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Am J Physiol Heart Circ Physiol 292: H348-H359, 2007. First published September 1, 2006; doi:10.1152/ajpheart.00512.2006
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Role of PKC in the novel synergistic action of urotensin II and angiotensin II and in urotensin II-induced vasoconstriction

Yan-Xia Wang,1 Ying-Jiong Ding,1 Yi-Zhun Zhu,2,3 Ying Shi,1 Tai Yao,1 and Yi-Chun Zhu1

1Department of Physiology and Pathophysiology, Shanghai Medical College, and 2Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, People's Republic of China; and 3Department of Pharmacology, National University of Singapore, Singapore

Submitted 19 May 2006 ; accepted in final form 25 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The intracellular signaling of human urotensin II (hU-II) and its interaction with other vasoconstrictors such as ANG II are poorly understood. In endothelium-denuded rat aorta, coadministration of hU-II (1 nM) and ANG II (2 nM) exerted a significant contractile effect that was associated with increased protein kinase C (PKC) activity and phosphorylation of PKC-{alpha}/betaII and myosin light chain, whereas either hU-II or ANG II administered alone at these concentrations had no statistically significant effect. This synergistic effect was abrogated by the PKC inhibitor chelerythrine (10 and 30 µM), the selective PKC-{alpha}/betaII inhibitor Gö-6976 (0.1 and 1 µM), the hU-II receptor ligand urantide (30 nM and 1 µM), or the ANG II antagonist losartan (1 µM). Moreover, in endothelium-intact rat aorta, the synergistic effect of hU-II and ANG II was not exerted any longer, and this synergistic effect was unmasked by pretreatment of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. hU-II (10 nM) alone caused a long-lasting increase in phospho-PKC-{theta}, phospho-myosin light chain, and PKC activity, which was associated with long-lasting vasoconstriction. These changes were prevented by chelerythrine. Methoxyverapamil-thapsigargin treatment reduced the hU-II-induced vasoconstriction by ~50%. The methoxyverapamil-thapsigargin-resistant component of hU-II-induced vasoconstriction was dose-dependently inhibited by chelerythrine. In conclusion, hU-II induces a novel PKC-dependent synergistic action with ANG II in inducing vasoconstriction. PKC-{alpha}/betaII is probably the PKC isoform involved in this synergistic action. Nitric oxide produced in the endothelium probably masks this synergistic action. The long-lasting vasoconstriction induced by hU-II alone is PKC dependent and associated with PKC-{theta} phosphorylation.

protein kinase C; vascular smooth muscle cells

THE HOMOLOG OF THE FISH PEPTIDE urotensin II (U-II) has been cloned in several mammalian species, including humans. The orphan G protein-coupled receptor 14 has been identified as U-II receptor (1), which is now nominated as UT receptor. U-II has recently been identified as the most powerful vasoconstrictor with a potency that is 8- to 110-fold greater than endothelin-1 in isolated aorta of the rats (11). The mRNA transcripts for U-II were detected in the kidney and right atrium but to a lesser extent in the vasculature in humans (28). When examined by immunohistochemical staining, U-II was detected in the endothelium of normal human blood vessels from various organs (heart, kidney, placenta, adrenal, thyroid, and umbilical cord), human kidney epithelial cells (27, 39), rat pancreas (40), and human urine (28). UT receptors were identified in cardiomyocytes (17), vascular smooth muscle cells (VSMCs) (1), endothelial cells (1), spinal cord (10), and endocrine tissue (9).

Moreover, the linkage between U-II and cardiovascular diseases has been suggested by several experimental and clinical studies. U-II stimulation increased the level of procollagens and fibronectin mRNA transcripts in neonatal cardiac fibroblasts (17). The expression of U-II has been shown to be upregulated in the myocardium (13) and plasma (36) of patients with congestive heart failure, as well as in infarcted heart of the rat (43), the plasma of hypertensive patients (8), and atherosclerotic lesions of human aorta (4). However, a few independent groups did not observe positive correlation between plasma U-II levels and congestive heart failure (14, 21). To date, the implied role of U-II in disease states is postulated largely on the basis of changes in the expression levels of this peptide (and/or its receptor) in the disease processes.

U-II has been shown to effectively contract the isolated arteries from rats (15), rabbits (12), dogs (12), pigs (12), nonhuman primates (1), and humans (26). On the other hand, the reported depressor and regionally selective vasodilator effects (3, 12, 26, 48) of U-II endow its vasoactive effects with complexity. Interestingly, exogenously administered U-II induced a vasoconstrictor response in skin microvasculature in patients with chronic heart failure (25) and essential hypertension (41), in contrast to its vasodilator effects in normal subjects, suggesting differential roles of U-II in cardiovascular physiology and pathophysiology. In cardiovascular diseases, the expression of many vasoconstrictors such as ANG II (34), endothelin (2), catecholamines (44), thromboxane A2 (29), and serotonin (23) has been shown to be upregulated. These studies give rise to the hypothesis that the interaction between U-II and other vasoactive substances may be crucial in modulating the vasoactive effect of U-II under a certain disease status.

