Activation of Endothelin ETA Receptors Induces Phosphorylation of α1b-Adrenoreceptors in Rat-1 Fibroblasts

Vázquez‐Prado, José; Medina, Luz del Carmen; García‐Sáinz, J. Adolfo

doi:10.1074/jbc.272.43.27330

Cited by 62 publications

(112 citation statements)

References 48 publications

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“…Although our data do not support a specific role of PKC "feedback" in agonistinduced desensitization of ␣ 1a ARs, this second messenger-dependent kinase may play a role in modulating the ability of ␣ 1a ARs to signal following stimulation of other PLC-coupled receptors within the same cell. This hypothesis is supported by recent demonstrations that stimulation of endothelin ET A receptors or bradykinin B 2 receptors in rat-1 fibroblasts leads to a marked increase in phosphorylation of stably expressed ␣ 1b ARs (32,33).…”

Section: Discussionsupporting

confidence: 68%

Acute Agonist-mediated Desensitization of the Human α1a-Adrenergic Receptor Is Primarily Independent of Carboxyl Terminus Regulation

Price¹,

Morris²,

Biswas³

et al. 2002

Journal of Biological Chemistry

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Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on ␣ 1a -adrenergic receptor (␣ 1a AR) signaling and function. Regulatory mechanisms are likely complex since 12 distinct human ␣ 1a AR carboxyl-terminal splice variants have been isolated. After determining the predominance of the ␣ 1a-1 AR isoform in human heart and prostate, we stably expressed an epitope-tagged ␣ 1a-1 AR cDNA in rat-1 fibroblasts and subsequently examined regulation of signaling, phosphorylation, and internalization of the receptor. Human ␣ 1a AR-mediated inositol phosphate signaling is acutely desensitized in response to both agonist and phorbol 12-myristate 13-acetate (PMA) exposure. Concurrent with desensitization, ␣ 1a ARs in 32 P ilabeled cells are rapidly phosphorylated in response to both NE and PMA stimulation. Despite the ability of PKC to desensitize ␣ 1a ARs when directly activated with PMA, inhibitors of PKC have no effect on agonist-mediated desensitization. In contrast, involvement of GRK kinases is suggested by the ability of GRK2 to desensitize ␣ 1a ARs. Internalization of cell surface ␣ 1a ARs also occurs in response to agonist stimulation (but not PKC activation), but is initiated more slowly than receptor desensitization. Significantly, deletion of the ␣ 1a AR carboxyl terminus has no effect on receptor internalization or either agonist-induced or GRK-mediated receptor desensitization. Because mechanisms underlying acute agonist-mediated regulation of human ␣ 1a ARs are primarily independent of the carboxyl terminus, they may be common to all functional ␣ 1a AR isoforms.␣ 1a -Adrenergic receptors (␣ 1a ARs) 1 are G protein-coupled receptors (GPCR) that mediate sympathetic nervous system responses such as smooth muscle contraction and myocardial inotropy (1). NE stimulation of ␣ 1 ARs predominantly activates G q and results in membrane polyphosphoinositide hydrolysis by activation of phospholipase C␤; the resultant second messengers IP 3 and DAG mobilize intracellular calcium and activate protein kinase C (PKC), respectively (2). Three ␣ 1 AR subtypes (␣ 1a , ␣ 1b , and ␣ 1d ) have been cloned and pharmacologically characterized in several expression systems (for review, see Ref.2). Clinically, activation of ␣ 1a ARs has been implicated in the dynamic component of benign prostatic hyperplasia leading to bladder outlet obstruction and in the development of myocardial hypertrophy (3-5). Notwithstanding the importance of ␣ 1a ARs in several pathophysiological states, surprising little is known about mechanisms underlying ␣ 1a AR expression and function. Transcriptional mechanisms unique to ␣ 1a ARs have been shown to be important in maintaining full ␣ 1 AR responsiveness to agonist in rat neonatal myocytes where long term (24 -72 h) NE stimulation leads to up-regulation ␣ 1a AR mRNA and receptor protein expression, concurrent with down-regulation of ␣ 1b and ␣ 1d AR subtypes (5, 6). While important, such long-term studies do not examine...

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Section: Discussionsupporting

confidence: 68%

Acute Agonist-mediated Desensitization of the Human α1a-Adrenergic Receptor Is Primarily Independent of Carboxyl Terminus Regulation

Price¹,

Morris²,

Biswas³

et al. 2002

Journal of Biological Chemistry

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“…It is possible that this phosphorylation relates to homologous or heterologous desensitization of the TP by other platelet agonists (32,41). Examples of heterologous phosphorylation of GPCRs include endothelin-dependent phosphorylation of ␣ 1B -adrenoreceptors (42) and thrombin-dependent phosphorylation of the prostacyclin receptor (43).Our results suggest that I-BOP, thrombin, and PMA-induced TP␣ phosphorylation were dependent on PKC because specific PKC inhibitors suppressed TP␣ phosphorylation.…”

Section: Discussionsupporting

confidence: 47%

Phosphorylation of the Thromboxane Receptor α, the Predominant Isoform Expressed in Human Platelets

Habib¹,

FitzGerald²,

Maclouf³

1999

Journal of Biological Chemistry

124

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A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, ␣ and ␤. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoformspecific antibodies permitted detection of TP␣, but not TP␤, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50 -60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid (ϳ30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TP␣ phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TP␣ phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF 2␣ -III induced platelet shape change but not TP␣ phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TP␣ was blocked by protein kinase C inhibitors. TP␣ was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF 2␣ -III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TP␣ in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TP␣.

