Development and pharmacological characterization of "caged" urotensin II analogs

Urotensin-II (U-II) is a cyclic 11-amino acid peptide known as a potent mammalian vasoconstrictor. To study some purported intracellular actions of U-II, masked analogs of this peptide, becoming biologically active only upon UV exposure, were developed. Those analogs described as "caged" were derivatized with a photolabile 4,5-dimethoxynitrobenzyl group on the side chain of Lys-8 or Tyr-9. Both caged analogs of U-II showed a major decrease in their affinity towards the UT receptor. Nevertheless, upon UV irradiation, the native and biologically active U-II peptide was recovered. Thus, this work describes the development of new "caged" U-II derivatives and demonstrates that vasoactivity of U-II can be controlled by masking and unmasking two key residues.     

Localization of urotensin-II immunoreactivity in normal human kidneys and renal carcinoma.

Human urotensin-II (U-II) is a cyclic 11-amino-acid residue peptide with a wide range of vasoactive properties dependent on the anatomic site and the species studied. The purpose of this study was to determine the localization of human U-II in normal human kidneys and in renal carcinoma. Normal human kidneys (n=11) and eight cases of clear-cell carcinoma were immunostained with a polyclonal antibody to human U-II. In normal human kidneys, U-II was mostly present in the epithelial cells of tubules and ducts, with greater intensity in the distal convoluted tubules. Moderate U-II immunoreactivity was seen in the endothelial cells of renal capillaries, but only focal immunoreactivity was found in the endothelial cells of the glomeruli. No staining was found in the veins. All tumors expressed moderate U-II immunoreactivity in the cancer cells and vasculature. Here we demonstrate abundant expression of U-II in normal human kidneys and renal carcinoma. These findings suggest that the vasoactive and growth-mediator peptide U-II may contribute to the pathophysiology of the human renal system.     

Effect of human urotensin-II infusion on hemodynamics and cardiac function

Recent studies have shown that the vasoactive peptide urotensin-II (U-II) exerts a wide range of action on the cardiovascular system of various species. In the present study, we determined the in vivo effects of U-II on basal hemodynamics and cardiac function in the anesthetized intact rat. Intravenous bolus injection of human U-II resulted in a dose-dependent decrease in mean arterial pressure and left ventricular systolic pressure. Cardiac contractility represented by +/-dP/dt was decreased after injection of U-II. However, there was no significant change in heart rate or diastolic pressure. The present study suggests that upregulation of myocardial U-II may contribute to impaired myocardial function in disease conditions such as congestive heart failure.     

Hybridization dynamics between Colorado's native cutthroat trout and introduced rainbow trout

Newly formed hybrid populations provide an opportunity to examine the initial consequences of secondary contact between species and identify genetic patterns that may be important early in the evolution of hybrid inviability. Widespread introductions of rainbow trout (Oncorhynchus mykiss) into watersheds with native cutthroat trout (Oncorhynchus clarkii) have resulted in hybridization. These introductions have contributed to the decline of native cutthroat trout populations. Here, we examine the pattern of hybridization between introduced rainbow trout and 2 populations of cutthroat trout native to Colorado. For this study, we utilized 7 diagnostic, codominant nuclear markers and a diagnostic mitochondrial marker to investigate hybridization in a population of greenback cutthroat trout (Oncorhynchus clarkii stomias) and a population of Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus). We infer that cutthroat-rainbow trout hybrid swarms have formed in both populations. Although a mixture of hybrid genotypes was present, not all genotype combinations were detected at expected frequencies. We found evidence that mitochondrial DNA introgression in hybrids is asymmetric and more likely from rainbow trout than from cutthroat trout. A difference in spawning time of the 2 species or differences in the fitness between the reciprocal crosses may explain the asymmetry. Additionally, the presence of intraspecific cytonuclear associations found in both populations is concordant with current hypotheses regarding coevolution of mitochondrial and nuclear genomes.     

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.     

