Sensitivity and specificity of microRNA-122 for liver disease in dogs.

BACKGROUND: Current tests for diagnosing liver disease in dogs are sub-optimal. MicroRNA-122 (miR-122) is a sensitive and specific biomarker of liver injury in humans and rodents. Circulating miR-122 could have utility in identifying dogs with liver disease. OBJECTIVE: Establish the reference interval for miR-122 in healthy dogs and determine performance in a range of dog breeds with liver disease and control animals with non-liver disease. ANIMALS: Stored serum from 120 healthy dogs, 100 dogs with non-liver diseases, and 30 dogs with histologically confirmed liver disease was analyzed. METHODS: Retrospective study. Medical records of dogs with liver disease, non-liver disease and healthy dogs were reviewed. Serum miR-122 concentrations were measured by PCR and compared with the characteristics of the dogs and their conventional clinical measurements. RESULTS: In healthy dogs the 2.5th, 50th, and 97.5th quartiles of miR-122 were 110 (90% CI 80-114), 594 (505-682), and 3312 (2925-5144) copies/µL, respectively. There was no difference between healthy dogs and dogs with non-liver disease (median ± IQR: healthy dogs 609 [327-1014] copies/µL; non-liver disease 607 [300-1351] copies/µL). miR-122 was higher in dogs with liver disease (11 332 [4418-20 520] copies/µL, P < .001 compared to healthy dogs). miR-122 identified dogs with liver disease with high accuracy (receiver operating characteristic area under curve for comparison with healthy dogs: 0.93 [95% CI 0.86-0.99]). The upper limit of normal for healthy dogs (3312 copies/µL) had a sensitivity of 77% and specificity of 97% for identifying liver disease. CONCLUSION AND CLINICAL IMPORTANCE: Liver disease can be sensitively and specifically diagnosed in dogs by measurement of miR-122.

Attending a social event and consuming alcohol is associated with changes in serum microRNA: a before and after study in healthy adults.

PURPOSE: Circulating microRNAs represent a reservoir for biomarker discovery. Our objective was to profile the change in human circulating microRNA associated with recreational use of alcohol at a social event. MATERIAL AND METHODS: Blood was collected from healthy volunteers (N = 16) before and after recreational consumption of alcohol (ethanol). Biochemistry, hematology and ethanol measurements were performed. The change in the serum small RNA fraction was quantified by RNA sequencing. RESULTS: Blood ethanol was undetectable at study entry in all subjects [<10 mg/dL]. After consuming alcohol the median concentration was 89 mg/dL [IQR: 71-138. Min-max 20-175]. There were no changes in biochemistry and hematology parameters. Serum RNA sequencing identified 1371 small RNA species (1305 microRNAs). There were significant increases [adjusted p-value <0.05, fold increase 2 or more] in 265 microRNAs, around a fifth of the total [median fold increase 2.3 [IQR: 2.1-2.5; Max: 3.7]]. miR-185-5p decreased following alcohol exposure [adjusted p-value <0.05, fold decrease 2 or more]. CONCLUSIONS: The microRNA composition of human serum is dynamic and environmental factors may have a significant impact. Within its context of use the fold change 'signal' of a microRNA must be large enough to negate the risk of false results due to background 'noise'.

Long-term adverse effects of paracetamol - a review.

Paracetamol (acetaminophen) is the most commonly used drug in the world, with a long record of use in acute and chronic pain. In recent years, the benefits of paracetamol use in chronic conditions has been questioned, notably in the areas of osteoarthritis and lower back pain. Over the same period, concerns over the long-term adverse effects of paracetamol use have increased, initially in the field of hypertension, but more recently in other areas as well. The evidence base for the adverse effects of chronic paracetamol use consists of many cohort and observational studies, with few randomized controlled trials, many of which contradict each other, so these studies must be interpreted with caution. Nevertheless, there are some areas where the evidence for harm is more robust, and if a clinician is starting paracetamol with the expectation of chronic use it might be advisable to discuss these side effects with patients beforehand. In particular, an increased risk of gastrointestinal bleeding and a small (~4 mmHg) increase in systolic blood pressure are adverse effects for which the evidence is particularly strong, and which show a degree of dose dependence. As our estimation of the benefits decreases, an accurate assessment of the harms is ever more important. The present review summarizes the current evidence on the harms associated with chronic paracetamol use, focusing on cardiovascular disease, asthma and renal injury, and the effects of in utero exposure.

