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Keywords:

  • Urotensin-II;
  • [125I]-U-II;
  • orphan receptor;
  • human;
  • cardiovascular tissue;
  • human brain;
  • radioligand binding;
  • vasoconstriction;
  • in vitro pharmacology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • We have determined the distribution of receptors for human urotensin-II (U-II) in human and rat CNS and peripheral tissues.

  • In rat, [125I]-U-II binding density was highest in the abducens nucleus of brainstem (139.6±14 amol mm−2). Moderate levels were detected in dorsal horn of spinal cord and lower levels in aorta (22.5±6 amol mm−2).

  • In human tissues density was highest in skeletal muscle and cerebral cortex (∼30 amol mm−2), with lower levels (<15 amol mm−2) in kidney cortex and left ventricle. Little binding was identified in atria, conducting system of the heart and lung parenchyma.

  • Receptor density was less in human coronary artery smooth muscle (14.6±3 amol mm−2, n=10) than rat aorta with no significant difference between normal and atherosclerotic vessels.

  • In human skeletal muscle [125I]-U-II bound to a single receptor population with KD=0.24±0.17 nM and Bmax=1.97±1.1 fmol mg−1 protein (n=4).

  • U-II contracted human coronary, mammary and radial arteries, saphenous and umbilical veins with sub-nanomolar EC50 values. U-II was 50 times more potent in arteries and <10 times more potent in veins than endothelin-1 (ET-1). The maximum response to U-II (∼20% of control KCl) was significantly less than to ET-1 (∼80% KCl). In contrast, in rat aorta, U-II and ET-1 were equipotent with similar maximum responses.

  • This is the first report of high affinity receptors for [125I]-U-II in human CNS and peripheral tissues. This peptide produces potent, low efficacy, vasoconstriction in human arteries and veins. These data suggest a potential role for U-II in human physiology.

British Journal of Pharmacology (2000) 131, 441–446; doi:10.1038/sj.bjp.0703601


Abbreviations:
DCM

dilated cardiomyopathy

ET-1

endothelin-1

GPCR

G-protein coupled receptor

IHD

ischaemic heart disease

[125I]-U-II

monoiodinated urotensin-II

U-II

urotensin-II

UT-II

human urotensin-II receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Urotensin II (U-II) was characterized over 30 years ago as one of several peptides present in the urophysis of teleost fish (see Bern et al., 1995). This dodecapeptide originally isolated and sequenced from goby Gillichthys mirabilis (Pearson et al., 1980), shares some structural homology with somatostatin-14. Several forms of U-II have been identified in species of teleost, but all contain a conserved hexapeptide cyclic sequence within the C-terminal region that confers the majority of the biological activity (Itoh et al., 1987). In teleosts U-II produces general smooth muscle contraction, including vasoconstriction, plays a role in osmoregulation and acts as a prolactin inhibitory factor (see Bern et al., 1995).

Effects of fish U-II, usually goby, were subsequently reported in mammalian systems. Initial observations were of relaxation of the mouse anococcygeus muscle (Gibson et al., 1984) but in rat isolated blood vessels both endothelium-dependent vasorelaxation and endothelium-independent vasoconstriction have been detected (Gibson, 1987). Responses appear to be variable and highly dependent on vascular bed, although robust contraction to U-II was obtained in rat thoracic aorta (Itoh et al., 1987; 1988; Ames et al., 1999). Despite the ability to contract some arteries, infusion of goby U-II into rats produced a reduction in arterial blood pressure, with heart rate unaffected (Gibson et al., 1986; Hasegawa et al., 1992). Given the effects of fish U-II on mammalian tissues, it is not surprising that U-II was isolated and sequenced from frog brain (Conlon et al., 1992) and subsequently cDNAs were characterized encoding frog (Coulouarn et al., 1998), mouse, rat (Coulouarn et al., 1999) and human (Coulouarn et al., 1998) U-II precursors.

