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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana (D.R.K., V.A.K.); and Zealand Pharma A/S, Glostrup, Denmark (C.T., E.M., M.M.V., C.Q., J.S.P.)
Received for publication
January 10, 2005
Accepted
April 18, 2005.
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Abstract |
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; Reinscheid et al., 1995). N/OFQ binds selectively to and is the endogenous ligand for the N/OFQ peptide (NOP) receptor (previously known as opioid receptor-like 1) (Mollereau et al., 1994; Lachowicz et al., 1995; Meunier et al., 1995; Reinscheid et al., 1995; Cox et al., 2000). Although many studies have focused on the effects of N/OFQ in the CNS, this peptide also produces important physiological responses when administered into the periphery. In particular, N/OFQ exerts prominent effects on the neural and humoral mechanisms that control arterial blood pressure and fluid and electrolyte balance. Thus, N/OFQ relaxes vascular smooth muscle (Gumusel et al., 1997; Armstead, 1999; Champion et al., 1999) and when administered as an intravenous (i.v.) bolus to anesthetized (Champion and Kadowitz, 1997; Bigoni et al., 1999) or conscious (Giuliani et al., 1997; Madeddu et al., 1999; Kapusta, 2000) rats or mice, this peptide produces dose-dependent hypotensive and bradycardic responses. In addition, we have demonstrated that the continuous i.v. infusion of N/OFQ at low doses produces a marked diuresis and antinatriuresis without concurrent cardiovascular depressor responses that otherwise result from the i.v. bolus injection of N/OFQ (Kapusta et al., 1997; Kapusta, 2000).
The ability of peripherally administered N/OFQ to elicit a selective water diuresis (i.e., aquaresis) is of significance because it suggests that peripherally acting NOP receptor compounds could be useful for the management of water-retaining states associated with hyponatremia. However, the enzymatic instability of endogenous N/OFQ makes this peptide unsuitable for clinical use. To circumvent this problem, we developed a novel NOP receptor ligand that was created by applying the structure-inducing probe (SIP) technology (Larsen and Holm, 1998; Larsen, 1999; Larsen et al., 2001; Kapusta et al., 2002) to the compound Ac-RYYRWK-NH2, a purported NOP receptor partial agonist (Dooley et al., 1997; Kapusta et al., 2005). The principle of the SIP technology is that a short (5–10-amino acid) sequence of certain charged amino acids (e.g., lysine or asparagine) forms an 
-helical structure, which when attached to the C or N terminus of another peptide may twist the whole peptide into an -helix and prevent enzymatic degradation (Larsen et al., 2001; Larsen, 2003).
In this study, we present in vitro and in vivo data on the pharmacologic behavior and biological activity of the NOP receptor ligand called ZP120 (Ac-RYYRWKKKKKKK-NH2), which was created by conjugation of a SIP sequence consisting of six lysines to the NOP receptor ligand Ac-RYYRWK-NH2 (Kapusta et al., 2002). Ac-RYYRWK-NH2 was chosen as the parent compound based on the results of investigations in which we demonstrated that the NOP receptor partial agonist Ac-RYYRWK-NH2 produced a modest water diuresis after i.v. bolus injection in conscious rats (Kapusta et al., 2005). In addition to this compound's agonist-like effects on kidney function, we also showed that Ac-RYYRWK-NH2 (and other ligands historically classified as NOP receptor partial agonists; Calo' et al., 2000) behaved as a weak partial agonist, eliciting a slight reduction in mean arterial pressure without affecting heart rate. However, Ac-RYYRWK-NH2 itself also antagonized the profound hypotensive and bradycardic responses produced by i.v. bolus injection of the native ligand N/OFQ (Kapusta et al., 2005). Thus, considering the mixed behavior of Ac-RYYRWK-NH2, the present investigation was performed to investigate whether addition of the SIP moiety used to form ZP120 affected the pharmacodynamic profile of the hexapeptide in different in vitro and in vivo preparations. For this purpose, we first compared the effects of Ac-RYYRWK-NH2 with that of ZP120 in cellular (cAMP assay) and tissue (mouse vas deferens contraction) assay systems. In addition, studies were performed in conscious rats to compare the cardiovascular and renal responses produced by the peripheral administration (i.v. infusion and i.v. bolus injection dose-response studies) of ZP120 and the parent compound Ac-RYYRWK-NH2. Finally, in each system additional studies were performed to compare how Ac-RYYRWK-NH2 or ZP120 pretreatment modified the biological responses to the endogenous ligand N/OFQ.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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7580 cells/cm2) and grown for 3 days before use under the same culture conditions as mentioned above. hNOP Membrane Receptor Binding Assay. hNOP membrane receptors (10 µg of protein/assay) were incubated for 60 min at 25°C in a total volume of 100 µl of binding buffer (50 mM HEPES, 1 mM EDTA, 10 mM MgCl2, pH 7.4) supplemented with 1% bovine serum albumin (to avoid ligand depletion) together with 1.2 nM [3H]N/OFQ either alone (total binding), in the presence of 1 µM N/OFQ (nonspecific binding) or at five concentrations of ZP120 or Ac-RYYRWK-NH2.