Circulating acetaminophen metabolites are toxicokinetic biomarkers of acute liver injury.

Acetaminophen (paracetamol-APAP) is the most common cause of drug-induced liver injury in the Western world. Reactive metabolite production by cytochrome P450 enzymes (CYP-metabolites) causes hepatotoxicity. We explored the toxicokinetics of human circulating APAP metabolites following overdose. Plasma from patients treated with acetylcysteine (NAC) for a single APAP overdose was analyzed from discovery (n = 116) and validation (n = 150) patient cohorts. In the discovery cohort, patients who developed acute liver injury (ALI) had higher CYP-metabolites than those without ALI. Receiver operator curve (ROC) analysis demonstrated that at hospital presentation CYP-metabolites were more sensitive/specific for ALI than alanine aminotransferase (ALT) activity and APAP concentration (optimal CYP-metabolite receiver operating characteristic area under the curve (ROC-AUC): 0.91 (95% confidence interval (CI) 0.83-0.98); ALT ROC-AUC: 0.67 (0.50-0.84); APAP ROC-AUC: 0.50 (0.33-0.67)). This enhanced sensitivity/specificity was replicated in the validation cohort. Circulating CYP-metabolites stratify patients by risk of liver injury prior to starting NAC. With development, APAP metabolites have potential utility in stratified trials and for refinement of clinical decision-making.

Renal extracellular vesicles: from physiology to clinical application.

Urinary extracellular vesicles (uEVs) are released from all regions of the kidney's nephron and from other cells that line the urinary tract. Extracellular vesicles retain proteomic and transcriptomic markers specific to their cell of origin and so represent a potential reservoir for kidney disease biomarker discovery. Exosomes, a subtype of uEVs, are distinguished from other vesicles by features related to their biogenesis within cells: mature multi-vesicular bodies fuse with the cellular membrane to liberate exosomes into the extracellular space. uEVs represent a novel cell signalling mechanism because they can be shuttled to a recipient cell and, through a number of proposed mechanisms, affect the recipient cell's proteome and function. Here we review the current evidence for uEV signalling along the nephron, their role in health and disease of the kidney, and their potential for clinical translation as biomarkers and therapeutics.

Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury.

Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury by profiling the circulating miRNome in patients with acetaminophen overdose. Plasma miRNAs were quantified in age- and sex-matched overdose patients with (N = 27) and without (N = 27) organ injury (APAP-TOX and APAP-no TOX, respectively). Classifier miRNAs were tested in a separate cohort (N = 81). miRNA specificity was determined in non-acetaminophen liver injury and murine models. Sensitivity was tested by stratification of patients at hospital presentation (N = 67). From 1809 miRNAs, 75 were 3-fold or more increased and 46 were 3-fold or more decreased with APAP-TOX. A 16 miRNA classifier model accurately diagnosed APAP-TOX in the test cohort. In humans, the miRNAs with the largest increase (miR-122-5p, miR-885-5p, miR-151a-3p) and the highest rank in the classifier model (miR-382-5p) accurately reported non-acetaminophen liver injury and were unaffected by kidney injury. miR-122-5p was more sensitive than ALT for reporting liver injury at hospital presentation, especially combined with miR-483-3p. A miRNA panel was associated with human kidney dysfunction. In mice, miR-122-5p, miR-151a-3p and miR-382-5p specifically reported APAP toxicity - being unaffected by drug-induced kidney injury. Profiling of acetaminophen toxicity identified multiple miRNAs that report acute liver injury and potential biomarkers of drug-induced kidney injury.

Exosome isolation: a microfluidic road-map.