The human homologue of fish U-II (Figure 1) is an eleven amino acid peptide (Coulouarn et al., 1998; Ames et al., 1999) that retains the highly conserved cyclic region of fish and frog homologues (see Davenport & Maguire, 2000). Using dot blot analysis of human tissues, the highest level of pre-pro-U-II mRNA expression was identified in spinal cord and medulla oblongata, with in situ hybridization studies implicating a sub-population of motor neurones and motor nuclei of the brainstem, including the abducens nucleus. In agreement with the molecular studies U-II-like immunoreactivity localized to motor neurones in the spinal cord of several species (Coulouarn et al., 1998; Ames et al., 1999) suggesting that U-II may modulate skeletal muscle activity and the receptor would be expected to be expressed in this tissue. Lower levels of mRNA were also detected in a number of peripheral tissues, including kidney (Coulouarn et al., 1998, Nothacker et al., 1999). The receptor mediating the actions of U-II was unknown, but was possibly one of the many orphan G-protein coupled receptors (GPCR) so far identified (see Marchese et al., 1999). These included rat GPR14 (SENR) which had some sequence homology with the somatostatin sst4 and μ-opioid receptors (Marchese et al., 1995; Tal et al., 1995). Recent reports, published within weeks of each other, have identified human U-II as a ligand for GPR14 (Ames et al., 1999; Liu et al., 1999; Mori et al., 1999; Nothacker et al., 1999). Ames et al., (1999) additionally identified a human GPCR with 75% identity to rat GPR14 which when expressed in HEK-293 cells in a calcium-mobilization assay, was activated by human U-II. Functional data from this group in isolated blood vessels from the cynomolgous monkey were remarkable. Human U-II was 10 times more potent than endothelin-1 (ET-1) with an extremely slowly developing response. However, although U-II contracted all arteries tested with sub-nanomolar EC50 values, no response was obtained from venous tissues. The effects of systemic administration of U-II in the anaesthetized monkey were dramatic, resulting in markedly increased total peripheral resistance, reduced myocardial contractility and at the highest concentration, death. The changes in ECG in these animals were reminiscent of those that occur during myocardial ischaemia.

image

Figure 1. Deduced amino acid sequence of human U-II indicating the position of the iodinated tyrosine residue. Shaded residues are those that are conserved across all known species homologues of the peptide.

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U-II clearly has potent cardiovascular effects in non-human primates but there is only one report of U-II effects in human vasculature. MacLean et al. (2000) have recently observed variable responses of human small pulmonary arteries to U-II, only in the presence of the NOS inhibitor L-NAME. The receptor distribution for U-II in human tissues has not been characterized. We have therefore localized binding sites for the novel radioligand human [125I]-U-II in human and rat tissues where molecular studies have predicted the human UT-II and GPR14 receptors are expressed. We have quantified the ability of human U-II to constrict human arteries and veins from a range of vascular beds and compared this to rat aorta that is known to respond to human U-II (Nothacker et al., 1999). The magnitude of these responses was compared to endothelin-1 (Yanagisawa et al., 1988) the most potent vasoconstrictor of human blood vessels yet described (Maguire & Davenport, 1995).

Preliminary data have been presented to the British Pharmacological Society (Kuc et al., 2000, in press; Maguire & Davenport, 2000, in press).

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Tissue collection

Human coronary artery, left ventricle and right atria were obtained from 25 patients transplanted for dilated cardiomyopathy (DCM), ischaemic heart disease (IHD) or whose hearts were not suitable for further transplantation (normal). Saphenous vein, mammary artery and radial artery were from 18 patients receiving coronary artery by-pass grafts. Skeletal muscle was removed with the mammary artery during these operations. Umbilical veins were from four cords obtained following normal deliveries. Histologically normal sections of kidney were obtained from four patients undergoing nephrectomy for non-obstructive carcinoma. Macroscopically normal cerebral cortex was obtained from three patients treated surgically for gliomas or epilepsy. Normal lung tissue was from three patients undergoing lobectomy for carcinoma. All human tissues were collected with local ethical committee approval.

Rat tissues (thoracic aorta, spinal cord and brain) were from seven female Sprague-Dawley rats (300–350 g). Animals were killed by an overdose of sodium pentobarbitone followed by exsanguination.