The binding reaction was stopped by vacuum filtration on a Packard cell harvester onto 96-well UniFilter GF/C presoaked at least 30 min before use in 0.5% polyethyleneimine followed by three washes with ice-cold binding buffer. Filters were dried for 90 min at 60°C before adding 50 µl of scintillation fluid (Ultima Gold catalog 6013329; PerkinElmer Life and Analytical Sciences). The filter-bound radioactivity (membrane-bound) was measured in a Top-Count (PerkinElmer Life and Analytical Sciences) scintillation counter. Binding was corrected for protein in each test sample, and the protein content was measured according to Lowry et al. (1951).
hNOP Receptor-Mediated Inhibition of Forskolin-Induced cAMP Formation. On the day of analysis, growth medium was removed, and the hNOP-transfected HEK293 cells were washed twice in Dulbecco's phosphate-buffered saline containing 6 mM glucose at 37°C. Cells were then incubated at 37°C for 20 min in the same medium supplemented with 1 µM forskolin and 2 mM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and increasing concentrations of ZP120 or Ac-RYYRWK-NH2. The reaction was stopped by addition of 20 µl of ice-cold 0.50 M HCl and incubation on ice for further 20 min. Twenty microliters of the acid extract was used for determination of cAMP by the FlashPlate technique, and another 20 µl was used for determination of protein content. Experiments were run in duplicate.
Mouse Vas Deferens. All animal procedures for mouse vas deferens studies followed the guidelines for the care and handling of laboratory animals established by the Danish government. The organs were prepared essentially as described by Hughes et al. (1975). In short, NMRI albino male mice (Taconic M&B A/S, Ry, Denmark) were sacrificed by cervical dislocation, and vas deferens were carefully dissected free from adhering fat, connective tissue, and blood vessels. Fine polyester threads were tied at both ends of the vas deferens, which hereafter were rapidly removed. The vas deferens were placed in an organ bath of 10 ml containing a modified Krebs' solution (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM glucose, and 0.50 mM CaCl2) maintained at 37°C and continuously gassed with carbogen (95% O2 + 5% CO2). The organs were attached to a force transducer (type 372; Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten, Germany) with a resting tension of 1 g, and the tissue was continuously stimulated through two silver electrodes with supramaximal voltage (40 V) rectangular pulses of 1-ms duration and 10-Hz frequency with the train stimulation repeated once every minute. The electrically evoked contractions (twitches) were recorded, and the analog signal was digitized via a 12-bit data acquisition board (model DT321; Data Translation, Inc., Marlboro, MA) and sampled at 1000 Hz using the Notocord HEM 3.1 (Notocord Systems, Croissy sur Seine, France).
Initially, concentration-response curves with N/OFQ were performed both cumulatively and noncumulatively with washing after each addition of peptide, but no difference in reactivity of the tissue was found. Cumulative concentration-response curves were generated for N/OFQ (n = 42), Ac-RYYRWK-NH2 (n = 14), and ZP120 (n = 8) in the dose range 0.1 to 300 nM. To minimize a time-dependent loss of responsiveness of the organs, each cumulative curve was not allowed to exceed 40 min. After the generation of a curve, the tissue was superfused with fresh Krebs' solution, and the signal was allowed to stabilize 15 to 20 min before the next addition of substance. Antagonist actions were examined by adding the antagonist 5 to 10 min before starting the cumulative addition of agonist. The action of the nonselective opioid receptor antagonist naloxone (1000 nM) was examined on responses to N/OFQ (n = 4), Ac-RYYRWK-NH2 (n = 4), and ZP120 (n = 6). The antagonistic properties of Ac-RYYRWK-NH2 (30 nM, n = 4) and ZP120 (10 nM, n = 4; and 100 nM, n = 4) were examined against N/OFQ.
