In vitro sepsis up-regulates Nociceptin/Orphanin FQ receptor expression and function on human T- but not B-cells.

BACKGROUND AND PURPOSE: In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ-NOP system in freshly isolated volunteer human B- and T-cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis. EXPERIMENTAL APPROACH: B- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQATTO594 , N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25-plex assay format. Cells were challenged with LPS/PepG. KEY RESULTS: CD19-positive B-cells bound N/OFQATTO594 ; they also contain N/OFQ. Stimulation with CXCL13/IL-4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL-4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM-CSF release in an N/OFQ sensitive manner. CD3-positive T-cells did not bind N/OFQATTO594 ; they did contain N/OFQ. Stimulation with CXCL12/IL-6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQATTO594 binding. In LPS/PepG-treated cells, N/OFQ reduced migration to CXCL12/IL-6. LPS/PepG increased GM-CSF release in an N/OFQ sensitive manner. CONCLUSIONS AND IMPLICATIONS: We suggest both a constitutive and sepsis-inducible N/OFQ-NOP receptor autocrine regulation of B- and T-cell function, respectively. These NOP receptors variably inhibit migration and reduce GM-CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments.

Effect of a continuous perineural levobupivacaine infusion on pain after major lower limb amputation: a randomised double-blind placebo-controlled trial.

OBJECTIVES: Randomised controlled trial of the effect of a perineural infusion of levobupivacaine on moderate/severe phantom limb pain 6 months after major lower limb amputation. SETTING: Single-centre, UK university hospital. PARTICIPANTS: Ninety patients undergoing above-knee and below-knee amputation for chronic limb threatening ischaemia under general anaesthesia. Exclusion criteria were patients having surgery under neuraxial anaesthesia; inability to operate a patient-controlled analgesia device or complete a Visual Analogue Scale; amputation for trauma or malignancy; or contraindication to levobupivacaine. INTERVENTIONS: Either levobupivacaine 0.125% or saline 0.9% (10 mL bolus, infusion of 8 mL/hour for 96 hours) via a sciatic or posterior tibial nerve sheath catheter placed under direct vision during surgery. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was the presence of phantom limb pain, residual limb pain and phantom limb sensations up to 6 months after amputation. Secondary outcome measures included early postoperative pain and morphine requirements after surgery. RESULTS: Data from 81 participants were analysed; 6-month follow-up data were available for 62 patients. Pain and morphine requirements varied widely before and after amputation in both groups. The incidences of moderate/severe phantom limb pain, residual limb pain and phantom limb sensations were low from 6 weeks with no significant differences between groups in phantom limb pain at rest (OR 0.56, 95% CI 0.14 to 2.14, p=0.394) or movement (OR 0.58, 95% CI 0.15 to 2.21, p=0.425) at 6 months. Early postoperative pain scores were low in both groups with no between-group differences in residual limb pain or phantom limb sensations (rest or movement) at any time point. High postoperative morphine consumption was associated with worsening phantom limb pain both at rest (-17.51, 95% CI -24.29 to -10.74; p<0.001) and on movement (-18.54, 95% CI -25.58 to -11.49; p<0.001). The incidence of adverse effects related to the study was low in both groups: postoperative nausea, vomiting and sedation scores were similar, and there were no features of local anaesthetic toxicity. CONCLUSIONS: Long-term phantom limb pain, residual limb pain and phantom limb sensations were not reduced significantly by perineural infusion of levobupivacaine, although the study was underpowered to show significant differences in the primary outcome. The incidence of phantom limb pain was lower than previously reported, possibly attributable to frequent assessment and early intervention to identify and treat postoperative pain when it occurred. There were large variations in postoperative pain scores, high requirements for analgesics before and after surgery and some problems maintaining recruitment and long -term follow-up. Knowledge of these potential problems should inform future research in this group of patients. Further work should investigate the association between perioperative morphine requirements and late phantom limb pain. TRIAL REGISTRATION NUMBERS: EudraCT 2007-000619-27; ISRCTN68691928.

In vitro sepsis induces Nociceptin/Orphanin FQ receptor (NOP) expression in primary human vascular endothelial but not smooth muscle cells.

