 12, 21) was studied with the above antibodies. No qualitative change in the nature of Gi alpha species was observed during gestation: Gi1 alpha remained undetectable, Gi2 alpha and Gi3 alpha were both present on days 12 and 21. Of significance was the increase (160%) in the amount of Gi2 alpha at midgestation (day 12) compared to days 0 and 21. A different pattern was observed with Gi3 alpha, which decreased with advancing gestation (day 0 greater than 12 greater than 21). Immunodetection of beta subunits of G proteins indicated the presence of a 35/36 kDa doublet on days 0, 12 and 21, with an increase at midgestation. The simultaneous increase in Gi2 alpha and beta subunits may provide an explanation for the previously demonstrated alteration in adenylate cyclase stimulability detected at midgestation.

Animals↗
PubMed1991

Robustness of G proteins in Alzheimer's disease: an immunoblot study.

Many of the neurotransmitter systems that are altered in senile dementia of the Alzheimer type are known to mediate their effects via G proteins, yet the integrity of guanine nucleotide-binding proteins (G proteins) in Alzheimer's diseased brains has received minimal investigation. The aim of this study was to establish whether the level of G alpha subunits of five G proteins was altered in Alzheimer's disease. We used immunoblotting (Western blotting) to compare the amounts of Gi1, Gi2, GsH (heavy molecular weight), GsL (light molecular weight), and Go in the frontal cortex and hippocampus, two regions severely affected by the disease, and the cerebellum, which is less severely affected. The number of senile plaques was also quantified. We report that there was no significant difference in the level of these G alpha subunits between Alzheimer's diseased and age-matched postmortem brains. These results suggest that alterations in the amount of G protein alpha subunits are not a feature of Alzheimer's disease.

Alzheimer Disease↗
PubMed1991

Molecular interaction of the human alpha 2-C10-adrenergic receptor, when expressed in Rat-1 fibroblasts, with multiple pertussis toxin-sensitive guanine nucleotide-binding proteins: studies with site-directed antisera.

DNA encoding the human alpha 2-C-10-adrenergic receptor was transfected into Rat-1 fibroblasts by CaPO4 precipitation, and clones expressing the receptor were isolated and expanded. One clone (1C) expressing high levels of the receptor was studied in order to determine the contacts between this receptor and guanine nucleotide-binding proteins (G proteins) mediating second messenger signaling. The alpha 2-adrenergic agonist UK 14304 stimulated high affinity GTPase activity in membranes from these cells. Incubation of these membranes with Protein A-purified fractions from an antiserum able to identify the carboxyl-terminal decapeptide common to Gi1 alpha and Gi2 alpha was partially able to prevent agonist stimulation of high affinity GTPase activity. Similar results were produced with an antiserum that identifies the carboxyl-terminal decapeptide of Gi3 alpha. In contrast, equivalent fractions of antisera that identify the carboxyl-terminal decapeptides of Go alpha and Gs alpha did not inhibit receptor stimulation of high affinity GTPase activity. Coincubation of the membranes from the cells with Protein A-purified fractions from the anti-Gi1 alpha + Gi2 alpha antiserum and the anti-Gi3 alpha antiserum produced greater inhibition of UK14304-stimulated GTPase activity than did either of the two antisera in isolation. These data show direct interaction of the human alpha 2-C10-adrenergic receptor, when expressed in this clone of Rat-1 fibroblasts, with multiple pertussis toxin-sensitive G proteins and demonstrate that a single receptor has the physical capacity to interact functionally with more than a single pertussis toxin-sensitive G protein in a native membrane. Furthermore, because the two antisera were able to inhibit receptor stimulation of high affinity GTPase activity to similar degrees, the G protein pools identified by these antisera must contribute similar amounts of the total receptor activation of pertussis toxin-sensitive G proteins in these cells.

Animals↗
PubMed1990

Gs alpha mediates epidermal growth factor-elicited stimulation of rat cardiac adenylate cyclase.