In Vivo Studies
Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing between 275 and 300 g, were used in these studies. Rats were housed in groups of five or fewer under a 12-h light/dark cycle until the day of the experiments. All rats were fed with normal sodium diets (sodium content, 163 mEq/kg) and were allowed tap water ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals and were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee.
Surgery. On the experimental day, rats were anesthetized with methohexital sodium (Brevital, 20 mg/kg, i.p., supplemented with 10 mg/kg, i.v., as needed). Polyethylene catheters (PE-10 tubing attached to PE-50; BD Biosciences, Sparks, MD) were then implanted into the left femoral artery and vein for recording of arterial pressure and infusion of isotonic saline, respectively. Through a suprapubic incision, a flanged polyethylene cannula (PE-240; BD Biosciences) was inserted into the urinary bladder. The bladder catheter was then exteriorized and secured by suturing to adjacent muscle, subcutaneous tissue, and skin. After surgical preparation, rats were placed in rat holders to minimize movement after recovery from anesthesia and to permit steady-state urine collections. An i.v. infusion of isotonic saline (55 µl/min) was then started and continued for the duration of the experiment. At 4 to 6 h after recovery and the start of isotonic saline infusion, the arterial catheter was flushed and attached to a pressure transducer (model P23Db; Statham, Oxnard, CA), and a collection beaker was placed under the bladder catheter. Heart rate was derived from the pulse pressure with a tachygraph (model 7 P4H; Grass Instruments, Quincy, MA), and arterial pressure and heart rate were recorded on a Grass model 7 polygraph and stored on a computer using Acknowledge version 4.7.2 software from BIOPAC Systems, Inc. (Goleta, CA).
Intravenous Infusion Studies. After stabilization of urine flow rate and urinary sodium excretion, urine was collected during a 20-min control period. After this, the i.v. infusate was switched to a solution of isotonic saline alone (time control group; n = 6) or saline containing ZP120 (0.1, 1.0, 10, or 100 nmol/kg/min; n = 6/group). For comparison, a separate group of rats (n = 6) received a continuous i.v. infusion of the parent hexapeptide Ac-RYYRWK-NH2 (1 nmol/kg/min). Immediately after the start of vehicle/drug infusion, experimental urine samples (10-min consecutive periods) were taken for 80 min.
Intravenous Bolus Injection Studies. Conscious rats were continuously infused i.v. with isotonic saline (55 µl/min) for the duration of the study. After collection of baseline control (20-min) measurements for each cardiovascular and renal parameter, ZP120 was injected as an i.v. bolus in the following doses: 10 (n = 6), 30 (n = 4), 100 (n = 6), 300 (n = 4), or 1000 nmol/kg (n = 4). Control rats (n = 8) received an i.v. bolus injection of isotonic saline vehicle (200 µl). For comparative purposes, a study was also performed to determine the cardiovascular and renal responses produced by the i.v. bolus injection of the unconjugated hexapeptide Ac-RYYRWK-NH2 (100 nmol/kg; n = 6) in conscious rats. Immediately after bolus drug/vehicle administration, experimental urine samples (10-min consecutive periods) were taken for 80 min.
Antagonist Studies. Experiments were performed to examine whether the i.v. bolus pretreatment of ZP120 blocked or attenuated the cardiovascular depressor responses to a subsequent i.v. bolus N/OFQ challenge. For these studies, separate groups of rats received an i.v. bolus pretreatment of saline (n = 6), ZP120 (100 nmol/kg; n = 6), or Ac-RYYRWK-NH2 (100 nmol/kg; n = 6). After measurement of cardiovascular and renal excretory function for the 2-h duration, each group then received an i.v. bolus injection of 100 nmol/kg N/OFQ, and the subsequent changes in heart rate and mean arterial pressure were examined for the next 15 min. In pilot studies, it was demonstrated that the bradycardic and hypotensive responses to 100 nmol/kg, i.v., N/OFQ were not blocked 2 h after pretreatment with 100 nmol/kg Ac-RYYRWK-NH2. Thus, for studies with this hexapeptide, rats were pretreated with i.v. bolus Ac-RYYRWK-NH2 for 10 min, and then the cardiovascular responses to i.v. bolus N/OFQ were examined.