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24-48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1µM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1µM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

The effect of constitutive pigmentation on the measured emissivity of human skin.

BACKGROUND: The measurement of body temperature has become commonplace in the current COVID-19 pandemic. Body temperature can be measured using thermal infrared imaging, a safe, non-contact method that relies on the emissivity of the skin being known to provide accurate readings. Skin pigmentation affects the absorption of visible light and enables us to see variations in skin colour. Pigmentation may also affect the absorption of infrared radiation and thus affect thermal imaging. Human skin has an accepted emissivity of 0.98 but the effect of different skin pigmentation on this value is not known. In this study, we investigated the influence of different skin pigmentation on thermal emissivity in 65 adult volunteers. METHODS: A reference object of known emissivity (electrical tape) was applied to participant's skin on the inner upper arm. Tape and arm were imaged simultaneously using a thermal infrared camera. The emissivity was set on the camera to the known value for electrical tape. The emissivity was altered manually until the skin temperature using thermal imaging software was equal to the initial tape temperature. This provided the calculated emissivity value of the skin. Participants were grouped according to skin pigmentation, quantified using the Fitzpatrick skin phototyping scale and reflectance spectrophotometry. Differences in emissivity values between skin pigmentation groups were assessed by one-way ANOVA. RESULTS: The mean calculated emissivity for the 65 participants was 0.972 (range 0.96-0.99). No significant differences in emissivity were observed between participants when grouped by skin pigmentation according to the Fitzpatrick scale (p = 0.859) or reflectance spectrophotometry (p = 0.346). CONCLUSION: These data suggest that skin pigmentation does not affect thermal emissivity measurement of skin temperature using thermal infrared imaging. This study will aid further research into the application of thermal infrared imaging as a screening or bedside diagnostic tool in clinical practice.

Pathophysiological and clinical considerations in the perioperative care of patients with a previous ischaemic stroke: a multidisciplinary narrative review.

With an ageing population and increasing incidence of cerebrovascular disease, an increasing number of patients presenting for routine and emergency surgery have a prior history of stroke. This presents a challenge for pre-, intra-, and postoperative management as the neurological risk is considerably higher. Evidence is lacking around anaesthetic practice for patients with vascular neurological vulnerability. Through understanding the pathophysiological changes that occur after stroke, insight into the susceptibilities of the cerebral vasculature to intrinsic and extrinsic factors can be developed. Increasing understanding of post-stroke systemic and cerebral haemodynamics has provided improved outcomes from stroke and more robust secondary prevention, although this knowledge has yet to be applied to our delivery of anaesthesia in those with prior stroke. This review describes the key pathophysiological and clinical considerations that inform clinicians providing perioperative care for patients with a prior diagnosis of stroke.

Ex vivo modelling of the formation of inflammatory platelet-leucocyte aggregates and their adhesion on endothelial cells, an early event in sepsis.

Septicaemia is an acute inflammatory reaction in the bloodstream to the presence of pathogen-associated molecular patterns. Whole blood stimulation assays capture endotoxin-induced formation of aggregates between platelets and leucocytes using flow cytometry. We wanted to assess extent of spontaneous aggregate formation in whole blood stimulation assays and compare the effects of endotoxin and heat-killed, clinically relevant, bacterial pathogens on aggregate formation and then on adhesion of aggregates to TNFα-stimulated endothelial cells. We found that endotoxin (from Escherichia coli or Salmonella enteritidis) was not a suitable stimulus to provoke platelet-leucocyte aggregates in vitro, as it did not further increase the extent of aggregates formed spontaneously in stasis of hirudin-anticoagulated blood. Specifically, whole blood samples stimulated with or without LPS produced aggregates with a mean surface area of 140.97 and 117.68 µm2, respectively. By contrast, incubation of whole blood with heat-killed Klebsiella pneumoniae or Staphylococcus aureus produced significantly enhanced and complex cellular aggregates (with a mean surface area of 470.61 and 518.39 µm2, respectively) which adhered more frequently to TNFα (and free fatty acid)-stimulated endothelial cells. These were reliably captured by scanning electron microscopy. Adhesion of cellular aggregates could be blocked by incubation of endothelial cells with a commercial P-selectin antibody and an angiopoietin-2 ligand trap. In conclusion, we have developed an in vitro method that models the acute inflammatory reaction in whole blood in the presence of sepsis-relevant bacterial pathogen surfaces.

