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1University of Sheffield, Academic Anaesthesia Unit and Microcirculation Research Group, Royal Hallamshire Hospital, Sheffield; 4University of Leicester, Division of Anaesthesia, Critical Care, and Pain Management, Department of Cardiovascular Sciences, Leicester Royal Infirmary, Leicester United Kingdom; 2Department of Pharmaceutical Sciences and Biotechnology Center and 3Department of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center, University of Ferrara, Ferrara, Italy
Submitted 12 April 2007 ; accepted in final form 28 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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200 µm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10–5 M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: –6.9 ± 3.8%; N/OFQ: 32.6 ± 8.4%; pyrilamine: 31.5 ± 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.
nociceptin/orphanin FQ; UFP-101; microcirculation; arteriole; nociceptin/orphanin FQ peptide receptor; ORL-1; permeability; vasodilatation; leukocyte
60% homology with the classical opioid peptides (excluding endomorphins) but lacks the NH2-tyrosine residue required for agonist activity at µ (MOP), 
, and 
receptors (30). Systemic N/OFQ administration (iv) causes both bradycardia and hypotension in anesthetized rodents (12, 20, 23). It follows that N/OFQ is a vasodilator (12–14), causing decreased perfusion pressure in the dennervated rat hindquarter vascular bed (14), but the effects on smaller arterioles and venules are poorly understood. Within the mesenteric microcirculation, there are two mechanisms that may serve as markers of inflammation: changes in postcapillary venule permeability because of the formation of gaps between vascular endothelial cells, and leukocyte-endothelial interactions. Vasodilation, macromolecular leak, and increased leukocyte endothelial interactions all occur in response to the inflammatory mediator histamine (1, 39, 43). Similarly, N/OFQ-NOP increases vascular permeability in vivo, as measured by the area (mm2) of Evans blue dye in extravascular tissues following injection in the tail vein (26). Furthermore, N/OFQ stimulates neutrophil chemotaxis, with neutrophils expressing the mRNA for the N/OFQ-NOP receptor on their surface (18, 37). They also secrete N/OFQ, suggesting that neutrophils are potentially an important source of this peptide (18).
The mechanisms of direct N/OFQ vasodilation and inflammation, however, remain uncertain. Previous studies using isolated mesenteric arteries (200 µm) demonstrated that vasodilation was not induced via muscarinic or traditional opioid receptors, calcitonin gene-related peptide, nitric oxide, prostaglandins, or ATP-dependent K+ channels (12–14). Nevertheless, in vitro N/OFQ stimulates the release of histamine from mast cells via NOP receptors present on their surface (26, 43), and N/OFQ injected intrathecally increased the concentration of histamine in the cerebrospinal fluid (42). It is possible therefore, that mesenteric microvascular responses to N/OFQ in vivo are mediated by histamine.
[Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) is a peptide antagonist for the NOP receptor (10, 11, 29). UFP-101 binds to the NOP receptor, expressed in Chinese hamster ovary cells in vitro, with 3,000-fold selectivity compared with classical opioid receptors (10) and does not inhibit binding of classical opiates such as [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin, which binds to MOP receptors (15). The ability of UFP-101 to antagonize physiological responses to N/OFQ in vivo is less certain, but, following intravenous administration, UFP-101 inhibits hypotension to N/OFQ (23, 28). The endothelium of larger vessels may express NOP receptors, since mRNA for NOP has been identified within sections of rat aorta (21). NOP receptors have not yet been characterized within the mesenteric microcirculation; however, inhibition of N/OFQ actions by UFP-101 could suggest a role for NOP in mediating microvascular vasodilation and inflammation.