Analytical Techniques for in Vivo Studies. Urine volume was determined gravimetrically. Urinary sodium and potassium concentrations were measured by flame photometry (model 943; Instrumentation Laboratories, Lexington, MA). Urine osmolality was measured by the vapor pressure method (Wescor 5500; Wescor Inc., Logan, UT). Osmolar clearance (Cosm) was measured as Cosm = (V Uosm)/Posm, where Uosm and Posm are urine and plasma osmolality, respectively, and V is urine flow rate. Free-water clearance (CH2O) was measured as CH2O = V – Cosm, where V is urine flow rate and Cosm is osmolar clearance.
Drugs
N/OFQ, the unconjugated hexapeptide Ac-RYYRWK-NH2, and the SIP-modified hexapeptide ZP120 were synthesized by Zealand Pharma A/S (Glostrup, Denmark). In brief, the peptide was synthesized by stepwise addition of the appropriate amino acids to a growing peptide chain, attached to solid support (poledimethylacrylamide resin). Once the peptide was fully assembled, it was detached from the solid support with a side chain group deprotection (cleavage step), purified using preparative high-performance liquid chromatography, and converted to chloride salt. The purity (>95%) and identity of the peptide was confirmed by mass spectrophotometry. Drug doses were corrected for content of counter-ions and water in the peptide batches. Thus, relative peptide content of each peptides was N/OFQ, 76.03%; Ac-RYYRWK-NH2, 74.74%; and ZP120, 63.45%.
Data Analysis
All data are expressed as means ± standard error of the mean of n experiments. One-way classified in vitro data were statistically analyzed using Student's t test for unpaired data or one-way analysis of variance (ANOVA). Post hoc comparison was performed using Fisher's least significant difference test. Data from in vivo studies (Figs. 3 and 4; Tables 3 and 4) were analyzed statistically (SigmaStat 3.0) by using a two-way ANOVA with repeated measures for main effects and interactions. Post hoc multiple comparison tests were performed with the Bonferroni test for both pairwise comparisons and comparisons versus a control group. A nonrepeated one-way ANOVA and post hoc Bonferroni multiple comparison test were used for pairwise comparison of peak changes (
, Fig. 5) in cardiovascular function produced by nociceptin (control) versus ZP120 or Ac-RYYRWK-NH2 administered alone or in combination (e.g., pretreatment) with nociceptin. Statistical significance was defined as P < 0.05. In the displacement experiments on hNOP membrane receptors, concentration of ligands producing 50% inhibition of specific binding (IC50) was corrected for the competing mass of radioligand using the Cheng-Prusoff equation to yield Ki values. Curve fitting was performed using SigmaPlot 2001 for Windows (Systat Software, Inc., Richmond, CA). The agonist potencies were expressed as pEC50, which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. The Emax is the maximal effect that an agonist can elicit in a given tissue/preparation. In the electrically stimulated MVD tissue, the Emax of agonists is expressed as percentage of inhibition of the control twitch. Antagonist potencies are expressed in terms of pA2, which is the negative logarithm to base 10 of the antagonist molar concentration that makes in necessary to elicit the original submaximal response. In this study, we used pA2 values as evaluated by Schild analysis as a measure of antagonistic properties of ligands (e.g., ZP120) in the mouse vas deferens assay. cAMP data were calculated as a percent effect of 10 nM N/OFQ since this concentration of N/OFQ gives 100% inhibition of the increase in cAMP induced by 1 µM forskolin.
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Results |
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Effects on Forskolin-Induced cAMP Formation on HEK293 Cells Transfected with hNOP. N/OFQ, the hexapeptide Ac-RYYRWK-NH2, and ZP120 inhibited forskolin-stimulated production of cAMP in HEK293 cells transfected with the hNOP receptor with similar EC50 and maximal efficacy (Table 1).
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MVD. As demonstrated by others, we found that N/OFQ is a potent inhibitor of electrically induced contractions in the MVD preparation (Berzetei-Gurske et al., 1996; Calo' et al., 1996; Rizzi et al., 2002) (Fig. 1). In comparison with N/OFQ, ZP120 behaved as a partial agonist displaying a lower Emax than N/OFQ (Fig. 1). To determine whether the response was mediated through classicopioid receptors, both N/OFQ and ZP120 were also tested in the MVD preparation in the presence of the competitive nonselective opioid receptor inhibitor naloxone. As shown in Fig. 1, both compounds evoked essentially identical concentration-response curves in the presence and absence of 1000 nM naloxone. The EC50 and Emax values for Ac-RYYRWK-NH2 and ZP120 in the MVD preparation are summarized in Table 2. As illustrated, Ac-RYYRWK-NH2 and ZP120 showed partial agonist behavior in this bioassay as indicated by the reduced maximal inhibitory responses relative to N/OFQ (Table 2).
