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Sökning: WFRF:(Leeb Lundberg L. M.Fredrik)

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1.
  • Leeb-Lundberg, L. M.Fredrik, et al. (författare)
  • Covalent labeling of the cerebral cortex α1-adrenergic receptor with a new high affinity radioiodinated photoaffinity probe
  • Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier. - 0006-291X. ; 115:3, s. 946-951
  • Tidskriftsartikel (refereegranskat)abstract
    • A novel high affinity radioiodinated photoaffinity probe, 4-amino-6,7-dimethoxy-2[4-[5(3-[125I]iodo-4-azidophenyl)pentanoyl]-1-piperazinyl]- quinazoline, structurally related to the potent α1-adrenergic antagonist prazosin, was developed and used to covalently label the rat cerebral cortex α1-adrenergic receptor. In the absence of light, this ligand binds to cortex plasma membranes with a dissociation constant of 308 pM and with a maximal number of binding sites of 200 fmol/mg protein. Upon photolysis, the ligand incorporates irreversibly into plasma membrane proteins. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at Mr = 79,000. The covalent incorporation into the peptide at Mr = 79,000 can be inhibited by several adrenergic receptor ligands with a typical α1-adrenergic receptor specificity and stereoselectivity.
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2.
  • Fathy, Dana B., et al. (författare)
  • Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists. Effect of basal desensitization
  • Ingår i: Journal of Biological Chemistry. - : ASBMB. - 0021-9258. ; 274:42, s. 29603-29606
  • Tidskriftsartikel (refereegranskat)abstract
    • In this report, we show that desensitization regulates ligand- independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.
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3.
  • Leeb, Tosso, et al. (författare)
  • The sixth transmembrane domains of the human B1 and B2 bradykinin receptors are structurally compatible and involved in discriminating between subtype-selective agonists
  • Ingår i: Journal of Biological Chemistry. - : ASBMB. - 0021-9258. ; 272:1, s. 311-317
  • Tidskriftsartikel (refereegranskat)abstract
    • In order to investigate the molecular basis for the ability of the human B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype- selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchanged. The pharmacological profiles of the constructs were analyzed by radioligand binding in particulate preparations of transiently transfected HEK293 cells using the agonist [3H]des-Arg10-kallidin and the antagonist (3H]NPC17731. The ability of these constructs to transmit an intracellular signal was measured in transiently transfected A10 cells, a vascular smooth muscle cell line, by single cell Ca2+ imaging. Substitution of B1 TM-VI into the B2 receptor (B2(B1VI)) dramatically reduced the affinity of the B2-selective agonist BK, whereas the affinity of the B2-selective antagonist NPC17731 was unaltered. High affinity BK binding was fully regained when two residues, Tyr259 and Ala263, near the extracellular surface of TM-VI in B2(B1VI), were replaced with the corresponding residues in the wild-type B2 receptor, which are Phe259 and Thr263. The construct B1(B2VI), produced by substitution of B2 TM-VI into the B1 receptor, did not support high affinity binding of the B1-selective agonist des-Arg10-kallidin. In contrast to BK and des-Arg10-kallidin, the binding of the less subtype-selective agonist kallidin showed little sensitivity to TM-VI exchange. These results show that TM-VI in the human B1 and B2 BK receptor subtypes, although only 36% identical, are structurally compatible. Furthermore, this domain contributes significantly to the ability of these receptors to discriminate between the subtype-selective agonists BK and des-Arg10-kallidin.
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4.
  • Mathis, Sandra A., et al. (författare)
  • B1 and B2 kinin receptors mediate distinct patterns of intracellular Ca2+ signaling in single cultured vascular smooth muscle cells
  • Ingår i: Molecular Pharmacology. - : American Society for Pharmacology and Experimental Therapeutics. - 0026-895X. ; 50:1, s. 128-139
  • Tidskriftsartikel (refereegranskat)abstract
    • Stimulation of B1 and B2 kinin receptors on cultured rabbit superior mesenteric artery smooth muscle cells with des-Arg9-bradykinin (DBK) and bradykinin (BK), respectively, results in significantly different patterns of intracellular Ca2+ mobilization. Single-cell fluorescence imaging of Fura- 2-loaded cells revealed that although both DBK and BK initially triggered similar rapid increases in cytosolic free Ca2+, the DBK response was biphasic and sustained, whereas the BK response was transient. The DBK response was maintained for ≤20 min with the second phase characterized by an elevated plateau and/or base-line oscillations. The BK response was limited to an initial transient peak with the exception of a few cells, which after a prolonged latency period, exhibited weak but regular base-line oscillations. The initial BK- and DBK-stimulated rises in cytosolic free Ca2+ were dependent on the release of Ca2+ from intracellular stores that seemed to be common for the two agonists. On the other hand, the continuation of the sustained phase of the DBK response required the influx of extracellular Ca2+, as well as continuous receptor occupancy by the agonist. Stimulation of cells with DBK followed by washing and restimulation with the same agonist within ≤2 min resulted in a second B1 receptor response that was not significantly different from the first response. In contrast, the same protocol with BK yielded a dramatically decreased second B2 receptor response. This attenuation did not seem to be due to a lack of Ca2+ in the agonist-sensitive intracellular stores because DBK elicited a full response after BK stimulation. This study shows that in single cultured RSMA smooth muscle cells, agonist stimulation of B1 receptors generates a sustained intracellular Ca2+ signal, whereas stimulation of B2 receptors promotes rapid and homologous desensitization, resulting in a transient Ca2+ signal. These distinct receptor-specific patterns of Ca2+ mobilization imply significantly different roles for B1 and B2 kinin receptors in vascular smooth muscle cells.
