Detection of influenza A(H3N2) virus in children with suspected mumps during winter 2014/15 in England.

Influenza A(H3N2) virus was detected in oral fluid from 16/107 children (aged 2 to 12 years) with a clinical diagnosis of mumps, who were sampled between December 2014 and February 2015 in England, during the peak of the 2014/15 influenza season. Sequence analysis of an A(H3N2) virus from a child with suspected mumps showed the virus was similar to other circulating A(H3N2) viruses detected in winter 2014/15, which were antigenically drifted from the A(H3N2) vaccine strain.

Vaccine effectiveness of 2011/12 trivalent seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the United Kingdom: evidence of waning intra-seasonal protection.

The 2011/12 season was characterised by unusually late influenza A (H3N2) activity in the United Kingdom (UK). We measured vaccine effectiveness (VE) of the 2011/12 trivalent seasonal influenza vaccine (TIV) in a test-negative case­control study in primary care. Overall VE against confirmed influenza A (H3N2) infection, adjusted for age, surveillance scheme and month, was 23% (95% confidence interval (CI): -10 to 47). Stratified analysis by time period gave an adjusted VE of 43% (95% CI: -34 to 75) for October 2011 to January 2012 and 17% (95% CI: -24 to 45) for February 2012 to April 2012. Stratified analysis by time since vaccination gave an adjusted VE of 53% (95% CI: 0 to 78) for those vaccinated less than three months, and 12% (95% CI: -31 to 41) for those vaccinated three months or more before onset of symptoms (test for trend: p=0.02). For confirmed influenza B infection, adjusted VE was 92% (95% CI: 38 to 99). A proportion (20.6%) of UK influenza A(H3N2) viruses circulating in 2011/12 showed reduced reactivity (fourfold difference in haemagglutination inhibition assays) to the A/Perth/16/2009 2011/12 vaccine component, with no significant change in proportion over the season. Overall TIV protection against influenza A(H3N2) infection was low, with significant intraseasonal waning.

Evaluation of influenza virus antiviral susceptibility testing in Europe: results from the first external quality assessment exercise.

BACKGROUND: The first antiviral susceptibility testing external quality assessment (EQA) was held for European influenza reference laboratories during winter 2010/11. OBJECTIVES: To assess European network influenza antiviral susceptibility testing capability and provide participants with an independent performance evaluation. STUDY DESIGN: The EQA panel contained ten coded specimens of inactivated human influenza A and B viruses with reduced susceptibility to neuraminidase inhibitors (NAI), or adamantanes. Twenty-four laboratories from 19 member states of the WHO European region analysed the panel using phenotypic (determination of 50% inhibitory concentration (IC(50)) values by neuraminidase (NA) enzyme inhibition assay) and/or genotypic methods. RESULTS: All 24 laboratories returned genotypic data for A(H1N1)pdm09 influenza virus, 18 (75%) for former seasonal A(H1N1), 16 (67%) for A(H3N2) and 15 (63%) for influenza B virus, correctly identifying NAI or adamantane reduced susceptibility-associated substitutions in the NA (mean 84%; range 52-100%) or M2 (mean 85%; range 73-94%), respectively. Thirteen laboratories (54%) returned phenotypic NAI susceptibility data. Despite inter-laboratory and inter-assay IC(50) value variation, all 13 laboratories correctly identified oseltamivir reduced susceptibility/resistance in pure preparations of A(H1N1) oseltamivir-resistant viruses. However, only 11 (85%) identified oseltamivir reduced susceptibility/resistance in a mixture of A(H1N1)pdm09 oseltamivir-sensitive/-resistant viruses. Furthermore, 3 laboratories (23%) considered oseltamivir-sensitive influenza B virus reduced susceptible/resistant. CONCLUSIONS: Detection of NA-H275Y in A(H1N1) viruses was achieved by most laboratories. IC(50) values and interpretation thereof varied for a sensitive/resistant virus mixture and for influenza B virus. The results of this exercise will assist harmonisation of antiviral susceptibility testing, interpretation and reporting within the European network through targeted training.