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Additional studies were performed to assess antagonistic properties of ZP120 at the NOP receptor. These studies involved the addition of selected concentrations of ZP120 in the MVD preparation 5 min before the cumulative addition of N/OFQ (Fig. 2). Note that after pretreatment of MVD tissue with 100 nM ZP120, the subsequent addition of N/OFQ elicited an increase in basal contraction above that for ZP120 alone. Despite this effect, as shown in Fig. 2, both 10 and 100 nM concentrations of ZP120 competitively antagonized the effect of N/OFQ on electrically induced contractions of the MVD. ZP120 produced a parallel rightward-shift in the concentration-response curves for N/OFQ with IC50 values of 5.5, 100, and 3400 nM, corresponding to the curves with 0, 10, and 100 nM ZP120 as antagonist, respectively. Within this concentration range, the slope of the linear Schild curve was of unity. The pA2 value of ZP120 was determined to be 9.5, which reflects a potent antagonistic action of ZP120 on the NOP receptor. The antagonistic property of Ac-RYYRWK-NH2 was tested at one concentration (30 nM; n = 4), and these experiments demonstrated that Ac-RYYRWK-NH2 also inhibits N/OFQ-induced relaxation. Based on these data, the pA2 of Ac-RYYRWK-NH2 was estimated to be 7.8, but it should be noted that this estimate is based on only one concentration of the antagonist.
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Table 3 depicts the peak changes in heart rate, mean arterial pressure, urine flow rate, urinary sodium excretion, and urinary potassium excretion produced by increasing i.v. infusion doses of ZP120 in conscious rats. Compared with respective predrug control values for each group, the i.v. infusion doses of 0.1, 1.0, and 10 nmol/kg/min ZP120 did not alter heart rate. In contrast, each of these doses elicited a slight, but statistically significant reduction in mean arterial pressure. In each group, the blood pressure-lowering effect was slow in onset (5–10 min) and persisted over the duration of the 80-min infusion (see Fig. 3 for time course of the 1 nmol/kg/min ZP120 dose). Although the largest infusion dose of ZP120 tested (100 nmol/kg/min) markedly reduced mean arterial pressure (control, 117 ± 3 mm Hg; ZP120 20 min, 66 ± 2 mm Hg), this hypotensive response did not elicit a baroreflex-induced tachycardia and instead produced marked bradycardia. The i.v. infusion of ZP120 also produced a marked increase in urine flow rate and decrease in urinary sodium and potassium excretion (i.e., ZP120 produced a solute-free water diuresis). An increase (P < 0.05) in urine output occurred at doses of 0.1, 1.0, and 10 nmol/kg/min, but the 1.0 nmol/kg/min dose of ZP120 produced the highest magnitude increase (control, 58 ± 7 µEq/min; ZP120 40 min, 169 ± 23 µEq/min) and duration of diuresis. Concurrent with the increase in urine flow rate, i.v. infusion of ZP120 (1.0, 10, and 100 nmol/kg/min) evoked significant reductions in urinary sodium and potassium excretion. In contrast, the lowest dose of ZP120 tested (0.1 nmol/kg/min) produced a selective aquaretic response with no changes in urinary electrolyte excretion, whereas the suprapharmacological dose level was identified at the highest dose (100 nmol/kg/min) as demonstrated by a complete shutdown of renal excretory function. Intravenous infusion of isotonic saline vehicle failed to alter any cardiovascular or renal parameter over the course of the experiment (80 min; data not shown). At all the doses tested, the i.v. infusion of ZP120 did not produce any apparent behavioral/CNS (e.g., agitation, gnawing, licking, and exploratory) or sedative/catatonic effects over the course of the experimental protocol.
