Urotensin II Receptor Research Articles

Urantide alleviates monocrotaline induced pulmonary arterial hypertension in Wistar rats

Pulmonary arterial hypertension (PAH) is a serious disorder with poor prognosis. Urotensin II (UII) has been confirmed to be powerful vasoconstrictor than endothelin-1, which may play an important role in PAH development. The aim of this study is to observe the effects of urantide, a UII receptor antagonist， on monocrotaline (MCT) induced PAH in rats. 60 male Wistar rats were divided into six groups. For early treatment experiment, rats were divided into normal control group, MCT 4w model group (MCT + saline × 3wks from the 8th day of MCT injection) and urantide early treatment group (MCT + urantide 10 μg/kg/d × 3wks, 1 week after MCT injection once). For late treatment experiment, rats were divided as controls, MCT 6w model group (MCT + saline × 2wks, 4 weeks after MCT injection once) and urantide late treatment group (MCT + urantide 10 μg/kg/d × 2wks, 4 weeks after MCT injection once). At the end of experiments, mean pulmonary arterial pressures (mPAP) and mean blood pressure (MBP) of rats in each group were measured by catheterization. Right ventricular weight ratio was also weighed. Relaxation effects of urantide on intralobar pulmonary arterial rings of normal control and MCT 4w model rats were investigated. Pulmonary artery remodeling was detected by hematoxylin and eosin (HE) staining and immunohistochemistry analysis. Serum nitric oxide (NO) levels in all six groups were assayed by ELISA kits. Urantide markedly reduced the mPAP levels of MCT induced PAH in both early and late treatment groups. It didn’t change the MBP. Urantide dose-dependently relaxed the pulmonary arterial rings of normal control and MCT 4w model rats. Moreover, N G-Nitro- l-arginine Methyl Ester ( l-NAME) blocked the dilation response induced by urantide. In addition, urantide inhibited the pulmonary vascular remodeling remarkably. Serum NO level elevated in both early and late treatment rats with urantide infusion. These results suggest that urantide effectively alleviated MCT induced rats PAH may through relaxing pulmonary arteries and inhibiting pulmonary vascular remodeling. NO pathway might be one of the mechanisms in urantide induced pulmonary artery dilation. Thus, it is expected that urantide may be a novel therapy for PAH.

Potent cardiovascular effects of homologous urotensin II (UII)-related peptide and UII in unanesthetized eels after peripheral and central injections

We cloned cDNAs encoding urotensin II (UII)-related peptide (URP) and UII in Japanese eel, Anguilla japonica, the former being the first such cloning in teleost fishes. Unlike the exclusive expression of UII in the urophysis, the URP gene was expressed most abundantly in the brain (medulla oblongata) followed by the urophysis. Peripheral injections of URP into eels increased blood pressure by 16.1 ± 0.8 mmHg at 0.1 nmol/kg in ventral aortic blood pressure (P(VA)) and with similar potency and efficacy to that of UII (relative potency of URP to UII = 0.83). URP/UII and ANG II preferentially acted on the branchial and systemic circulations, respectively, and the duration of effect was distinct among the three peptides in the order of UII (60 min) >URP (30 min) >ANG II (14 min) in P(VA). Urantide, a mammalian UII receptor antagonist, inhibited the URP effect (-63.6 ± 5.2%) to a greater extent than for UII (-39.9 ± 5.0%). URP and UII constricted isolated eel branchial and systemic arteries, showing their direct actions on the vascular smooth muscle. Central injection of URP increased blood pressure by 12.3 ± 0.8 mmHg at 50 pmol/eel in P(VA) and with similar efficacy but less potency (relative potency = 0.47) and shorter duration compared with UII. The central actions of URP/UII were more potent on the branchial circulation than on the systemic circulation, again opposite the effects of ANG II. The similar responses to peripheral and central injections suggest that peripheral hormones may act on the brain. Taken together, in eels, URP and UII are potent cardiovascular hormones like ANG II, acting directly on the peripheral vasculature, as well as a central vasomotor site, and their actions are mediated to different degrees by the UII receptor.

