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Receptor binding of fish neuropeptide urotensin II (UII) was characterized in membranes isolated from major rat arteries. Monoiodinated UII radioligand (125I-UII) was prepared and purified using high-pressure liquid chromatography (HPLC). The contractile potency of iodinated UII (I-UII) on rat thoracic aorta strips was somewhat lower than that of native UII. The binding of 125I-UII to the membrane preparations of rat thoracic aorta was saturable, specific and time-dependent. Scatchard analysis indicated a single population of binding sites with an apparent dissociation constant of 5.9 x 10(-9) M. The calculated maximal number of binding sites was about 155 fmol/mg protein. The specific binding to the membrane preparations from the abdominal aorta and mesenteric artery was about 27 and 8%, respectively, of those in the thoracic aorta, which corresponds to the order of contractile potency of UII on rat blood vessels: thoracic aorta greater than abdominal aorta greater than mesenteric artery. The displacement of 125I-UII binding by the UII peptide or its fragments (UII-(5-12), UII-(6-12) and UII-(6-11] were also comparable to their contractile effects on rat thoracic aorta strips (UII greater than UII-(5-12) greater than UII-(6-12) much greater than UII-(6-11]. These results suggest that the fish neuropeptide, UII, can induce contraction of rat vascular tissue by interacting with its functional receptors.

Publisher Summary This chapter discusses the urophysiology and the caudal neurosecretory system of fishes, chemistry of urotensin II, immunocytochemistry of the caudal neurosecretory system, biological activities of urotensin II in teleosts, and effects of urotensin II in mammals. The caudal system in teleosts is heavily innervated, consistently by aminergic input and occasionally by cholinergic and peptidergic pathways from the brain. The complexity and intensity of the innervation shows some important physiological role for this system; yet, it has been difficult to ascribe a definite functional contribution to the caudal system and its hormones—the urotensins. The urophysis contains and presumably releases a variety of substances: (1) urotensin I—a 41-amino acid peptide which is homologous and analogous to mammalian corticotropin-releasing factors (CRFs); (2) urotensin II—a 12-amino acid peptide which is partially homologous and partially analogous to somatostatin-14; (3) urotensin IV—indistinguishable from arginine vasotocin and present in only a few teleost species; (4) urophysins—putative carrier proteins, cysteine-free, analogous to the neurophysins and with unknown biogenetic relationships to the urotensins; and (5) acetylcholine—the highest concentrations of this substance reported for any tissue but of unknown significance in this locale.

Urotensin II, a peptide hormone from the caudal neurosecretory system of the teleost, Gillichthys mirabilis, was isolated by using classical chromatographic techniques and high-performance liquid chromatography (HPLC). Direct microtechniques for sequence determination were used to establish its structure. Urotensin II from Gillichthys is a 1363-dalton dodecapeptide with the amino acid sequence Ala-Gly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. This sequence is homologous with somatostatin in positions 1 and 2 and 7-9. The sequence has been verified by the production of a bioactive synthetic urotensin II. The possible chemical and physiological significance of its homology to somatostatin is discussed.

NEW FINDINGS What is the central question of this study? Urotensin II is upregulated in patients in the later stages of chronic kidney disease (CKD), particularly in individuals requiring dialysis. Could treatment with a urotensin II receptor antagonist slow progression of renal disease? What is the main finding and its importance? In the rat, expression of urotensin II and its receptor increased, extending into cortical structures as CKD progressed towards end-stage renal failure. Subchronic treatment with a urotensin receptor antagonist slowed but did not prevent progression of CKD. This suggests that urotensin II contributes to the decline in renal function in CKD. ABSTRACT Elevated serum and urine urotensin II (UII) concentrations have been reported in patients with end-stage chronic kidney disease (CKD). Similar increases in UII and its receptor, UT, have been reported in animal models of CKD, but only at much earlier stages of renal dysfunction. The aim of this study was to characterize urotensin system expression as renal disease progresses to end-stage failure in a ⅚ subtotal nephrectomy (SNx) rat model. Male Sprague-Dawley rats underwent SNx or sham surgery and were killed at 8 weeks postsurgery [early (E)] or immediately before end-stage renal failure [30 ± 3 weeks postsurgery; late (L)]. Systolic blood pressure, urinary albumin:creatinine ratio and glomerulosclerosis index were all increased in SNx-E rats compared with sham-E by 8 weeks postsurgery. These changes were associated with an increase in renal immunoreactive UII staining but little change in UT expression. As CKD progressed to end-stage disease in the SNx-L group, markers of renal function deteriorated further, in association with a marked increase in immunoreactive UII and UT staining. Subchronic administration of a UT antagonist, SB-611812, at 30 mg kg-1 day-1 for 13 weeks, in a separate group of SNx rats resulted in a 2 week delay in the increase in both systolic blood pressure and urinary albumin:creatinine ratio observed in vehicle-treated SNx but did not prevent the progression of renal dysfunction. The urotensin system is upregulated as renal function deteriorates in the rat; UT antagonism can slow but not prevent disease progression, suggesting that UII plays a role in CKD.