This study utilized intravital microscopy (IVM) to investigate our hypothesis that microvascular responses to N/OFQ involved mast cell release of histamine, using the histamine H1 receptor antagonist pyrilamine (mepyramine), H2 antagonist ranitidine, and mast cell stabilizer cromolyn. Fluorescently labeled albumin [fluorescein isothiocyanate (FITC)-BSA] was used to determine the effects of N/OFQ on arteriolar and venular diameter (vessels <50 µm) and macromolecular leak within the rat mesentery (4, 8), and early leukocyte activation (rolling and adhesion to the venular endothelium) was also assessed. Furthermore, UFP-101 was used in vivo to determine the role of NOP in the histamine pathway mediating responses to N/OFQ. Isolated vessel (200 µm) responses to N/OFQ were also studied using pressure myography to gain further mechanistic insight into this pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male Wistar rats weighing between 220 and300 g (n = 100) were obtained from Sheffield Field Laboratories. Animals were exposed to a 12:12-h light-dark cycle in a humidity- and temperature-controlled environment while allowed access to water ad libitum. All procedures were performed under the Home Office Animal Procedures Act (1986), Project Licence nos. 50/2110 and 40/2813. Award of these project licences involved rigorous ethical and stastical review of protocols by both the University of Sheffield and the United Kingdom Home Office. At the end of the procedures, animals were killed humanely by cervical dislocation.
Drugs and Solutions
Peptides (N/OFQ and UFP-101) were prepared as previously described (19), but for all in vitro experiments and in vivo experiments utilizing histamine antagonists (pyrilamine and ranitidine; Sigma) and cromolyn (Sigma), N/OFQ was obtained from Sigma (catalog no. 04011). FITC was conjugated on celite (10%, A1628; Sigma, Dorset, UK) to BSA (98%, A7030; Sigma). This was achieved by combining 340 mg of FITC with 2 g BSA as previously described (7). The FITC-BSA solution was stored in 0.5-ml aliquots at –20°C until required.
Protein A-labeled polystyrene fluorescent microspheres (100 ml; F13081, Molecular Probes Europe) were added to 4.9 ml of BlockAid (B107101; Molecular Probes) and incubated without light at room temperature for 30 min. PBS (5 ml) was then added and centrifuged for 15 min at 1,000 g. The supernatant was removed, and 5 ml of PBS were added and centrifuged again, after which the pellet was resuspended in 300 µl of PBS. No more than 1 wk later, on the day of use, 100 µl of vortexed bead solution were added to 1.9 ml of PBS (7).
The constituents of HEPES-buffered phosphate saline (HEPES-PBS) solution were as follows: 0.2884 g/l MgSO4, 0.245 g/l CaCl2, 2.383 g/l HEPES, 8.2983 g/l NaCl, 0.3504 g/l KCl, and 0.1606 g/l KH2PO4. On the day of use, 0.99 g/l of D(+)-glucose was added to the solution, and the pH was adjusted to 7.4 (6). N/OFQ (prepared by R. Guerrini and G. Calo or Sigma) was diluted in distilled water to make 100x stock solutions and stored in aliquots at –25°C until the day of use. Where appropriate, dilutions of all stocks (10–2 to 10–9 M) were made with HEPES-PSS.
IVM
Surgical procedures. Rats were briefly anesthetized with halothane, and a pediatric butterfly needle was inserted in the ventral tail vein (not heparinized) to provide induction and maintenance of anesthesia with 30 mg/kg and 40–90 mg·kg–1·h–1 thiopental, respectively (Intra-Vital Sodium, Rhone-Poulenc Rourer, West Malina, UK), as previously described (4).
A tracheotomy was performed to allow adequate ventilation, and body temperature was maintained at 37°C throughout the experiment (Heating pads; Cole Palmer). The left carotid artery was cannulated with polyethylene tubing (0.58 mm ID x 0.965 mm OD) for computerized monitoring and continuous recording of mean arterial blood pressure by WINDAQ (DI-400; DATAQ Instruments, Akron, OH). The peak changes in blood pressure were used for measurements, which always occurred within 0–60 s. The right jugular vein was cannulated (polyethylene, 0.58 mm ID, 0.965 mm OD) for injections of either saline or fluorescent microspheres.
