Figure 1.
Characterisation of FITC-N/OFQ (F-N/OFQ) at recombinant NOP and classical opioid receptors on CHO cells.
A: Binding affinity of F-N/OFQ and a range of opioid receptor subtype selective reference compounds. F-N/OFQ shows high selectivity for NOP over classical opioid receptors (mean (range) of up to 4 experiments.) *curves did not saturate Ki estimated between 1 and 10µM. B: Both N/OFQ and F-N/OFQ behave as full agonists on recombinant human NOP. These data are stimulation factor = agonist stimulated specific binding / basal specific binding (mean +SEM, n=8).
Figure 2.
Representative images of rat mesenteric arterioles and venules 5 seconds after injection of 200 nM kg-1 FITC-N/OFQ into the mesenteric artery with (C,D) and without (A,B) N/OFQ (added 15 sec prior to FITC-N/OFQ and labelled T15) competing for the same NOP receptor site.
The areas highlighted by arrows indicate binding of FITC-N/OFQ to NOP (FITC-N/OFQ-NOP) and thus sparse distribution of NOP receptors on the endothelium in this animal.
Figure 3.
The decay of FITC fluorescence (gray level) in the endothelium of rat mesenteric arterioles and venules over 60 seconds in response to 200 nM kg-1 FITC-N/OFQ (F-N/OFQ), with (T-15, open circles) and without (T0, closed circles) 200nM.kg-1 N/OFQ (n=5 animals).
Values are median, with upper and lower error bars representing the 75th and 25th percentiles respectively. The time courses were significantly different.
Figure 4.
The effect of UFP-101 on cardiovascular variables: mean arterial pressure (MAP, mmHg; upper panel) and heart rate (beats per minute, bpm; lower panel) were measured in anesthetised rats at baseline (T0), this being 24 hours after i.v. injection with 1 mg kg-1 + 0.5 mg kg-1 (LPS, n=6; LPS + UFP-101, n=6) or saline (control, n=6; UFP-101, n=6).
Measurements were repeated 40 minutes after baseline (T40) in response to i.v. injection of 150 nM kg-1 UFP-101 (LPS + UFP-101, UFP-101 groups) or saline (control; LPS, groups). Values are median, with open bars representing the 50th-75th percentile, grey bars the 25th-50th percentile and upper and lower error bars representing the maximum and minimum respectively.
Figure 5.
The effect of UFP101 on microvessel diameters in vivo: arteriole (upper panel) and venule (lower panel) diameters were measured in anesthetised rats at baseline (T0), this being 24 hours after i.v. injection with 1 mg kg-1 + 0.5 mg kg-1 (LPS, n=6; LPS + UFP-101, n=6) or saline (control, n=6; UFP-101, n=6).
Measurements were repeated 40 minutes after baseline (T40) in response to i.v. injection of 150 nM kg-1 UFP-101 (LPS + UFP-101, UFP-101 groups) or saline (control; LPS, groups). Values are median, with open bars representing the 50th-75th percentile, grey bars the 25th-50th percentile and upper and lower error bars representing the maximum and minimum respectively.
Figure 6.
The effect of UFP-101 on macromolecular leak and leukocyte rolling in vivo: macromolecular leak (percentage change in interstitial FITC-BSA fluorescence from baseline, upper panel) and leukocyte rolling (per minute, baseline and after treatment, lower panel) in post capillary venules (<40 µm) within the anesthetised rat mesentery preparation.
Measurements were taken in response to i.v. injection of 150 nM kg-1 UFP-101 (LPS + UFP-101 and UFP-101 groups) or saline (control and LPS groups). Values are median, with open bars representing the 50th-75th percentile, grey bars the 25th-50th percentile and upper and lower error bars representing the maximum and minimum respectively. *increased compared to control.