Cross talk of intracellular signaling pathways is probably the underlying mechanism of the interaction between U-II and other vasoactive substances (48). Nevertheless, the intracellular signaling of U-II remains largely unknown. In isolated rabbit thoracic aorta, the contractile effect of human U-II (hU-II) was significantly inhibited by 2-nitro-4-carboxyphenyl-N,N,-diphenylcarbamate, a phospholipase C inhibitor, but not by indomethacin, a cyclooxygenase inhibitor. Meanwhile, hU-II increased phosphoinositide hydrolysis, and this effect was also inhibited by the phospholipase C inhibitor, suggesting a role of the phospholipase C-dependent inositol phosphate pathway in mediating hU-II-induced vasoconstriction (37). In isolated rat aortic rings, hU-II increased cytosolic Ca2+ levels. Complete inhibition of the cytosolic Ca2+ increase could only prevent a part of the hU-II-induced vasoconstriction. Likewise, protein kinase C (PKC) inhibitor Gö-6976, MEK inhibitor U-0126, p38 MAPK inhibitor SB-203580, and myosin light chain (MLC) kinase inhibitor wortmannin partially inhibited the U-II-induced vasoconstriction, suggesting the involvement of multiple signaling pathways (42).

The present study aimed to investigate the purported interaction between hU-II and ANG II in inducing the contraction of arterial vascular smooth muscles, as well as the intracellular signaling of hU-II in the presence or absence of ANG II.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Measurement of vasoconstriction. Male Sprague-Dawley rats weighing 200–250 g were obtained from the Department of Experimental Animals, Chinese Academy of Sciences. The investigation conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health, and was approved by the Ethic Committee of Experimental Research, Fudan University Shanghai Medical College. After chloral hydrate overdose, animals were killed; an ~12-mm length of proximal descending thoracic aorta was isolated at a point immediately distal to the left subclavian bifurcation. No more than four 3-mm-length rings were isolated from any given rat. Endothelium-denuded arterial rings were placed in oxygenated (95% O2-5% CO2) Krebs solution (in mmol/l: 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 11 D-glucose) (44) and equilibrated for 1 h at 37°C under a resting tension of 2 g before all subsequent manipulations. In concentration-response studies, hU-II or ANG II (1 pM to 1 µM) was administered at 5-min intervals. To block the vasoconstrictor effect of hU-II, urantide (30 nM and 1 µM) or chelerythrine (30 µM) (47) was administered 30 min in advance. To further elucidate the role of Ca2+ in hU-II-induced vasoconstriction, the voltage-gated Ca2+ channel inhibitor methoxyverapamil (20 µM) (31) and Ca2+ store-depleting agent thapsigargin (2 µM) (32) were used to suppress the agonist-induced rise in intracellular Ca2+ concentration ([Ca2+]i). To block the vasoconstrictor effect of ANG II, losartan (1 µM) (19) was administered 30 min in advance. To assess the synergistic action of hU-II and ANG II, hU-II (1 nM) was administered 20 min before ANG II (2 nM) administration. To investigate the mechanisms of the synergistic action of hU-II and ANG II, losartan (1 µM) (19), urantide (30 nM and 1 µM), chelerythrine (10 and 30 µM) (47), or Gö-6976 (0.1 and 1 µM) (24) was administered 30 min before hU-II administration. Additional experiments were performed to examine the effect of a mechanical increase in resting tension on the contractile response to 2 nM ANG II using phenylephrine (0.1 µM) to induce a similar increase in resting tension, which was induced by 1 nM hU-II. Responses were normalized to that induced by 100 mM KCl.

For Western blot analysis, the aortic rings were quickly removed from the organ bath, snapped in liquid nitrogen, and homogenized in SDS sample buffer containing 0.05 mol/l Tris·HCl, 2% SDS, 10% glycerol, and proteinase inhibitor cocktail (Roche, Mannheim, Germany).

Cell culture. Aortic VSMCs of the rat were obtained from the American Type Culture Collection (no. CRL-2018, SV402T-SMC clone HEP-SA). Human VSMCs were isolated from umbilical arteries as previously described (5) and identified by immunocytochemical staining with {alpha}-smooth muscle actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were incubated in DMEM supplemented with 10% FBS and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) in a humidified atmosphere of 95% air-5% CO2 at 37°C. Cells at ~80% confluence in culture wells were made quiescent by incubation with 0.4% FBS medium for 24–48 h before treatment and were incubated for 0–60 min with hU-II (100 nM). In some experiments, VSMCs were pretreated with various inhibitors for 30 min before hU-II treatment.