Rapid generation of a well-matched vaccine seed from a modern influenza A virus primary isolate without recourse to eggs.

Most influenza vaccines are produced in chicken eggs but recent human influenza strains often do not grow well in this substrate. The PER.C6 cell line is an alternative platform for vaccine production. Here we demonstrate that PER.C6 cells faithfully propagate recent clinical isolates, without selecting for mutations in the HA gene. PER.C6 cells support the rescue of recombinant influenza viruses from cDNA. We used sequence data from a surveillance programme to generate a PR8-based seed virus with the HA and NA of a contemporary circulating H3N2 human strain, A/England/611/07 (E611) that did not itself grow in eggs. We engineered mutations that affected receptor-binding, G186V or L194P, into the E611 HA gene. Whilst the L194P mutation conferred efficient growth in eggs, G186V did not. The L194P mutation was also spontaneously selected during egg propagation of E611/PR8 7:1 recombinant virus. This suggests generation of a single recombinant vaccine seed might satisfy manufacturers that utilize either eggs or cells for vaccine production.

Microfiltration of gluten processing streams from corn wet milling.

In corn wet milling, dry matter can be separated from liquids in process streams with centrifuges or vacuum belt filtration (VBF). Because separations usually are not complete, dry matter can be lost in the liquid streams (overflow from the gluten thickener centrifuge and filtrate from VBF). This represents a loss of nutrients, especially protein, to low valued coproducts and reduces quality of water for recycling within the process. The objective was to compare microfiltration of light and heavy gluten process streams to conventional separation methods. Batches of light and heavy gluten were obtained from a wet mill plant and processed by microfiltration. Samples of permeate and concentrate from microfiltration were analyzed and compared to corresponding streams from wet milling. Microfiltration of light gluten resulted in concentrate and permeate streams similar in composition to conventionally processed light gluten using a centrifuge, suggesting that microfiltration is as effective as centrifugation in partitioning solids and water in light gluten. Dewatering of heavy gluten found that conventional VBF caused dry matter concentrations in gluten cake to be higher than concentrate from microfiltration. Permeate from microfiltration of heavy gluten had higher concentrations of ash and lower soluble nitrogen than filtrate from VBF. Microfiltration was able to remove more ash from concentrate, which may improve the value of wet milling coproducts. These data demonstrated microfiltration has potential for separation of light and heavy gluten streams, but more data are needed on effectiveness and practicality.

Control of myocardial oxygen consumption in transgenic mice overexpressing vascular eNOS.

