Differential levels of “urotensin-II-like” activity determined by radio-receptor and radioimmuno-assays

Peptides 25 (2004) 1339–1347

Differential levels of “urotensin-II-like” activity determined by radio-receptor and radioimmuno-assays Nambi Aiyar a,∗ , Brian Guida a , Zhaohui Ao a , Jyoti Disa a , Diane Naselsky a , David J. Behm a , Jui-Lan Su b , Frederick C. Kull Jr. b , Stephen A. Douglas a a

Department of Vascular Biology and Thrombosis (UW2510), Cardiovascular and Urogenital Center of Excellence for Drug Discovery (CEDD), GlaxoSmithKline, P.O. Box 1539, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA b Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA Received 24 November 2003; received in revised form 20 May 2004; accepted 20 May 2004 Available online 1 July 2004

Abstract Plasma and urinary levels of “urotensin(U)-II-like” substances determined in healthy human volunteers were 12.4 ± 0.6 ng/ml and 2.2 ± 0.3 ng/ml by RIA, an order of magnitude lower than that seen by RRA, 167.5 ± 9.5 ng/ml and 65.2 ± 4.3 ng/ml. HPLC demonstrated the existence of at least three prominent activity peaks in plasma and urine, the more hydrophobic of which did not co-elute with U-II, degradation products or URP. RRA and RIA recognized these peaks with contrasting efficacy. As such, published levels of “U-II-like” activity should be interpreted with caution until a better understanding is obtained regarding what species specific RIA and RRA assay reagents interact with. © 2004 Elsevier Inc. All rights reserved. Keywords: Urotensin-II; UT receptor; Vasoactive peptide; Radioimmunoassay; Radioreceptor assay; Hypertension

1. Introduction Human urotensin-II (hU-II), a cysteine disulfide bridged undecapeptide derived from the precursor polypeptide preprourotensin-II (preproU-II), mediates its biological effects by interacting with a specific plasma membrane G-protein coupled receptor, GPR14, recently renamed UT by the International Union of Pharmacology [1,4]. Although U-II exerts a broad spectrum of biological actions in mammals, diverse responses purported to influence cardiorenal, pulmonary (bronchoconstriction), central nervous system (locomotion) and endocrine (insulin secretion) function [5], the majority of research has focused on the actions of this peptide/receptor system within the cardiovasculature. However, despite a flurry of contemporary research in this area, the precise (patho) physiological function(s) of U-II remains to be fully elucidated. One aspect of delineating the putative role of U-II in the etiology of cardiovascular disease involves investigating changes in U-II expression in the normal and disease state. Over the last several years, numerous reports have ∗

Corresponding author. Tel.: +1 610 270 5004; fax: +1 610 270 5080. E-mail address: nambi [email protected] (N. Aiyar).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.05.015

appeared in the scientific literature detailing levels of “U-II-like” immunoreactivity in plasma from healthy volunteers using both RIA and ELISA (anti-U-II polyclonal antibodies [pAbs]). Further to this, a growing body of evidence suggests that such levels are increased in patients with diverse diseases including renal [23] and heart failure [3,11,14], portal hypertension [9], diabetes mellitus [24], and essential hypertension [13]. Strikingly, however, the levels of “U-II-like” immunoreactivity reported in such studies (determined using pAbs from a variety of commercial and academic sources) can differ by in excess of three orders of magnitude, a dynamic range unlikely to reflect simple patient/disease heterogeneity. For example, whereas Richards et al. [14], Totsune et al. [24], Wilkinson et al. [25], and Thompson et al. [22] reported “U-II-like immunoreactivity” levels in low pM range (2, 4, 12, and 21 pM [2.6–22.3 pg/ml], respectively) using antisera/pAbs from Phoenix Pharmaceuticals, Peptide Research Institute, University of Manchester, and Phoenix GmbH Europe. In contrast, however, Dschietzig et al. [6] Lapp et al. [11], Heller et al. [9], and Stangl et al. [17] recorded significantly higher values of 0.06, 1, 2, and 9 nM, respectively (80–12.4 ng/ml) using antisera/ELISA reagents developed by the German Heart Institute and Immunodiagnostik AG.