Urotensin-II receptor antagonism does not improve renal haemodynamics or function in rats with endotoxin-induced acute kidney injury

1. Urotensin-II (U-II) is a vasoactive peptide that influences renal haemodynamics and kidney function. The aim of the present study was to examine the effects of the selective U-II receptor antagonist, urantide, on renal haemodynamics, oxygenation and function in endotoxaemic rats. 2. Endotoxaemia was induced in Sprague-Dawley rats by an intraperitoneal dose of lipopolysaccharide (LPS; Escherichia coli O127:B8, 7.5 mg/kg). At 16 h after endotoxin was given, renal clearance experiments were carried out in thiobutabarbital anaesthetized rats. Group 1, sham-saline; group 2, sham-urantide; group 3 LPS-saline; and group 4, LPS-urantide received isotonic saline or urantide (0.2 mg/kg bolus intravenously, followed by an infusion of 1.2 mg/kg/h throughout) after baseline measurements. Kidney function, renal blood flow (RBF), and cortical and outer medullary perfusion (laser-Doppler flowmetry) and oxygen tension (Clark-type microelectrodes) were analysed during 2 h of drug administration. 3. At baseline, endotoxaemic rats showed approximately 50% reductions in glomerular filtration rate (GFR) and RBF (P < 0.05), a decline in cortical and outer medullary perfusion and pO(2) (P < 0.05), and a significant increase in mean arterial pressure (MAP; P < 0.05) compared with saline-injected controls. In sham animals, urantide in a dose that did not significantly influence MAP or RBF, increased GFR (P < 0.05 time × treatment interaction) and filtration fraction (P < 0.05 treatment effect). However, urantide had no statistically significant effects on any of the investigated variables in endotoxaemic rats. 4. These findings show that U-II, through the UT receptor, does not contribute to abnormalities in renal haemodynamics and function in endotoxaemic rats.

E0159 Osteopontin is involved in urotensin II induced migration of adventitial fibroblasts from rat aorta

BackgroundRecent studies suggest osteopontin (OPN) plays a critical role in the progression of vascular remodelling, and that Urotensin II (UII) is a potent vasoconstrictor and stimulator of cellular migration. The...

E0156 Urotensin II promotes monocyte chemoattractant protein-1 expression in aortic adventitial fibroblasts of rat

BackgroundRecent studies reported that vascular adventitial fibroblasts (AFs) are involved in the development of vascular inflammatory diseases, such as atherosclerosis. Urotensin II (UII), a potent vasoconstrictive peptide, could stimulate phenotype...

Expression of urotensin II and its receptor in human lung adenocarcinoma A549 cells and the effect of urotensin II on lung adenocarcinoma growth in vitro and in vivo

Urotensin II (UII), originally identified from fish urophysis, is a potent vasoactive peptide and an endogenous ligand for an orphan G protein-coupled receptor GPR14, now named as urotensin II receptor (UT-R). In this study, we investigated the mRNA and protein expressions of UII and its receptor (UT-R) in human lung adenocarcinoma A549 cells, and the effect of exogenous UII on the proliferation of A549 cells in vitro and in vivo. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis showed that both mRNAs and proteins of UII and UT-R were obviously expressed in human lung adenocarcinoma A549 cells. Immunohistochemical analysis showed that UII peptide was mainly expressed in the cyto-plasm, and UT-R protein was expressed on the cytomembrane and also in the cytoplasm. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) analysis demonstrated that treatment with different concentrations of human UII (10(-9), 10(-8), 10(-7) and 10(-6) M) for 48 h significantly increased the number of A549 cells. The effect of UII at the concentration of 10(-7) M on the proliferation of A549 cells is most pronounced. Nude mice bearing human lung adenocarcinoma A549 cells treated with UII showed a significant increase in tumor volume and tumor weight compared with control group. These findings suggest that UII may contribute to the pathogenesis of human lung adenocarcinoma as an autocrine/paracrine growth stimulating factor.

Urotensin II receptor antagonist attenuates monocrotaline-induced cardiac hypertrophy in rats