Our objective was to investigate the potential role of selective endothelial nitric oxide (NO) synthase (eNOS) overexpression in coronary blood vessels in the control of myocardial oxygen consumption (MVO2). Transgenic (Tg) eNOS-overexpressing mice (eNOS Tg) (n=22) and wild-type (WT) mice (n=24) were studied. Western blot analysis indicated greater than sixfold increase of eNOS in cardiac tissue. Echocardiography in awake mice indicated no difference in cardiac function between WT and eNOS Tg; however, systolic pressure in eNOS Tg mice decreased significantly (126 +/- 2.3 to 109 +/- 2.3 mmHg; P <0.05), whereas heart rate (HR) was not different. Total peripheral resistance (TPR) was also decreased (9.8 +/- 0.8 to 7.6 +/- 0.4 4 mmHg.ml(-1).min; P <0.05) in eNOS Tg. Furthermore, female eNOS Tg mice showed even lower TPR (7.2 +/- 0.4 mmHg.ml(-1).min) compared with male eNOS mice (8.6 +/- 0.5, mmHg.ml.min(-1); P <0.05). Left ventricular slices were isolated from WT and eNOS Tg mice. With the use of a Clark-type oxygen electrode in an airtight bath, MVO2 was determined as the percent decrease during increasing doses (10(-10) to 10(-4) mol/l) of bradykinin (BK), carbachol (CCh), forskolin (10(-12) to 10(-6) mol/l), or S-nitroso-N-acetyl penicillamine (SNAP; 10(-7) to 10(-4) mol/l). Baseline MVO2 was not different between WT (181 +/- 13 nmol.g(-1).min(-1)) and eNOS Tg (188 +/- 14 nmol.g(-1).min(-1)). BK decreased MVO2 (10(-4) mol/l) in WT by 17% +/- 1.1 and 33% +/- 2.7 in eNOS Tg (P < 0.05). CCh also decreased MVO2, 10(-4) mol/l, in WT by 20% +/- 1.7 and 31% +/- 2.0 in eNOS Tg (P <0.05). Forskolin (10(-6) mol/l) or SNAP (10(-4) mol/l) also decreased MVO2 in WT by 24% +/- 2.8 and 36% +/- 1.8 versus eNOS 31% +/- 1.8 and 37% +/- 3.5, respectively. N-nitro-L-arginine methyl ester (10(-3) mol/l) inhibited the MVO2 reduction to BK, CCh, and forskolin by a similar degree (P <0.05), but not to SNAP. Thus selective overexpression of eNOS in cardiac blood vessels in mice enhances the control of MVO2 by eNOS-derived NO.

Characterization of light gluten and light steep water from a corn wet milling plant.

The primary commodity of corn wet milling is starch, but two coproducts (corn gluten feed, CGF and corn gluten meal, CGM) also are produced. CGM and CGF are marketed as animal foodstuffs and are important economically; however, variation in composition reduces quality. There are few data on the effect of composition of the parent process streams, light steep water (LSW) and light gluten (LG), respectively, on composition of CGF and CGM. The objective was to characterize LG and LSW. Samples of LG and LSW were collected: (1) hourly for one day, (2) every 3 h for 3 days, and (3) daily for 3 weeks. Dry matter, N and ash were determined. Variation in composition of LG and LSW was greatest during longer periods of time (days and weeks) rather than shorter (hourly or every 3 h). There was significant variation in DM (solids) content, which directly affected the concentration of other components. Variation in N (protein) of LG and LSW accounted for much of the variation in CGF and CG. Processes that modify processing and reduce variation could increase the quality of CGF and CGM.

Characterization of gluten processing streams.

Corn gluten meal (CGM) is a major coproduct of corn wet milling; it has value because of high protein. However, variation in composition and high P content reduce market value. Data that characterize gluten streams would be helpful in identifying key processing steps that could be modified to improve the quality of CGM and increase processing efficiency. Few data are published in the literature on the detailed composition of gluten processing streams. The objective was to characterize the gluten process streams in a corn wet milling plant. Samples were obtained from one plant over a six month period and analyzed for dry matter (DM), total N (protein), ash and elements. DM and macroelement content of the streams were increased significantly during processing. Ash, priority pollutant elements and microelement concentrations were low and of little concern. About 38% of the N (protein) in light gluten was not recovered in the CGM; most of this was lost at the gluten thickener step into the gluten thickener overflow. Much of the P also was removed at this step. Modification of the gluten thickener overflow to increase N and reduce P could make CGM a more valuable coproduct and improve processing efficiency.

Myocardial glucose uptake is regulated by nitric oxide via endothelial nitric oxide synthase in Langendorff mouse heart.