A midline abdominal incision was made, and the distal ileum was exteriorized. The animal was positioned on its left side, and the mesentery was placed on a glass microscope slide mounted on Perspex pillars on a Perspex board (4). The mesentery was kept moist throughout the experiment with regular applications of saline warmed to 37°C, and the moisture was retained by surrounding the preparation with saline-soaked gauzes and covering with Saran wrap (Dow Chemical; see Ref. 4).
Equipment.
The animal, warming pad, and Perspex board were transferred to the modified stage of an Ophtiphot-2 Nikon microscope (Nikon, Kingston, UK). This was equipped with a tungsten lamp for transmitted light microscopy. A filter cube interspersed in the path of the mercury arc lamp allowed a controlled amount of blue light (460–490 nm) on the area of interest (2 mm2). The images were viewed with a x20 objective (0.40 numerical aperture; Nikon), monitored using a CCD camera (KP-161; Hitachi), and displayed on a high-resolution monitor (PVM-1443; Sony). Images were recorded on to CD-RW using a CD/DVD Video Data Recorder (VDR-3000; Holdan) for later off-line computerized image analysis (Capiscope; KK Technology) using a frame grabber card (Matrox Meteor II). One to two areas of interest containing arterioles (15–40 µm in diameter) and venules (20–50 µm in diameter) were selected during the equilibration period and then recorded for 1 min using transmitted light (leukocyte-endothelial interactions) and 30 s using epi-illumination (diameter, leak, or velocity) immediately following drug administration throughout the experiment. All microvascular responses were therefore measured within the first 70 s following administration of saline or N/OFQ.
Data acquisition and analysis.
VESSEL DIAMETER.
Vessel diameter was measured using FITC-BSA and epi-illumination. Capiscope was calibrated with a micrometer specifically designed for the aforementioned camera and monitor, and three lines were drawn across the vessel at the same anatomical position at each time point to produce a median value in microns. An intraobserver error of <2% was observed previously with this technique and software (5). One arteriole and one venule were studied in each animal.
MACROMOLECULAR LEAK.
Macromolecular leak was also measured using FITC-BSA and epi-illumination. Capiscope assigned an integer value to the brightness of the interstitial fluorescence based on an arbitrary gray scale ranging from 0 (black) to 255 (white). The fluorescent light intensity thus measured is proportional to the amount of leak (24). Three small boxes (2 mm2 on screen) were placed at three equidistant sites, at alternating sides and adjacent to (1–2 µm) a 100-µm length of venule, selected at the beginning of experimentation, to produce a median of three values.
LEUKOCYTE ROLLING AND ADHESION.
Leukocyte rolling and adhesion were measured in postcapillary venules. Because the mesentery is extremely thin, these could be observed clearly with transmitted light. A straight length of venule without branches was selected, and the numbers of adherent leukocytes was determined per 100 µm by counting the number of stationary leukocytes that remained firmly adherent for >30 s. In each animal, the same venule was assessed for the numbers of leukocytes rolling along the endothelial wall, counted as the numbers of leukocytes passing a predetermined site over a 1-min period.
VELOCITY AND BLOOD FLOW.
Velocity and blood flow were determined following administration of 1 µm Protein A-labeled fluorescently labeled microspheres (yellow-green, 505/515 nm). At 25 frames/s, the distance microspheres moved over time, also within a straight length of vessel without branches, could thus be computed using Capiscope. Velocity (µm/s) was measured over three to four frames for arterioles and in <10 frames for venules. Ten centerline velocity measurements were made, and the three fastest were used to calculate mean values to ensure centerline measurements were used in a two-dimensional analysis system (31). Mean velocity (Vf) was calculated from centerline velocity requiring multiplication by an empirical factor of 0.625, as described earlier (27). Blood flow (ml/min) was then calculated using the circular cross-sectional area (an assumption of this model but applied to all vessels) multiplied by the mean blood flow velocity: 
x (diameter/2)2 x Vf (5, 24). For determining flow, diameter was measured in the same vessel at the time of velocity measurement using transmitted light, which allowed assessment of lumen diameter.