Non-native species limit stream restoration benefits for brook trout

Success of stream restoration can be difficult to define because many interacting abiotic and biotic factors across spatio-temporal scales can have measurable effects. Consequently, failure in habitat restoration to achieve targeted biological goals may reflect interactions of habitat restoration with unaccounted risks that have yet to be addressed on the landscape. This is particularly true within invaded landscapes, where habitat restoration can benefit non-native competitors as much as the native fishes for which restoration is designed. We tested for interacting effects of a reach scale habitat restoration effort and non-native trout competition on habitat use by a brook trout (Salvelinus fontinalis) metapopulation within a productive main stem corridor of the Shavers Fork watershed, West Virginia. We used a joint species occupancy model within a BACI sampling design to show that brook trout occupancy of main stem habitat was highest post-restoration within restored sampling reaches, but this benefit to native brook trout was conditional on brown trout (Salmo trutta) not being present within the main stem habitat. Collectively these results indicate that habitat restoration was only beneficial for native brook trout when non-native trout were absent from the restored sampling area. Proactive approaches to restoration will be integral for supporting resilient ecosystems in response to future anthropogenic threats (e.g. climate change), and we have shown that such actions will only be successful if non-native competitors do not also benefit from the restoration actions.     

Trophic relationships of nonnative brown trout, Salmo trutta, and native Bonneville cutthroat trout, Oncorhynchus clarkii utah, in a northern Utah, USA river

Nonnative trout invasions have caused the widespread decline of cutthroat trout populations in western North America. In contrast to other nonnative salmonids, the role of nonnative brown trout in native cutthroat trout decline is poorly understood. Specifically, the level of ecological similarity that occurs between these species and the importance of other trophic mechanisms (e.g., predation) in their interactions are key uncertainties. We evaluated the trophic relationships of brown trout and cutthroat trout in a northern Utah river using a combination of diet and stable isotope analyses. We compared the dietary habits of these two species using multiple and complementary measures. Based on both stomach contents and δ¹³C signatures, we found that these species consumed a similar and opportunistic diet (i.e., they were nonselective in their foraging patterns). However, at most sizes, brown trout ingested larger prey—including fishes—and occupied a higher relative trophic position (i.e., δ¹⁵N) than cutthroat trout. Overall, these results demonstrate a high degree of dietary similarity and therefore strengthen earlier conclusions regarding interspecific competition between these two species. Our study, when considered alongside the work of others, suggests there is potential for predatory interactions between these species (i.e., brown trout preying on small cutthroat trout). We believe that future research on brown trout–cutthroat trout interactions should consider predatory effects in greater detail.

Pro-angiogenic activity of Urotensin-II on different human vascular endothelial cell populations

Urotensin-II (U-II), along its receptor UT, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. In the present study, human vascular endothelial cells (EC) from one arterious and three venous vascular beds were used to investigate in vitro their heterogeneity in terms of expression of U-II and UT and of angiogenic response to the peptide. Real-time PCR and immunocytochemistry demonstrated the expression of UT, as mRNA and protein, in all the EC populations investigated. U-II, on the contrary, was detectable only in EC from aorta and umbilical vein. U-II did not affect the proliferation rate of adult human EC, but induced a moderate proliferative effect on EC from human umbilical vein. When tested in the Matrigel assay, however, all EC exhibited a strong angiogenic response to the peptide, comparable to that of fibroblast growth factor-2 (FGF-2) and it was not associated to an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. The angiogenic effect of U-II was abolished by the UT antagonist palosuran. Overall, these data suggest that U-II, in addition to the well known role in the regulation of cardiovascular function, also exert a specific angiogenic activity.     