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Contrast these estimations with those of Matsushita et al. [13] who reported that “U-II-like” immunoreactivity in human plasma was “undetectable” (i.e. <40 pM [<50 pg/ml]) using antiserum from Peptide Institute Inc. and a clear disparity is apparent. Such differences have, by and large, been ignored in the literature, an issue compounded by the fact that many of the pAbs/assays used have been poorly characterized in the conventional sense of scientific peer review. The disparities in plasma “hU-II-like” levels seen between published studies are unlikely to be accounted for by differences in subject cohorts, assay format, extraction methods, etc. It is of note, therefore, that anti-human-U-II pAbs have been shown recently to interact with more than simply the mature hU-II isopeptide. For example, anti-hU-II pAbs recognize both “mature” human U-II and its prepropeptide isoform [16]. Indeed, multiple “U-II-like” immunoreactive fragments have been described in culture media conditioned by adrenocortical carcinoma cells [21] and in plasma samples from congestive heart failure patients [15]. More recently, a novel “urotensin-related peptide” (URP; A[CFWKYC]V) has been described in mammals, a biologically active peptide derived from a distinct gene product [20]. Since this peptide shares the “CFWKYCV” heptapeptide core sequence absolutely conserved among mammalian U-II isopeptides, one would predict that URP would also be recognized to a greater or lesser extent by U-II pAbs/antisera (since such reagents were raised against antigens containing this motif, e.g. KLH-conjugated mature hU-II). As such, it seems increasingly apparent that it is inappropriate to assume a priori that “U-II-like” immunoreactivity signals are derived from an interaction with a single immunoreactive species, namely hU-II It seems reasonable, therefore, to question whether such observations underlie the discrepancies in plasma “U-II-like” levels reported in man [5]. Since pAbs are, in general, available only in limited supply and are “contaminated” with non-specific Abs (i.e. those not directed toward the specific antigen of interest), the present study describes the use of a more specific anti-hU-II monoclonal antibody (mAb) to examine levels of immunoreactive U-II in human plasma and urine by RIA. In addition, the present study also describes the development of a supplementary radioreceptor assay (RRA), an analytical approach that does not rely on the utilization of antisera and one that is designed to compare and contrast “immunoreactive” U-II levels in plasma and urine with levels of “biologically active” material (since mAbs are also subject to some of the same potential for “promiscuity” that is inherent to pAbs, e.g. recognition of U-II propeptides, proteolytic fragments, etc.). In performing comparisons between RIA and RRA analysis, it was also acknowledged that additional materials of biological significance could be present in human plasma and urine, hitherto uncharacterized entities distinct from “mature hU-II” that were capable of interacting with the UT receptor (but perhaps not with the anti-hU-II mAb). Indeed, as will be described, the presence

of multiple “immunoreactive/bioactive” species was demonstrated by rp-HPLC. Furthermore, such species appeared to be distinct from hU-II, URP and a selection of putative hU-II putative “metabolites” and, to a greater or lesser extent, were detected by RRA but not by RIA (e.g. “Peak B” in urine). Indeed, the RRA appears to offer the advantage of measuring, in addition to the mature peptide, all “bioactive” materials present in plasma and urine, e.g. isoforms and metabolites that interact specifically with the U-II receptor. Such species are of “pharmacological significance” as they have the potential to contribute to the biological functions of the UT receptor system, e.g. vasoconstriction. As such, subsequent analytical studies will be required to characterize such putative factor(s). While the present study does not provide any insight into the nature of these species, the present findings suggest that caution should be employed when attempting to interpret published data reporting plasma “U-II-like” levels, etc. data generated using available pAbs/mAbs.

2. Materials and methods 2.1. Reagents Human U-II, porcine U-IIA and U-IIB isopeptides, human hU-II[4–11], hU-II[5–11] [(Cys5,10 )Acm]hU-II, prepro hU-II[86–99] and prepro hU-II[102–125] were either synthesized at GlaxoSmithKline (King of Prussia, PA) or were purchased from California Peptide Research Inc. (Napa, CA). Rat and mouse U-II isoforms, “urotensin-related peptide” (URP) and urotensin-I were purchased from Phoenix Pharmaceuticals Inc. (Mountain View, CA) whereas endothelin-1, angiotensin-II, calcitonin gene-related peptide, adrenomedullin, vasoactive intestinal peptide and somatostatin-14 were purchased from Bachem (Torrance, CA). Monoiodinated human U-II ([125 I]Tyr9 ; specific activity 2000 Ci/mmol) was custom labeled through Amersham (Chicago, IL). All other reagents were of analytical grade. 2.2. Sample collection Venous blood (∼10 ml), obtained from 15 healthy subjects (11 male, 4 female, ages 22–50 years), was collected in potassium EDTA tubes containing trasylol. Samples were immediately centrifuged (2000 × g for 15 min at 4 ◦ C) and the plasma removed and stored at −20 ◦ C until processed (within 2 weeks of collection). A similar protocol was used for urine (∼5 ml), which was obtained from seven healthy individuals (males, ages 20–60 years). 2.3. Extraction procedure RIA and RRA were performed for plasma and urine samples with and without extraction (see below). Samples, which did not undergo extraction, were used by diluting 1:10 for plasma and 1:5 for urine in RIA buffer for