The microcirculation and its measurement in sepsis.

The microcirculation describes the smallest elements of the cardiovascular conducting system and is pivotal in the maintenance of homeostasis. Microcirculatory dysfunction is present early in the pathophysiology of sepsis, with the extent of microcirculatory derangement relating to disease severity and prognosis in ICU patients. However, at present microcirculatory function is not routinely monitored at the bedside. This article describes the pathophysiology of microcirculatory derangements in sepsis, methods of its measurement and evidence to support their clinical use.

Post-carotid Endarterectomy Hypertension. Part 2: Association with Peri-operative Clinical, Anaesthetic, and Transcranial Doppler Derived Parameters.

OBJECTIVE/BACKGROUND: The first paper in this series observed that pre-operative baroreceptor dysfunction and poorly controlled hypertension were independently predictive for identifying patients who went on to require treatment for post-endarterectomy hypertension (PEH). The second paper examines the influence of intra-operative patient, transcranial Doppler (TCD) ultrasound, and anaesthetic variables on the incidence of PEH. METHODS: In total, 106 patients underwent carotid endarterectomy (CEA) under general anaesthesia. Systolic blood pressure (SBP) changes, anaesthetic and vasoactive agents, analgesia, and post-operative pain scores, as well as TCD derived changes in middle cerebral artery (MCA) velocity during surgery were recorded. Patients who met pre-existing unit criteria for treating PEH after CEA (SBP > 170 mmHg without symptoms or SBP > 160 mmHg with headache/seizure/neurological deficit) were treated according to an established and validated protocol. RESULTS: In total, 40/106 patients (38%) required treatment for PEH following CEA (26 in theatre recovery [25%], 27 back on the vascular surgery ward [25%]), whereas seven (7%) had SBP surges > 200 mmHg on the ward. Patients requiring treatment for PEH had significantly higher pre-induction SBP (174 ± 21 mmHg vs. 153 ± 21 mmHg; p < .001), the greatest decreases in SBP after induction of anaesthesia (median decrease 100 ± 32 mmHg vs. 83 ± 24 mmHg; p = .01) and were significantly more likely to experience moderate/severe pain scores post-operatively (p = .003). Logistic regression analysis of the pre- and intra-operative data revealed that higher pre-induction mean SBP and lower pre-operative (impaired) BRS were the only independent predictors of PEH. CONCLUSION: This analysis of intra-operative variables has demonstrated that patients with poorly controlled and/or labile hypertension at induction of general anaesthesia were those at greatest risk of requiring treatment for PEH in the post-operative period after CEA. No other variables, including use of vasopressors, treatment of hypotension, anaesthetic agents, or changes in MCA velocity after clamp release and restoration of flow were able to predict who might go on to require treatment for PEH. Identification of at-risk individuals and aggressive blood pressure control in the post-operative period remains the mainstay of treatment.

Properdin levels in human sepsis.

Properdin is a normal serum protein that increases the production of complement activation products by binding C3b integral to convertase complexes and amplifying their activity at the site of activation. Thereby, it not only can aid in the resolution of infection but also contribute to tissue damage. In human sepsis, circulating complement C3 concentrations are decreased, though C3 is described as a positive acute phase reactant. However, properdin levels in human sepsis have not been reported. In this study, serum from 81 critically ill patients (predominately abdominal and respiratory sepsis) were analyzed for properdin levels at defined points of their stay in the intensive care unit (ICU) and compared with 61 age and sex-matched healthy volunteers. Properdin concentrations were significantly decreased in patients with sepsis on admission to ICU, but increased after clinical recovery to exceed levels observed in healthy volunteers. Properdin concentrations at ICU admission were decreased in non-survivors of sepsis compared to survivors, but this did not correlate with APACHE II score. However, pathologically low properdin levels (<7 µg/ml) were related to increased duration of treatment.

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system.