Activation of trout adipose tissue lipoprotein lipase by trout apoproteins

In rainbow trout (Salmo gairdnerii) lipoprotein profiles change during the annual sexual cycle. Among other factors, lipoprotein lipase (LPL) activity might play a role. This enzyme is activated by trout serum suggesting the existence of a cofactor corresponding to apoprotein CII in this species. In the present study, we determined more accurately some characteristics of the enzyme activity inhibited by 0.3 M NaCl. Trout serum and high density lipoproteins (HDL) activated both rat and trout adipose tissue LPLs. A fraction of apo HDL obtained by gel filtration also activated the enzyme. The mean Mr was 10,000. Isoelectric focusing of the same fraction gave several bands of proteins with apparent pI in the range of 4.2-4.9. These results show that in trout, LPL is activated by a cofactor similar to that in mammals, the apo CII. In addition, a fraction mainly containing apo AI (+ traces of apo C) activated trout LPL and reinforced the activation by apo CII. These findings suggest that trout apo AI may promote the activating effect of apo CII on trout LPL.     

Interspecific association of brown trout (Salmo trutta) with non‐native brook trout (Salvelinus fontinalis) at the fry stage

The introduction of non‐native brook trout (Salvelinus fontinalis) in Europe has led to displacement and decreasing populations of native brown trout (Salmo trutta). Some studies have found that brown trout shift to a diet niche similar to brook trout when the two species live in sympatry, which conflicts with the competitive exclusion principle. A change in feeding niche may be a sign of early interspecific association and social learning, leading to behavioral changes. As a first step to address this possibility, it is essential to assess the interspecific association between the species during the early ontogenetic life stages. In this study, we therefore assess whether juvenile brown trout associate with non‐native juvenile brook trout to the same extent as with conspecifics by setting up two experiments: (i) a binomial choice test allowing visual and chemical cues to estimate the species specificity of group preference, and (ii) an association test without physical barriers to estimate the degree of association of a focal brown trout with a group of either conspecifics or heterospecifics. In experiment (1), we found that focal juvenile brown trout preferred to associate with the stimuli groups and did not discriminate either against conspecific or heterospecific groups. Furthermore, more active individuals showed stronger preference for the stimuli group than less active ones, regardless of species. In experiment (2), we found that brook trout groups had a tighter group structure than brown trout groups, and that focal brown trout showed stronger association with brook trout than with brown trout. These results indicate that brown trout may associate with brook trout at an early life stage, which would allow for interspecific social learning to occur. Future studies should look closer into causes and consequences of interspecific association and social learning, including potential effects on the phenotype selection in brown trout populations.     

Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp

Glucokinase (GK) plays a central role in glucose homeostasis in mammals. The absence of an inducible GK has been suggested to explain the poor utilization of dietary carbohydrates in rainbow trout. In this context, we analyzed GK expression in three fish species (rainbow trout, gilthead seabream, and common carp) known to differ in regard to their dietary carbohydrate tolerance. Fish were fed for 10 wk with either a diet containing a high level of digestible starch (>20%) or a diet totally deprived of starch. Our data demonstrate an induction of GK gene expression and GK activity by dietary carbohydrates in all three species. These studies strongly suggest that low dietary carbohydrate utilization in rainbow trout is not due to the absence of inducible hepatic GK as previously suggested. Interestingly, we also observed a significantly lower GK expression in common carp (a glucose-tolerant fish) than in rainbow trout and gilthead seabream, which are generally considered as glucose intolerant. These data suggest that other biochemical mechanisms are implicated in the inability of rainbow trout and gilthead seabream to control blood glucose closely.     

Expression of urotensin-II in human coronary atherosclerosis

The vasoactive peptide urotensin-II (U-II) is best known for its ability to regulate peripheral vascular and cardiac contractile function in vivo, and recent in vitro studies have suggested a role for the peptide in the control of vascular remodeling by inducing smooth muscle proliferation and fibroblast-mediated collagen deposition. Therefore, U-II may play a role in the etiology of atherosclerosis. In the present study we sought to determine the expression of U-II in coronary arteries from patients with coronary atherosclerosis and from normal control subjects, using immunohistochemistry and in situ hybridization. In normal coronary arteries, there was little expression of U-II in all types of cells. In contrast, in patients with coronary atherosclerosis, endothelial expression of U-II was significantly increased in all diseased segments (P < 0.05). Greater expression of U-II was noted in endothelial cells of lesions with subendothelial inflammation or fibrofatty lesion compared with that of endothelial cells underlined by dense fibrosis or minimal intimal thickening. Myointimal cells and foam cells also expressed U-II. In most diseased segments, medial smooth muscle cells exhibited moderate expression of U-II. These findings demonstrate upregulation of U-II in endothelial, myointimal and medial smooth muscle cells of atherosclerotic human coronary arteries, and suggest a possible role for U-II in the pathogenesis of coronary atherosclerosis.     