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5.
  • Mårtensson, Ulrika E A, et al. (författare)
  • Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.
  • 2009
  • Ingår i: Endocrinology. - 1945-7170. ; 150:2, s. 687-98
  • Tidskriftsartikel (refereegranskat)abstract
    • In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.
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6.
  • Cotecchia, Susanna, et al. (författare)
  • Phorbol ester effects on α1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells
  • Ingår i: Life Sciences. - : Elsevier. - 0024-3205. ; 37:25, s. 2389-2398
  • Tidskriftsartikel (refereegranskat)abstract
    • Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on α1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to α1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate α1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating α1-adrenergic receptor functional properties.
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7.
  • De Weerd, Willem F.C., et al. (författare)
  • Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled gα subunits Gα(q) and Gα(i) in caveolae in DDT1 MF-2 smooth muscle cells
  • Ingår i: Journal of Biological Chemistry. - : ASBMB. - 0021-9258. ; 272:28, s. 17858-17866
  • Tidskriftsartikel (refereegranskat)abstract
    • In this report, we show that the vasoactive peptide agonist bradykinin (BK) when bound to B2 BK receptors on DDT1 MF-2 smooth muscle cells promotes the recruitment and sequestration of the occupied receptors and the receptor- coupled G-protein α subunits Gα(q) and Gα(i) in caveolae. Association of ligand receptor complexes and Gα subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae. Caveolin and Gα subunits were monitored by immunoblotting, whereas receptors were monitored as ligand receptor complexes formed by labeling receptors with the agonist BK or the antagonist NPC17731 prior to cell disruption and caveolae enrichment. These complexes were detected with radioligand and by immunoblotting with BK antibodies. A direct interaction of Gα subunits with caveolin was also indicated by their co- immunoprecipitation. Immunoelectron microscopy revealed that the enriched caveolin, Gα subunits, and BK receptor complexes were present in structures of 0.1-0.2 μm. At 4 °C, BK and NPC17731 receptor complexes were detected in caveolae, and both complexes were sensitive to acid washing prior to cell disruption and caveolae enrichment. Elevation of the temperature to 37 °C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling) or 20 min (single-round labeling), and the complexes became acid-resistant. These conditions also increased the amount of Gα(q) and Gα(i) in caveolae with a maximal response at 5-10 min. In contrast, the NPC17731 receptor complexes remained acid-sensitive and dissociated at this temperature, and antagonists did not increase the amount of Gα subunits in caveolae. These results show that some agonists that act through G-protein-coupled receptors promote the association of their receptors and receptor-coupled Gα subunits with caveolae.
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8.
  • Hembree, Timothy N., et al. (författare)
  • Regulation of bradykinin B2 receptors by the ras oncogene : Evidence for multiple mechanisms
  • Ingår i: Journal of Cellular Physiology. - : John Wiley and Sons Inc.. - 0021-9541. ; 169:2, s. 248-255
  • Tidskriftsartikel (refereegranskat)abstract
    • The objective of this study was to probe the molecular mechanisms underlying the increase in sensitivity of cells to bradykinin (BK) following expression of a transforming Ha-ras oncogene. We used native NIH3T3 fibroblast (3T3) cells and 3T3 cells transfected with a glucocorticoid-sensitive oncogenic Ha-ras construct (DT cells). DT cells incubated in the presence of 1 μM dexamethasone (DEX) for 24 hr expressed a relatively high level of membrane-bound Ha-Ras protein, BK B2 receptor mRNA, and B2 receptor binding as determined by Western blotting with anti-Ha-Ras antibodies, reverse transcriptase polymerase chain reaction using B2 receptor-specific primers, and specific [3H]BK binding, respectively. BK also stimulated a significant B2 receptor-mediated increase in [3H]thymidine incorporation in the cells both alone and in synergy with epidermal growth factor. In the absence of DEX, the DT cells expressed a considerably lower but yet clearly significant level of Ha-Ras. Under this condition, receptor mRNA and receptor binding remained maximally expressed. On the other hand, BK was unable to stimulate any increase in [3H]thymidine incorporation. In contrast to DT cells, no Ha-Ras, receptor mRNA, receptor binding, or BK-stimulated, B2 receptor-mediated [3H]thymidine incorporation was detected in 3T3 cells (+/- DEX). However, BK stimulated a transient increase in the level of intracellular free Ca2+ in the 3T3 cells indicating that these cells express a small number of functional B2 receptors. In all, these results show that oncogenic Ha-Ras regulates the sensitivity of 3T3 cells to BK through at least two different mechanisms. One mechanism occurs at a relatively low level of Ha-Ras and involves an increase in B2 receptor mRNA and expressed B2 receptor levels, and another mechanism occurs at a relatively high level of Ha-Ras and involves an increase in B2 receptor-mediated mitogenic signaling.