Exosomes, first isolated 30 years ago, are nanoscale vesicles shed by most types of cells. The nucleic acid rich content of these nanoparticles, floating in virtually all bodily fluids, has great potential for non-invasive molecular diagnostics and may represent a novel therapeutic delivery system. However, current isolation techniques such as ultracentrifugation are not convenient and do not result in high purity isolation. This represents an interesting challenge for microfluidic technologies, from a cost-effective perspective as well as for enhanced purity capabilities, and point-of-care acquisition and diagnosis. In this frontier review, we present the current challenges, comment the first microfluidic advances in this new field and propose a roadmap for future developments. This review enables biologists and clinicians familiar with exosome enrichment to assess the performance of novel microfluidic devices and, equally, enables microfluidic engineers to educate themselves about this new class of promising biomarker-rich particles and the challenges arising from their clinical use.

Impact of reducing the threshold for acetylcysteine treatment in acute paracetamol poisoning: the recent United Kingdom experience.

BACKGROUND: On 3 September 2012, the licensed indication for acetylcysteine was changed in the United Kingdom (UK) so that all patients with a plasma paracetamol concentration above a "100 mg/L" (4 h post ingestion) nomogram treatment line after an acute paracetamol (acetaminophen) overdose should be treated. This is a lower threshold than that used in the United States, Canada, Australia, and New Zealand. Here we report the impact of this change in the UK on the management of patients with acute overdose in different paracetamol concentration ranges. METHODS: This is a cohort study, consisting of a retrospective analysis conducted on prospectively collected audit data in three UK hospitals. Following appropriate ethical and data protection authority approval, data for patients presenting within 24 h of an acute timed single paracetamol overdose were extracted. Numbers of admissions and use of antidote in relation to different paracetamol concentration bands (< 100 mg/L; 100-149 mg/L; 150-199 mg/L; and ≥ 200 mg/L at 4 h) were analyzed for one-year periods before and after the change. RESULTS: Comparing the year before with the year after the change, there was no change in the numbers of patients presenting to hospital within 24 h of acute timed paracetamol overdose (1246 before and 1251 after), but more patients were admitted (759 before and 849 after) and treated with acetylcysteine (389 before and 539 after). Of the 150 additional patients treated with acetylcysteine in the year following the change, 114 (76%, 95% CI: 68.4-82.6) were in the 100-149 group and 9 (6.0%, 95% CI: 2.8-11.1) in the 150-199 group. CONCLUSIONS: Changes to national guidelines for managing paracetamol poisoning in the UK have increased the numbers of patients with acute overdose treated with acetylcysteine, with most additional treatments occurring in patients in the 100-149 mg/L dose range, a group at low risk of hepatotoxicity and higher risk of adverse reactions.

The predictive value of hospital admission serum alanine transaminase activity in patients treated for paracetamol overdose.

BACKGROUND: Paracetamol is a major cause of poisoning. Treatment decisions are predominately based on the dose ingested and a timed blood paracetamol concentration because most patients present to hospital soon after overdose, before hepatotoxicity can be confirmed/excluded using serum alanine transaminase (ALT). Nonetheless, ALT is measured at hospital presentation; we investigated its value in predicting hepatotoxicity. METHODS: From March 2011 to May 2012, patients admitted to the Royal Infirmary of Edinburgh for paracetamol overdose treatment were identified. We determined the value of admission ALT (below or above our upper limit of normal-50 IU/l) at predicting three endpoints: 1-doubling of ALT; 2-peak ALT >1000 IU/l; 3-peak international normalized ratio (INR) >2. RESULTS: From 500 patients, 410 met the entry criteria; 264 presented within 8 h of overdose, 54 between 8 and 24 h, 53 after 24 h and 39 were staggered ingestions. Admission ALT was increased in 71. For endpoint 1 (ALT doubling), the positive predictive value (PPV) of admission ALT was 19% [95% confidence interval (CI) 12-30] with a negative predictive value (NPV) of 98% (95% CI 96-99); endpoint 2 (ALT >1000 IU/l: PPV 23% (95% CI 14-34) and NPV 100% (95% CI 99-100) and for endpoint 3 (INR >2): PPV 14% (95% CI 7-25) and NPV of 100% (95% CI 99-100). The NPV remained high when only late presenters were included. CONCLUSION: Admission ALT within the normal range has a high NPV and could be used, alone or in combination with newer biomarkers, to identify lower risk patients at hospital presentation.