In general, i.v. bolus ZP120 (Table 4) produced changes in mean arterial pressure and renal excretory function (diuresis, antinatriuresis, and antikaliuresis) that were qualitatively similar to the dose-response effects elicited by the i.v. infusion of this drug (Tables 3 and 4). During i.v. bolus administration, the dose level that produced selective aquaresis was not identified, but the suprapharmacological dose level was determined at 1000 nmol/kg, i.v. Both the magnitude and duration of the diuresis produced by increasing i.v. bolus doses of ZP120 (Table 4) were similar to those elicited by the i.v. infusion of the drug (Table 3). One point, however, is that although the 1000 nmol/kg, i.v., bolus dose produced marked peak cardiovascular depressor and antidiuretic (control, 55 ± 4 µl/min; 20-min nadir, 5 ± 1 µl/min) responses, the levels for heart rate, mean arterial pressure, and urine flow rate again returned to control levels by the end of the 80-min protocol (80 min; 56 ± 16 µl/min), with a diuresis occurring thereafter (personal observation). At all doses tested, the i.v. bolus injection of ZP120 did not produce any apparent behavioral/CNS (e.g., agitation, gnawing, licking, and exploratory) or sedative/catatonic effects over the course of the experimental protocol. Compared with 100 nmol/kg Ac-RYYRWK-NH2, i.v. bolus ZP120 (100 nmol/kg) produced a greater magnitude and duration increase in urine flow rate and decrease in urinary sodium excretion but comparable changes in mean arterial pressure (slight hypotension) and urinary potassium excretion (antikaliuresis) (data not shown).
Figure 4 depicts the time-course changes in free-water clearance for groups of rats treated with ZP120 or Ac-RYYRWK-NH2. After either i.v. bolus injection (100 nmol/kg, i.v., of both peptides) or during i.v. infusion (1 nmol/kg/min of both peptides), both compounds produced a significant increase in free-water clearance, thus demonstrating the aquaretic profile of these ligands. In each case, however, the effects of ZP120 on free-water clearance were of markedly greater magnitude and duration. Importantly, the increase in free water clearance was of faster onset after i.v. bolus administration (within 10–20 min) than after i.v. infusion (within 20–40 min).
In separate studies, the cardiovascular responses to an i.v. bolus N/OFQ (100 nmol/kg) challenge were examined in groups of rats that had been pretreated with 100 nmol/kg, i.v., bolus ZP120 (2-h pretreatment) or Ac-RYYRWK-NH2 (10-min pretreatment). In pilot studies, it was shown that when 100 nmol/kg, i.v., Ac-RYYRWK-NH2 was administered either 60 or 30 min before the N/OFQ challenge, this hexapeptide had no effect or only partially blunted, respectively, the cardiovascular depressor responses to i.v. N/OFQ. As demonstrated in Fig. 5, when the pretreatment time was further shortened to 10 min, i.v. bolus Ac-RYYRWK-NH2 only partially inhibited the bradycardia and hypotensive response to i.v. bolus N/OFQ. In contrast, even 2 h after the initial i.v. bolus pretreatment injection with ZP120, the hypotensive and bradycardic response to i.v. bolus N/OFQ was abolished (longer pretreatment times not tested). In other pilot studies (data not shown), we have also observed that the cardiovascular depressor responses produced by i.v. bolus 100 nmol/kg N/OFQ are abolished in rats that have been infused with ZP120 (1 nmol/kg/min; other doses and infusion times not tested).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The findings of the present investigations demonstrate that the SIP-modified hexapeptide ZP120 is also a functionally selective NOP receptor partial agonist with a cellular/tissue-dependent pharmacological profile; i.e., in different in vitro/in vivo preparations, this compound produces a variable pharmacological profile from full agonist (inhibition of stimulated cAMP production in cells transfected with the hNOP receptor; aquaresis in vivo), partial agonist (submaximal inhibition of electrically evoked twitch in MVD assay; subtle hypotensive response in vivo), to antagonist (competitive blockade of N/OFQ's action in the MVD assay; blockade of the N/OFQ-evoked bradycardia and hypotension in vivo) behavior. Moreover, the biological activity (including water diuresis) of ZP120 is significantly increased above that of the parent hexapeptide, Ac-RYYRWK-NH2.
Ac-RYYRWK-NH2, the parent compound used in this study, was identified as one of five hexapeptides with high affinity for the NOP receptor from a combinatorial library containing more than 52 million hexapeptides (Dooley et al., 1997). All five peptides were characterized as partial agonists in three different assays: stimulation of [35S]guanosine 5'-O-(3-thio)triphosphate binding, inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells transfected with NOP, and inhibition of electrically induced contractions in the MVD. Partial agonist behavior of these hexapeptides has been described by several groups (Dooley et al., 1997; Berger et al., 2000; Calo' et al., 2000; Rizzi et al., 2002; McDonald et al., 2003; Corbani et al., 2004).