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2mg/kg produced a profound response within 5h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25mg/kg resulted in marked lethargy before 24h. Splenic interleukin-1ß mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1ß and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose-response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis.

Nociceptin system as a target in sepsis?

The nociceptin system comprises the nociceptin receptor (NOP) and the ligand nociceptin/orphanin FQ (N/OFQ) that binds to the receptor. The archetypal role of the system is in pain processing but the NOP receptor is also expressed on immune cells. Activation of the NOP receptor is known to modulate inflammatory responses, such as mast-cell degranulation, neutrophil rolling, vasodilation, increased vascular permeability, adhesion molecule regulation and leucocyte recruitment. As there is a loss of regulation of inflammatory responses during sepsis, the nociceptin system could be a target for therapies aimed at modulating sepsis. This review details the known effects of NOP activation on leucocytes and the vascular endothelium and discusses the most recent human and animal data on the role of the nociceptin system in sepsis.

The Nociceptin/Orphanin FQ system is modulated in patients admitted to ICU with sepsis and after cardiopulmonary bypass.

BACKGROUND AND OBJECTIVES: Nociceptin/Orphanin FQ (N/OFQ) is a non-classical endogenous opioid peptide that modulates immune function in vitro. Its importance in inflammation and human sepsis is unknown. The objectives of this study were to determine the relationship between N/OFQ, transcripts for its precursor (pre-pro-N/OFQ [ppNOC]) and receptor (NOP), inflammatory markers and clinical outcomes in patients undergoing cardiopulmonary bypass and with sepsis. METHODS: A prospective observational cohort study of 82 patients admitted to Intensive Care (ICU) with sepsis and 40 patients undergoing cardiac surgery under cardiopulmonary bypass (as a model of systemic inflammation). Sixty three healthy volunteers, matched by age and sex to the patients with sepsis were also studied. Clinical and laboratory details were recorded. Polymorph ppNOC and NOP receptor mRNA were determined using quantitative PCR. Plasma N/OFQ was determined using ELISA and cytokines (TNF- α, IL-8, IL-10) measured using radioimmunoassay. Data from patients undergoing cardiac surgery were recorded before, 3 and 24 hours after cardiopulmonary bypass. ICU patients with sepsis were assessed on Days 1 and 2 of ICU admission, and after clinical recovery. MAIN RESULTS: Plasma N/OFQ concentrations increased (p<0.0001) on Days 1 and 2 of ICU admission with sepsis compared to matched recovery samples. Polymorph ppNOC (p= 0.019) and NOP mRNA (p<0.0001) decreased compared to healthy volunteers. TNF-α, IL-8 and IL-10 concentrations increased on Day 1 compared to matched recovery samples and volunteers (p<0.0001). Similar changes (increased plasma N/OFQ, [p=0.0058], decreased ppNOC [p<0.0001], increased IL-8 and IL-10 concentrations [both p<0.0001]) occurred after cardiac surgery but these were comparatively lower and of shorter duration. CONCLUSIONS: The N/OFQ system is modulated in ICU patients with sepsis with similar but reduced changes after cardiac surgery under cardiopulmonary bypass. Further studies are required to clarify the role of the N/OFQ system in inflammation and sepsis, and the mechanisms involved.

Human peripheral blood mononuclear cells produce pre-pro-nociceptin/orphanin FQ mRNA.

BACKGROUND: Peripheral blood mononuclear cells (PBMC) transcribe mRNA for the nonclassical opioid nociceptin/orphanin FQ (N/OFQ) receptor (NOP). We probed for the N/OFQ precursor, pre-pro-N/OFQ (ppN/OFQ). METHODS: Using PBMC from 10 healthy volunteers we probed for ppN/OFQ using polymerase chain reaction (PCR) based experimental paradigms. RESULTS: In gel-based PCR, we detected amplicons consistent with ppN/OFQ mRNA in all samples. This was confirmed in quantitative real-time PCR with cycle thresholds (representing quantity of mRNA) of 30.91 +/- 0.18 (n = 10). CONCLUSIONS: These data indicate that PBMCs transcribe ppN/OFQ which, coupled with NOP expression, suggest NOP may be involved in the autoregulation of PBMCs.