Although the role of nitric oxide (NO) in the modulation of vascular tone has been studied and well understood, its potential role in the control of myocardial metabolism is only recently evident. Several lines of evidence indicate that NO regulates myocardial glucose metabolism; however, the details and mechanisms responsible are still unknown. The aim of this study was to further define the role of NO in the control of myocardial glucose metabolism and the nitric oxide synthase (NOS) isoform responsible using transgenic animals lacking endothelial NOS (ecNOS). In the present study, we examined the regulation of myocardial glucose uptake using isometrically contracting Langendorff-perfused hearts from normal mice (C57BL/6J), mice with defects in the expression of ecNOS [ecNOS (-/-)], and its heterozygote [ecNOS (+/-)], and wild-type mice [ecNOS (+/+)] (n=6, respectively). In hearts from normal mice, little myocardial glucose uptake was observed. This myocardial glucose uptake increased significantly in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Similarly, in the hearts from ecNOS (-/-), glucose uptake was much greater than in normal mice, whereas myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice was not different from normal mice. In addition, myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice increased significantly in the presence of L-NAME. At a workload of 800 g. beats/min, L-NAME increased glucose uptake from 0.1+/-0.1 to 3+/-0.4 microg/min x mg in ecNOS (+/-) mice and from 0.2+/-0.1 to 2.7+/-0.7 microg/min x mg in ecNOS (+/+) mice. Furthermore, in the hearts from ecNOS (-/-) mice, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog or S-nitroso-N-acetylpenicillamine (SNAP), a NO donor essentially shut off glucose uptake, and in hearts from ecNOS (+/-) mice, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), an inhibitor of cGMP, increased the glucose uptake significantly. These results indicate clearly that cardiac NO production regulates myocardial glucose uptake via a cGMP-dependent mechanism and strongly suggest that ecNOS plays a pivotal role in this regulation. These findings may be important in the understanding of the pathogenesis of the diseases such as ischemic heart disease, heart failure, diabetes mellitus, hypertension, and hypercholesterolemia, in which NO synthesis is altered and substrate utilization by the heart changes.

Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption.

Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O2 consumption and to further examine the role of nitric oxide (NO) in the control of mitochondrial respiration. Left ventricle O2 consumption in mice with defects in the expression of eNOS [eNOS (-/-)] and inducible NOS [iNOS (-/-)] was measured with a Clark-type O2 electrode. The rate of decreases in O2 concentration was expressed as a percentage of the baseline. Baseline O2 consumption was not significantly different between groups of mice. Bradykinin (10(-4) mol/L) induced significant decreases in O2 consumption in tissues taken from iNOS (-/-) (-28+/-4%), wild-type eNOS (+/+) (-22+/-4%), and heterozygous eNOS(+/-) (-22+/-5%) but not homozygous eNOS (-/-) (-3+/-4%) mice. Responses to bradykinin in iNOS (-/-) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (-2+/-5%, -3+/-2%, and -6+/-5%, respectively, P<0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4) mol/L), which releases NO spontaneously, induced decreases in myocardial O2 consumption in all groups of mice, and such responses were not affected by L-NAME. In addition, pretreatment with bacterial endotoxin elicited a reduction in basal O2 consumption in tissues taken from normal but not iNOS (-/-)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O2 consumption and modulation of mitochondrial respiration by NO may have an important role in pathological conditions such as endotoxemia in which the production of NO is altered.

Role of nitric oxide in the control of renal oxygen consumption and the regulation of chemical work in the kidney.