Experimental protocol. Following surgery, there was a 30-min stabilization period, followed by a 30-min equilibration period with IVM observation, before the following experimental manipulations.
UFP-101 EXPERIMENTS (N = 36).
Animals were allocated to two experimental groups to measure diameter, macromolecular leak, and leukocyte endothelial interactions (rolling and adhesion; n = 18). This required administration of FITC-BSA (0.2 ml/100 g, i.e., 0.02 g/100 g) in the carotid artery following the stabilization period. The second group involved measurement of lumen diameter and centerline velocity (n = 18). This required administration of 1 µm fluorescent microspheres (0.1 ml/100 g: 1 x 10–6 beads) in the jugular vein following the equilibration period (t = 0) and every 10 min thereafter (t = 10–40).
In both groups, animals were entered into the following experimental protocols: 1) control (n = 6), 2) N/OFQ (n = 6), or 3) UFP-101 (n = 6). Controls received a bolus injection of saline (0.2 ml/100 g) at t = 0, 10, 20, 30, and 40 min. The N/OFQ group received saline at t = 0 min followed by increasing doses of N/OFQ at t = 10 [0.6 nmol/kg (0.25 nmol)], t = 20 [3 nmol/kg (1.25 nmol)], t = 30 [15 nmol/kg (6.25 nmol)], and t = 40 [60 nmol/kg (25 nmol)]. The UFP-101 group received saline at t = 0 min followed by UFP-101 at t = 10 [30 nmol/kg (12.5 nmol)] and t = 30 [150 nmol/kg (62.5 nmol)] and N/OFQ 10 min later at t = 20 [3 nmol/kg (1.25 nmol)] and t = 40 [15 nmol/kg (6.25 nmol)] to determine whether UFP-101 was having an antagonistic effect on N/OFQ actions. These doses are based upon those shown to decrease blood pressure in guinea pigs following intravenous bolus administration (23).
HISTAMINE EXPERIMENTS (N = 39).
Vessel diameter, macromolecular leak, and leukocyte endothelial interactions (rolling and adhesion) were assessed (t = 0–50 min) following surgery and a 30-min stabilization plus a 30-min equilibration period.
Animals were allocated into one of the following experimental groups: 1) controls (n = 6), 2) N/OFQ (n = 6), 3) pyrilamine + N/OFQ (n = 6), 4) ranitidine + N/OFQ (n = 6), or 5) cromolyn + N/OFQ (n = 6). Controls received a bolus injection of saline (0.2 ml/100 g) at t = 0, 10, 20, 30, 40, and 50 min. The N/OFQ group received saline at t = 0 min followed by increasing doses of N/OFQ at t = 10 [0.3 nmol/kg (0.13 nmol)], t = 20 [0.6 nmol/kg (0.25 nmol)], t = 30 [3 nmol/kg (1.25 nmol)], t = 40 [15 nmol/kg (6.25 nmol)] and t = 50 [60 nmol/kg (25 nmol)] (21). The cromolyn group received a continuous infusion of cromolyn (1 mg·kg–1·min–1) for 5 min before each dose of N/OFQ, following precisely the protocol previously described by Blom et al. (3). The pyrilamine and ranitidine groups received saline at t = 0 min, the appropriate antagonist (1 mg/kg iv) at t = 10, and again 0.3–60 nmol/kg N/OFQ at t = 20–70 min. Preliminary experiments were also performed (n = 9) to ensure that changes in blood pressure and microvascular responses occurred to 0.1–20 µg/kg histamine (e.g., 20 µg/ml histamine: 35 mmHg decrease in mean arterial pressure, 36% increase in arteriolar diameter, 107-unit increase on gray scale, 9 adherent leukocytes/ 100 µm length of venule, n = 3) and that these responses could be inhibited 1 mg/kg pyrilamine and ranitidine (<20 mmHg mean arterial pressure decrease, <3% dilation, <5 change in gray level, <4 adherent leukocytes, n = 6).