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RIA and RRA. Extracted plasma and urine samples were used without further dilution. When required, plasma and urine samples were subjected to acid/acetone–petroleum ether extraction using a method described previously [19]. Briefly, samples were mixed with acid–acetone (acetone, 1 N HCl and water in the ratio 40:1:5) in a ratio of 2:1 and vortexed followed by centrifugation (2000 × g for 20 min at 4 ◦ C). The supernatant was mixed with petroleum ether (10 ml), vortexed and the upper layer discarded. Samples were washed an additional two times and the lower phase dried in a SC210A Speed Vac® plus ThemoSavant. All samples were subsequently reconstituted in 250 ␮l RIA buffer for the assay. The efficiency of the extraction method was evaluated by adding either [125 I]hU-II (∼25,000 cpm) or a known amount of synthetic hU-II to plasma or urine. Recovery was estimated by counting the extracted material in a Packard Cobra gamma counter (Packard Instrument Company, Meriden, CT) for [125 I]hU-II recovery and RIA was used to estimate the recovery of synthetic hU-II. The recovery was 70–80%. RIA and RRA were performed for plasma samples with and without extraction. The reconstituted plasma and urine samples after extraction were used without further dilution in the assay. Unextracted samples were used for the assay after 1:10 dilution for plasma and 1:5 dilution for urine in RIA buffer. Aliquot size for RIA was 100 ␮l and for RRA was 20 ␮l. 2.4. Radioimmunoassay The RIA incubation mixture (1 ml/tube) consisted of 100 ␮l hU-II standard or plasma/urine sample, 100 ␮l [125 I]hU-II (∼25,000 cpm), 700 ␮l standard buffer (50 mM phosphate buffer [pH7.4] containing 10 ␮M Na-EDTA and 0.1% BSA) and 100 ␮l diluted (1:300,000 final dilution) mAb 11–17 [18]. The mixture was incubated for 16 h (4 ◦ C) at the end of which bound and free ligands were separated by addition of 0.25 ml secondary goat anti-mouse antibody (BioMagnectic Goat Antimouse IgG; Qiagen). All assays were performed in duplicate.

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assay plate was sealed and binding was allowed to proceed at room temperature for 60 min on an orbital shaker. Plates were then spun at 2000 × g (10 min) and cell bound radioactivity was determined on a Top Count scintillation counter (Packard Instrument Company). Assays were performed in duplicate. 2.6. Reverse phase-HPLC Extracted human plasma (40 ml) or urine (60 ml) samples were injected onto semi-preparative column (Symmetry C18 5 ␮m 19 mm × 100 mm column [Walters Corporation, Milford, MA]) equilibrated with 0.1% trifluroacetic acid/water at a flow rate of 10 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 80% over 90 min using a linear gradient. Absorbance was monitored (214/280 nm) and fractions collected every 30 s. Samples were evaporated (SpeedVac) over night and reconstituted in assay buffer prior to evaluation by RIA and RRA. 2.7. Data analysis Graph PAD Prism (Graph PAD Software, San Diego, CA) was used for the analysis of sigmoidal dose response curves obtained in RIA and RRA studies and linear regression analysis was used to assess the correlation between variables. All data are expressed as mean ± S.E.M.

3. Results The anti-hU-II mAb 11–17 (final dilution of 1:300,000) exhibited sub-nM affinity for hU-II in the RIA (0.9 ± 0.1 nM association constant [Ka ]; Fig. 1). As predicted, mAb 11–17 also recognized U-II isopeptides from other non-primate species (goby, rat, mouse and pig) with

2.5. Radioreceptor assay The [125 I]hU-II radioreceptor assay (scintillation proximity assay [SPA]) was performed at room temperature using membranes prepared from HEK-293 cells stably transfected with monkey UT receptor [7]. Wheatgerm agglutinin-SPA beads (Amersham, Chicago, IL) suspended in binding buffer (25 mM Tris–HCl, [pH 7.4], 5 mM MgCl2 and 0.1% BSA) to give a stock of 50 mg/ml and stored at 4 ◦ C. At the time of assay, SPA beads (12.5 ␮g/ml) and HEK-293-monkey UT membranes (50 ␮g/well) were pre-coupled by gentle shaking (room temperature for 60 min). The binding assay consisted of 140 ␮l SPA–membrane complex, 20 ␮l [125 I]hU-II (300 pM) and 20 ␮l hU-II (5–15 ng) or plasma/urine sample. The assay was carried out using 96-well plates (Packard Optiplate, Packard Instrument Company, Meriden, CT). The