Urotensin II (UII) is a vasoactive peptide with potent cardiovascular effects through a G protein-coupled receptor. Hypoxia stimulates the secretion of UII and atrial natriuretic peptide (ANP). However, the effect of UII on hypoxia-induced cardiac hypertrophy is still controversial. The present study was conducted to determine whether human UII (hUII)-mediated ANP secretion influences hypoxia-induced cardiac hypertrophy using in vitro and in vivo models. Hypoxia caused an increase in ANP secretion and a decrease in atrial contractility in isolated perfused beating rat atria. hUII (0.01 and 0.1 nM) attenuated hypoxia-induced ANP secretion without changing the atrial contractility, and the hUII effect was mediated by the UII receptor signaling involving phospholipase C, inositol 1,3,4 trisphosphate receptor, and protein kinase C. Rats treated with monocrotaline (MCT, 60 mg/kg) showed right ventricular hypertrophy with increases in pulmonary arterial pressure and its diameter and plasma levels of UII and ANP that were attenuated by the pretreatment with an UII receptor antagonist, urantide. An acute administration of hUII (5 μM injection plus 2.5 μM infusion for 15 min) decreased the plasma ANP level in MCT-treated rats but increased the plasma ANP level in MCT plus urantide-treated and sham-operated rats. These results suggest that hUII may deteriorate MCT-induced cardiac hypertrophy mainly through a vasoconstriction of the pulmonary artery and partly through the suppression of ANP secretion.

GSK1562590, a slowly dissociating urotensin‐II receptor antagonist, exhibits prolonged pharmacodynamic activity ex vivo

Recently identified antagonists of the urotensin-II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity. The pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays. GSK1440115 (pK(i)= 7.34-8.64 across species) and GSK1562590 (pK(i)= 9.14-9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA(2)= 5.59-7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pK(b)= 8.93-10.12 across species), but a competitive antagonist in monkey arteries (pK(b)= 8.87-8.93). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT. Whereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.

Ligand-Supported Purification of the Urotensin-II Receptor

A crucial limitation for structural and biophysical analysis of G protein-coupled receptors (GPCRs) is the inherent challenge of purifying and stabilizing these receptors in an active (agonist-bound) conformation. Peptide ligands, such as the vasoactive, cyclic hormone urotensin-II (U-II), may provide new purification tools, via high affinity, pseudo-irreversible binding suitable for ligand-based affinity purification. We show that the U-II receptor (UT) is resistant to desensitization as a result of low phosphorylation and diminished endocytosis. UT also displays an unusual proclivity to remain active with vasoconstriction sustained despite extensive washout of the ligand. To exploit these properties for ligand-supported purification, we modified the U-II ligand by attaching a biotin moiety and spacer arm to the N terminus, creating a novel affinity ligand (Bio-U-II) to interface with streptavidin media. Bio-U-II bound to UT with pharmacological properties analogous to those of the unmodified U-II ligand (high-affinity, pseudo-irreversible binding). The prebinding of Bio-U-II to UT (before exposure to detergent) facilitated specific capture of UT by stabilizing the receptor structure during solubilization with detergent. Solubilization of UT with the most compatible detergent, n-dodecyl β-d-maltoside, was dependent on the critical micelle concentration, and Gα(q/11) protein was copurified with captured Bio-U-II-UT complexes. Furthermore, captured Bio-U-II-UT complexes were resistant to dissociation at elevated temperatures, suggesting that UT is relatively thermostable, making it an ideal candidate for future structural and biophysical studies. This work demonstrates the utility of pseudo-irreversible ligands to support the purification of a GPCR during detergent extraction, resulting in the first successful purification of the UT.

A rat brain atlas of urotensin-II receptor expression and a review of central urotensin-II effects

Urotensin-II (U-II) is an 11-amino acid cyclic peptide which exerts its actions through a G(q) protein-coupled receptor, UT. Much of the research focus of U-II is as a peptide of the periphery, particularly cardiovascular. Despite this, U-II was originally identified as a neuropeptide, and its expression is broad throughout the central nervous system. This brief review article catalogs the known sites of expression of UT within the CNS in the form of a diagrammatic rat brain atlas. Furthermore, the functional consequences of UT activation within specific brain regions are discussed along with the likely actions of synthetic UT ligands. Areas of high, medium, and low expression include the arcuate, paraventricular, and pedunculopontine tegmental nuclei, respectively. In the arcuate and paraventricular nuclei, where expression is high and moderate, U-II produces a pressor/tachycardic response in the former and a weaker response in the latter. Based on the known pharmacology of UT ligands (and assuming density is the primary determinant of efficacy in this case), we predict a weak response in the arcuate nucleus and possible antagonism of endogenous U-II response in the paraventricular nucleus by a low-efficacy partial agonist. These predicted responses lend themselves to relatively simple experimental verification.