In an earlier study we demonstrated that epidermal growth factor (EGF) increases the cellular accumulation of cAMP in perfused rat hearts by stimulating the cardiac adenylate cyclase via a stimulatory GTP-binding protein (Nair, B. G., Rashed, H. M., and Patel, T. B. (1989) Biochem. J. 264, 563-571). Employing antiserum, CS1, generated against a synthetic decapeptide RMHLRQYELL representing the carboxyl terminus of Gs alpha, the involvement of Gs in mediating the effects of EGF on cardiac adenylate cyclase was further investigated. The CS1 antiserum specifically recognized two forms, (52 and 40 kDa) of Gs alpha in rat cardiac membranes; the 52 kDa being the predominant species. In functional assays of adenylate cyclase activity, the CS1 antiserum did not alter either aluminum fluoride- or forskolin-stimulated adenylate cyclase activity. Similarly, basal adenylate cyclase activity in the absence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was also not altered by the CS1 antiserum. However, as compared with controls performed in the presence of non-immune serum, preincubation of cardiac membranes with the CS1 antiserum resulted in a concentration-dependent inhibition of Gpp(NH)p-, isoproterenol-, and EGF-stimulated activities. In experiments which monitored Gi function as the ability of different G(pp)NHp, (-)N6-(R-phenylisopropyl)adenosine and carbachol to inhibit forskolin-stimulated adenylate cyclase, CS1 antiserum by inhibiting Gs, increased the apparent activity of Gi. Overall, our data demonstrate that the CS1 antiserum can specifically inhibit Gs function and therefore the stimulation of adenylate cyclase by agonists whose actions are mediated by Gs. In this respect, the data presented here demonstrate that Gs is the G-protein involved in mediating EGF-elicited stimulation of cardiac adenylate cyclase. Additionally, the finding that CS1 antiserum can overcome the effects of Gpp(NH)p on Gs, but not Gi, suggests that the carboxyl-terminal region of Gs alpha is important in the interactions with GTP or its analogs.

Adenylyl Cyclases↗
PubMed1990

Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes.

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Prostaglandin E1-mediated, cyclic AMP-independent, down-regulation of Gs alpha in neuroblastoma x glioma hybrid cells.

Treatment of neuroblastoma x glioma hybrid, NG108-15, cells with prostaglandin E1, which in these cells activates adenylate cyclase, produced a marked (50%) reduction in immunologically detectable levels of Gs alpha associated with the plasma membrane. This effect was dependent both on the time of treatment and on the concentration of the receptor ligand used and did not involve a translocation of Gs alpha from the membrane to the cytoplasm of the cells. Both the 45- and 42-kDa forms of Gs alpha which are expressed by these cells were reduced in levels by treatment with the agonist but the greater effect was on the more prevalent 45-kDa polypeptide. By contrast, treatment of the cells with forskolin over the same period did not produce a reduction in levels of Gs alpha, indicating that the effect of prostaglandin E1 was independent of cAMP production. Prostaglandin E1-mediated down-regulation of Gs alpha levels was not produced at the transcriptional level as amounts of mRNA encoding Gs alpha were not reduced by treatment of the cells with agonist. Further, treatment of NG108-15 cells with cycloheximide, throughout the time period required to produce maximal prostaglandin E1-dependent down-regulation of Gs alpha, demonstrated that complete suppression of de novo protein synthesis could not mimic the effect of prostaglandin E1 and hence even complete inhibition of transcription of the Gs alpha gene and/or translation of pre-existing mRNA could not account for these results. Prostaglandin E1 treatment of the cells had no effect on steady-state levels of the alpha subunits of the pertussis toxin-sensitive G-proteins, Gi2, Gi3, Go, which are expressed by these cells or on the level of G-protein beta subunit. Fluoride stimulation of adenylate cyclase activity in membranes of S49 cyc- cells following addition of sodium cholate extracts of membranes of prostaglandin E1-treated NG108-15 cells was only some 50% as effective as with equivalent extracts from untreated cells. These results provide evidence for a novel mechanism of receptor-mediated control of the stimulation of adenylate cyclase, involving reduction in the steady-state amounts of Gs alpha.

Adenosine Deaminase↗