Increased expression of urotensin II-related peptide and its receptor in kidney with hypertension or renal failure

Urotensin II-related peptide (URP) is a novel vasoactive peptide that shares urotensin II receptor (UT) with urotensin II. In order to clarify possible changes of URP expression in hypertension and chronic renal failure (CRF), the expressions of URP and UT were studied by quantitative RT-PCR and immunohistochemistry in kidneys obtained from spontaneous hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and WKY with CRF due to 5/6 nephrectomy. Expression levels of URP mRNA and UT mRNA were significantly higher in the kidneys obtained from SHR compared with age-matched WKY (at 5-16 and 16 weeks old, respectively). A dissection study of the kidney into three portions (inner medulla, outer medulla and cortex) showed that the expression levels of URP mRNA and UT mRNA were highest in the inner medulla and the outer medulla, respectively, in both SHR and WKY. The expression levels of URP and UT mRNAs were greatly elevated in the remnant kidneys of CRF rats at day 56 after nephrectomy, compared with sham-operated rats (about 6.5- and 11.9-fold, respectively). Immunohistochemistry showed that URP immunostaining was found mainly in the renal tubules, vascular smooth muscle cells and vascular endothelial cells. UT immunoreactivity was localized in the renal tubules and vascular endothelial cells. These findings suggest that the expressions of URP and UT mRNAs in the kidney are enhanced in hypertension and CRF, and that URP and its receptor have important pathophysiological roles in these diseases.     

Cardiovascular effects of native and non-native urotensin II and urotensin II-related peptide on rat and salmon hearts

Urotensin II (UII) was first discovered in the urophyses of goby fish and later identified in mammals, while urotensin II-related peptide (URP) was recently isolated from rat brain. We studied the effects of UII on isolated heart preparations of Chinook salmon and Sprague–Dawley rats. Native rat UII caused potent and sustained, dose-dependent dilation of the coronary arteries in the rat, whereas non-native UII (human and trout UII) showed attenuated vasodilation. Rat URP dilated rat coronary arteries, with 10-fold less potency compared with rUII. In salmon, native trout UII caused sustained dilation of the coronary arteries, while rat UII and URP caused significant constriction. Nω-nitro-_l-arginine methyl (l-NAME) and indomethacin significantly attenuated the URP and rat UII-induced vasodilation in the rat heart. We conclude that UII is a coronary vasodilator, an action that is species form specific. We also provide the first evidence for cardiac actions of URP, possibly via mechanisms common with UII.     

Upregulation of angiotensin AT1a receptors mRNA in the heart and renal medulla after myocardial infarction in rats.

The myocardial infarct causes prolonged activation of the renin-angiotensin system and profoundly influences cardiac performance and renal excretory capabilities. The aim of the present study was to determine whether the myocardial infarct is also associated with an altered expression of AT1a receptors (AT1aR) mRNA in the heart and the kidney. To this end male Sprague-Dawley rats were subjected either to the left coronary artery ligation or to the sham surgery. Four weeks after the surgery the animals were sacrificed. In 11 infarcted and 10 sham-operated rats expression of AT1aR mRNA in the walls of the left and right ventricle of the heart, and in the renal cortex and renal medulla was determined by semiquantitative PCR method. In another group of 10 infarcted and 14 sham-operated rats the diameter of cardiomyocytes in the left and right cardiac ventricle was determined. The size of the infarct in the rats used for mRNA determination and for morphometric measurements was equal to 29.4 +/- 1.8% and to 31.0 +/- 1.2 % of the left ventricular wall, respectively. Expression of AT1aR mRNA was significantly greater in the left (P< 0.01) and right ventricle (P<0.03) of the heart in the infarcted than in the sham operated rats. AT1aR mRNA expression was also significantly greater (P<0.02) in the renal medulla of the infarcted rats than in the renal medulla of the sham operated rats whereas no significant difference was found in the renal cortex. The myocardial infarct was associated with a significant increase of diameter of cardiomyocytes of the left ventricle of the heart (P< 0.0001), however there was no significant correlation between changes in AT1aR mRNA expression and diameter of cardiomyocytes. The results provide evidence that the myocardial infarct results in significant and prolonged upregulation of AT1a receptors mRNA expression in the heart and in the medullary region of the kidney.     