Urotensin II induction of adult cardiomyocytes hypertrophy involves the Akt/GSK-3β signaling pathway

Urotensin II (UII) a potent vasoactive peptide is upregulated in the failing heart and promotes cardiomyocytes hypertrophy, in particular through mitogen-activated protein kinases. However, the regulation by UII of GSK-3beta, a recognized pivotal signaling element of cardiac hypertrophy has not yet been documented. We therefore investigated in adult cardiomyocytes, if UII phosphorylates GSK-3beta and Akt, one of its upstream regulators and stabilizes beta-catenin, a GSK-3beta dependent nuclear transcriptional co-activator. Primary cultures of adult rat cardiomyocytes were stimulated for 48h with UII. Cell size and protein/DNA contents were determined. Phosphorylated and total forms of Akt, GSK-3beta and the total amount of beta-catenin were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the chemical phosphatidyl-inositol-3-kinase inhibitor, LY294002, and urantide, a competitive UII receptor antagonist. UII increased cell size and the protein/DNA ratio, consistent with a hypertrophic response. UII also increased phosphorylation of Akt and its downstream target GSK-3beta. beta-Catenin protein levels were increased. All of these effects of UII were prevented by LY294002, and urantide. The UII-induced adult cardiomyocytes hypertrophy involves the Akt/GSK-3beta signaling pathways and is accompanied by the stabilization of the beta-catenin. All these effects are abolished by competitive inhibition of the UII receptor, consistent with new therapeutic perspectives for heart failure treatment.

Urotensin-II Receptor Modulators as Potential Drugs

Amino acidresidues 5-10 of human U-II are contained in a disulfide-linked cyclic array, [CFWKYC], which is highly species-con-served,whereastheN-terminalregionfromotherspeciesdiffersin length and constitution (Figure 1). The first U-II peptide (a12-mer) was isolated from the urophysis of the goby fish(Gillichthys mirabilis) as a smooth muscle contractile sub-stance.

The pro-angiogenic activity of urotensin-II on human vascular endothelial cells involves ERK1/2 and PI3K signaling pathways

Human vascular endothelial cells express the urotensin-II (U-II) receptor and exhibit a strong in vitro angiogenic response to the peptide. Thus, in the present study an in vitro model, based on human umbilical vein endothelial cells (HUVEC) cultured on Matrigel, was used to characterize more in detail the signaling pathways that control the pro-angiogenic action of U-II. The activation of the U-II receptor (UT) was associated with an increase of intracellular calcium concentration. Both calcium rise and pro-angiogenic effect of the peptide can be blocked by U73122, a selective inhibitor of phospholipase-C, indicating that the signal transduction from UT mainly involves the phospholipase-C/IP 3 pathway. As far as the downstream signaling pathways are concerned, western blot analyses and experiments with specific inhibitors indicated that the U-II-induced self-organization of the cells into capillary-like structures was PKC dependent and involved the activation of the ERK1/2, but not p38-MAPK, transduction pathway. Interestingly, the pharmacological inhibition of PI3K (obtained with LY294002), hindered the capacity of U-II to induce a proangiogenic effect on HUVEC, suggesting that PI3K-dependent pathways also play a role in regulating the process.

Effect of the urotensin-II receptor antagonist palosuran on secretion of and sensitivity to insulin in patients with Type 2 diabetes mellitus.

To investigate the effects of palosuran, a nonpeptidic, potent and selective antagonist of the urotensin-II receptor, on insulin and glucose regulation in 20 diet-treated patients with Type 2 diabetes mellitus in a double-blind, placebo-controlled, randomized, crossover, proof-of-concept study. After 4 weeks' oral treatment with 125 mg palosuran or placebo b.i.d.,effects on insulin secretion and sensitivity and blood glucose levels were assessed by means of a hyperglycaemic glucose clamp, meal tolerance test, homeostasis model assessment-insulin resistance score, and daily self-monitoring of blood glucose. Plasma concentrations of palosuran were determined for 12 h on the last day of intake. Palosuran did not affect second-phase insulin response (primary end-point) during the hyperglycaemic glucose clamp in comparison with placebo [paired difference of -1.8 microU ml(-1), 95% confidence interval (CI) -7.8, 4.2]. Likewise, no effects of palosuran were detected on the first-phase insulin response, or on insulin secretion and blood glucose levels during the meal tolerance test or on homeostasis model assessment-insulin resistance score. No clinically significant effects on daily blood glucose profiles were observed during the study. Geometric mean C(max) and AUC(tau) (95% CI) and median t(max) (range) in this patient population were 180 ng ml(-1) (125, 260), 581 ng.h ml(-1) (422, 800) and 3.0 h (0.67, 4.3), respectively. The results of this study indicate that antagonism of the urotensin-II system does not influence insulin secretion or sensitivity or daily blood glucose levels in diet-treated patients with Type 2 diabetes.