The caudal neurosecretory system: control and function of a novel neuroendocrine system in fish.

The caudal neurosecretory system (CNSS) of fish was first defined over 70 years ago yet despite much investigation, a clear physiological role has yet to be elucidated. Although the CNSS structure is as yet thought to be confined to piscine species, the secreted peptides, urotensins I and II (UI and UII), have been detected in a number of vertebrate species, most recently illustrated by the isolation of UII in humans. The apparent importance of these peptides, suggested by their relative phylogenetic conservation, is further supported by the complex control mechanisms associated with their secretion. The CNSS in teleosts is known to receive extensive and diverse innervation from the higher central nervous system, with evidence for the presence of cholinergic, noradrenergic, serotonergic, and peptidergic descending inputs. Recent observations also suggest the presence of glucocorticoid receptors in the flounder CNSS, supporting previous evidence for a possible role as a pituitary-independent mechanism controlling cortisol secretion. The most convincing evidence as to a physiological role for the CNSS in fish has stemmed from the direct and indirect influence of the urotensins on osmoregulatory function. Recent advances allowing the measurement of circulating levels of UII in the flounder have supported this. In addition, there is evidence to suggest some seasonal variation in peptide levels supporting the notion that the CNSS may have an integrative role in the control of coordinated changes in the reproductive, osmoregulatory and nutritional systems of migratory euryhaline species.     

Sympatric relationship between redband trout and non-native brook trout in the Southeastern Oregon Great Basin

Brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss) have been widely introduced outside their respective ranges within North America causing declines and displacement of native trout. Yet, successful coexistence of native and non-native trout has received little attention. Here we evaluated the effect of introduced brook trout on the size and density of native redband trout in two invaded sub-basins in southeastern Oregon. In a multi-year study, we investigated whether habitat and fish communities differed between streams and stream reaches where redband trout were allopatric versus where redband trout were sympatric with brook trout. We hypothesized that redband trout would be less dense and have smaller total length in sympatry with brook trout than in allopatry, but that total trout density would not differ. We investigated whether differences in habitat existed between sympatric and allopatric locations that would indicate differentiation in site level habitat preferences for each trout species. We found that sympatric locations had more wood but similar fish community structure. Mean length and densities of redband trout were higher at allopatric locations. However, in most years at sympatric locations total trout density was twice that of allopatric redband trout sites. Using comparable data from an eastern United States system where brook trout are native, sympatric sites had lower densities of brook trout; however, total trout density did not differ. We conclude that invading trout negatively impact native trout densities; but in southeastern Oregon system the negative impact is minimized.     

Alien invasions in aquatic ecosystems: Toward an understanding of brook trout invasions and potential impacts on inland cutthroat trout in western North America