In the present investigation and in a previous study performed only with ZP120 (Rizzi et al., 2002), we confirmed that Ac-RYYRWK-NH2 and ZP120 were partial agonists in the MVD assay with similar pEC50 values (9.0 and 8.6, respectively), but with each of these compounds inhibiting electrically evoked contractions with substantially lower maximal effect, higher potency, and slower onset of action than N/OFQ. The inhibitory effects of ZP120 and Ac-RYYRWK-NH2 on electrically induced contractions were not antagonized by the nonselective opioid receptor antagonist naloxone. In further support that ZP120 is a selective NOP receptor ligand, Rizzi et al. (2002) also found that the effects of ZP120 in the MVD assay were not modified by 1 µM naloxone, but they were prevented by the selective NOP receptor antagonist CompB. However, in addition to their partial agonist activity, both ZP120 and Ac-RYYRWK-NH2 also behaved as antagonists in this tissue. Thus, in the MVD assay both peptides blocked the inhibitory effects of the native ligand N/OFQ on electrically induced contractions, but ZP120 was approximately 50-fold more potent as an antagonist than Ac-RYYRWK-NH2. In contrast to findings in the MVD assay, we found instead that N/OFQ, ZP120 and Ac-RYYRWK-NH2 each behaved as full agonists in inhibiting forskolin-stimulated cAMP accumulation in HEK293 cells transfected with NOP receptors. Furthermore, in this cell culture system ZP120 and Ac-RYYRWK-NH2 displaced [3H]N/OFQ with pKi values (both peptides pKi = 9.6) in accord to that reported for N/OFQ in the literature (Reinscheid et al., 1995; Corbani et al., 2004). Finally, in previous binding studies we showed that ZP120 is a highly selective NOP receptor ligand that has no or only negligible binding to more than 50 receptors and ion channels (Hadrup et al., 2004).
It is clear that the variable pharmacological behavior of ZP120 (and the parent hexapeptide) is not limited to isolated tissue or cellular systems. This was demonstrated by the finding that in conscious rats, ZP120 evoked a marked increase in urine flow rate and decrease in urinary sodium and potassium excretion. Alternatively, in regard to blood pressure, dose-response studies showed that over the range of doses tested ZP120 (i.v. bolus or i.v. infusion) acted as a partial agonist, with this compound only producing a subtle reduction in mean arterial pressure that was slow in onset and unlike the profound and immediate bradycardic and hypotensive response elicited by i.v. bolus N/OFQ. But simultaneously, ZP120 acted as an antagonist in the circulation, because when administered in an aquaretic dose, ZP120 had negligible effects on heart rate and mean arterial pressure, but it prevented the characteristic and marked bradycardia/hypotensive responses to a subsequent i.v. bolus N/OFQ challenge. Of interest, ZP120 did not produce any apparent CNS or behavioral effects after i.v. administration of even very high doses, which in agreement with our other studies with different NOP receptor ligands (Kapusta et al., 2005), suggesting that ZP120 is likely to mediate its renal excretory responses via a peripheral (e.g., kidneys) site of action (Hadrup et al., 2004).
Although of significant interest, the reasons for the different maximal efficacy and pharmacologic behavior of ZP120 (and Ac-RYYRWK-NH2) in different test systems are not known. As discussed (Kapusta et al., 2005), it has been suggested that in different cell/tissue test systems, ligands classified as NOP receptor partial agonists may behave as either full/partial agonists, or antagonists (Calo' et al., 2000), at least in part due to differences in NOP receptor density and coupling efficiency (Berger et al., 2000; McDonald et al., 2003) and species differences (Burnside et al., 2000).
In addition to examining potential changes in pharmacological behavior in vitro, a major focus of this investigation was to examine how the addition of the SIP moiety to Ac-RYYRWK-NH2 (i.e., to form ZP120) affected this hexapeptide's ability to alter cardiovascular and renal function in vivo in conscious rats. Compared with the responses elicited by the unconjugated hexapeptide Ac-RYYRWK-NH2, an equivalent i.v. bolus or i.v. infusion dose of ZP120 markedly enhanced the renal responses producing a greater magnitude and sustained duration diuresis, antinatriuresis, antikaliuresis, and increase in free-water clearance (Figs. 3 and 4). The enhanced renal excretory responses produced by ZP120 relative to Ac-RYYRWK-NH2 are consistent with a SIP-induced increased stability of the compound in an enzymatic environment. Although this premise still has to be confirmed with in vivo plasma half-life data, note that i.v. bolus pretreatment of rats with ZP120 prevented the cardiovascular depressor responses to i.v. bolus N/OFQ for a substantially greater time (2 h; longer times not tested) than did Ac-RYYRWK-NH2 (10 min), thus providing additional support for increased metabolic stability and biological activity of ZP120.