Human peripheral blood mononuclear cells express nociceptin/orphanin FQ, but not mu, delta, or kappa opioid receptors.

BACKGROUND: Expression of opioid receptors on peripheral blood mononuclear cells (PBMC) is controversial. These receptors are currently classified as classical (MOP/mu/mu, DOP/delta/delta and KOP/kappa/kappa) and nonclassical NOP (nociceptin/orphanin FQ; N/OFQ). METHODS: In this volunteer study we probed for the expression of both classical and nonclassical opioid receptors using 1) radioligand binding, 2) specific antibody binding, and 3) polymerase chain reaction-based experimental paradigms. RESULTS: Membranes prepared from PBMC from healthy volunteers did not bind either [3H]diprenorphine (a nonselective radioligand for classical opioid receptors) or [3H]N/OFQ. There was significant concentration-dependent binding of each radioligand to control tissues expressing recombinant MOP and NOP. In addition, using fluorescence-activated cell sorting paradigms, there was no binding of fluorescent naloxone or either of two MOP antibodies to whole PBMC, though fluorescent naloxone did bind to recombinant MOP (as a positive control). Using primers specific for classical and nonclassical opioid receptors, and RNA extracted from the PBMC of 10 healthy volunteers, we were also unable to detect MOP, DOP, and KOP transcripts. In contrast, NOP was detected in all samples. CONCLUSIONS: Despite using several complementary experimental strategies, we failed to demonstrate protein for classical or nonclassical opioid receptors on PBMC from healthy volunteers. We detected NOP mRNA, suggesting low-density NOP expression on these immunocytes. It is possible that N/OFQ, produced by the PBMC itself, may be involved in the control of immune function.

Cell and tissue responses of a range of Urotensin II analogs at cloned and native urotensin II receptors. Evidence for coupling promiscuity.

Urotensin II (U-II) is the peptide ligand for the G-protein-coupled U-II receptor (UT). U-II has been dubbed "the most potent vasoconstrictor identified to date". However, in vivo studies with this system are hampered by the paucity of available ligands. Here, we characterise Chinese hamster ovary (CHO) cells expressing the human UT receptor in the following assays; (1) [(125)I]U-II binding, (2) GTPgamma[(35)S] binding, (3) cAMP formation, and (4) intracellular Ca(2+). We assess activity of 9 U-II analogues using these paradigms and examine their ability to contract isolated rat aorta. CHO(hUT) cells bound [(125)I]U-II with a B (max) and K (d) of 1,110+/-70 fmol/mg protein and 742 pM, respectively. hU-II stimulated GTPgamma[(35)S] binding (pEC(50) 8.38), optimal at low (0.1 muM) GDP concentrations. The hU-II GTPgamma[(35)S] response was partially PTx sensitive and there was a potent (pEC(50) 9.23) low efficacy ( approximately 20% inhibition) coupling to adenylyl cyclase. In CHO(hUT) cells hU-II stimulates calcium release from intracellular stores (pEC(50) 8.80) and calcium influx in a PTx-insensitive manner. In our structure-activity relationship study most ligands acted as full agonists. However, urantide behaved as a partial agonist (pEC(50) 7.67/pK(B) 7.55) in GTPgamma[(35)S] binding, a full agonist (pEC(50) 8.11) for increases in intracellular Ca(2+) and a competitive antagonist in the rat aorta bioassay (pK(B) 8.59). Collectively, these data show promiscuity at high expression and indicate the need for careful multi-assay evaluation of novel U-II analogues. Further modification of urantide, in order to eliminate residual agonist activity and to identify novel ligands for in vivo cardiovascular studies are clearly warranted.

In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen5,DTrp7,Dab8]urotensin II(4-11) (UFP-803).