Setting the stage for acute-on-chronic kidney injury.

Acute-on-chronic kidney disease will be familiar to many nephrologists. Hsu et al. quantify the risk of acute-on-chronic disease across the stages of preexisting chronic kidney disease. Their study demonstrates the valuable insights that large epidemiological studies can bring to the field of acute kidney injury.

Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure.

Sepsis is one of the common causes of acute renal failure (ARF). The objective of this study was to identify new biomarkers and therapeutic targets. We present a new rat model of sepsis-induced ARF based on cecal ligation and puncture (CLP). We used this model to find urinary proteins which may be potential biomarkers and/or drug targets. Aged rats were treated with fluids and antibiotics after CLP. Urinary proteins from septic rats without ARF and urinary proteins from septic rats with ARF were compared by difference in-gel electrophoresis (DIGE). CLP surgery elevated interleukin (IL)-6 and IL-10 serum cytokines and blood nitrite compared with sham-operated rats. However, there was a range of serum creatinine values at 24 h (0.4-2.3 mg/dl) and only 24% developed ARF. Histology confirmed renal injury in these rats. Forty-nine percent of rats did not develop ARF. Rats without ARF also had less liver injury. The mortality rate at 24 h was 27% but was increased by housing the post-surgery rats in metabolic cages. Creatinine clearance and urine output 2-8 h after CLP was significantly reduced in rats which died within 24 h. Using DIGE we identified changes in a number of urinary proteins including albumin, brush-border enzymes (e.g., meprin-1-alpha) and serine protease inhibitors. The meprin-1-alpha inhibitor actinonin prevented ARF in aged mice. In summary, we describe a new rat model of sepsis-induced ARF which has a heterogeneous response similar to humans. This model allowed us to use DIGE to find changes in urinary proteins and this approach identified a potential biomarker and drug target - meprin-1-alpha.

Sepsis-induced organ failure is mediated by different pathways in the kidney and liver: acute renal failure is dependent on MyD88 but not renal cell apoptosis.

Toll-like receptors (TLRs) are important in sepsis. Myeloid differentiation factor 88 (MyD88) is a key molecule involved in signal transduction by multiple TLRs. The objective of this study was to investigate the contribution of TLR4 and MyD88 to acute renal failure (ARF) induced by polymicrobial sepsis. Liver dysfunction and apoptosis in the spleen contribute to sepsis severity after cecal ligation and puncture (CLP). Therefore, we also investigated liver injury and splenic apoptosis. We used a mouse model of sepsis-induced ARF using CLP to generate polymicrobial sepsis. Despite fluid and antibiotic resuscitation the mice developed multi-organ failure, including ARF, which resembles human sepsis. We investigated the role of the TLR4 receptor by comparing C3H/HeJ mice (which lack TLR4) with C3H/He0UJ normal controls. The role of MyD88 was investigated by comparing MyD88 knockout mice (MyD88(-/-)) with wild-type controls. Following CLP, mice lacking TLR4 and wild-type mice both developed comparable ARF. However, MyD88(-/-) mice did not develop ARF compared to wild-type controls. In contrast, MyD88(-/-) mice developed liver injury comparable to wild type. After CLP, MyD88(-/-) mice had significantly reduced apoptosis in the spleen compared with wild type. Apoptosis was not detected in the kidney of wild-type or MyD88(-/-) mice after CLP. In summary, ARF induced by polymicrobial sepsis is dependent on MyD88, but not TLR4. The absence of MyD88 dissociates ARF from liver injury; liver injury is MyD88-independent. There was MyD88-dependent apoptosis in the spleen, but no apoptosis in the kidney. MyD88 may be a good drug target for some, but not all, organ dysfunctions following sepsis.

