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Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart.

ABSTRACT: BACKGROUND AND PURPOSE During the past decade, a few GPCRs have been characterized at the nuclear membrane where they exert complementary physiological functions. In this study, we investigated (1) the presence of a functional urotensin-II (U-II) receptor (UT) in rat heart nuclear extracts and (2) the propensity of U-II and U-II-related peptide (URP) to cross the plasma membrane in a receptor-independent manner. EXPERIMENTAL APPROACH Biochemical and pharmacological methods including competitive binding assays, photoaffinity labelling, immunoblotting as well as de novo RNA synthesis were used to characterize the presence of functional UT receptors in rat heart nuclei. In addition, confocal microscopy and flow cytometry analysis were used to investigate the cellular uptake of fluorescent U-II and URP derivatives. KEY RESULTS The presence of specific U-II binding sites was demonstrated in rat heart nuclear extracts. Moreover, such subcellular localization was also observed in monkey heart extracts. In vitro transcription initiation assays on rat, freshly isolated, heart nuclei suggested that nuclear UT receptors are functional, and that U-II, but not URP, participates in nuclear UT-associated gene expression. Surprisingly, hU-II and URP efficiently crossed the plasma membrane in a receptor-independent mechanism involving endocytosis through caveolin-coated pits; this uptake of hU-II, but not that of URP, was dependent on extracellular pH. CONCLUSION Our results suggest that (1) U-II and URP can differentially modulate nuclear UT functions such as gene expression, and (2) both ligands can reach the internal cellular space through a receptor-independent mechanism.

SUBMITTER: Doan ND

PROVIDER: S-EPMC3415652 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart.

Doan N D ND Nguyen T T M TT Létourneau M M Turcotte K K Fournier A A Chatenet D D

British journal of pharmacology 20120501 1

BACKGROUND AND PURPOSE During the past decade, a few GPCRs have been characterized at the nuclear membrane where they exert complementary physiological functions. In this study, we investigated (1) the presence of a functional urotensin-II (U-II) receptor (UT) in rat heart nuclear extracts and (2) the propensity of U-II and U-II-related peptide (URP) to cross the plasma membrane in a receptor-independent manner. EXPERIMENTAL APPROACH Biochemical and pharmacological methods including competitive ...[more]

PMID: 22044114

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Project description:Urotensin-II (U-II) has been shown to be the most potent mammalian vasoconstrictor known. Thus, a U-II antagonist might be of therapeutic value in a number of cardiovascular disorders. However, interspecies variability of several nonpeptidic ligands complicates the interpretation of in vivo studies of such antagonists in preclinical animal disease models. ACT058362 is a selective antagonist for the human U-II receptor (hUT2R) with a reported K(d) value of approximately 4 nM in a molecular binding assay, but it is reported to bind weakly to rat UT2R (rUT2R), with a K(d) value of approximately 1 500 nM. In contrast, the arylsulphonamide SB706375 is a selective antagonist against both hUT2R (K(d)= approximately 9 nM) and rUT2R (K(d)= approximately 21 nM). To understand the species selectivity of the UT2R, we investigated the binding site of ACT058362 and SB706375 in both hUT2R and rUT2R to explain the dramatically lower (approximately 400-fold) affinity of ACT058362 for rUT2R and the similar affinity (approximately 10 nM) of SB706375 for both UT2Rs. These studies used MembStruk and MSCDock to predict the UT2R structure and the binding site of ACT058362 and SB706375. Based on binding energies, we found two binding modes each with D130(3.32) as the crucial anchoring point (Ballesteros-Weinstein numbering given in superscript). We predict that ACT058362 (an aryl-amine-aryl or ANA ligand) binds in the transmembrane (TM) 3456 region, while SB706375 (an aryl-aryl-amine or AAN ligand) binds in the TM 1237 region. These predicted sites explain the known differences in binding of the ANA ligand to rat and human receptors, while explaining the similar binding of the AAN compound to rat and human receptors. Moreover the predictions explain currently available structure-activity relationship (SAR) data. To further validate the predicted binding sites of these ligands in hUT2R and rUT2R, we propose several mutations that would help define the structural origins of differential responses between UT2R of different species, potentially indicating novel UT2R antagonists with cross-species high affinity.

Project description:New findingsWhat is the central question of this study? Urotensin II is upregulated in patients in the later stages of chronic kidney disease (CKD), particularly in individuals requiring dialysis. Could treatment with a urotensin II receptor antagonist slow progression of renal disease? What is the main finding and its importance? In the rat, expression of urotensin II and its receptor increased, extending into cortical structures as CKD progressed towards end-stage renal failure. Subchronic treatment with a urotensin receptor antagonist slowed but did not prevent progression of CKD. This suggests that urotensin II contributes to the decline in renal function in CKD.AbstractElevated serum and urine urotensin II (UII) concentrations have been reported in patients with end-stage chronic kidney disease (CKD). Similar increases in UII and its receptor, UT, have been reported in animal models of CKD, but only at much earlier stages of renal dysfunction. The aim of this study was to characterize urotensin system expression as renal disease progresses to end-stage failure in a ⅚ subtotal nephrectomy (SNx) rat model. Male Sprague-Dawley rats underwent SNx or sham surgery and were killed at 8 weeks postsurgery [early (E)] or immediately before end-stage renal failure [30 ± 3 weeks postsurgery; late (L)]. Systolic blood pressure, urinary albumin:creatinine ratio and glomerulosclerosis index were all increased in SNx-E rats compared with sham-E by 8 weeks postsurgery. These changes were associated with an increase in renal immunoreactive UII staining but little change in UT expression. As CKD progressed to end-stage disease in the SNx-L group, markers of renal function deteriorated further, in association with a marked increase in immunoreactive UII and UT staining. Subchronic administration of a UT antagonist, SB-611812, at 30 mg kg-1 day-1 for 13 weeks, in a separate group of SNx rats resulted in a 2 week delay in the increase in both systolic blood pressure and urinary albumin:creatinine ratio observed in vehicle-treated SNx but did not prevent the progression of renal dysfunction. The urotensin system is upregulated as renal function deteriorates in the rat; UT antagonism can slow but not prevent disease progression, suggesting that UII plays a role in CKD.

Dataset Information

Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart.

Publications

Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart.

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