Pressure Myography
Surgical procedures. Rats were deeply anaesthetized with halothane (3–4%, inhalation; Concord Pharmaceuticals) and killed humanely by schedule 1 methods to enable removal of the small intestine and adjoining mesentery through a midline laparotomy incision. The tissue was then transferred onto a dissecting dish and maintained in HEPES-PSS solution at 4°C. With the use of fine scissors, a second-order mesenteric artery (37) was then dissected free from surrounding adipose tissue for observation using myography.
Equipment and data analysis.
Vessels were carefully transferred to the vessel chamber of the pressurized myograph and mounted on glass cannulas (Living Systems Instrumentation, Burlington, VT), ensuring that no leakage occurred, i.e., maintenance of pressure at 60 mmHg without further flow in the vessel. As previously described, vessels were allowed to stabilize, first for 30 min at a low flow rate (0.6 ml/min) that did not exceed physiological values (6) and then for 50 min with an intraluminal pressure of 60 mmHg. The vessel chamber was maintained at 37 ± 0.5°C, and the arteries were pressurized at 60 mmHg throughout the duration of the experimental period. Arteries (200 µm) were viewed with a black and white CCD camera (Sony) and displayed on a monitor (Costar). Luminal diameters were measured using a video dimension analyzer, and data are displayed 2 min after application of each drug concentration (Living Systems Instrumentation).
Experimental protocol.
INTRALUMINAL ADMINISTRATION.
Following stabilization, a cumulative concentration-response to the thromboxane A2 agonist U-46619 (10–11 to 10–6 M) was performed to determine the EC80 (mean ± SE), i.e., the concentration of U-46619 in each vessel that induced 80% maximal constriction. This concentration was then added to the organ bath. Either 1) HEPES-PSS (n = 6), 2) 10–5 M N/OFQ (n = 6), or 3) 10–5 M N/OFQ + 10–5 M pyrilamine (n = 6) was then added to the perfusate and allowed to flow through the vessel at a rate of 15 µl/min. The vessel was then repressurized, and the intraluminal diameter (vasodilator response) was recorded and expressed as a percentage change from baseline 10 min later. In preliminary experiments, it was ensured that dilation to histamine (5 x 10–5 M) was inhibited by the dose of pyrilamine used (controls: 82.7 ± 5.8%, pyrilamine: 8.7 ± 4.6%, n = 6).
EXTRALUMINAL ADMINISTRATION.
Following preconstriction with the EC80 of U-46619 a cumulative dose response to N/OFQ (1 x 10–12 to 1 x 10–5 M, added to the bath every 5 min) was performed (n = 7). In experiments where vessels responded (n = 2), the dose response to N/OFQ was repeated in the presence of 10–5 M pyrilamine. Data are expressed as the dose required to achieve the maximal percentage change from baseline (Emax). All seven vessels were viable (see below); therefore, we could not continue the experiment to obtain n = 6 "responding" vessels.
In both intraluminal and extraluminal experiments at the end of the protocol, the organ bath was washed out and ACh (10–5 M) was added to preconstricted vessels to ensure that endothelial integrity had been maintained.
Flow Cytometry: CD11b Expression
Leukocyte isolation and labeling. Aliquots (50 µl) of whole blood obtained from animals at the end of the UFP-101 experiments (controls and N/OFQ, n = 12) were incubated with either 10 µl of FITC-labeled mouse antibody (Ab) against rat CD11b (MCA275FT; Serotec, Oxford, UK), 10 µl of FITC-labeled mouse anti-rat IgG Ab (MCA1209F; Serotec) or with no Ab, as described previously (8).
