scholarly journals Up-regulation of the levels of expression and function of a constitutively active mutant of the hamster α1B-adrenoceptor by ligands that act as inverse agonists

1997 ◽  
Vol 325 (3) ◽  
pp. 733-739 ◽  
Author(s):  
Tae Weon LEE ◽  
Susanna COTECCHIA ◽  
Graeme MILLIGAN
Keyword(s):  
Phospholipase D ◽  
Wild Type ◽  
Adrenergic Agonist ◽  
Inverse Agonists ◽  
Agonist Stimulation ◽  
And Function ◽  
Type Receptor ◽  
Stimulation Of ◽  
Constitutively Active

The α1-adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at the wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expressing the CAM α1B-adrenoceptor with phentolamine resulted in a marked up-regulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC50 of approx. 40 nM. Such an up-regulation could be produced with a range of other ligands generally viewed as α1-adrenoceptor antagonists but equivalent treatment of cells expressing the wild-type α1B-adrenoceptor was unable to mimic these effects. After sustained treatment of the CAM α1B-adrenoceptor expressing cells with phentolamine, basal PLD activity was increased and phenylephrine was now able to stimulate PLD activity to greater levels than in vehicle-treated CAM α1B-adrenoceptor-expressing cells. The EC50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulation of basal PLD activity, a range of α1-antagonists were shown to possess the characteristics of inverse agonists of the CAM α1B-adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.

Phospholipase D is activated by G protein and not by calcium ions in vascular smooth muscle

1996 ◽  
Vol 270 (3) ◽  
pp. H1031-H1037
Author(s):  
E. F. LaBelle ◽  
R. M. Fulbright ◽  
R. J. Barsotti ◽  
H. Gu ◽  
E. Polyak
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
G Protein ◽  
Phospholipase D ◽  
Pertussis Toxin ◽  
Alpha Toxin ◽  
Tail Artery ◽  
Agonist Stimulation ◽  
Rat Tail ◽  
Stimulation Of

We assessed the sensitivity of phospholipase D (PLD) activity in vascular smooth muscle to cytosolic Ca2+ by increasing cytosolic Ca2+ levels independently of agonist stimulation. When rat tail artery was preloaded with the Ca2+ indicator fluo 3 pentaacetoxymethyl ester, the addition of high extracellular K+, caffeine, or norepinephrine rapidly enhanced cytosolic Ca2+ levels. Neither increased extracellular K+ nor caffeine addition increased phosphatidylethanol production, indicating that cytosolic Ca2+ elevation alone did not stimulate PLD. In contrast, norepinephrine stimulated phosphatidylethanol production in this tissue. In strips of tail artery permeabilized with alpha-toxin and incubated in solutions containing free Ca2+ concentrations observed during physiological stimulation (pCa 6.4), PLD was not stimulated, whereas incubation with guanosine 5'-O-(3-thiotriphosphate) at pCa 7.0 activated this enzyme. Aluminum fluoride (AlF4-) stimulated PLD, and this activity was insensitive to pertussis toxin after stimulation by either norepinephrine or AlF4-. These results indicate that PLD in vascular smooth muscle is activated by norepinephrine via stimulation of a pertussis toxin-insensitive G protein and not via an increase in intracellular Ca2+ levels.

scholarly journals A constitutively active mutant of the α1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins Gqα and G11α than the wild-type receptor

1996 ◽  
Vol 320 (1) ◽  
pp. 79-86 ◽  
Author(s):  
Tae Weon LEE ◽  
Alan WISE ◽  
Susanna COTECCHIA ◽  
Graeme MILLIGAN
Keyword(s):  
G Proteins ◽  
Cell Lines ◽  
Adrenergic Receptor ◽  
Inositol Phosphate ◽  
Wild Type ◽  
Down Regulation ◽  
Eta Receptor ◽  
Type Receptor ◽  
In Cells ◽  
Constitutively Active

Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα1B-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins Gq and G11 in cells expressing either the wild-type or the CAMα1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of Gqα/G11α degradation between cells expressing the wild-type or the CAMα1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gq and G11.

scholarly journals Evidence for Rho-mediated Agonist Stimulation of Phospholipase D in Rat1 Fibroblasts

1996 ◽  
Vol 271 (22) ◽  
pp. 13135-13139 ◽  
Author(s):  
Kenneth C. Malcolm ◽  
Cassondra M. Elliott ◽  
John H. Exton
Keyword(s):  
Phospholipase D ◽  
Agonist Stimulation ◽  
Stimulation Of

scholarly journals An Effector Domain Mutant of Arf6 Implicates Phospholipase D in Endosomal Membrane Recycling

2006 ◽  
Vol 17 (1) ◽  
pp. 327-335 ◽  
Author(s):  
Olivera A. Jovanovic ◽  
Fraser D. Brown ◽  
Julie G. Donaldson
Keyword(s):  
Phospholipase D ◽  
Aluminum Fluoride ◽  
Membrane Recycling ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Endosomal Membrane ◽  
Endosomal Membranes ◽  
Stimulation Of ◽  
In Cells

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent α1-subunit of the Na+ pump

2006 ◽  
Vol 290 (4) ◽  
pp. R1142-R1150 ◽  
Author(s):  
Pedro Gomes ◽  
Patrício Soares-da-Silva
Keyword(s):  
Atpase Activity ◽  
Wild Type ◽  
Transfected Cells ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Functional Classes ◽  
A Cell ◽  
And Function ◽  
Α1 Subunit ◽  
Stimulation Of

Vectorial Na+ reabsorption across the proximal tubule is mediated by apical entry of Na+, primarily via Na+/H+ exchanger isoform 3 (NHE3), and basolateral extrusion via the Na+ pump (Na+-K+-ATPase). We hypothesized that regulation of Na+ reabsorption should involve not only the activity of the basolateral Na+-K+-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na+-K+-ATPase, opossum kidney (OK) cells were transfected with the rodent Na+-K+-ATPase α1-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na+-K+-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na+-K+-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC50 values for ouabain: the first value was similar to the IC50 of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum α1-isozymes. It is shown that transfection of OK cells with Na+-K+-ATPase increased Na+-K+-ATPase and NHE3 activities. This was associated with overexpression of the Na+-K+-ATPase α1-subunit and NHE3 in transfected OK cells. The abundance of the Na+-K+-ATPase β1-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na+-K+-ATPase in cells transfected with the rodent Na+ pump α1-subunit cDNA is expected to stimulate apical Na+ influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.

scholarly journals Interactions of the α2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants

1997 ◽  
Vol 321 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Alan WISE ◽  
Marie-Ange WATSON-KOKEN ◽  
Stephen REES ◽  
Melanie LEE ◽  
Graeme MILLIGAN
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Resistant Mutant ◽  
Cysteine Residue ◽  
Gtpase Activity ◽  
Wild Type ◽  
High Affinity ◽  
Agonist Stimulation ◽  
Stimulation Of

The α2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of ‘Gi-like’ pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine α2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1α, Gi2α and Gi3α or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The α2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5ƀ-[γ-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein α-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1α, Gi2α and Gi3α resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys → Gly mutations of Gi1α, Gi2α and Gi3α were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1a was correlated highly both with the level of expression of this G-protein and with the level of expression of the α2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys → Gly mutants of Gi1α, Gi2α and Gi3α required 10Ő15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the α2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

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