Measurement of [Ca2+]i. For measurement of [Ca2+]i in VSMCs, the cells were loaded with 2 mM fluo 3-AM at 37°C. Measurements were performed with an MRC 600 confocal imaging system and laser scanning confocal microscope (Radiance 2000; Bio-Rad, Hertfordshire, UK). Briefly, VSMCs were seeded onto glass coverslips and incubated with fluo 3-AM at 37°C under an atmosphere of 5% CO2. After a loading period of 30 min, the cells were washed and placed on the stage of the microscope. The fluorescence in the cell was excited at 488 nm by an argon-ion laser, and emission at wavelengths between 515 and 545 nm was detected by a photomultiplier. Peak [Ca2+]i was determined from the average of three data points.

Western blot analysis. Cells and tissue specimens were homogenized in SDS sample buffer (Roche). Protein was extracted from the homogenates, and protein content was determined in the lysate supernatant. Each sample (10 µg) was separated by electrophoresis on a 7.5% or 15% polyacrylamide-SDS gel and transferred to polyvinylidene difluoride membrane (Gelman-Pall, Ann Arbor, MI). The membranes were incubated with the primary antibodies against various intracellular proteins. Antigen detection was performed with an ECL detection kit (Pierce, Rockford, IL). Phosphorylation of the PKC targets, i.e., (ser)PKC substrate and myristoylated alanine-rich protein kinase C substrate (MARCKS), was assessed as parameters of PKC activity by Western blot analysis. The following primary antibodies were used: anti-phospho-MLC (Ser19), anti-phospho-(ser)PKC substrate, anti-phospho-PKC-{alpha}/betaII (Thr638/641), anti-phospho-PKC-{delta} (Thr505), anti-phospho-PKC-{theta} (Thr538), anti-phosopho-PKC-{zeta}/{lambda} (Thr410/403), anti-phospho-MARCKS, and anti-beta-actin. All primary antibodies were obtained from Cell Signaling Technology (Beverly, MA) except the anti-beta-actin antibodies, which were from Santa Cruz Biotechnology.

To detect PKC activation and MLC phosphorylation involved in the synergistic action of hU-II and ANG II, hU-II (1 nM) was administered 20 min before ANG II (2 nM). Five minutes after ANG II administration, the aortic rings were quickly removed from the organ bath, snapped in liquid nitrogen, and homogenized for subsequent Western blot analysis.

Chemicals. hU-II was obtained from Bachem (Bubendorf, Switzerland). Chelerythrine, thapsigargin, methoxyverapamil, ANG II, NG-nitro-L-arginine methyl ester (L-NAME), and Gö-6976 were obtained from Sigma (St. Louis, MO). Losartan was provided by MSD (Whitehouse Station, NJ). Urantide was custom synthesized by GL Biochem (Shanghai, China). DMSO was from Wako (Richmond, VA). Fluo 3-AM was from Calbiochem (La Jolla, CA). Methoxyverapamil and chelerythrine were dissolved in DMSO. All cell culture reagents were obtained from Sigma.

Statistics. All values are expressed as means ± SE, and n represents the number of samples of cell cultures or animals from which blood vessels were isolated. Statistical comparisons were performed by one-way ANOVA, followed by the Student-Newman-Keuls test. A P value of <0.05 was taken as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Synergistic action of hU-II and ANG II in contracting the vascular smooth muscle through a PKC-dependent mechanism. Either hU-II or ANG II administered alone resulted in a dose-dependent contraction in endothelium-denuded thoracic aorta of the Sprague-Dawley rat with EC50 values of 5.13 ± 1.65 and 3.65 ± 0.99 nM, respectively (Fig. 1A; n = 8 in each group). The Hill slope of the ANG II curve (1.2 ± 0.04) was steeper than that of the hU-II curve (0.6 ± 0.04) (P < 0.05). From the results depicted in Fig. 1A, an hU-II concentration of 1 nM was chosen to assess the putative synergistic action of hU-II with ANG II of increasing concentrations (1, 2, and 3 nM). On the other hand, because a single administration of hU-II caused a slow-developing and long-lasting increase in vascular tension and ANG II induced a fast-developing and transient vasoconstriction (Fig. 1B), hU-II was administered 20 min before ANG II administration to assess the interaction between these two peptides. Cumulative administration of ANG II (1, 2, and 3 nM) induced a concentration-dependent contractile response in the presence or absence of hU-II (1 nM). At concentrations of 2 and 3 nM, ANG II produced a greater contractile response in vessels pretreated with hU-II than vessels pretreated with vehicle (Fig. 1, C and D; n = 6 in each group). The putative synergistic action of hU-II and ANG II was further studied at concentrations of 1 and 2 nM for hU-II and ANG II, respectively. At the above-mentioned respective concentrations for hU-II and ANG II, neither of these two peptides administered alone was effective in causing statistically significant contraction of the endothelium-denuded aorta compared with the vehicle-treated control, although a trend toward an increase in vascular tension was observed in vessels treated with hU-II alone (Fig. 1, E and F). Interestingly, ANG II (2 nM) elicited a significantly increased constriction of the vessels pretreated with hU-II (1 nM) compared with either control or the vessels treated with ANG II alone (P < 0.05; Fig. 1, E and F). Moreover, this synergistic action was prevented by the ANG II AT1-receptor antagonist losartan (1 µM), the UT-receptor ligand urantide (30 nM and 1 µM), the PKC inhibitor chelerythrine (10 and 30 µM) or the selective PKC-{alpha}/betaII inhibitor Gö-6976 (0.1 and 1 µM) (Fig. 2), suggesting a PKC-dependent cross talk between the intracellular signaling pathways mediated by the AT1 receptor and UT receptor in arterial smooth muscle.