Saturation analysis of human [125I]-urotensin II binding

Saturation binding experiments were carried out on cryostat-cut sections (20 μm) of normal human skeletal muscle. Sections were pre-incubated for 1 h in 20 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl2 and 0.2% BSA and then incubated with increasing concentrations (7.8 pM–4 nM) [125I]-U-II for 1 h. Non-specific binding was determined using human U-II (1 μM). Sections were washed for 10 min in 50 mM Tris-HCl, pH 7.4 at 4°C, and counted for radioactive content. Data were analysed using the iterative, non-linear curve-fitting programmes EBDA and LIGAND (Munson & Rodbard, 1980) in the KELL package (Elsevier Biosoft, Cambridge, U.K.) to determine values of equilibrium dissociation constant (KD) and receptor density (Bmax). Protein concentration/tissue section was determined using a DC protein assay kit (Bio-Rad, Herts, U.K.).

Autoradiographical visualization of [125I]-urotensin II binding

Autoradiography was carried out using cryostat-cut sections of rat thoracic aorta, spinal cord and brain and human tissues including coronary artery, left ventricle, right atria, kidney, cerebral cortex and skeletal muscle. Sections were pre-incubated as described above and then incubated with 0.25 nM [125I]-U-II for 1 h. Dried sections were apposed, with standards, to radiation sensitive film for 3 days. The resulting autoradiograms were analysed using computer-assisted densitometry (Davenport et al, 1995). Binding densities (amol mm−2±s.e.mean) were compared using Student's 2-tailed t-test or one-way analysis of variance as appropriate using Minitab statistical package (Minitab Inc., PA, U.S.A.). The level of significance was set at 5% (P<0.05).

In vitro pharmacology

Human and rat arteries and veins were cleaned of connective tissue, cut into 4-mm rings and their luminal surface rubbed gently with a metal seeker to remove the endothelium (confirmed by the absence of staining with rabbit antiserum to human von Willebrand factor as previously described (Davenport et al., 1996)). Vessels were set up for isometric force recordings in 5-ml organ baths containing oxygenated Kreb's solution (37°C) and allowed to equilibrate for 1 h. Contractile responses were then recorded to 100 mM KCl at incrementally increasing levels of basal tension until no further increase in KCl response was obtained. This determined the optimum resting tension for each preparation and was followed by a further 30 min equilibration period. Cumulative concentration response curves were then constructed to ET-1 (10−10–3×10−7 M) or to U-II (10−13–10−7 M). In some experiments, ET-1 (1–3×10−7 M) was added at the end of the U-II curve and then all experiments were terminated by addition of 100 mM KCl to determine the maximum possible response for each preparation. Agonist responses were subsequently expressed as a percentage of this KCl maximum.

Data were analysed using the iterative curve-fitting programme Fig P (Biosoft, Cambridge, U.K.) to give values of pD2 (negative log10 of the molar concentration producing 50% of the maximum response) and Emax (maximum agonist response as a percentage of the terminal KCl response). All data were expressed as mean±s.e.mean and n values refer to the number of patients or rats from whom tissue was obtained. pD2 and Emax values were compared using Student's 2-tailed t-test with a significance value of 5% (P<0.05).

Modified Kreb's solution had the following composition (mM): NaCl, 90; KCl, 5; MgSO4.7H2O, 0.5; Na2HPO4, 1; NaHCO3, 45; CaCl2, 2.25; glucose, 10; Na pyruvate, 5; fumaric acid, 5; L-glutamic acid, 5.

Materials

Human urotensin-II (Glu-Thr-Pro-Asp-[Cys-Phe-Trp-Lys-Tyr-Cys]-Val) and endothelin-1 were purchased from Peptide Institute (Osaka, Japan). Iodinated human urotensin-II (2000 Ci mmol−1) was from Amersham Pharmacia Biotech (Amersham, U.K.). All other reagents were from standard commercial sources and of Analar grade.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Radioligand saturation binding and autoradiography

Human tissues were selected for autoradiographical analysis based on the reported expression of mRNA encoding either U-II peptide or receptors. Using a fixed concentration of [125I]-U-II the highest density of receptors in the peripheral tissues analysed was present in skeletal muscle (31.9±9 amol mm−2, n=5; Figure 2) and this was used for further characterization by saturation analysis. [125I]-U-II bound with a single high affinity (KD=0.24±0.17 nM, n=4) and a Bmax of 1.97±1.1 fmol mg−1 to sections of human skeletal muscle. Hill slopes were close to unity (nH=1.02±0.03) and a one site fit was preferred over a two site model.

image

Figure 2. Relative densities of [125I]-U-II binding determined autoradiographically in 20-μm sections of rat and human tissues. Data are mean±s.e.mean. n-values are given in parentheses.

