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Biomedical subjects

G Milligan

Publications and source records attributed to G Milligan.

At least 127 records · Page 7Linked to original sources
PubMed1995

Bradykinin excites rat sympathetic neurons by inhibition of M current through a mechanism involving B2 receptors and G alpha q/11.

Bradykinin (BK) is a peptide mediator released in inflammation that potently excites sympathetic neurons. We have studied the mechanism of this excitation in dissociated rat sympathetic neurons and found that at low nanomolar (EC50 = 0.9 nM) concentrations, BK inhibited the M-type K+ current IK(M). Studies with the selective antagonist Hoe140 revealed that this effect was mediated via the B2 receptor subtype, and mRNA encoding this receptor was identified in these neurons by RT-PCR. IK(M) inhibition was unaffected by Pertussis toxin or microinjection of antibodies to G alpha o but was selectively inhibited by microinjection of antibodies to G alpha q/11. Thus, BK is the most potent M current inhibitor yet described in mammalian neurons, and BK inhibition of M current is mediated by a G protein pathway similar to that activated by muscarinic acetylcholine receptors.

Animals↗
PubMed1995

The dynamic role of palmitoylation in signal transduction.

Guanine nucleotide binding protein (G protein)-linked receptors, the alpha-subunits of heterotrimeric G proteins and members of the Src family of nonreceptor tyrosine kinases are among many polypeptides that are posttranslationally modified by the addition of palmitate, a long-chain fatty acid. Attachment of palmitate to these proteins is dynamic and may be regulated by their activation. The presence of palmitate appears to play a key role in the membrane localization of either the entire polypeptide or parts of it, and may regulate the interactions of these polypeptides with other proteins.

Amino Acid Sequence↗
PubMed1995

Expression of the human beta 2-adrenoceptor in NCB20 cells results in agonist activation of adenylyl cyclase and agonist-mediated selective down-regulation of Gs alpha.

Murine neuroblastoma x embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human beta 2-adrenoceptor under the control of a beta-actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the beta-adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild-type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein alpha subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane-associated levels of Gs alpha in membranes of clone L9 cells and a 50% reduction in Gs alpha levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gs alpha in wild-type NCB20 cells, and such treatment had no effect on the levels of other G protein alpha subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half-maximal loss of Gs alpha in clone D1 was achieved within 1-2 h of addition of agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1995

Melatonin receptors couple through a cholera toxin-sensitive mechanism to inhibit cyclic AMP in the ovine pituitary.

The nature of melatonin receptor-G-protein coupling in ovine pars tuberalis (PT) cells of the pituitary was addressed using cholera (CTX) and pertussis (PTX) toxins. ADP-ribosylation of ovine PT membrane proteins using 32P-NAD in the presence of CTX radiolabelled several substrates including 44, 51, and 60 kD proteins. Each were clearly distinct from the 40 kD substrate radiolabelled in the presence of PTX. Acute incubation of PT membranes with either toxin reduced the number of high affinity binding sites for 125I-MEL, although the magnitude of the inhibition was much greater for CTX (56%) than for PTX (20%). A CTX-sensitive component also mediates the inhibition of forskolin-stimulated cyclic AMP accumulation as pre-treatment of PT cells with CTX (5 micrograms/ml) for 16 h blocked this response. Gs alpha is a major substrate for ADP-ribosylation by CTX, and 16 h pre-treatment of PT cells with CTX (5 micrograms/ml) caused a down-regulation of Gs alpha. Northern analysis showed only one major transcript of Gs alpha of about 2 kb, which would encompass all of the known splice variants of the Gs gene. Screening of a cDNA library from ovine PT for Gs-related genes and sequencing of clones, combined with RT-PCR of PT mRNA, revealed no novel products. On this basis it is concluded that the CTX substrate is unlikely to be a novel splice variant or related gene product of the Gs class of G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1995

Involvement of G-protein alpha il subunits in activation of G-protein gated inward rectifying K+ channels (GIRK1) by human NPY1 receptors.

This study investigated the type of G-protein alpha subunit(s) that human neuropeptide Y (NPY)1 receptors preferentially utilize when activating G-protein gated K+ currents. Two electrode voltage-clamp recordings were made from Xenopus oocytes that had been injected with cDNAs encoding either human NPY1 or D2(short) dopamine receptors, and GIRK1 a cloned rat brain K+ channel. These receptors were also co-injected with G-protein alpha i1, alpha i2, alpha i3 and alpha o1 subunits to determine which subunit(s) modulate the efficiency of signal transduction. In NPY1 receptor injected cells neuropeptide Y (100 nM) caused a 53 +/- 10 nA inward current (n = 14; EC50 = 3 nM) and this effect was blocked by pertussis toxin (500 ng ml-1 24 h). Activation of GIRK1 currents by neuropeptide Y was selectively potentiated by alpha i1 subunit cDNA whereas coupling dopamine of D2 receptors to this channel was not.

Animals↗
PubMed1995

Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor.

Many agonist ligands are known experimentally to display a range of efficacies and potencies in different tissues and preparations. To analyze the role of the levels of receptor expression and availability in the intrinsic activities and potencies of agonists, the function of a number of beta-adrenoceptor ligands was examined in clones of neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor, as well as after treatment of these cell lines with the irreversible beta-adrenoceptor antagonist bromoacetyl alprenolol menthane (BAAM). Clone beta N22 expressed approximately 10-fold higher levels of the receptor than did clone beta N17. In measurements of agonist stimulation of adenylyl cyclase activity in membranes of these cells or agonist stimulation of the formation of the complex of Gs alpha and adenylyl cyclase, which acts as the high affinity binding site for [3H]forskolin in whole cells, a series of beta-adrenoceptor agonists, including dichloroisoprenaline, ephedrine, dobutamine, and salbutamol, displayed higher intrinsic activity and showed concentration-response curves that were substantially to the left (lower EC50 values) in clone beta N22, compared with clone beta N17. Treatment of clone beta N22 cells with varying concentrations of BAAM reduced the intrinsic activity of these ligands and shifted the concentration-response curves for these agents to the right. In clone beta N22 cells and membranes, reduction in the observed intrinsic activity for ephedrine required elimination of a smaller fraction of the beta 2-adrenoceptor reserve than for salbutamol and reduction in the effect of the full agonists isoprenaline and epinephrine was noted only with high fractional elimination of the receptor pool. The effect of isoprenaline was substantially reduced, however, by BAAM treatment of clone beta N17 cells, where the beta 2-adrenoceptor number approached extremely low levels. Analysis of the data using the formalisms of Whaley et al. [Mol. Pharmacol. 45:481-489 (1994)] showed that prediction of alterations in agonist potency with receptor number for full agonists can be adequately extended to partial agonists.

Adenylyl Cyclases↗