Figure 1
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  		Fig. 1. Synergistic action of human urotensin II (hU-II) and ANG II in contracting endothelium-denuded thoracic aorta of the Sprague-Dawley rat through a protein kinase C (PKC)-dependent mechanism. A: concentration-response curves showing the dose-dependent vasoconstrictor effects of hU-II and ANG II in endothelium-denuded rat thoracic aorta (n = 8 in each group). B: traces showing vasoconstriction induced by a single administration of hU-II (10 nM) or ANG II (100 nM). C and D: representative traces (C) and bar graph (D) showing ANG II (1, 2, and 3 nM)-induced contraction in the presence or absence of hU-II (1 nM) (n = 6 in each group). E and F: representative traces (E) and bar graph (F) showing the synergistic action of hU-II (1 nM) and ANG II (2 nM) in the presence or absence of losartan (1 µM), urantide (30 nM and 1 µM), chelerythrine (10 and 30 µM), or phenylephrine (0.1 µM) (n = 11 in the control group, n = 11 in the hU-II + ANG II group, and n = 6 in each of the other groups). Values shown in D and F are means ± SE. *P < 0.05 in D; #P < 0.05 vs. all other groups in F.

 

Figure 2
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  		Fig. 2. Synergistic action of hU-II and ANG II in contracting endothelium-denuded thoracic aorta of the Sprague-Dawley rat through a PKC-{alpha}/betaII-dependent mechanism. A and B: representative traces (A) and bar graph (B) showing the synergistic action of hU-II (1 nM) and ANG II (2 nM) in the presence or absence of the selective PKC-{alpha}/betaII inhibitor Gö-6976 (0.1 and 1 µM; n = 6 in each group). Values in B are means ± SE. *P < 0.05 vs. each of the other groups in B.

 
Because mechanical increases in the resting tension may influence the contractile response to ANG II, phenylephrine was employed to create a control group with increased resting tension. Phenylephrine (0.1 µM) induced an increase in resting tension comparable with that induced by 1 nM hU-II but had no effect on the vasoactive effect of 2 nM ANG II, excluding the possibility that the synergistic interaction between hU-II and ANG II was simply induced by the hU-II-induced increase in resting tension.

Role of endothelium in the synergistic action of hU-II and ANG II. hU-II (1 nM) did not potentiate the vasoconstrictive effect of ANG II (2 nM) any longer in endothelium-intact aortic rings isolated from the Sprague-Dawley rat (Fig. 3). Pretreatment with the nitric oxide synthase inhibitor L-NAME uncovered the synergistic action of hU-II and ANG II, suggesting a role of endothelial nitric oxide in masking the synergistic action of hU-II and ANG II (Fig. 3).


Figure 3
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  		Fig. 3. Interaction of hU-II and ANG II in endothelium-intact thoracic aorta of the Sprague-Dawley rat. Representative traces (A) and bar graph (B) show interaction of hU-II (1 nM) and ANG II (2 nM) in inducing vasoconstriction in endothelium-intact aortic tissues of the Sprague-Dawley rat in the absence or presence of NG-nitro-L-arginine methyl ester (L-NAME; 100 µM) (n = 6 in each group). Values shown in B are means ± SE. *P < 0.05 vs. each of the other groups in B.

 
Synergistic action of hU-II and ANG II in inducing PKC activation and MLC phosphorylation. The synergism of hU-II and ANG II was also observed in their actions on the intracellular molecules PKC and MLC in endothelium-denuded aortic tissue of the Sprague-Dawley rat. Phosphorylation of the PKC targets, i.e., (ser)PKC substrate, was assessed as parameters of PKC activity. When administered alone, neither hU-II (1 nM) nor ANG II (2 nM) caused significant increase in PKC activity (Fig. 4A) and MLC phosphorylation (Fig. 4B) in endothelium-denuded thoracic aorta. Coadministration of hU-II (1 nM) and ANG II (2 nM) produced an apparent increase in PKC activity (Fig. 4A) and MLC phosphorylation (Fig. 4B), indicating a synergistic action of hU-II and ANG II on the intracellular molecules PKC and MLC.