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In human cardiovascular tissue, binding was detected to vessels, including the media (smooth muscle layer) of epicardial coronary arteries (14.6±3 amol mm−2, n=10) and to myocytes in the left ventricle (9.4±4 amol mm−2, n=4) (Figure 2). There was no difference in the density of medial U-II receptors between normal (14.2±5 amol mm−2, n=4), DCM (12.6±4 amol mm−2, n=3) and atherosclerotic IHD (16.9±6 amol mm−2, n=3) arteries. Little or no specific binding (<5 amol mm−2) was detectable in the atria or to the conducting system of the human heart. In other peripheral tissues low densities were detected in kidney cortex (14.5±4 amol mm−2, n=3), but no binding was detected in lung parenchyma although low levels localized to pulmonary vessels of varying diameter (consistent with the constrictor response reported by MacLean et al., 2000) and bronchioles present within the lung sections (<20 amol mm−2). Binding was below the level for detection in fat. In human cerebral cortex densities were comparable to the highest levels in the peripheral tissues (32.6±13 amol mm−2, n=3) (Figure 2). Quantitative autoradiography using rat tissues indicated low density of specific binding (22.5±6 amol mm−2, n=5) localized to smooth muscle of aorta. In neuronal tissues, moderate levels (85±14 amol mm−2) were found in dorsal root ganglion of spinal cord with highest binding density observed in the abducens nucleus of the brain (139.6±14 amol mm−2, n=4) (Figures 2 and 3).

image

Figure 3. Autoradiographical localization of [125I]-U-II binding to 20-μm coronal sections (Bregma −10.3) of rat brain showing (A) total binding to abducens nuclei and (B) non-specific binding in adjacent section. Scale bar=2 mm.

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In vitro pharmacology

In rat the response to U-II in aorta was confined to an approximately 2-cm segment of thoracic vessel proximal to the carotid bifurcation of the aortic arch. Consistent responses were obtained to U-II in the first four aortic rings (4 mm) taken from this region, with loss of maximum response in more distal segments (Figure 4). No response was obtained in abdominal aorta (data not shown). U-II contracted proximal thoracic aorta from six out of seven rats with a pD2 value in responders of 8.87±0.28 and an Emax of 68.25±11.59% KCl 100 mM. These were not significantly different from data for ET-1; pD2=8.80±0.31, Emax=92.90±6.93% KCl 100 mM (n=6) (Table 1; Figure 5A).

image

Figure 4. Example of response to U-II in consecutive 4-mm rings of rat thoracic aorta: effect of distance of aortic ring from carotid bifurcation on maximum U-II response.

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Table 1. Comparison of vasoconstrictor activity of endothelin-1 and urotensin-II in rat aorta and human arteries and veinsThumbnail image of
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Figure 5. (A) Concentration-response curves to ET-1 and U-II in endothelium-denuded rat thoracic aorta. Data points are mean±s.e.mean, n=6. (B) Concentration-response curves to ET-1 (n=9) and U-II (n=6) in endothelium-denuded human coronary artery as an example of the relative response of the two peptides in human arteries and veins. Data points are mean±s.e.mean.