Equipment and data analysis. Leukocyte CD11b expression was analyzed on a FACSscan flow cytometer (BD Biosciences) using an FL1 detector (488 nm excitation wavelength). CELLQUEST software was used for data acquisition and analysis of 5,000 cells, with results expressed as mean channel fluorescence, which is a measure of CD11b expression. Populations of neutrophils were gated, and their fluorescent intensity was analyzed, since in rats 89% of rolling leukocytes detected using IVM were found to be polymorphonuclear (PMN, i.e., granulocytes) or monomorphonuclear (lymphocytes and monocytes), and of these 94–100% were PMNs (39, 41). Baseline mean channel fluorescent indexes were consistent but higher (218.8 ± 15.6) than our previous in vivo studies measuring cd11b from animals without FITC-BSA (8). Therefore, to account for any possible FITC-BSA interference, the Geo mean fluorescent index was used, i.e., test mean fluorescence divided by the mean fluorescence of the control (containing serum with FITC-BSA but no CD11b Ab).
Statistical Analysis
Data are presented as means ± SE with n = 6 animals used in each group as determined using a 80% power calculation with a predicted change of 15% and 95% Confidence interval. Within-group analysis was performed using a one-way repeated-measures ANOVA over time on ranks, followed by post hoc analysis with a Wilcoxon test for nonparametric data to identify points of significance compared with baseline. Between-group analysis was performed using a Mann-Whitney U-test. Results were considered statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Arterial blood pressure. N/OFQ (0.3–15 nmol/kg) caused a dose-dependent decrease in mean arterial pressure (Fig. 1, P < 0.05). In all animals, mean arterial pressure returned to a stable baseline within 3 min after administration of N/OFQ. UFP-101 alone had no effect on blood pressure (baseline, 118.5 ± 16.0 mmHg; 30 nmol/kg, 109.3 ± 14.0 mmHg; 150 nmol/kg, 102.9 ± 13.0 mmHg) but prevented decreases in blood pressure to 3 and 15 nmol/kg N/OFQ added 10 min later (Fig. 1); however, pyrilamine, ranitidine, and cromolyn had no effect on hypotension (Fig. 1).
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Intraluminal administration of N/OFQ. Preconstricted arteries (EC80 U-46619: 2.9 ± 0.26 x 10–7 M, no significant difference between groups) dilated in response to N/OFQ (controls: –6.9 ± 3.8%, n = 6; N/OFQ: 32.6 ± 8.4%, n = 6), but this dilation was not inhibited by pyrilamine (31.5 ± 6.8%, n = 6). At the end of the study, all vessels responded to ACh (controls: 101.9 ± 10.8%; N/OFQ: 96.2 ± 2.0%; pyrilamine: 96.6 ± 2.9%), confirming that endothelial integrity had been maintained.
Extraluminal administration of N/OFQ. Preconstricted arteries (EC80 U-46619: 2 x 10–7 M) demonstrated dose-dependent dilation to N/OFQ, which was inhibited by pyrilamine (Emax: N/OFQ, 27.2 and 26.9%; pyrilamine, –4.7 and –0.6%, n = 2). At the end of the study, these vessels responded to ACh (Emax: 104.2 and 110.9%, n = 2). However, the majority of vessels also preconstricted with U-46619 (EC80: 6.4 x 10–7 M) did not respond to N/OFQ (Emax: 5.0 ± 2.7%, n = 5); hence, the effect of pyrilamine could not be evaluated. All vessels responded to ACh (Emax: 94.0 ± 9.2%, n = 5).
Flow Cytometry: CD11b Expression
There was increased expression of CD11b on circulating neutrophils collected from animals receiving N/OFQ, since Geo mean fluorescent indexes were 2.06 ± 0.38 in controls and 4.19 ± 0.13 in the N/OFQ group (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Our study initially confirmed and extended understanding of the hypotensive effects of intravenously administered N/OFQ (23, 28), which has previously been correlated with inhibition of the sympathetic nervous system and decreased circulating norepinephrine concentrations (20, 23). UFP-101 is a NOP selective antagonist (10, 29) that, in agreement with previous studies, we found to inhibit hypotension to N/OFQ in vivo (23). Furthermore, we observed that pyrilamine, ranitidine, and cromolyn had no effect on hypotension to N/OFQ, indicating that, despite the described direct microvascular role, histamine did not contribute to neural mechanisms regulating blood pressure in response to N/OFQ in vivo.