Experience from case studies of biologicalinvasions in aquatic ecosystems has motivated aset of proposed empirical rules forunderstanding patterns of invasion and impactson native species. Further evidence is neededto better understand these patterns, andperhaps contribute to a useful predictivetheory of invasions. We reviewed the case ofbrook trout (Salvelinus fontinalis)invasions in the western United States andtheir impacts on native cutthroat trout (Oncorhynchus clarki). Unlike many biologicalinvasions, a considerable body of empiricalresearch on brook trout and cutthroat trout isavailable. We reviewed life histories of eachspecies, brook trout invasions, their impactson cutthroat trout, and patterns and causes ofsegregation between brook trout and cutthroattrout. We considered four stages of theinvasion process: transport, establishment,spread, and impacts to native species. Most ofthe research we found focused on impacts. Interspecific interactions, especiallycompetition, were commonly investigated andcited as impacts of brook trout. In many casesit is not clear if brook trout invasions have ameasurable impact. Studies of speciesdistributions in the field and a variety ofexperiments suggest invasion success of brooktrout is associated with environmental factors,including temperature, landscape structure,habitat size, stream flow, and humaninfluences. Research on earlier stages ofbrook trout invasions (transport,establishment, and spread) is relativelylimited, but has provided promising insights. Management alternatives for controllingbrook trout invasions are limited, and actions tocontrol brook trout focus on direct removal,which is variably successful and can haveadverse effects on native species. Themanagement applicability of research has beenconfounded by the complexity of the problem andby a focus on understanding processes atsmaller scales, but not on predicting patternsat larger scales. In the short-term, animproved predictive understanding of brooktrout invasions could prove to be most useful,even if processes are incompletely understood. A stronger connection between research andmanagement is needed to identify more effectivealternatives for controlling brook troutinvasions and for identifying managementpriorities.     

Microsatellite DNA data point to extensive but incomplete admixture in a marble and brown trout hybridisation zone

Marble trout, endemic to the Adriatic drainage basin, is severely threatened by hybridisation with non-native brown trout. In the present study, we analysed 12 microsatellite DNA loci to assess genetic population structure and differentiation between sympatric phenotypic marble and brown trout at nine sampling sites in the upper Etsch/Adige River system. F _ST and AMOVA analyses revealed significant genetic differentiation between marble and brown trout samples. Thus, admixture between brown and marble trout appears to be incomplete. However, factorial correspondence analysis depicted marble trout, Atlantic brown trout and intermediate genotypes. Bayesian-based individual assignment tests identified indigenous marble trout at five sampling sites. In four other samples no ‘pure’ marble trout were detected. Bidirectional, first-generation hybridisation, involving both sexes of both parental species was observed. In locations where ‘pure’ marble trout still exist, post-F1 hybridisation appears to be directed towards brown trout. This has likely slowed the rate of hybridisation between the two trout species and the decline of relic marble trout populations. Based on these results, restoration management actions are proposed, such as the abandonment of brown trout stocking activities, sharper angling policies, establishment of indigenous marble trout breeding strains and the elaboration of a conservation priority list.     

Organization and chromosomal location of the major histone cluster in brown trout,Atlantic salmon and rainbow trout

The major histone cluster (hisDNA) was mapped by fluorescent in situ hybridization (FISH) to mitotic chromosomes of Atlantic salmon, brown trout, and rainbow trout. The data reveal that in the three species hisDNA is tandemly repeated in a single locus. Southern blots of genomic DNA indicate that these clusters are representative of the vast majority of the histone genes in these species. Similar reiteration values were found among the three species. Genetic variability in the hisDNA was found only in brown trout for an EcoRI site.     

Habitat shifts in rainbow trout: competitive influences of brown trout

Summary We compared habitat use by rainbow trout sympatric (three streams) and allopatric (two streams) with brown trout to determine whether competition occurred between these two species in the southern Appalachian Mountains. We measured water depth, water velocity, substrate, distance to overhead vegetation, sunlight, and surface turbulence both where we collected trout and for the streams in general. This enabled us to separate the effects of habitat availability from possible competitive effects. The results provided strong evidence for asymmetrical interspecific competition. Habitat use varied significantly between allopatric and sympatric rainbow trout in 68% of the comparisons made. Portions of some differences refelected differences in habitats available in the several streams. However, for all habitat variables measured except sunlight, rainbow trout used their preferred habitats less in sympatry with brown trout than in allopatry if brown trout also preferred the same habitats. Multivariate analysis indicated that water velocity and its correlates (substrate particle size and surface turbulence) were the most critical habitat variables in the interaction between the species, cover in the form of shade and close overhead vegetation was second most important, and water depth was least important.