Fig. 1. hU-II standard curve in the radioimmunoassay using mAb 11–17 (Scatchard plot analysis of the binding of U-II to mAb 11–17 is shown in the inset where bound [B] and free [F] ligand were calculated from the above data and B/F plotted against B to estimate Ka ).

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Fig. 2. Competition curve of hU-II for U-II isoforms in the RIA. [125 I]hU-II and various concentrations of human U-II (䊐), rat U-II (䉱), porcine U-II-A (䉬), porcine U-II-B (䊊) and mouse U-II (䉲)were incubated with (mAb 11–17) for 16 h at 4 ◦ C.

similarly high affinity (0.3–1.1 nM Ki ’s; Fig. 2, Table 1). The mAb retained significant affinity (0.2–1.3 nM Ki ’s; Table 1) for the truncated peptides hU-II[4–11] and hU-II[5–11], analogs which retain the cyclic hexapeptide core sequence (“CFWKYC”) common to all native, mature U-II isoforms. Indeed, as predicted, mAb 11–17 also exhibited high affinity (1.9 ± 0.3 nM Ki ) for the recently identified cyclic “urotensin-related peptide”, URP (A[CFWKYC]V). In contrast, however, the affinity of mAb 11–17 for the linearized peptide [(Cys5,10 )Acm]hU-II, an analog that lacks the cyclized structure characteristic of the native U-II isopeptides, Table 1 Relative affinities of U-II isopeptides and analogs and other vasoactive peptides in the U-II RIA Competing ligand

Ki (nM)

U-II isopeptides Human U-II Goby U-II Rat U-II Mouse U-II Porcine U-IIA Porcine U-IIB

0.96 1.10 0.75 0.40 1.03 0.32

± ± ± ± ± ±

0.18 0.20 0.15 0.10 0.25 0.10

Human U-II “analogs” hU-II[4–11] hU-II[5–11] hU-II[5–10] ([Cys5,10 ]Acm)hU-II Urotensin-II-related peptide (URP) Pro hU-II[86–99] Pro hU-II[102–125]

1.26 ± 0.15 0.20 ± 0.05 9.20 ± 1.40 11.78 ± 2.10 1.90 ± 0.30 >1,000 >1,000

“Non U-II” peptides Urotensin-I Somatostatin-14 Endothelin-1 Vasoactiveintestinal peptide Adrenomedullin Calcitonin gene-related peptide

>10,000 >10,000 >10,000 >10,000 >10,000 >10,000

All values are expressed as mean ± S.E.M. (n = 3).

was significantly attenuated. The “specificity” of the antiserum was further demonstrated when mAb 11–17 was shown to exhibit greater than 10,000-fold lower affinity for unrelated (i.e. pharmacologically distinct) peptides such as urotensin-I, endothelin-1, angiotensin-II, calcitonin gene-related peptide, adrenomedullin and somatostatin-14 (Table 1). Using mAb 11–17, the half-maximal inhibition of [125 I]hU-II by hU-II in the RIA format was 3.0 ± 0.2 ng/ml and measured in the linear range 1.0–10.0 ng/ml (Fig. 3a). Repeated RIA measurements (n = 8) of one selected plasma sample yielded an intra-assay variance of 10.5%. Inter assay coefficients of variation for the RIA was 16.5%. The standard curve for RRA (Fig. 3b), constructed using [125 I]hU-II and monkey UT expressed in HEK-293-cells, revealed a half-maximal inhibition of [125 I]hU-II by hU-II at 1.6 ± 0.1 ng/assay (and measured in the linear range, 0.2–7.5 ng/assay). Intra- and inter-assay coefficients of variation for the RRA were 11.8% and 18.2%, respectively. To estimate the total amount of “U-II-like” immunoreactivity in human plasma and urine, samples were evaluated both with and without being subjected to acid–acetone extraction [19]. The recovery of extraction, evaluated through by adding a known amount of radioiodinated hU-II or a known amount of synthetic hU-II to plasma or urine, was approximately 70–80%. RIA analysis demonstrated that levels of “U-II-like” substances in plasma and urine from healthy human volunteers were 12.4 ± 0.6 ng/ml and 2.2 ± 0.3 ng/ml without extraction and 3.6 ± 0.4 ng/ml and 0.6 ± 0.1 ng/ml following extraction, respectively (Table 2). However, RRA analysis revealed that corresponding plasma and urine levels of “U-II-like” substances were greater than an order of magnitude higher than that seen by RIA (167.5 ± 9.5 ng/ml and 65.2 ± 4.3 ng/ml without extraction and 79.0 ± 7.4 ng/ml and 27.5 ± 2.4 ng/ml following extraction, respectively). Thus, irrespective of whether either plasma or urine was extracted (∼40-fold lower, respectively) or not (∼15-fold lower, respectively), RIA values were strikingly and consistently at least an order of magnitude lower than those values determined by RRA. Furthermore, there as a more profound Table 2 Plasma and urine “U-II like species” concentration in healthy subjects as determined by radioimmuno (RIA) and radio-receptor (RRA) assay RIA (ng/ml)