Tonin in rat heart with experimental hypertrophy

The present study was undertaken to determine tonin expression and activity in rat heart presenting isoproterenol-induced hypertrophy. Renin, angiotensin-converting enzyme (ACE), and angiotensinogen (AG) expression were also determined. Wistar rats were treated with isoproterenol for 7 days (5 mg x kg(-1) x day(-1) sc). For untreated animals, the levels of tonin-specific activity in the atrium were 2.6- and 5.5-fold higher than those of the left and right ventricle, respectively. After treatment, the levels of tonin-specific activity increased twofold in the atrium but did not change in the ventricles. Renin expression was not detectable in these structures, and ACE expression levels did not change with treatment. AG expression was detected in the left ventricle at very low levels compared with the atrium and increased significantly only in the hypertrophied atrium (1.8-fold). Tonin mRNA was not detected in the ventricle but was found at low levels in the atrium, which increased after isoproterenol treatment. Our results permit us to conclude that tonin may play a role in the process of heart hypertrophy in the rat.     

Increase in cardiac P2X1-and P2Y2-receptor mRNA levels in congestive heart failure.

We wanted to study the expression of P2-receptors at the mRNA-level in the heart and if it is affected by congestive heart failure (CHF). To quantify the P2 receptor mRNA-expression we used a competitive RT-PCR protocol which is based on an internal RNA standard. The P2 receptor mRNA-expression was quantified in hearts from CHF rats and compared to sham-operated rats. Furthermore, the presence of receptor mRNA was studied in the myocardium from patients with heart failure. In the sham operated rats the G-protein coupled P2Y-receptors were expressed at a higher level than the ligand gated ion-channel receptor (P2X1). Among the P2Y-receptors the P2Y6-receptor was most abundantly expressed (P2Y6 > P2Y1 > P2Y2 = P2Y4 > P2X1). A prominent change was seen for the P2X1- and P2Y2-receptor mRNA levels which were increased 2.7-fold and 4.7-fold respectively in the myocardium from the left ventricle of CHF-rats. In contrast, the P2Y1-, P2Y4- and P2Y6-receptor mRNA levels were not significantly altered in CHF rats. In human myocard the P2X1-, P2Y1-, P2Y2-, P2Y6- and P2Y11-receptors were detected by RT-PCR in both right and left atria and ventricles, while the P2Y4-receptor band was weak or absent. In conclusion, most of the studied P2-receptors were expressed in both rat and human hearts. Furthermore, the P2X1- and P2Y2-receptor mRNA were upregulated in CHF, suggesting a pathophysiological role for these receptors in the development of heart failure.     

Immunolocalization of urotensin II and its receptor in human adrenal tumors and attached non-neoplastic adrenal tissues

Urotensin II (UII), first identified from goby urophysis, is a potent vasoactive peptide hormone and an endogenous ligand for an orphan G protein-coupled receptor GPR14, now named urotensin II receptor (UT-R). In addition to its vascular actions, UII has been shown to have mitogenic effects on tumor growth and some regulatory effects on adrenal steroidogenesis. In the present study, we examined expression of UII and UT-R in human adrenal tumors and attached non-neoplastic adrenal tissues by immunohistochemistry. Both UII and UT-R were immunolocalized in tumor cells of all adrenal tumors examined: 8 cases of cortisol-producing adenomas, 8 cases of aldosterone-producing adenomas, 2 cases of non-functioning adenomas, 17 cases of adrenocortical carcinomas, and 8 cases of pheochromocytomas. In attached adrenals, immunoreactivity for UII was detected in medulla, but much weaker in the cortex than in cortical tumors, suggesting that expression of UII was up-regulated in neoplastic adrenocortical tissues. No significant differences were found in the degree of immunoreactivity for UT-R between the tumors and the attached adrenal tissues. The present study showed that both UII and UT-R were expressed in the adrenal tumors and attached non-neoplastic adrenal tissues, and suggests possible roles of UII and UT-R in tumor growth and/or secretory activities of these tumors.     

Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain

Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.     

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 and urotensin II-related peptide (URP) in cardiac ischemia-reperfusion injury

Circulating urotensin II (UII) concentrations and the tissue expression of its cognate receptor (UT) are elevated in patients with cardiovascular disease (CVD). The functional significance of elevated plasma UII levels in CVD is unclear. Urotensin-related peptide (URP) is a paralog of UII in that it contains the six amino acid ring structures found in UII. Although both peptides are implicated as bioactive factors capable of modulating cardiovascular status, the role of both UII and URP in ischemic injury is unknown. Accordingly, we provide here the first report describing the direct cardiac effects of UII and URP in ischemia-reperfusion injury. Isolated perfused rat hearts were subjected to no-flow global ischemia for 45 min after 30min preconditioning with either 1nM rUII or 10nM URP. Both rUII- and URP-induced significant vasodilation of coronary arteries before (both P<0.05) and after ischemia (both P<0.05). Rat UII alone lowered contractility prior to ischemia (P=0.053). Specific assay of perfusate revealed rUII and URP both significantly inhibited reperfusion myocardial creatine kinase (CK) release (P=0.012 and 0.036, respectively) and atrial natriuretic peptide (ANP) secretion (P=0.025). Antagonism of the UT receptor with 1muM palosuran caused a significant increase in perfusion pressure (PP) prior to and post-ischemia. Furthermore, palosuran significantly inhibited reductions in both PP and myocardial damage marker release induced by both rUII and URP. In conclusion, our data suggests rUII and URP reduce cardiac ischemia-reperfusion injury by increasing flow through the coronary circulation, reducing contractility and therefore myocardial energy demand, and inhibiting reperfusion myocardial damage. Thus, UII and URP present as novel peptides with potential cardioprotective actions.     