Figure 4
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  		Fig. 4. Synergistic action of hU-II and ANG II in inducing PKC activation and myosin light chain (MLC) phosphorylation. ANG II (2 nM) caused apparent PKC activation (A) and MLC phosphorylation (B) in the presence of hU-II (1 nM) in endothelium-denuded thoracic aorta of the Sprague-Dawley rat. When administered alone, neither hU-II (1 nM) nor ANG II (2 nM) caused significant increase in PKC activity (A) and MLC phosphorylation (B). Shown are representative blots and values enumerated by densitometry of 6 experiments. Values in B are means ± SE. *P < 0.05 vs. each of other groups in B.

 
Synergistic action of hU-II and ANG II in selective phosphorylation of the PKC isoform {alpha}/betaII. To detect the PKC isoforms involved in the synergistic contractile effects of hU-II and ANG II, endothelium-denuded aortic tissues treated with a combination of hU-II (1 nM) and ANG II (2 nM) were quickly removed from the organ bath and snapped in liquid nitrogen when a significant contractile response was observed. Phosphorylation of PKC isoforms was detected by Western blot analysis with homogenates of the aortic tissues of the Sprague-Dawley rat. PKC-{alpha}/betaII was selectively phosphorylated by combined treatment of hU-II (1 nM) and ANG II (2 nM) but not by hU-II (1 nM) or ANG II (2 nM) alone (Fig. 5, A and B). On the other hand, hU-II administered alone at a higher concentration (10 nM) resulted in time-dependent (5, 15, and 30 min) selective phosphorylation of PKC isoforms {alpha}/betaII and {theta} (Fig. 5, C and D). Worthy of notice is that the time course of hU-II (10 nM)-induced phosphorylation of PKC-{theta} (Fig. 5D) is associated with the long-lasting pattern of hU-II (10 nM)-induced vasoconstriction (Fig. 1B). However, hU-II administered alone at a lower concentration (1 nM) had no significant effect on phosphorylation of the PKC isoforms at corresponding time points (Fig. 5, E and F).


Figure 5
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  		Fig. 5. Synergistic interaction between hU-II and ANG II in inducing a selective phosphorylation of PKC-{alpha}/betaII in endothelium-denuded thoracic aorta of the Sprague-Dawley rat. When administered alone, neither hU-II (1 nM) nor ANG II (2 nM) caused apparent phosphorylation of any PKC isoforms. A combined administration of hU-II (1 nM) and ANG II (2 nM) caused selective phosphorylation of PKC-{alpha}/betaII. Shown are representative blots (A) and values enumerated by densitometry (B) of 6 experiments. hU-II administered alone at a higher concentration (10 nM) resulted in time-dependent (5, 15, and 30 min) selective phosphorylation of PKC isoforms {alpha}/betaII and {theta} (C and D). However, hU-II administered alone at a lower concentration (1 nM) had no significant effect on phosphorylation of the PKC isoforms at corresponding time points (E and F). Shown are representative blots (A, C, and E) and values enumerated by densitometry (B, D, and F) of 6 experiments. Data are means ± SE. *P < 0.05 vs. control (B); #P < 0.05 vs. 0 min (D).

 
Phosphorylation of MLC in VSMCs and aortic tissues induced by hU-II. Homogenates of cultured VSMCs or aortic tissues were analyzed by Western blot analysis. hU-II caused a dose-dependent increase in MLC phosphorylation in cultured aortic VSMCs of the Sprague-Dawley rat (Fig. 6A). A single administration of hU-II (100 nM for cultured cells and 10 nM for aortic tissues) caused MLC phosphorylation for at least 30 min in cultured rat aortic (Fig. 6B) and human umbilical (Fig. 6C) VSMCs and in endothelium-denuded thoracic aorta of the Sprague-Dawley rat (Fig. 6D). In cultured aortic VSMCs of the Sprague-Dawley rat, hU-II-induced MLC phosphorylation was prevented by urantide at concentrations of 30 and 300 nM but not at 3 nM (Fig. 6E). In endothelium-denuded thoracic aorta of the Sprague-Dawley rat, hU-II-induced MLC phosphorylation was prevented by urantide at concentrations of 30 nM and 1 µM (Fig. 6F). These data suggested a role of MLC in mediating intracellular signaling after UT receptor stimulation.