We also report here N/OFQ-induced dilation of isolated mesenteric arterioles in vitro (200 µm), as previously described (12, 13), and for the first time dilation of smaller mesenteric microvessels (<50 µm) in vivo, although dilation of cerebral microvessels has previously been shown (2). The magnitude of dilation, although small (15%), was significant. It was also similar between in vivo and in vitro studies, despite the experimental limitations of comparing different models. Both types of vessels were used because the small vessels studied using IVM could not be mounted on the pressure myograph and the larger vessels used for myography were obscured by fatty tissue in vivo. Combined, however, N/OFQ-induced dilation of small vessels in vivo and isolated arteries perfused free of blood, neither of which receive significant extrinsic neural control (19), imply that mesenteric vasodilation to N/OFQ must involve a direct or nonneural mechanism that is also independent of blood-borne factors (19). In vivo we demonstrated that the mechanism of vasodilation to N/OFQ involved mast cell release of histamine, since pyrilamine, ranitidine, and cromolyn all inhibited the response.
Pyrilamine also caused constriction (and decreased leak), which could be due to the fact that surgery alone may induce a slight inflammatory response and loss of vascular tone, such that a histamine antagonist is able to induce a measurable response. In addition, histamine receptors show spontaneous activity in the absence of any stimuli, thus pyrilamine, by acting as an inverse agonist, could induce an opposite, i.e., constrictor, effect (34, 38).
N/OFQ is known to cause the release of histamine from mast cells via NOP receptors located on their surface (26, 37, 43). Our studies using cromolyn confirmed that histamine released from mast cells in response to N/OFQ then stimulates H1 and H2 receptors found on vascular smooth muscle to induce vasodilation (1, 34). NOP must also be involved in this pathway, since we further determined that intravenous UFP-101 inhibited vasodilation to N/OFQ in vivo. Therefore, NOP-dependent release of histamine from mast cells in vivo could cause local dilation to N/OFQ via histamine receptors, and we have attempted to summarize this new pathway in Fig. 6. Nevertheless, other mediators are released from mast cells, including serotonin and cytokines, and their role in microvascular responses to N/OFQ have yet to be explored.
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Our other in vitro studies also elucidated that extraluminally administered N/OFQ caused vasodilation. However, in isolated arterioles that responded (n = 2), vasodilation was inhibited by pyrilamine; hence, this histamine-dependent mechanism modeled our in vivo findings (Fig. 6). Nevertheless, the majority of vessels did not respond to N/OFQ (n = 5), despite having a viable endothelium (responded to ACh). One possible explanation is that arterioles that did respond to N/OFQ did so because connective tissue had not been completely removed from outside the vessel during the dissection process, leaving intact mast cells.
N/OFQ increased macromolecular leak from postcapillary mesenteric venules in vivo, since we measured increased fluorescence of FITC-BSA in the interstitium following intravenous administration. It was unlikely that N/OFQ caused loss of endothelial integrity due to changes in microvascular pressure or flux, since there were no significant changes in blood flow accompanying the response. UFP-101, pyrilamine, ranitidine, and cromolyn all inhibited this permeability change in response to N/OFQ. Therefore, taken together with previous findings, similar to vessel diameter, N/OFQ regulates vessel integrity in the mesentery via NOP and mast cell release of histamine in vivo (Fig. 6). This is in agreement with previous studies in other organs, since N/OFQ increased leak of Evans blue dye (1.5 mg/kg) in the extravascular tissue of rat skin was inhibited by pyrilamine (26). We also report for the first time that UFP-101 is an effective antagonist within the microcirculation in vivo and inhibits macromolecular leak to N/OFQ. We must be extremely cautious in extrapolating our findings at this stage, but, combined with known analgesic properties (11), NOP antagonists could therefore be an exciting new class of therapeutically useful antagonists for diseases involving inflammation of small blood vessels, e.g., sepsis.