Urotensin II-related peptide, the endogenous ligand for the urotensin II receptor in the rat brain

Urotensin II (UII) is a piscine neuropeptide originally isolated from the teleost urophysis. The existence of UII in mammals has been demonstrated by cloning of the mammalian orthologs of UII precursor protein genes. While rat and mouse orthologs have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack the typical processing sites in the amino-terminal region of the mature peptides. A novel peptide, UII-related peptide (URP), was discovered by monitoring UII-immunoreactivity in the rat brain, and its amino acid sequence was determined to be ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and showed that the sequences of mouse and human URP peptides are identical to that for rat URP. URP was found to bind and activate the human or rat urotensin II receptors [GPR14, UT receptor (UTR)] and showed a hypotensive effect when administrated to anesthetized rats. The prepro-URP gene is expressed in several rat tissues, although with lower levels than the prepro-UII gene and, in the human, is expressed comparably to prepro-UII in several tissues except the spinal cord. These results suggest that URP is the endogenous and functional ligand for urotensin II receptor in the rat and mouse, and possibly in the human.     

Regulation of the (pro)renin-renin receptor in cardiac remodelling

The (pro)renin-renin receptor [(P)RR] was discovered as an important novel component of the renin-angiotensin system (RAS). The functional significance of (P)RR is widely studied in renal and vascular pathologies and has sparked interest for a potential role in cardiovascular disease. To investigate the role of (P)RR in cardiac pathophysiology, we aimed to assess (P)RR regulation in adverse cardiac remodelling of the failing heart. In particular, we evaluated the expression of (P)RR in different models of heart failure and across different species. Significantly increased levels of (P)RR mRNA were found in post-myocardial infarcted (MI) hearts of rats (1.6-fold, P < 0.05) and mice (5-fold, P < 0.01), as well as in transgenic rats with overexpression of the mouse renin gene (Ren2) (2.2-fold, P < 0.01). Moreover, we observed a strong increase of (P)RR expression in hearts of dilated cardiomyopathy (DCM) patients (5.3-fold, P < 0.001). Because none of the tested commercially available antibodies appeared to detect endogenous (P)RR, a (P)RR-specific polyclonal antibody was generated to study (P)RR protein levels. (P)RR protein levels were significantly increased in the post-MI rat heart (1.4-fold, P < 0.05) as compared to controls. Most interestingly in DCM patients, a significant 8.7-fold (P < 0.05) increase was observed. Thus, protein expression paralleled gene expression. These results demonstrate that (P)RR expression is strongly up-regulated both in rodent models of heart failure and in the failing human heart, hinting to a potential role for (P)RR in cardiac pathophysiology.     

Localization of the insulin-like growth factor system in a rat model of heart failure induced by myocardial infarction.

Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (MI) in the rat. The left anterior descending coronary artery was ligated in adult female Sprague-Dawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after MI. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to MI and consequent failure.     

Tissue specific expression of rat peptidylglycine alpha-amidating monooxygenase activity and mRNA

The tissue specific expression of peptidylglycine alpha-amidating monooxygenase [(PAM) EC 1.14.17.3], an enzyme which catalyzes the formation of amidated bioactive peptides from their glycine-extended precursors, was examined in adult rat. Soluble and membrane-associated PAM enzymatic activities were determined, and the levels and size classes of PAM mRNA were examined by Northern blot analysis. PAM specific activity varied 1000-fold in the tissues examined, with highest levels in heart atrium, pituitary and salivary glands, and hypothalamus. The fraction of total PAM activity that was membrane associated varied from approximately 70% in heart atrium to 10% in neurointermediate pituitary lobe and thyroid gland. Levels of PAM mRNA varied over 300-fold. In the heart atrium, PAM mRNA accounts for more than 0.1% of the mRNA. For many tissues the ratio of total PAM specific activity to PAM mRNA levels was similar; however, PAM activity was higher than expected from mRNA levels in the salivary glands and lower than expected in several tissues, including heart ventricle. Three major size classes of PAM mRNA were identified among the tissues. Use of RNAse H indicated that differences in size were not due to the length of the poly(A) tail. The heart and central nervous system expressed PAM mRNA of the 4.2 kilobase (kb) and 3.8 kb size classes, while the remaining tissues expressed predominantly 3.8 kb and 3.6 kb classes; few tissues contained only one size class of PAM mRNA. The two major forms of PAM mRNA in adult heart atrium differ by the presence or absence of a 315 nucleotide segment in the protein coding region. Using a cDNA probe from within this segment, the 4.2 kb and 3.8 kb size classes of PAM mRNA in the central nervous system appeared to resemble those in the heart atrium. In the remaining tissues, a subset of PAM mRNAs in the 3.8 kb and 3.6 kb size classes hybridized with this probe, suggesting that additional forms of PAM mRNA are present.     