RRA (ng/ml)

RRA:RIA ratio

Plasma Without extraction With extraction Without extraction: with extraction ratio

12.4 ± 0.6 2.2 ± 0.3 5.6

167.5 ± 9.5 79.0 ± 7.4 2.1

13.5 35.9

Urine Without extraction With extraction Without extraction: with extraction ratio

3.6 ± 0.4 0.6 ± 0.1 6.0

65.2 ± 4.3 27.4 ± 2.4 2.4

18.1 45.9

All values are expressed as mean ± S.E.M. (n = 5).

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Fig. 3. Standard curves of: (a) hU-II in the RIA (with mAb 11–17) and (b) displacement of [125 I]hU-II by hU-II (RRA using membranes from HEK-293 cells transfected with monkey recombinant UT).

loss in “U-II-like” signal as a result of acid–acetone extraction observed using the RIA methodology (27–29% of unextracted values) compared to the RRA approach (42–47% of unextracted values). As such, the RIA and RRA methods provided estimates of “U-II-like” activity that differed dramatically. In human plasma, RIA values correlated significantly with those values determined by RRA irrespective of whether samples were extracted or not (Table 3; Figs. 4 and 5). This was, however, only the case for unextracted

urine. Irrespective of whether RRA or RIA methods were used, estimates of “U-II-like” activity in extracted human plasma did not correlate with those values estimated in unextracted plasma (although they did for RIA, but not RRA, for estimates of U-II in extracted/unextracted urine). Depending on the detection method employed (RRA versus RIA), reverse-phase HPLC fractionation of acid–acetone extracted human plasma (Fig. 6a) and urine (Fig. 6b)

Fig. 4. Correlation between “U-II-like” activity in human plasma determined by RIA and RRA: (a) without extraction and (b) following acid–acetone extraction. Correlation of plasma “U-II-like” activity for (c) RRA and (d) RIA in plasma with and without acid–acetone extraction.

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Fig. 5. Correlation between “U-II-like” activity in human urine determined by RIA and RRA: (a) without extraction and (b) following acid–acetone extraction. Correlation of plasma “U-II-like” activity for (c) RRA and (d) RIA in urine with and without acid–acetone extraction.

produced multiple “immunoreactive” (detected by RIA using an anti-hU-II mAB) and/or “bioactive” (detected by RRA using the monkey UT receptor) peaks. At least three peaks were identified with approximate retention times of (a) 29 min (“Peak A”; a sharp, well-defined peak recognized by both RIA and RRA in plasma and urine, eluting at 34% acetonitrile), (b) 33–50 min (“Peak B”; a broad peak

Table 3 Correlation between levels of “U-II like” activity in extracted and unextracted human plasma and urine as determined by radioimmuno- (RIA) and radioreceptor- (RRA) assay without and with extraction (normal, healthy volunteers) Correlation coefficient r

P-value

Plasma RRA vs. RIA without extraction RRA vs. RIA with extraction RRA without vs. with extraction RIA without vs. with extraction

0.57 0.46 0.14 0.32

0.04 0.01 0.72 0.32

Urine RRA vs. RIA without extraction RRA vs. RIA with extraction RRA without vs. with extraction RIA without vs. with extraction