The novel urotensin-II (U-II) receptor (UT) ligand, [Pen(5),DTrp(7),Dab(8)]U-II(4-11) (UFP-803), was pharmacologically evaluated and compared with urantide in in vitro and in vivo assays. In the rat isolated aorta, UFP-803 was inactive alone but, concentration dependently, displaced the contractile response to U-II to the right, revealing a competitive type of antagonism and a pA(2) value of 7.46. In the FLIPR [Ca(2+)](i) assay, performed at room temperature in HEK293(hUT) and HEK293(rUT) cells, U-II increased [Ca(2+)](i) with pEC(50) values of 8.11 and 8.48. Urantide and UFP-803 were inactive as agonists, but antagonized the actions of U-II by reducing, in a concentration-dependent manner, the agonist maximal effects with apparent pK(B) values in the range of 8.45-9.05. In a separate series of experiments performed at 37 degrees C using a cuvette-based [Ca(2+)](i) assay and CHO(hUT) cells, urantide mimicked the [Ca(2+)](i) stimulatory effect of U-II with an intrinsic activity (alpha) of 0.80, while UFP-803 displayed a small (alpha=0.21) but consistent residual agonist activity. When the same experiments were repeated at 22 degrees C (a temperature similar to that in FLIPR experiments), urantide displayed a very small intrinsic activity (alpha=0.11) and UFP-803 was completely inactive as an agonist. In vivo in mice, UFP-803 (10 nmol kg(-1)) antagonized U-II (1 nmol kg(-1))-induced increase in plasma extravasation in various vascular beds, while being inactive alone. In conclusion, UFP-803 is a potent UT receptor ligand which displays competitive/noncompetitive antagonist behavior depending on the assay. While UFP-803 is less potent than urantide, it displayed reduced residual agonist activity and as such may be a useful pharmacological tool.

Cough and glottic-stop reflex sensitivity in health and disease.

STUDY OBJECTIVES: Little is known about the normal ranges and repeatability of cough reflex sensitivity measurements, or the relationship of cough reflex sensitivity to other upper airway reflexes in subjects with chronic dry cough. We set out to define the normal range of cough reflex sensitivity and its repeatability in health and disease, and to assess its relationship to the glottic-stop reflex. DESIGN: Prospective, cross-sectional study. SUBJECTS AND METHODS: We measured capsaicin cough reflex sensitivity in 134 healthy subjects and 88 patients with respiratory disease, and assessed the repeatability over 2 weeks in a subgroup of individuals (healthy subjects, 15; chronic cough patients, 15). In another subgroup (healthy patients, 16; chronic cough patients, 14), we measured the sensitivity of the glottic-stop reflex (using inhaled ammonia). RESULTS: Capsaicin cough sensitivity varied widely in the population of healthy subjects, and there was considerable overlap of cough reflex sensitivity between healthy control subjects and patients with cough. The intraclass correlation coefficients for repeatability of cough sensitivity (concentration of capsaicin that causes two coughs, and concentration of capsaicin that causes five coughs) were 0.89 and 0.88, respectively. Patients with chronic cough had a significantly more sensitive glottic-stop reflex than healthy subjects (glottic-stop sensitivity threshold, 483 ppm vs 1,029 ppm, respectively; p = 0.01), and there was a significant positive correlation between glottic-stop and cough reflex sensitivity (r = 0.5; p < 0.01). CONCLUSIONS: We have shown a wide variation of cough reflex sensitivity in healthy subjects, although the measurement does have good 2-week repeatability. There was a reasonably close relationship between cough sensitivity and glottic-stop reflex sensitivity, indicating either that the cough reflex and the glottic-stop reflex share a common pathway or that subjects who have a chronic cough have a global abnormality of upper airway reflexes.

Urantide mimics urotensin-II induced calcium release in cells expressing recombinant UT receptors.

Urotensin-II is the natural ligand of the UT receptor. This novel system is involved in the regulation of cardiovascular functions. Recently, a urotensin-II analog ([Pen5,DTrp7,Orn8]urotensin-II(4-11)) named urantide, has been proposed as a selective and potent UT receptor antagonist. In order to pharmacologically characterize this new compound, urantide was tested on the native UT receptors of the rat aorta and on the human recombinant receptors expressed in CHO cells (CHO(hUT)). Indeed, urantide behaves as a competitive, potent (pA2 8.24), and pure antagonist in the rat aorta bioassay, while as an agonist (pEC50 8.11) in a calcium mobilization assay performed in CHO(hUT) cells. Urantide should be considered a low efficacy partial agonist.