As illustrated in Tables 3 and 4, we completed a full dose-response evaluation of ZP120 after both i.v. infusion and i.v. bolus injection. With both dosing regimens, at the lowest doses ZP120 produced sodium-potassium-sparing aquaresis associated with a slight lowering of blood pressure, but without reflex tachycardia. The renal no-effect dose level of ZP120 was not established in these studies. However, the suprapharmacological dose level was identified after i.v. infusion (100 nmol/kg/min) and i.v. injection (1000 nmol/kg), and it was defined as the dose that produced complete shutdown of renal excretory function secondary to a marked decrease in arterial pressure falling below the autoregulatory range for maintenance of renal hemodynamics. Based on these findings, the maximal tolerated doses of ZP120 were 10 nmol/kg/min, i.v., infusion and 300 nmol/kg, i.v., bolus. Thus, the therapeutic index defined as the ratio between the toxic dose relative to the minimal diuretic dose for i.v. bolus and i.v. infusion of ZP 120 was estimated to be at least 1000 for i.v. infusion and 100 for i.v. bolus, respectively. Moreover, it should be added that although 1000 nmol/kg, i.v., bolus ZP120 caused significant cardiovascular depressor responses and marked antidiuresis, these responses were transient and followed by restoration of systemic cardiovascular function and urine flow rate with onset of diuresis becoming apparent after completion of the 80-min protocol (our personal observation).
In conclusion, the unique pharmacological profile of a sodium-potassium-sparing aquaretic with mild vasodilatory properties in absence of reflex tachycardia makes ZP120 an interesting new drug candidate for acute treatment of critical water-retaining conditions with pulmonary congestion and/or hyponatremia/hypokalemia. In the present study, we demonstrated that i.v. infusion of 1 nmol/kg/min produced a maximal diuretic response, and using this dose, we recently reported that ZP120 inhibits approximately 40% of water reabsorption in the distal nephron and increases fractional water excretion to approximately 6% of glomerular filtration rate in absence of changes in glomerular filtration rate or renal blood flow (Hadrup et al., 2004). ZP120 induces a rapid down-regulation of AQP2 expression in the collecting duct. Therefore, it is expected that this powerful new aquaretic principle may act in synergy with existing diuretics with other tubular sites of action (Hadrup et al., 2004). Whether using a partial agonist rather than a full NOP receptor agonist presents any clinical advantages is not known. Corbani et al. (2004) recently examined the effect of full agonists, partial agonists, and antagonists on hNOP internalization, and they demonstrated that N/OFQ and the full agonist Ro64-6198 induced rapid internalization of the hNOP receptor, whereas antagonists and the partial agonist Ac-RYYRWR-NH2 had little effect on internalization. From these intriguing observations by Corbani et al. (2004), the marked and sustained renal and cardiovascular responses to ZP120 may not only be related to improved pharmacokinetic properties but also to the fact that partial agonists are less likely to induce tolerance, and thus they may present improved pharmacodynamic properties relative to full hNOP receptor agonists.
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ABBREVIATIONS: N/OFQ, nociceptin/orphanin FQ; NOP, nociceptin/orphanin FQ peptide; CNS, central nervous system; SIP, structure-inducing probe; ZP120, Ac-RYYRWKKKKKKK-NH2; hNOP, human nociceptin/orphanin FQ peptide receptor; MVD, mouse vas deferens; HEK, human embryonic kidney; ANOVA, analysis of variance; CompB, 1-[(±)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; Ro64-6198, (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one.
Address correspondence to: Dr. Daniel R. Kapusta, Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. E-mail: dkapus{at}lsuhsc.edu
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) or ZP120 (n = 6; 
) in conscious rats. Urine samples were collected during control (C, 20 min) and immediately after start of i.v. drug infusion (arrow), denoted as time periods 10 to 80 min (consecutive 10-min periods). HR, heart rate; MAP, mean arterial pressure; V, urine flow rate; UNaV, urinary sodium excretion; UKV, urinary potassium excretion. *, P < 0.05, significantly different than respective group control value. 
, P < 0.05, significantly different between groups.
