Jyoti Disa

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14

Urotensin-II (U-II) is a vasoactive ‘somatostatin-like’ cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 (refs 4, 5) and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

Urotensin‐II (U‐II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U‐II/UT receptor system. The goal of this study was to clone mouse and primate preproU‐II and UT for pharmacological profiling. Monkey and mouse preproU‐II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU‐II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U‐II/UT receptor mRNA in extra‐vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U‐II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654±154 and 214±65 pM and Bmax of 1011±125 and 497±68 fmol mg−1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U‐II isopeptides to these receptors (Ki=0.8 – 3 nM). Fluorescein isothiocyanate (FITC) labelled U‐II, bound specifically to HEK‐293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U‐II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2±0.8 and 1.1±0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2±1.8 and 0.9±0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1α, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.

Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors

This investigation was undertaken to study the mechanisms of calcitonin gene-related peptide (CGRP)-mediated desensitization using recombinant porcine CGRP receptors stably expressed in human embryonic kidney (HEK-293) cells. Pretreatment of these cells with human alphaCGRP resulted in an approximately 60% decrease in CGRP-stimulated adenylyl cyclase activity and an approximately 10-fold rightward shift in the dose-response curve of CGRP. This effect was rapid (t(1/2) approximately 5 min) and was accompanied by a significant decrease in [125I]CGRP binding to membrane preparations from CGRP-pretreated cells. In contrast, CGRP pretreatment had no effect on isoproterenol- or forskolin-stimulated adenylyl cyclase activity in these cells. The potential involvement of protein kinase A or protein kinase C in CGRP-mediated desensitization was studied using selective inhibitors or activators of these kinases. Pretreatment of the cells with forskolin (adenylyl cyclase activator) or phorbol dibutyrate (protein kinase C activator) had no effect on CGRP-mediated adenylyl cyclase activity and did not influence CGRP-mediated desensitization. However, pretreatment of the cells with 2-(8-[(dimethylamino)methyl]-6,7,8, 9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methylindol-3-yl)m aleimide hydrochloride (Ro 32-0432) (a potent inhibitor of protein kinase C) resulted in significant attenuation of CGRP-mediated desensitization with an IC(50) approximately 3 microM. To establish whether this effect might be due to inhibition of other protein kinases by Ro 32-0432, its effect was tested against several G protein-coupled receptor kinases (GRKs). Ro 32-0432 was found to inhibit GRK2, GRK5, and GRK6 with IC(50) values of 29, 3.6, and 16 microM, respectively, suggesting that its effect on CGRP-mediated desensitization might be a result of GRK inhibition. To further test this hypothesis, as well as the potential GRK specificity, the cells were treated with antisense oligonucleotides to GRK2, GRK5, and GRK6. While GRK2 and GRK5 antisense nucleotides had no effect on CGRP-mediated desensitization, the GRK6 antisense nucleotide treatment significantly reversed CGRP-mediated desensitization. These results suggest the involvement of GRK6 in CGRP-mediated desensitization in HEK-293 cells.

Calcitonin gene-related peptide receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+ signaling pathways.

Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological properties including potent vasodilating activity. Recently, we reported the cloning of complementary DNAs (cDNAs) encoding the human and porcine CGRP receptors which share significant amino acid sequence homology with the human calcitonin receptor, a member of the recently described novel subfamily of G-protein-coupled 7TM receptors. Activation of this family of receptors has been shown to result in an increase in intracellular cAMP accumulation and calcium release. In this study, we demonstrate that HEK-293 cells expressing recombinant CGRP receptors (HEK-293HR or PR) respond to CGRP with increased intracellular calcium release (EC50 = 1.6 nM) in addition to the activation of adenylyl cyclase (EC50 = 1.4 nM). The effect of CGRP on adenylyl cyclase activation and calcium release was inhibited by CGRP (8-37), a CGRP receptor antagonist. Both effects were mediated by cholera toxin-sensitive G-proteins, but these two signal transduction pathways were independent of each other. While cholera toxin pretreatment of HEK-293PR cells resulted in permanent activation of adenylyl cyclase, the same pretreatment resulted in an inhibition of CGRP-mediated [Ca2+]i release. Pertussis toxin was without effect on CGRP-mediated responses. In addition, CGRP-mediated calcium release appears to be due to release from a thapsigargin-sensitive intracellular calcium pool. These results show that the recombinant human as well as porcine CGRP receptor can independently increase both cAMP production and intracellular calcium release when stably expressed in the HEK-293 cell line.