0.40 0.01 0.27 0.70

0.04 0.84 0.12 0.01

eluting at 46–52% acetonitrile, most prominent in urine extracts and recognized with greater efficacy by RRA than RIA, Fig. 6b) and (c) 54 min (“Peak C”; recognized with equal efficiency by both RRA and RIA, eluting at 58% acetonitrile). The more hydrophobic peaks B and C did not appear to correspond to known U-II peptides/analogs. For example, mature hU-II and the truncated hU-II[4–11] fragment possessed identical retention times of 29 min (and, therefore, corresponded to “Peak A”). The truncated U-II analogs hU-II[5–10] and hU-II[5–11] had marginally shorter retention times (28 and 28.5 min, respectively) than hU-II. Similarly, urotensin-related peptide (URP) also had a retention time close to that seen with mature hU-II, eluting from the column 60 s after mature hU-II (30 min retention time). As such, none of the endogenous proteolytic hU-II fragments or isoforms exhibited retention times consistent with either Peak “B” or “C”.

4. Discussion Urotensin-II, a potent mammalian vasoconstrictor [1], is purported to regulate cardiovascular homeostasis [5,12]. In order to define the contributions of this peptide to the

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Fig. 6. Reverse phase-HPLC profile of “U-II-like” activity in the acid–acetone extracts of human (a) plasma and (b) urine using RRA (closed square) and RIA (open square). The elution position of standard hU-II (Peak A, 29 min retention time) is shown by arrow (total recovery from the column was 60–70% and 70–75%, respectively).

etiology of conditions such as hypertension, heart failure, etc. there is a need to be able to measure accurately levels biologically active “U-II-like” substances in pertinent biological fluids. As reported in the present study, several antibody-based assays have been developed to this end [5], assays sufficiently sensitive to measure U-II concentrations in ∼1 ml of human plasma in an affordable, reliable and reproducible manner. The levels of “U-II-like” immunoreactivity determined in the present study using extracted plasma samples from healthy volunteers (∼2.6 nM [3.6 ± 0.4 ng/ml] assessed using an anti-hU-II mAb) are similar to those reported in the literature (∼1–9 nM) using a commercial pAb from Immunodiagnostik [6,9,11]. Alarmingly, however, such values are several orders of magnitude higher than those levels (∼2–21 pM) recorded using antisera from alternate commercial and academic sources [14,22,23,25]. It is not the goal of the present study to say that one mAb or pAb is better than any other per se, rather it is to emphasize the discrepancy and underscore the fact that different assays/reagents utilized in this research field are not measuring exactly the same thing(s). While the present study offers only limited insight into this phenomenon, it illustrates that a better understanding of these types of assays is key if one is to be able to interpret clinical data generated using such reagents. Although U-II pAb-based RIAs and ELISAs have been available commercially for several years now, most are

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poorly characterized, at least in the conventional sense of scientific peer review. Based on recent reports in the literature, and the findings of the current study, it is becoming increasingly apparent that multiple “U-II-like” fragments are present in both human blood and urine. Such fragments: (a) cannot be considered as pharmacodynamically inert (i.e. factors such as urotensin-related peptide [URP] and hU-II[4–11], etc. all exhibit appreciable UT affinity) and (b) they appear to be differentially detected by RIA and RRA (e.g. “Peak B” in urine). For example, there is evidence in the literature to suggest that anti-human-U-II pAbs recognize both “mature” hU-II and its prepropeptide isoform [16] along with related, but distinct gene products such as urotensin-related peptide, URP [20]. Analysis of conditioned media [21] reveals that adrenocortical carcinoma cells secrete factor(s) that generate multiple “U-II-like” immunoreactive peaks upon reverse-phase-HPLC analysis [21]. Nothing is known about the molecular or pharmacodynamic properties of such factors and, as such, these observations clearly bring in question the ability to interpret published data unambiguously. Not surprisingly then, published estimates of “U-II-like“ activity have shown up to a 5000-fold variation in plasma “U-II-like” levels, e.g. 2 pg/ml [14] versus 12,400 pg/ml [17]. The cyclic hexapeptide region of U-II (“CFKWYC”) is fully conserved among U-II isopeptides from a variety of mammalian and non-mammalian species. It is also present in URP [20]. Accordingly, in addition to mature hU-II (Ka : 0.9 ± 0.1 nM), the mAb utilized for RIA in the present study displayed high affinity for all U-II isoforms studied (goby, rat, porcine and mouse). While such cross-species reactivity is of some practical utility (i.e. it facilitates the analysis of biological specimens from a variety of preclinical species), it suggests that antisera derived from hU-II-conjugated antigen will recognize the “CFWKYC” motif in peptides other than mature hU-II. In other words, anti-hU-II pAbs and mAbs will interact with: (a) U-II precursor peptides (namely preproU-II and related intermediates), (b) putative proteolytic fragments (e.g. hU-II[4–11]), (c) related, but distinct, gene products (e.g. URP) and (d) hitherto undefined factors with poorly characterized pharmacodynamic activities. Indeed, the present study demonstrates that the truncated peptide analogs hU-II[4–11] and [5–11], putative proteolytic hU-II fragments known to be equipotent with mature hU-II as spasmogens/UT ligands [8], are recognized by mAb 11–17 with an affinity comparable to mature hU-II. Similarly, URP also exhibited high affinity for the mAb. Presumably the extreme carboxyl-terminus of the U-II prepropeptide represents an exposed epitope with which the anti-hU-II pAbs and mAbs can interact with and as such pAbs and mAbs will interact with both mature U-II and its prepropeptide as suggested by Shenouda et al. [16]. Although the affinity of preproU-II for the UT receptor is unknown and the efficiency with which it can be extracted from blood/urine has not been reported, one cannot assume it is pharmacodynamically inert.