The peptide human urotensin-II (hUT-II) and its receptor have recently been cloned. The vascular function of this peptide in humans, however, has yet to be determined. Vasoconstrictor and vasodilator responses to hUT-II were investigated in human small muscular pulmonary arteries [∼170 μm internal diameter (ID)] and human abdominal resistance arteries (∼200 μm ID). Vasodilator responses were investigated in endothelin-1 (3 nM) precontracted vessels and, in the small pulmonary vessels, compared with the known vasodilators adrenomedullin, sodium nitroprusside, and acetylcholine. In human small pulmonary arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [−log M concentration causing 50% of the maximum vasodilator effect (pIC50) 10.4 ± 0.5; percentage of reduction in tone ( E max) 81 ± 8% (vs. 23 ± 11% in time controls), n = 5]. The order of potency for vasodilation was human urotensin-II = adrenomedullin (pIC50 10.1 ± 0.4, n = 6) > sodium nitroprusside (pIC50 7.4 ± 0.2, n = 6) = acetylcholine (pIC50 6.8 ± 0.3, n = 6). In human abdominal arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [pIC50 10.3 ± 0.7; E max96 ± 8% (vs. 43 ± 16% in time controls), n = 4]. This is the first report that hUT-II is a potent vasodilator but not a vasoconstrictor of human small pulmonary arteries and systemic resistance arteries.

Urp1 and Urp2 are two neuropeptides of the urotensin II family identified in teleost fish and mainly expressed in cerebrospinal fluid (CSF)-contacting neurons. It has been recently proposed that Urp1 and Urp2 are required for correct axis formation and maintenance. Their action is thought to be mediated by the receptor Uts2r3, which is specifically expressed in dorsal somites. In support of this view, it has been demonstrated that the loss of uts2r3 results in severe scoliosis in adult zebrafish. In the present study, we report for the first time the occurrence of urp2, but not of urp1 , in two tetrapod species of the Xenopus genus. In X. laevis , we show that urp2 mRNA-containing cells are CSF-contacting neurons. Furthermore, we identified utr4, the X. laevis counterparts of zebrafish uts2r3, and we demonstrate that, as in zebrafish, it is expressed in the dorsal somatic musculature. Finally, we reveal that, in X. laevis, the disruption of utr4 results in an abnormal curvature of the antero-posterior axis of the tadpoles. Taken together, our results suggest that the role of the Utr4 signalling pathway in the control of body straightness is an ancestral feature of bony vertebrates and not just a peculiarity of ray-finned fishes.

Despite being identified as the most potent receptor related to vasoconstriction, human urotensin-II receptor (hUT) has not been fully explored as a target for the treatment of cardiovascular diseases. In view of this and with an aim to identify precise structural requirements for binding of hUT antagonists, we endeavoured to develop, for the first time, multivariate QSAR models using chemometric methods like partial least squares (PLS) and feed-forward neural network (FFNN). A set of 48 pyrrolidine derivatives having hUT binding affinity was used for multivariate model development. The accuracy and predictability of the developed models was evaluated using crossvalidation. The PLS model showed good correlation between selected descriptors and Ki values (r(2) =0.745 and r(2) (CV) =0.773). However, the predictive performance of FFNN was better than the PLS technique with r(2) =0.810. The study clearly suggests the role of lipophilic and steric descriptors in the ligand-hUT interactions. The QSAR models generated can be successfully extended to predict the binding affinities and for the effective design of novel hUT antagonists.