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Involvement of kinins in hyperresponsiveness induced by platelet activating factor in the human nasal airway.

1. The aim of this study was to investigate the role of kinins in the development of nasal hyperresponsiveness induced by platelet activating factor (PAF) in normal human subjects. 2. Intranasal administration of PAF, 60 micrograms, induced an increased responsiveness to histamine, 200 micrograms per nostril, 6 h later. This effect was abolished by pretreatment with the bradykinin B2 receptor antagonists icatibant and [1-adamantaneacetyl-D-Arg0,Hyp3,beta-(2-thienyl)-Al a5,8,D-Phe7]-bradykinin ([Ad]-BK), both at 200 micrograms, every 2 h following PAF administration. 3. In a separate experiment, utilizing the same protocol, nasal lavage was used to measure the release of mediators into the nasal cavity following treatment with PAF. PAF increased the levels of eosinophil cationic protein (ECP) and kinin detected in the lavage samples, compared with a saline control. The levels of these mediators were reduced by pretreatment with either icatibant or [Ad]-BK. 4. Administration of lyso-PAF, 60 micrograms intranasally, did not cause a rise in kinin or ECP levels in nasal lavage fluid. 5. Exogenous bradykinin, 500 micrograms, or a saline control, applied topically to the nasal mucosa every 30 min for 2 h, failed to cause hyperresponsiveness to histamine. 6. We conclude that bradykinin itself does not cause hyperresponsiveness, but is involved in the hyperresponsiveness induced by PAF in the human nasal airway.

Characterization of the bradykinin receptor in the human nasal airway using the binding of [125I]-Hoe 140.

1. The aim of this study was to characterize the kinin receptor in the human nasal airway using [125I]-Hoe 140 binding to a membrane preparation from human nasal turbinates and to compare Ki values from binding displacement by antagonists with the functional effects of these drugs in vivo. We also investigated the effect of Hoe 140 ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin), on bradykinin release into the nasal airway. 2. In a membrane preparation from human nasal turbinates removed during surgery, [125I]-Hoe 140 labelled a single, saturable binding site. The equilibrium dissociation constant (at 20 degrees C) for [125I]-Hoe 140 binding to the receptor was 0.46 +/- 0.08 nM. The Bmax was 0.136 +/- 0.003 pmol mg-1 protein and the Hill coefficient was 1.01 +/- 0.07. 3. The association rate constant for [125I]-Hoe 140 binding to the receptor was 0.20 +/- 0.06 nM-1 min-1 and the dissociation rate constant was 0.14 +/- 0.01 min-1. These values were determined at 4 degrees C. The equilibrium dissociation constant calculated from these rate constants was 0.70 nM. 4. Bradykinin and the B2 receptor antagonists, NPC 567, NPC 17731, NPC 17761, [1-adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, WIN 64338 and Hoe 140 displaced [125I]-Hoe 140 binding: the Ki values from binding displacement are consistent with values expected from a B2 receptor. The B1 agonist, [des-Arg9]-bradykinin and the B1 antagonist, [des-Arg9]-Hoe 140 failed to displace [125I]-Hoe 140 binding at concentrations up to 1 microM. 5. The bradykinin antagonist, Hoe 140, 10 to 200 micrograms, given by intranasal aerosol, produced a dose-related inhibition of the reduction in minimal nasal cross-sectional area (Amin) induced by bradykinin in normal subjects and by house dust mite antigen in subjects with allergic rhinitis to house dust mite. Hoe 140, 10 to 200 micrograms, also caused a dose-related inhibition of the release of albumin into the nasal cavity following challenge with bradykinin. 6. [1-Adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, 30 to 200 micrograms, caused a dose-related inhibition of the reduction in Amin and the release of albumin into the nasal cavity induced by bradykinin. NPC 567 ([D-Arg0, Hyp3, D-Phe7]-bradykinin) failed to inhibit the reduction in Amin or the release of albumin into the nasal cavity at a dose of 10 mg. 7. Challenge of allergic subjects with house dust mite antigen caused a significant elevation of the bradykinin concentration in nasal lavage fluid and a reduction in Amin. Hoe 140, 100 micrograms, prevented the antigen-induced reduction in Amin and also abolished the antigen-induced increase of bradykinin in nasal lavage fluid. 8. We conclude that there is a B2 bradykinin receptor in the human nasal airway which mediates nasal blockage and plasma extravasation induced by either bradykinin or antigen challenge. It is possible that Hoe 140 inhibits kallikrein in the human nasal airway as well as blocking the B2 receptor.