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375

1 SB‐706375 potently inhibited [125I]hU‐II binding to both mammalian recombinant and ‘native’ UT receptors (Ki 4.7±1.5 to 20.7±3.6 nM at rodent, feline and primate recombinant UT receptors and Ki 5.4±0.4 nM at the endogenous UT receptor in SJRH30 cells). 2 Prior exposure to SB‐706375 (1 μM, 30 min) did not alter [125I]hU‐II binding affinity or density in recombinant cells (KD 3.1±0.4 vs 5.8±0.9 nM and Bmax 3.1±1.0 vs 2.8±0.8 pmol mg−1) consistent with a reversible mode of action. 3 The novel, nonpeptidic radioligand [3H]SB‐657510, a close analogue of SB‐706375, bound to the monkey UT receptor (KD 2.6±0.4 nM, Bmax 0.86±0.12 pmol mg−1) in a manner that was inhibited by both U‐II isopeptides and SB‐706375 (Ki 4.6±1.4 to 17.6±5.4 nM) consistent with the sulphonamides and native U‐II ligands sharing a common UT receptor binding domain. 4 SB‐706375 was a potent, competitive hU‐II antagonist across species with pKb 7.29–8.00 in HEK293‐UT receptor cells (inhibition of [Ca2+]i‐mobilization) and pKb 7.47 in rat isolated aorta (inhibition of contraction). SB‐706375 also reversed tone established in the rat aorta by prior exposure to hU‐II (Kapp∼20 nM). 5 SB‐706375 was a selective U‐II antagonist with 100‐fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (Ki/IC50>1 μM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin‐1 were unaltered by SB‐706375 (1 μM). 6 In summary, SB‐706375 is a high‐affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross‐species activity that will assist in delineating the pathophysiological actions of U‐II in mammals.

Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines.

In an effort to identify endogenous, native mammalian urotensin‐II (U‐II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU‐II binding sites. UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. [125I]hU‐II bound to a homogenous population of high‐affinity, saturable (Kd 67.0±11.8 pM, Bmax 9687±843 sites cell−1) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (15% dissociation 90 min). A lower density of high‐affinity U‐II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667±165 sites cell−1, Kd 74±8 pM). Consistent with the profile recorded in human recombinant UT‐HEK293 cells, [125I]hU‐II binding to SJRH30 cells was selectively displaced by both mammalian and fish U‐II isopeptides (Kis 0.5±0.1–1.2±0.3 nM) and related analogues (hU‐II[4‐11]>[Cys5,10]Acm hU‐II; Kis 0.4±0.1 and 864±193 nM, respectively). U‐II receptor activation was functionally coupled to phospholipase C‐mediated [Ca2+]i mobilization (EC50 6.9±2.2 nM) in SJRH30 cells. The present study is the first to identify the presence of ‘endogenous’ U‐II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU‐II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione¿, a PI3-kinase inhibitor, attenuated only calcitonin gene-related peptide-induced ERK and not P38 MAPK activation. Thus, these data suggest that activation of ERK by calcitonin gene-related peptide involves a H89-sensitive protein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene-related peptide involves only the H89 sensitive pathway and is independent of PI3 kinase. This also suggests that although both ERK and P38 can be activated by protein kinase A, the distal signaling components to protein kinase A in the activation of these two kinases (ERK and P38) are different.

Receptor activity modifying proteins interaction with human and porcine calcitonin receptor-like receptor (CRLR) in HEK-293 cells

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), two closely related peptides, initiate their biological responses through their interaction with calcitonin receptor-like receptor (CRLR). The CRLR receptor phenotype can be determined by coexpression of CRLR with one of the three-receptor activity modifying proteins (RAMPs). In this report, we characterized the pharmacological properties of the human or porcine CRLR with individual RAMPs transiently expressed in human embroynic kidney cell line (HEK-293). Characterization of RAMP1/human or porcine CRLR combination by radioligand binding ([125I] hαCGRP) and functional assay (activation of adenylyl cyclase) revealed the properties of CGRP receptor. Similarly characterization of RAMP2/human or porcine CRLR and RAMP3/human or porcine CRLR combination by radioligand binding ([125I]rADM) and functional assay (activation of adenylyl cyclase) revealed the properties of ADM (22–52) sensitive-ADM receptor. In addition, porcine CRLR/RAMP2 or 3 combination displayed specific high affinity [125I] hαCGRP binding also. Also, co-transfection of porcine CRLR with RAMPs provided higher expression level of the receptor than the human counterpart. Thus the present study along with earlier studies strongly support the role of RAMPs in the functional expression of specific CRLRs.

Quinine analogs as non-peptide calcitonin gene-related peptide (CGRP) receptor antagonists

Abstract A high-throughput screen identified quinine analog 1 as an antagonist of the human calcitonin generelated peptide (hCGRP) receptor. Thus, compound 1 displaces [125I]-CGRP from the hCGRP receptor and inhibits CGRP-mediated cAMP production with IC50 values of 5.9 ± 1.2 μM and 26 ± 5 μM, respectively. A limited structure-activity study of 1 is described.