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Unrelated peptides (urotensin-I, endothelin, somatostatin, etc.) did not interact with the mAb illustrating the defined SAR/specificity. Loss of the cyclic hexapeptide core structure, such as that seen in ([Cys5,10 ]Acm)hU-II (a linear ligand in which Cys5 –Cys10 cyclization is prevented by the presence of acetamidomethyl blocking groups) or the putative preprohU-II fragments (preprohU-II[86–99] and [102–125]) resulted in a loss in affinity for the mAb. As such, RIAs and ELISAs probably measure, to a greater or lesser extent, numerous U-II propeptide precursors and proteolytic fragments, multiple species likely subject to differential recovery during extraction (i.e. mAb 11–17 is recognizing a specific epitope). If the present estimate of plasma “U-II-like” activity is accurate, the data implies that U-II circulates in plasma at concentrations approaching two orders of magnitude above it’s affinity for the UT receptor. As such, the extent of UT receptor occupation by endogenous ligand would be high. While not unprecedented, this would distinguish urotensin-II from, for example, a vasoactive peptide such as endothelin-1 which typically circulates in plasma at concentrations two orders of magnitude below its EC50 as a vasoconstrictor. However, the assumption that mature hU-II alone accounts for the immunoreactive signal reported in the present study and others is, clearly, questionable and, as such, the extent of endogenous UT receptor occupancy remains to be determined. While specificity is generally high, RIA analysis of human plasma and urine is limited by the fact that the assay will only detect species that share a common immunoreactivity, i.e. those species that share a defined epitope recognized by the specific mAb 11–17. The possibility exists that additional “biologically active” species are present in human blood and urine and that such factors are expressed in significant concentrations. Such putative factors might not retain the “CFWKC” motif recognized by mAb 11–17 and other anti-hU-II pAbs but they might still exhibit affinity for the UT receptor. As such, the ability to detect the presence of any such factors is of significance since such species are likely to have a biological impact. In order to assess this possibility, RIA analysis was supplemented by the development of a specific RRA. Such an assay has not been described for the U-II/UT system to date. The RRA exploits ability of “U-II-like” species (i.e. those with detectable UT receptor affinity) to displace radioactive ligand from HEK-293 cell membranes expressing recombinant UT (in this case, the monkey isoform). Further, the RRA approach also has the advantage that it can be used to measure large number of samples in a high throughput screening format (in contrast to RIA, which requires overnight incubation, samples could be assayed and data generated within a few hours). Strikingly, the RRA was consistently found to detect levels of “U-II-like” activity 13- to 18-fold higher in both plasma and urine than that estimated by RIA. This difference was even starker when acid–acetone-extracted samples were evaluated, where RRA values were typically ∼40-fold