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All human blood vessels, arteries and veins, responded to ET-1 (Table 1). However, approximately 30% of coronary and mammary arteries were not contracted by any concentration of U-II tested, although ET-1 added after U-II contracted these vessels. For human arteries U-II was approximately 50 times more potent than ET-1, but because responses to U-II were very variable between individuals this difference only reached significance for coronary artery. In contrast the maximum response to U-II (approximately 20% of KCI response) was significantly lower than that achieved by ET-1 (approximately 80%) in all arteries tested (Figure 5B). The significant difference in maximum response between ET-1 and U-II was also observed in preparations of human veins. However, compared to arteries there was less than a 10 fold increase in potency of U-II compared to ET-1, which did not reach significance in either venous preparation (Table 1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This is the first report of the distribution of [125I]-U-II binding in human tissue. [125I]-U-II binding was saturable to human skeletal muscle with an affinity constant in the sub-nanomolar range. Hill slopes were close to unity indicating that [125I]-U-II bound with a single affinity, with no evidence for receptor sub-types. The KD for human [125I]-U-II, not previously determined in native human tissues, was within the range reported for goby [125I]-U-II binding to rat cardiac membranes (0.35 nM) or expressed human UT-II receptors (0.43 nM; Ames et al., 1999) and human [125I]-U-II binding to expressed rat GPR14 receptors (0.07 nM, Nothacker et al., 1999), under comparable binding conditions. The maximum density of receptors measured in human skeletal muscle using human [125I]-U-II was similar to that reported for goby [125I]-U-II in rat arteries or cardiac membranes, 2–20 fmol mg−1 (Itoh et al., 1988; Ames et al., 1999). These densities correspond to Bmax values in human arteries for other vasoactive peptides such as angiotensin II (Wharton et al., 1998), thromboxane A2 (Katugampola & Davenport, 2000, in press) and ET-1 (Davenport et al., 1995; Bacon et al., 1996). Our data are therefore consistent with [125I]-U-II binding to human UT-II receptor, the human homologue of rat GPR14.

Ames and colleagues (1999) have recently deduced the sequence of the human isoform of the rat GPR14 receptor. This human UT-II receptor (Davenport & Maguire, 2000) comprises 389 amino acids. Previous reports suggest that human UT-II receptor mRNA can be detected in tissue homogenates of human heart (cardiac myocytes in atria and ventricle) (Ames et al., 1999; Liu et al., 1999), skeletal muscle and bladder (Liu et al., 1999), pancreas, brain, arterial (but not venous) smooth muscle and endothelial cells (Ames et al., 1999). In agreement with these findings we detected highest levels of human [125I]-U-II binding in human skeletal muscle, compatible with the proposed distribution of U-II in motorneurones of the spinal cord. Although some binding was detected in human cerebral cortex it was not possible to investigate if motor nuclei of human brain express very high densities of receptors as seen in the rat abducens nucleus. Consistent with predictions from dot blot analysis we also localized low levels of receptors to coronary artery smooth muscle and kidney cortex. However we found very low levels of receptor expression in human left ventricle and could not detect specific binding in atria and conducting system of the human heart. This finding is perhaps not unexpected as Ames group used quantitative RT–PCR to detect the low levels of transcript present in human cardiovascular tissues. Our observation of binding of human [125I]-U-II to rat aorta, spinal cord and abducens nucleus is also consistent with previous reports of a neural and sensory localization of this receptor in rat (Tal et al., 1995; Liu et al., 1999; Nothacker et al., 1999).

In both rat and human nervous system prepro-U-II mRNA was consistently identified in spinal cord and motor nuclei (including the abducens nucleus) of the medulla oblongata (Coulouarn et al., 1998; 1999; Ames et al., 1999; Nothacker et al., 1999). However the source of U-II peptide interacting with the UT-II receptor in peripheral tissues is unclear and there is some discrepancy between molecular studies. Whilst Ames and colleagues (1999) reported U-II-like immunoreactivity in the human vasculature, with diffuse staining in the heart, mRNA encoding the peptide was not detected in homogenates of human tissue, including heart and aorta, by dot blot analysis. Nothacker et al. (1999) using similar techniques, also failed to detect message in most of the peripheral tissues they examined although they found high expression of human prepro-U-II mRNA in human kidney. Coulouarn et al. (1998) also reported highest expression of U-II mRNA in human spinal cord and medulla oblongata but found a more widespread distribution (by dot blot analysis) of human prepro-U-II mRNA in peripheral tissues, including the adrenal glands, kidney and spleen, which may be suggestive of some local synthesis and release. However, it is possible that mRNA may be below the level for detection or the peptide may circulate in the plasma and is synthesized some distance from target organs.