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9.
  • Kang, Dong Soo, et al. (författare)
  • Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G9/11-mediated phospholipase Cβ activity
  • Ingår i: Molecular Pharmacology. - : American Society for Pharmacology and Experimental Therapeutics. - 0026-895X. ; 62:2, s. 281-288
  • Tidskriftsartikel (refereegranskat)abstract
    • The human B1 bradykinin (BK) receptor (B1R) is more efficacious than the human B2 BK receptor (B2R) in both ligand-independent and agonist-dependent coupling to Gq/11-mediated phospholipase Cβ activity. In fact, B1R is constitutively active, whereas B2R exhibits little if any constitutive activity. To evaluate the role of the C-terminal domain in receptor Gq/11 coupling, we constructed chimeric and C-terminally truncated receptors. The slopes of the increase in basal and agonist-dependent cellular phosphoinositide hydrolysis as a function of receptor density in transiently transfected human embryonic kidney 293 cells provided parameters of receptor coupling. Exchanging the C-terminal domains between the two receptors revealed that these domains are largely responsible for the difference in coupling. B1R truncation showed that this receptor does not directly depend on the C-terminal domain for efficient coupling, although coupling is dramatically augmented by residues in the membrane-distal portion of the domain downstream from Tyr327. On the other hand, coupling of B2R is absolutely dependent on a membrane-proximal epitope in the C-terminal domain upstream from Lys315. This epitope is adjacent to a basic residue, Arg311, which exerts an inhibitory effect on coupling. Arg311 is not conserved in B1R, and complementary mutations in B2R and B1R showed that this residue, together with previously identified serines and threonines, acts to attenuate the coupling efficacy of B2R. Therefore, the C-terminal domain participates intimately in the efficacy of B1R and B2R Gq/11 coupling by contributing both positive and negative regulatory epitopes.
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10.
  • Leeb-Lundberg, L. M.Fredrik, et al. (författare)
  • Antagonists of bradykinin that stabilize a G-protein-uncoupled state of the B2 receptor act as inverse agonists in rat myometrial cells
  • Ingår i: Journal of Biological Chemistry. - : ASBMB. - 0021-9258. ; 269:42, s. 25970-25973
  • Tidskriftsartikel (refereegranskat)abstract
    • Several B2 bradykinin (BK) receptor-specific antagonists including HOE140, NPC17731, and NPC567 exhibited negative intrinsic activity, which was observed as a decrease in basal phosphoinositide hydrolysis in primary cultures of rat myometrial cells, and this response was opposite to that elicited by the agonist BK. The order of potency of the antagonists in attenuating basal activity was essentially the same as that in competing both [3H]BK and [3H]NPC17731 for binding to B2 receptors on both intact rat myometrial cells and bovine myometrial membranes. We previously proposed a three-state model for the binding of agonists to G-protein-coupled B2 receptors in bovine myometrial membranes (Leeb-Lundberg, L. M. F. and Mathis, S. A. (1990) J. Biol. Chem. 265, 9621-9627). This model was based on the ability of BK to promote the sequential formation of three receptor binding states where formation of the third, equilibrium state was blocked by Gpp(NH)p (guanyl-5'-yl imidodiphosphate) identifying it as the G-protein- coupled state of the receptor. Here, we show that, in contrast to BK, these antagonists bound preferentially to a G-protein-uncoupled state of the receptor. These results indicate that B2 receptor antagonists that stabilize a G-protein-uncoupled state of the receptor act as inverse agonists. Furthermore, these results provide strong evidence that endogenous G-protein- coupled receptors exhibit spontaneous activity in their natural environment in the absence of agonist occupancy.
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