Our results have also measured differences in the magnitude of macromolecular leak (and leukocyte-endothelial interactions) between UFP-101 and histamine in in vivo studies. We speculate that this is due to the use of N/OFQ synthesized in house for the first set of in vivo studies, as opposed to N/OFQ purchased from a commercial source (Sigma) for the in vivo histamine experiments and in vitro studies. Importantly, we demonstrate correlation between the peptides regarding patterns of response in vivo; therefore, the use of both peptides did not compromise our ability to investigate the aims of our study. However, the commercial product appeared to be more potent.
N/OFQ increased leukocyte rolling and adhesion in vivo. This agrees with previous in vitro studies reporting neutrophil chemotaxis to N/OFQ (18, 37). Indeed, the NOP receptor has been identified on the surface of circulating leukocytes in normal subjects (37). Using flow cytometry, we have confirmed the ability of N/OFQ to activate leukocytes, demonstrating that N/OFQ increases the expression of the 
2-integrin CD18/CD11b on the surface of circulating rat neutrophils, an adhesion molecule that facilitates the binding of leukocytes to the vascular endothelium via intercellular adhesion molecule-1 (33). We did not measure CD11b in the UFP-101 group, since the in vivo studies always necessitated that N/OFQ was added after UFP-101 to ensure the effectiveness of the antagonist.
Despite our observations with N/OFQ alone, UFP-101 did not prevent leukocyte activation to N/OFQ in vivo. Conversely, pyrilamine, ranitidine, and cromolyn inhibited N/OFQ-mediated rolling and adhesion. Therefore, N/OFQ appears to increase leukocyte rolling and adhesion via mast cell release of histamine, but this may not involve NOP. Hence, although the histamine-dependent mechanism of vasodilation and macromolecular leak to N/OFQ appears to be resolved, leukocyte-endothelial interactions will require further investigation. Other possible explanations for increased leukocyte-endothelial interactions include decreased velocity, which reduces dispersive forces at the vessel wall and allows greater binding of leukocytes to the vascular endothelium (27). In agreement with this, we observed corresponding decreases in blood flow velocity (not flow) with higher doses of N/OFQ.
This investigation demonstrated that blood flow within the mesenteric microcirculation decreased slightly in response to N/OFQ but did not reach significance compared with baseline because vasodilation was accompanied by a decrease in blood flow velocity. It is appreciated that some variability occurred between animals, as indicated by the difference between groups at t = 10 min in arterioles. However, despite changes in systemic blood pressure and vessel diameter to N/OFQ, it generally appeared that perfusion of this important splanchnic organ was maintained. During the microsphere experiments, it must be noted that there was decreased velocity with higher doses of N/OFQ (15 nmol/kg), despite the presence of UFP-101 (150 nmol/kg). We aimed for a 10-fold concentration difference between the agonist and antagonists (23). A higher concentration (>150 nmol/kg) of the antagonist UFP-101 may have inhibited the change in velocity to 15 nmol/kg N/OFQ; however, although we cannot make conclusions at this stage, it is also possible that the effects of N/OFQ at high doses are not NOP mediated.
In summary, N/OFQ causes dilation of small mesenteric arterioles and venules. N/OFQ also induces responses indicative of inflammation, namely increased leukocyte-endothelial interactions and macromolecular leak. UFP-101, pyrilamine, ranitidine, and cromolyn inhibited vasodilation and macromolecular leak to N/OFQ. Hence we conclude that N/OFQ-NOP-mediated release of histamine from mast cells acts on histamine receptors within the microvasculature to induce vasodilation and inflammation. N/OFQ-NOP therefore has an important role in microvascular inflammation, and in future studies this pathway may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist at the microvascular level.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* Z. L. S. Brookes and E. N. Stedman contributed equally to this study. 
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