It has been known for some time that fish U-II will contract rat arteries, particularly the thoracic aorta. We used this preparation as a control to compare the constrictor activity of human U-II to the potent vasoconstrictor ET-1 (Yanagisawa et al., 1988; Maguire & Davenport, 1995). We found that U-II and ET-1 were equipotent, with EC50 values of approximately 1.5 nM. This is consistent with that observed for human U-II in rat aorta of 2.4 nM reported by Nothacker and colleagues (1999). The maximum response to ET-1 was greater than that to U-II, but due to the greater variability of the U-II data this difference was not significant. Indeed one rat aorta failed to respond to U-II and we observed that only vessel rings removed from the portion of thoracic aorta proximal to the carotid bifurcation responded well, with a drastic reduction in efficacy seen in more distal tissue (Figure 4). As reported by others we found no response to U-II in rat abdominal aorta. As predicted by the lower receptor density in human coronary artery media compared to rat aorta the responses to U-II in human blood vessels were less robust. In human coronary, mammary and radial arteries U-II was approximately 50 times more potent than ET-1, however whereas all arteries contracted to ET-1 approximately 30% did not respond to U-II. This is likely to be due to the low receptor density and the greater variability of human compared to rat tissues. The low receptor density perhaps also may explain the low efficacy of U-II compared to ET-1 in these blood vessels. Ames et al. (1999) also reported U-II to be more potent than ET-1 in monkey arteries. However they found a very clear restriction of U-II-mediated vasoconstriction to arterial vessels with no response in venous tissue. In contrast we found that human U-II contracted human saphenous and umbilical vein and although all veins responded (in contrast to the arterial preparations), U-II was less than 10 times more potent than ET-1 in these vessels and the maximum response to U-II was again significantly attenuated compared to ET-1. Therefore in human blood vessels we find that human U-II is extremely potent, but has low efficacy which may reflect a low density of high affinity receptors localized to the vascular smooth muscle. The effect of U-II on human coronary artery is much less dramatic than that observed in monkey and it is possible therefore that the spectacular cardiac collapse elicited by U-II in vivo in this animal (Ames et al., 1999) would not be observed in man. Interestingly U-II-like immunoreactivity was reportedly present in atherosclerotic human coronary artery (Ames et al., 1999) suggesting the peptide may have a potential role in human cardiovascular disease. In our investigations we could detect no alteration in the density [125I]-U-II binding in the media of healthy compared to atherosclerotic epicardial coronary arteries. This does not however preclude an increase in the synthesis of the peptide in this disease.

It has been reported that in human pulmonary artery in vitro constrictor responses to human U-II were not observed in vessels with an intact endothelium but were observed in 3/10 adjacent rings treated with the NOS inhibitor L-NAME (MacLean et al., 2000). These authors suggested that U-II-mediated release of NO may physiologically antagonize its direct vasoconstrictor activity and that U-II constrictor activity may be of greater relevance in pathophysiological conditions in which endothelial dysfunction occurs. The more robust response to U-II observed in our preparations may reflect the absence of endothelium in these experiments and certainly endothelium-dependent relaxation to fish U-II has previously been reported in mammalian vascular smooth muscle (Gibson et al., 1987). However, the relative importance of human U-II as constrictor or dilator of the human vasculature and whether this is altered in disease remains to be determined.

In conclusion this is the first report of the localization of high affinity binding sites for the novel radioligand human [125I]-U-II in human and rat tissues. The receptor for this ligand was present in human arteries and veins, left ventricle, kidney cortex, skeletal muscle and cerebral cortex. Receptor density in these tissues was comparable to that in rat aorta but lower than detected in rat spinal cord and abducens nuclei of the rat brainstem. It will be of interest if high receptor densities localize to these same regions in man. In functional assays we find that U-II potently constricts both human arteries and veins suggesting that this peptide may behave as a ubiquitous vasoconstrictor, similar to ET-1. However, while U-II was more potent than ET-1, the magnitude of response to U-II was considerably less than this peptide. Intriguingly, greater densities of UT-II receptors were found in tissues such as human skeletal muscle with highest densities in discrete regions of rat brain and spinal cord suggesting that the peptide may have other and perhaps more important physiological roles in the periphery and central nervous system.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Supported by grants from the British Heart Foundation, Royal Society and Isaac Newton Trust. With thanks to Stephen Douglas and Eliot Ohlstein (SmithKline Beecham Pharmaceuticals, King of Prussia, PA, U.S.A.) for helpful discussion. We are grateful to Jean Chadderton and staff at Papworth and Addenbrooke's Hospitals and the Rosie Maternity Unit (Cambridge, U.K.) for their assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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