Nonpeptide Urotensin-II Receptor Antagonists: A New Ligand Class Based on Piperazino-Phthalimide and Piperazino-Isoindolinone Subunits

We have discovered two related chemical series of nonpeptide urotensin-II (U-II) receptor antagonists based on piperazino-phthalimide (5 and 6) and piperazino-isoindolinone (7) scaffolds. These structure types are distinctive from those of U-II receptor antagonist series reported in the literature. Antagonist 7a exhibited single-digit nanomolar potency in rat and human cell-based functional assays, as well as strong binding to the human U-II receptor. In advanced pharmacological testing, 7a blocked the effects of U-II in vitro in a rat aortic ring assay and in vivo in a rat ear-flush model. A discussion of U-II receptor antagonist pharmacophores is presented, and a specifically defined model is suggested from tricycle 13, which has a high degree of conformational constraint.

Urotensin II Receptor Knockout Mice on an ApoE Knockout Background Fed a High-Fat Diet Exhibit an Enhanced Hyperlipidemic and Atherosclerotic Phenotype

Expression of the vasoactive peptide Urotensin II (UII) is elevated in a number of cardiovascular diseases. Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis. UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice. We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor-mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.

Identification of new agonists of urotensin-II from a cyclic peptide library

Urotensin-II (UT-II) is thought to be involved in the regulation of cardiovascular homeostasis and pathology. A head-to-tail cyclic hexapeptide library based on UT-II sequence was designed, synthesized, and evaluated by the activity on the UT-II receptor (GPR-14). A new synthetic sequence, WK[Xaa] (Xaa: amino acid with aromatic side chain), was identified as a characteristic minimum fragment activating hUT-II receptor instead of the WK[Y] sequence. Compound 1 showed an agonistic activity with an EC 50 value of 6.94 nM. The conformational investigation suggested that 1 did not have typical secondary structure in the message sequence. Structural analyses may enable us to investigate the active conformation of UT-II and lead to the identification of new ligands for GPR-14.

Generation of novel, potent urotensin-II receptor antagonists by alkylation–cyclization of isoindolinone C3-carbanions

We report a facile alkylation–cyclization reaction involving the isoindolinone C3 position, which resulted in tricyclic derivatives 2 and 10 in 48% and 32% yields, respectively. These novel compounds possess potent urotensin-II receptor antagonist activity.

New Insight into the Binding Mode of Peptide Ligands at Urotensin-II Receptor: Structure−Activity Relationships Study on P5U and Urantide

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized several analogues of P5U and urantide in which the Asp(4) residue in N-terminus position was replaced with coded and noncoded amino acids. The replacement of the Asp(4) residue by Tic led to an analogue, compound 14, more potent as antagonist (pK(B) = 8.94) compared to urantide. Furthermore, a different SAR was observed for the P5U compared to the urantide analogues. NMR and docking studies revealed a different binding mode for the agonist and antagonist ligands which could explain the observed SAR.

Urotensin II Induced Signal Involved in Proliferation of Vascular Smooth Muscle Cells

Urotensin II receptor (UIIR), bound by the ligand Urotensin II (UII), generates second messengers IP3 and DAG, which stimulates the subsequent release of calcium (Ca2+) in vascular smooth muscle cells (VSMC). Ca2+ influx leads to the activation of Ca2+ dependent kinases via calmodulin binding, resulting in cellular proliferation. We hypothesize that UII signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/ CaM dependent kinases leading to cellular proliferation. UII exposure to Pac1 cell cultures increased intracellular Ca2+, which subsequently activated Ca2+/CaM dependent kinases (CaMKK &amp; CaMKI), ERK 1/ 2, and PKD (PKC‐µ). Pac1 and PAVSMC cells treated with UII resulted in increased proliferation as measured by 3H thymidine uptake. UII‐induced increase of 3H thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that UII stimulation of smooth muscle cells leads to cellular proliferation, utilizing signaling mechanisms involving Ca2+/CaM dependent kinases.