Developmental regulation of peptidylglycine alpha-amidating monooxygenase (PAM) in rat heart atrium and ventricle. Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.     

Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system

Redox imbalance elicited by oxidative stress contributes to pathogenic remodeling of ion channels that underlies arrhythmogenesis and contractile dysfunction in the failing heart. This study examined whether the expression of K(+) channels in the remodeled ventricle is controlled by the thioredoxin system, a principal oxidoreductase network regulating redox-sensitive proteins. Ventricular dysfunction was induced in rats by coronary artery ligation, and experiments were conducted 6-8 wk postinfarction. Biochemical assays of tissue extracts from infarcted hearts showed that thioredoxin reductase activity was decreased by 32% from sham-operated controls (P < 0.05), whereas thioredoxin activity was 51% higher postinfarction (P < 0.05). These differences in activities paralleled changes in protein abundance as determined by Western blot analysis. However, whereas real-time PCR showed thioredoxin reductase mRNA levels to be significantly decreased postinfarction, thioredoxin mRNA was not altered. In voltage-clamp studies of myocytes from infarcted hearts, the characteristic downregulation of transient-outward K(+) current density was reversed by exogenous pyruvate (5 mmol/l), and this effect was blocked by the specific inhibitors of the thioredoxin system: auranofin or 13-cis-retinoic acid. Real-time PCR and Western blot analyses of myocyte suspensions from infarcted hearts showed that pyruvate increased mRNA and protein abundance of Kv4.2 and Kv4.3 channel alpha-subunits as well as the accessory protein KChIP2 when compared with time-matched, untreated cells (P < 0.05). The pyruvate-induced increase in Kv4.x expression was blocked by auranofin, but the upregulation of KChIP2 expression was not affected. These data suggest that the expression of Kv4.x channels is redox-regulated by the thioredoxin system, which may be a novel therapeutic target to reverse or limit electrical remodeling of the failing heart.     

Urotensin-II receptor blockade with SB-611812 attenuates cardiac remodeling in experimental ischemic heart disease

It is now well established that urotensin-II (UII) levels are increased in several cardiovascular diseases. We previously demonstrated that UII and the UII receptor (UT) protein levels are significantly increased in the hearts of both humans and rats with congestive heart failure (CHF). We have also recently demonstrated that UII blockade, with a selective UII antagonist, improves heart function in a rat model of ischemic CHF. Here, we evaluated the attenuation of cardiac remodeling associated with UII antagonism in the same rat model of ischemic CHF. Animals were administered a specific UT receptor antagonist, SB-611812 (30 mg/kg/day, gavage), or vehicle 30 min prior to coronary artery ligation followed by daily treatment for 8 weeks. Myocardial interstitial fibrosis was analyzed by Masson's trichrome and picrosirius red staining. RT-PCR analysis was utilized for mRNA expression studies. We used Western blotting to assess levels of collagen types I and III. Mitogenic activity of UII on cultured neonatal cardiac fibroblasts was also evaluated. Following coronary ligation, SB-611812 significantly attenuated both myocardial and endocardial interstitial fibrosis, and reduced collagen type I:III ratio (P<0.01). UII induced proliferation of cardiac fibroblasts and this mitogenic effect was significantly inhibited with 1 microM of SB-611218 (P<0.05). We demonstrate here that selective blockade of UT reduces diastolic dysfunction by decreasing myocardial fibrosis post-coronary ligation in vivo, and inhibits UII-mediated fibroblast proliferation in vitro.     

Elevated plasma atrial natriuretic peptide in rats with myocardial infarcts

We measured atrial natriuretic peptide (ANP) plasma levels in rats with experimental heart failure caused by left coronary artery ligation. ANP levels were clearly higher in infarcted rats (409 +/- 59 pg/ml; mean +/- S.E.M.) than in sham-operated controls (39 +/- 6 pg/ml). Moreover, plasma ANP levels increased progressively with the severity of cardiac dysfunction and size of infarct. Increased release of ANP in post-infarction heart failure appears to be a meaningful compensatory response to control rising preload. Our results are in keeping with evidence from human studies showing increased plasma concentration of ANP in patients with congestive heart failure. This model is a useful tool to further explore the role of ANP in heart failure.