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“…The effect of LDTs on human a 1 -adrenoceptor subtypes was determined by measuring intracellular Ca 21 in rat-1 fibroblasts stably expressing a 1A -, a 1B -, or a 1D -adrenoceptors (Vázquez-Prado et al, 1997). These cells were cultured in high-glucose Dulbecco's modified Eagle's medium with L-glutamine supplemented with 10% fetal bovine serum, 300 mg/ml neomycin analog G418 sulfate, 100 mg/ml streptomycin, 100 U/ml penicillin, and 0.25 mg/ml amphotericin B, at 37°C, and under a 5% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

“…To confirm the antagonistic properties of LDT3 and LDT5 toward different human a 1 -adrenoceptor subtypes, we used a functional assay based on [Ca 21 ]i elevation in Rat-1 cells overexpressing human a 1A -, a 1D -, or a 1B -adrenoceptors (Vázquez-Prado et al, 1997). In Rat-1 cells expressing a 1D -adrenoceptors, stimulation with 100 mM phenylephrine typically induced a pronounced and transient increase in [Ca 21 ]i levels (Fig.…”

Section: Ldt3 Ltd5mentioning

confidence: 99%

New Multi-target Antagonists of 1A-, 1D-Adrenoceptors and 5-HT1A Receptors Reduce Human Hyperplastic Prostate Cell Growth and the Increase of Intraurethral Pressure

Nascimento-Viana

Carvalho

Nasciutti

et al. 2015

Journal of Pharmacology and Experimental Therapeutics

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Benign prostatic hyperplasia (BPH) is characterized by stromal cell proliferation and contraction of the periurethral smooth muscle, causing lower urinary tract symptoms. Current BPH treatment, based on monotherapy with a 1A -adrenoceptor antagonists, is helpful for many patients, but insufficient for others, and recent reports suggest that stimulation of a 1D -adrenoceptors and 5-hydroxytryptamine (serotonin) (5-HT) 1A receptors contributes to cell proliferation. In this study, we investigated the potential of three N-phenylpiperazine derivatives (LDT3, LDT5, and LDT8) as multi-target antagonists of BPH-associated receptors. The affinity and efficacy of LDTs were estimated in isometric contraction and competition-binding assays using tissues (prostate and aorta) and brain membrane samples enriched in specific on-or off-target receptors. LDTs' potency was estimated in intracellular Ca 21 elevation assays using cells overexpressing human a 1 -adrenoceptor subtypes. The antiproliferative effect of LDTs on prostate cells from BPH patients was evaluated by viable cell counting and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays. We also determined LDTs' effects on rat intraurethral and arterial pressure. LDT3 and LDT5 are potent antagonists of a 1A -, a 1D -adrenoceptors, and 5-HT 1A receptors (K i values in the nanomolar range), and fully inhibited phenylephrine-and 5-HT-induced proliferation of BPH cells. In vivo, LDT3 and LDT5 fully blocked the increase of intraurethral pressure (IUP) induced by phenylephrine at doses (ED 50 of 0.15 and 0.09 mg.kg 21 , respectively) without effect on basal mean blood pressure. LDT3 and LDT5 are multi-target antagonists of key receptors in BPH, and are capable of triggering both prostate muscle relaxation and human hyperplastic prostate cell growth inhibition in vitro. Thus, LDT3 and LDT5 represent potential new lead compounds for BPH treatment.

show abstract

Activation of Endothelin ETA Receptors Induces Phosphorylation of α1b-Adrenoreceptors in Rat-1 Fibroblasts

Cited by 62 publications

References 48 publications

Acute Agonist-mediated Desensitization of the Human α1a-Adrenergic Receptor Is Primarily Independent of Carboxyl Terminus Regulation

Acute Agonist-mediated Desensitization of the Human α1a-Adrenergic Receptor Is Primarily Independent of Carboxyl Terminus Regulation

Phosphorylation of the Thromboxane Receptor α, the Predominant Isoform Expressed in Human Platelets

New Multi-target Antagonists of 1A-, 1D-Adrenoceptors and 5-HT1A Receptors Reduce Human Hyperplastic Prostate Cell Growth and the Increase of Intraurethral Pressure

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