Figure 6
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  		Fig. 6. hU-II leads to MLC phosphorylation in cultured rat aortic and human umbilical vascular smooth muscle cells (VSMCs) and in endothelium-denuded aortic tissues of the Sprague-Dawley rat through UT receptors. Cell lysates and tissue homogenates were analyzed for MLC phosphorylation by Western blotting. A: Western blotting showing concentration-dependent MLC phosphorylation induced by hU-II in cultured rat VSMCs. B, C, and D: time courses of MLC phosphorylation induced by hU-II (100 nM) in cultured VSMCs of rat (B) and human (C) and in endothelium-denuded rat aortic tissue (D). hU-II-induced MLC phosphorylation in cultured rat VSMCs was prevented by the UT receptor ligand urantide at concentrations of 30 and 300 nM but not 3 nM (E). hU-II-induced MLC phosphorylation in endothelium-denuded rat thoracic aorta was prevented by urantide at concentration of 30 nM and 1 µM (F). Shown are representative blots and values enumerated by densitometry of 5 independent experiments for A, B, C, D, E, and F, respectively. Values are means ± SE. *P < 0.05 vs. control (A) or 0 min (B, C, and D); **P < 0.05 vs. control (E and F); #P < 0.05 vs. hU-II (E and F).

 
hU-II activates PKC through UT receptors. A single administration of hU-II (100 nM for the cells and 10 nM for the tissues) caused time-dependent PKC activation in cultured rat aortic VSMCs (Fig. 7A) and endothelium-denuded thoracic aorta of the Sprague-Dawley rat (Fig. 7B). hU-II-induced PKC activation in aortic tissue was prevented by urantide (1 µM) (Fig. 7C), supporting a role for PKC in mediating the intracellular signaling on UT receptor stimulation.


Figure 7
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  		Fig. 7. hU-II activates PKC through UT receptors. Cell lysates and tissue homogenates were analyzed for phospho-(ser)PKC substrates, phospho-myristoylated alanine-rich protein kinase C substrate (MARCKS), and beta-actin by Western blotting. A and B: time course of hU-II (100 nM)-induced PKC activation in cultured rat VSMCs (A) and endothelium-denuded rat thoracic aorta (B). C: hU-II (10 nM)-induced PKC activation was prevented by the UT receptor ligand urantide (1 µM) in endothelium-denuded thoracic aorta of the Sprague-Dawley rat. Shown are representative blots and values enumerated by densitometry of 5 independent experiments.

 
PKC is required for hU-II-induced MLC phosphorylation and vasoconstriction. In cultured rat aortic VSMCs, the PKC inhibitor chelerythrine (1 µM) was effective in abrogating hU-II (100 nM)-induced increase in PKC activity (Fig. 8A) and MLC phosphorylation (Fig. 8B). In endothelium-denuded thoracic aorta of the Sprague-Dawley rat, hU-II-induced MLC phosphorylation (Fig. 8C) and vasoconstriction (Fig. 8D) were also prevented by chelerythrine (30 µM). The cumulative concentration-response curve of hU-II-induced vasoconstriction was shifted rightward in a parallel manner by 30 nM urantide, and the effect of hU-II was almost abolished by 1 µM (Fig. 8D). Losartan (1 µM) had no effect on vasoconstriction induced by hU-II alone (Fig. 8D). ANG II-induced vasoconstriction was inhibited by losartan (1 µM) and chelerythrine (30 µM) but not by urantide (1 µM) (Fig. 8E). These data suggest that PKC is an upstream regulator of MLC phosphorylation in hU-II-induced arterial smooth muscle contraction. PKC is also involved in vasoconstriction induced by ANG II alone.


Figure 8
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  		Fig. 8. PKC is required for hU-II-induced MLC phosphorylation and vasoconstriction. In cultured rat aortic VSMCs, PKC inhibitor chelerythrine (1 µM) was effective in abrogating hU-II (100 nM)-induced increase in PKC activity (A) and MLC phosphorylation (B). In endothelium-denuded thoracic aorta of the Sprague-Dawley rat, hU-II-induced MLC phosphorylation (C) and vasoconstriction (D) were also prevented by chelerythrine (30 µM). The cumulative concentration-response curve of hU-II-induced vasoconstriction was shifted rightward in a parallel manner by urantide at a concentration of 30 nM, and the effect of hU-II was almost abolished by urantide at a concentration of 1 µM (D). Losartan (1 µM) had no effect on vasoconstriction induced by hU-II alone (D). ANG II-induced vasoconstriction was inhibited by losartan (1 µM) and chelerythrine (30 µM) but not by urantide (1 µM) (E). Shown in A, B, and C are representative blots and values enumerated by densitometry of 6 experiments. Data in D and E are means ± SE of 8 experiments for each group. *P < 0.05 vs. control (B and C); #P < 0.05 vs. hU-II (B and C); **P < 0.01 vs. hU-II + chelerythrine or hU-II + urantide (1 µM) (D); ##P < 0.05 vs. hU-II (D); ***P < 0.01 vs. ANG II + losartan or ANG II + chelerythrine (E).