higher in both fluids compared to RIA analysis. Notably, “U-II-like” levels were differentially affected following extraction (71–73% versus 53–58% reduction in activity in plasma and urine using RIA and RRA analysis, respectively). As such, it appears that there are species present in plasma and urine that possess significant biological activity but are not immunoreactive, i.e. they possess detectable UT receptor affinity do not interact readily with antisera generated against mature hU-II. Indeed, it could be argued perhaps that such species appear to be present in such fluids in the majority. This hypothesis would appear to be supported, at least in part, by the observations that reverse-phase HPLC fractionation of human plasma and urine reveals three prominent peaks upon subsequent RRA and RIA analysis. The profiles recorded differed from that observed following reverse phase-HPLC fractionation of culture media conditioned by adrenocortical carcinoma cells [21]. The HPLC peak that appeared with a retention time of 29 min (“Peak A”; 34% acteonitrile) was evident in both human plasma and urine and was readily detected by RIA and RRA. This fraction co-eluted with standard synthetic hU-II. “Peak B” was a much broader peak (∼33–50 min retention time; 46–52% acetonitrile). The total activity detected appeared to be much greater when RRA was used compared to RIA analysis in acid–acetone extracted urine (see Fig. 6b) i.e. several fractions were collected that possessed UT receptor affinity but were not immunoreactive i.e. species that bound to UT but appeared to lack the “CFWKYC” epitope common to U-II isoforms (inasmuch as mAb 11–17 did not recognize them). Furthermore, a third fraction of note was “Peak C”, a well defined peak of RRA and RIA activity identified in both extracted urine and plasma, with a retention time of 54 min (58% acetonitrile). The molecular nature of “Peaks B and C” is unknown at present. However, based on HPLC elution times, neither “Peak “B” nor “Peak “C” appear to be proteolytic fragments of U-II (at least not the [4–11], [5–11], [5–10] analogs) or URP [20]. The differential ability of published assays to detect such species could, at least in part, underlie the profound differences (in excess of 3 orders of magnitude) reported for “U-II-like” levels in human plasma (it seems implausible to explained such differences simply based on differences of subject selection or assay performance, etc.). Hypothetically, the presence of “additional peaks” in acid–acetone extracted human plasma and urine could result from the presence of species derived from the U-II prepropeptide. Such a phenomenon has been purported by Conlon et al. [2] following the fractionation of flounder urophysis extracts. Indeed, recently Russell et al. [15] proposed the presence a circulating “hU-II-like” immunoreactive species related to the preproU-II in the plasma of heart failure patients (since they could not detect the presence of mature U-II by liquid chromatography/mass spectrometry analysis). However, since (a) hU-II antisera is known to recognize both “mature” hU-II and its prepropeptide isoform [16] and (b) urinary Peak B was readily detected by

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RRA but not by RIA (at least in extracted urine), this peak with UT receptor affinity is thought unlikely to contain a precursor fragment of preproU-II. In conclusion, the present study contrasts “hU-II-like” levels in human plasma and urine by RIA (using mAb against human U-II) and RRA (using recombinant monkey UT receptor). The present data demonstrate the presence of multiple “U-II-like” activities in human plasma and urine, factors, which require further detailed characterization. The possibility exists that, under pathophysiological conditions, activation of the UT may involve multiple endogenous vasoactive species. Such findings demonstrate that caution must be employed when interpreting data generated with tool reagents (including commercial pAbs, etc.) that have not been characterized completely since their ability to recognize one or more potentially biologically active species (that is, plasma/urinary factors possessing significant UT receptor affinity) remains unknown. As such, it is possible that published levels of “U-II-like” factors present in plasma or urine are under/over estimated. Clearly, effort is needed to fully characterize physical nature and pharmacodynamic properties of such species and their interaction with assay reagents. References [1] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999;401:282–6. [2] Conlon JM, Arnold-Reed D, Balment RJ. Post-translational processing of prepro-urotensin II. FEBS Lett 1990;266:37– 40. [3] Douglas SA, Tayara L, Ohlstein EH, Halawa N, Giaid A. Congestive heart failure and expression of myocardial urotensin II. Lancet 2002;359:1990–7. [4] Douglas SA, Ohlstein EH. Urotensin receptors. In: Girdlestone, D, editor. The IUPHAR compendium of receptor characterization and classification. London: IUPHAR Media; 2000. p. 365–72. [5] Douglas SA. Human urotensin-II as a novel cardiovascular target: “heart” of the matter or simply a fishy “tail”. Curr Opin Pharmacol 2003;3:159–67. [6] Dschietzig T, Bartsch C, Pregla R, Zurbrugg HR, Ambruster FP, Richter C, et al. Plasma levels and cardiovascular gene expression of urotensin-II in human heart failure. Regul Pept 2002;110:33–8. [7] Elshourbagy NA, Douglas SA, Shabon U, Harrison S, Duddy G, Sechler JL, et al. Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey. Br J Pharmacol 2002;136:9–22. [8] Grieco P, Carotenuto A, Campiglia P, Zampelli E, Patacchini R, Maggi CA, et al. A new, potent urotensin II receptor peptide agonist containing a Pen residue at the disulfide bridge. J Med Chem 2002;45:4391–4.

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