The contribution of histamine to the action of bradykinin in the human nasal airway.

Bradykinin, 10 to 1000 micrograms given by aerosol into the nasal cavity of normal, healthy volunteers, produced a dose-related increase of nasal airway resistance. Bradykinin also reduced the minimal nasal cross-sectional area (Amin), increased albumin release into nasal lavage fluid and increased the symptoms of nasal inflammation. Pretreatment with cetirizine (10 mg orally) reduced the fall in Amin induced by bradykinin, 300 micrograms, but not by bradykinin, 100 micrograms. Pre-treatment of the subjects with the H1 histamine receptor antgonist cetirizine (10 mg, orally) or terfenadine (60 mg, orally) 3 h before bradykinin administration caused significant reduction of the bradykinin-induced increase in nasal airway resistance in the upper range of bradykinin doses (300-1000 micrograms) but not in the lower range (10-100 micrograms). Cetirizine reduced the albumin release into the nasal airway and the symptoms induced by bradykinin, 1000 micrograms. Following nasal challenge with bradykinin 300 micrograms or 1000 micrograms, no increase could be detected in the histamine content of nasal lavage fluid. Isolated human nasal cells released histamine in response to bradykinin, 33 and 100 microM, anti-IgE and calcium ionophore, A23187. We conclude that the actions of bradykinin in the human nasal airway are, in part, accounted for by the release of histamine.

Attenuation of human nasal airway responses to bradykinin and histamine by inhibitors of nitric oxide synthase.

1. The effects of inhibitors of nitric oxide synthase and local anaesthetics were studied on changes in human nasal airway patency and albumin extravasation in response to bradykinin and histamine, in vivo. 2. Compared with the action of the vasoconstrictor, ephedrine, 2.5 mumol, NG-nitro-L-arginine methyl ester (L-NAME), 1 mumol alone, did not change the resting value of the minimal cross-sectional area (A min) of the human nasal airway. L-NAME, 0.1 to 10 mumol, produced a dose-related inhibition of the reduction in A min caused by bradykinin, 300 micrograms. NG-monomethyl-L-arginine (L-NMMA), 1 mumol, similarly reduced the effect of bradykinin, 300 micrograms, on A min, but NG-nitro-D-arginine methyl ester (D-NAME), had no effect. L-NAME, 0.1 to 10 mumol, or L-NMMA, 10 mumol, failed to inhibit the effect of histamine, 300 micrograms on A min. 3. The inhibition by L-NAME, 1 mumol of the action of bradykinin, 300 micrograms on A min was maximal between 15 and 30 min after pretreatment with L-NAME. 4. L-NAME, 1 and 10 mumol, inhibited the extravasation of albumin into the nasal cavity induced by bradykinin, 300 micrograms, and also by histamine, 300 micrograms. D-NAME, 1 and 10 mumol had no effect on the extravasation of albumin in response to bradykinin or histamine. 5. L-Arginine, 30 mumol, reversed the effect of L-NAME, 1 mumol, on the bradykinin- and histamine-induced albumin extravasation into the nasal airway. 6. Local anaesthesia of the nasal airway with lignocaine, 10 mg, or benzocaine, 10 mg, failed to inhibit the reduction in A min or the albumin extravasation induced by either bradykinin, 300 micrograms, and histamine, 300 micrograms. 7. We conclude that the extravasation of plasma albumin caused by bradykinin and by histamine involves the generation of nitric oxide. The nasal blockage induced by bradykinin involves nitric oxide generation but the nasal blockage induced by histamine does not.