 
Role of calcium in hU-II-induced vasoconstriction. hU-II (100 nM) caused a significant increase in [Ca2+]i in rat aortic VSMCs as observed by confocal microscope (Fig. 9A). Methoxyverapamil-thapsigargin treatment reduced the hU-II-induced vasoconstriction by ~50% in endothelium-denuded thoracic aorta of the Sprague-Dawley rat. The methoxyverapamil-thapsigargin-resistant component of hU-II-induced vasoconstriction was dose-dependently inhibited by chelerythrine, with IC50 value of 7.98 µM (Fig. 9B; n = 6), suggesting Ca2+ sensitization of the contractile apparatus through the activation of PKC.


Figure 9
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  		Fig. 9. Role of calcium in hU-II-induced vasoconstriction. A: trace showing hU-II (100 nM)-induced increase in intracellular Ca2+ concentration in cultured rat aortic VSMCs measured with a confocal microscope (representative trace of 4 experiments). B: concentration-response curves showing the inhibitory effect of chelerythrine on hU-II (10 nM)-induced tension in the presence ({blacktriangleup}) or absence (bullet) of thapsigargin (TSG; 2 µM) and methoxyverapamil (20 µM) in endothelium-denuded thoracic aorta of the Sprague-Dawley rat. Values shown in B are means ± SE of 6 experiments. *P < 0.05 vs. control (B); **P < 0.01 vs. control (B).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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The role of U-II in cardiovascular physiology and diseases remains largely uncertain. Recent experimental and clinical studies have revealed increased expression of U-II and UT receptor in animals with experimentally induced myocardial infarction and heart failure and in patients with hypertension, atherosclerosis, and diabetic nephropathy, suggesting a potential role of U-II in both cardiovascular and renal diseases (22). On the other hand, numerous neurohumoral factors such as ANG II, endothelin, and catecholamines have been reported to be involved in these disease states (2, 30, 34). Therefore, it is important to know the interaction between U-II and other vasoactive factors under physiological and pathological conditions.

The present study demonstrates a synergistic action of hU-II and ANG II in contracting arterial smooth muscle. This is the first demonstration of a synergistic effect of hU-II with ANG II in vasoconstriction. In line with the present study, the synergistic effect of hU-II with mildly oxidized LDL (45) and serotonin (46) on VSMC proliferation has been reported.

The intracellular signaling of U-II-induced vasoconstriction remains largely unknown. Most published studies in this area have been performed in isolated aortic rings with synthetic inhibitors to distinguish the pathways involved. In isolated rabbit thoracic aorta, the contractile effect of hU-II was reported to be mediated by a phospholipase C-dependent-inositol trisphosphate pathway (37). In isolated rat aortic rings, Ca2+, PKC, ERK, MAPK, and MLC have been suggested to be involved in hU-II-induced vasoconstriction. The conclusion of this study (42) was based on the inhibitory effects of the synthetic inhibitors for the above-mentioned intracellular elements. Whether there was an activation of certain signaling elements, e.g., PKC, was not elucidated. Sauzeau et al. (38) reported an hU-II-induced RhoA activation. The RhoA inhibitor TAT-C3 and Rho-kinase inhibitor Y-27632 partially inhibited the hU-II-induced vasoconstriction (38). Herein, we report a selective phosphorylation of PKC isoforms {alpha}/betaII and {theta} and increased PKC activity induced by hU-II (10 nM) alone in endothelium-denuded rat thoracic aorta. Interestingly, the time course of hU-II-induced phosphorylation of PKC-{alpha}/betaII was rather transient (~5 min), whereas the phosphorylation of PKC-{theta} was long lasting (~30 min). Also, in rat aorta, Giebing et al. (16) recently suggested that the sustained contractile response to U-II is ascribed to the continuous externalization and recycle of UT receptors in VSMCs. Whether the long-lasting phosphorylation of PKC-{theta} observed in the present study contributes to the long-lasting effect of hU-II-induced vasoconstriction remains to be elucidated.

On the other hand, the synergistic action of hU-II (1 nM) and ANG II (2 nM) observed in the present study was associated with a selective phosphorylation of PKC-{alpha}/betaII. This synergistic effect on vasoconstriction was abrogated by either the PKC inhibitor chelerythrine, the selective PKC-{alpha}/betaII inhibitor Gö-6976, the ANG II AT1-receptor antagonist losartan, or the UT-receptor ligand urantide. These data suggest that PKC mediates the cross talk of the intracellular signaling pathways triggered by the UT receptor and AT1 receptor. Moreover, {alpha}/betaII is probably the PKC isoform involved in PKC-mediated cross talks of the signaling pathways of these two peptides.

Urantide has been used in the present study as a UT-receptor antagonist. This UT-receptor ligand has been shown to produce a concentration-related competitive inhibition of U-II-induced contraction in rat thoracic aorta (pKB = 8.3) without any agonistic effect up to a concentration of 1 µM (33). However, urantide has also been shown to act as a low-efficacy partial agonist in inducing Ca2+ release in Chinese hamster ovary cells overexpressing human UT receptors (6, 7). Although the efficacy of a UT-receptor ligand as a partial agonist may be overestimated in such a cell system expressing high levels of recombinant UT receptors, the partial agonistic effect of urantide suggests that this UT-receptor ligand is probably not the best tool to define receptor subtype.

PKC phosphorylation has been well established as an upstream regulator of MLC in inducing arterial smooth muscle contraction (18). In the present study, the vasoconstriction induced by hU-II alone or the combination of hU-II and ANG II was coupled with an increase in PKC activity and MLC phosphorylation. Meanwhile, inhibition of PKC diminished hU-II-induced MLC phosphorylation in VSMCs, suggesting a role of PKC in mediating hU-II (alone or synergistically with ANG II)-induced smooth muscle contraction through phosphorylation of MLC.

Intracellular Ca2+ has been shown to be involved in hU-II-induced vasoconstriction (38, 42). Sauzeau et al. (38) further demonstrated that the increase in [Ca2+]i sensitized the RhoA/Rho kinase pathways and the contractile response to hU-II in endothelium-denuded rat aortic rings (38) In the present study, hU-II-induced vasoconstriction was reduced by ~50% in the presence of the voltage-gated Ca2+ channel inhibitor methoxyverapamil and the Ca2+ store-depleting agent thapsigargin. Meanwhile, the methoxyverapamil-thapsigargin-resistant component of hU-II-induced vasoconstriction was concentration-dependently inhibited by chelerythrine. In cultured VSMCs, single-dose hU-II induced a transient [Ca2+]i rise. These results suggest that both an increase in [Ca2+]i and Ca2+ sensitization of PKC-dependent contractile apparatus are involved in hU-II-induced vasoconstriction. On the other hand, PKC may also mediate hU-II-induced smooth muscle contraction through a pathway independent of a rise in [Ca2+]i.

In the present study, the synergistic effect of U-II and ANG II was not present any longer in endothelium-intact aortic rings. Interestingly, pretreatment with L-NAME uncovered this synergistic effect in endothelium-intact aortic rings, suggesting a role of nitric oxide in blunting the synergistic effect of U-II and ANG II. Worthy of notice is that endothelium dysfunction has been well established in cardiovascular diseases (20); therefore, the synergistic effect of U-II and ANG II may be unmasked in these disease states. In line with this hypothesis, iontophoresis-administered hU-II (0.001, 1, and 100 nM) induced a vasoconstrictor response in skin microvasculature as assessed by laser-Doppler velocimetry in patients with either chronic heart failure (25) or essential hypertension (34), which is in sharp contrast to its vasodilator effect in normal subjects. The underlying mechanisms of the differential effects of hU-II on vascular tone in normal subjects and in patients with chronic heart failure and hypertension are interesting and remain to be clarified. It is possible, for example, that endothelium dysfunction, the diversity in the intracellular signaling pathways downstream of the UT receptor, and/or the interactions between hU-II and other vasoactive factors in disease states may modulate the vasoactive effect of hU-II (48). The present study provides direct evidence for a synergistic action of hU-II and ANG II through a PKC-dependent cross talk of the intracellular signaling pathways of the UT receptors and ANG II AT1 receptors. This synergistic action may amplify the vasoactive effects of hU-II and ANG II in diseases such as hypertension and heart failure. In support of this hypothesis, it has been demonstrated that plasma hU-II levels increased in patients with hypertension (8). In heart failure patients, both positive (30, 35, 36) and negative (21) correlations between plasma hU-II levels and the disease have been reported. Moreover, ANG II levels have been shown to be increased in hypertension (34) and heart failure (34). In patients with hypertension or heart failure, increased hU-II and ANG II may interact on each other and produce a stronger contractile effect than that produced by either of the peptides alone. In addition, endothelium dysfunction may uncover the interaction between hU-II and ANG II. On the other hand, the receptors of hU-II and ANG II on VSMCs may be upregulated in hypertension and heart failure (14, 43) and hence result in increased cross talks of the intracellular signaling pathways of these two peptides.

In conclusion, hU-II potentiates the vasoconstrictor effect of ANG II by a PKC-dependent cross talk of the intracellular signaling pathways of these two peptides. {alpha}/betaII is probably the PKC isoform involved in this synergism. Nitric oxide produced in the endothelium probably masks this synergistic action. The long-lasting vasoconstriction induced by hU-II alone is PKC dependent and associated with PKC-{theta} phosphorylation. PKC is an upstream regulator of MLC. [Ca2+]i sensitizes PKC-dependent vasoconstriction induced by hU-II.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Natural Science Foundation of China Grant 30470628 (Y.-C. Zhu).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-C. Zhu, Dept. of Physiology and Pathophysiology, Fudan Univ. Shanghai Medical College, 138 Yi Xue Yuan Road, Shanghai 200032, People's Republic of China (e-mail: yczhu{at}shmu.edu.cn)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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