Inhibition of NO synthesis has recently been shown to increase oxygen extraction in vivo, and NO has been proposed to play a significant role in the regulation of oxygen consumption by both skeletal and cardiac muscle in vivo and in vitro. It was our aim to determine whether NO also has such a role in the kidney, a tissue with a relatively low basal oxygen extraction. In chronically instrumented conscious dogs, administration of an inhibitor of NO synthase, nitro-L-arginine (NLA, 30 mg/kg i.v.), caused a maintained increase in mean arterial pressure and renal vascular resistance and a decrease in heart rate (all P<0.05). At 60 minutes, urine flow rate and glomerular flow rate decreased by 44+/-12% and 45+/-7%, respectively; moreover, the amount of sodium reabsorbed fell from 16+/-1.7 to 8.5+/-1.1 mmol/min (all P<0.05). At this time, oxygen uptake and extraction increased markedly by 115+/-37% and 102+/-34%, respectively (P<0.05). Oxygen consumption also significantly increased from 4.5+/-0.6 to 7.1+/-0.9 mL O2/min. Most important, the ratio of oxygen consumption to sodium reabsorbed increased dramatically from 0.33+/-0.07 to 0.75+/-0.11 mL O2/mmol Na+ (P<0.05), suggesting a reduction in renal efficiency for transporting sodium. In vitro, both a NO-donating agent and the NO synthase-stimulating agonist bradykinin significantly decreased both cortical and medullary renal oxygen consumption. In conclusion, NO plays a role in maintaining a balance between oxygen consumption and sodium reabsorption, the major ATP-consuming process in the kidney, in conscious dogs, and NO can inhibit mitochondrial oxygen consumption in canine renal slices in vitro.

Insulin during infancy attenuates insulin-induced hypoglycemia in adult male rats.

To examine whether insulin during infancy affects later glucose regulation, 60 rat pups of both sexes were injected daily on postnatal Days 9-20 with either insulin (2 or 8 U/kg), 2-deoxy-D-glucose (2DG; 200 or 400 mg/kg), or saline. Two hours postinjection on Day 15, pups given insulin were hypoglycemic and pups given 2DG were hyperglycemic; both groups were normoglycemic at 6 h. The two insulin doses produced similar long-term effects, as did the 2DG doses, so doses were combined to make single insulin and 2DG groups. On Day 44, baseline plasma glucose for rats given insulin or 2DG during infancy was below saline control levels (111.3, 114.5, and 120.7 mg/dL, respectively, p < 0.05). On Days 60 and 62, plasma glucose was assessed 2 h after injection of 3 U/kg insulin; food was allowed between the insulin injection and the glucose test on only one of these days. When food was present after injection of insulin, the early treatments did not influence either amount of food ingested or plasma glucose levels. When food was withheld, however, males given insulin before weaning maintained a higher plasma glucose than did males given 2DG or saline before weaning (67.4, 40.1, and 16.4 mg/dL, respectively, p < 0.01); females were unaffected by the early treatment. Postweaning body weights did not differ from control values for rats given early insulin or 2DG. On Day 72, there were no effects of early treatment on plasma insulin, plasma glucose, liver weight, or kidney weight. These results suggest that preweaning exposure to insulin attenuates the hypoglycemia of adult males given insulin without food.

Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension.

Monocrotaline (MCT)-induced pulmonary hypertension (PH) is associated with impaired endothelium-dependent nitric oxide (NO)-mediated relaxation. To examine the role of NO in PH, Sprague-Dawley rats were given a single subcutaneous injection of normal saline [control (C)], 80 mg/kg MCT, or the same dose of MCT and a continuous subcutaneous infusion of 2 mg.kg-1.day-1 of molsidomine, a NO prodrug (MCT+MD). Two weeks later, plasma NO3- levels, pulmonary arterial pressure (Ppa), ratio of right-to-left ventricular weights (RV/LV) to assess right ventricular hypertrophy, and pulmonary histology were evaluated. The plasma NO3- level in the MCT group was reduced to 9.2 +/- 1.5 microM (n = 12) vs. C level of 17.7 +/- 1.8 microM (n = 8; P < 0.02). In the MCT+MD group, plasma NO3- level was 12.3 +/- 2.0 microM (n = 8). Ppa and RV/LV in the MCT group were increased compared with C [Ppa, 34 +/- 3.4 mmHg (n = 6) vs. 19 +/- 0.8 mmHg (n = 8) and 0.41 +/- 0.01 (n = 9) vs. 0.25 +/- 0.008 (n = 8), respectively; P < 0.001]. In the MCT+MD group, Ppa and RV/LV were not different when compared with C [19 +/- 0.5 mmHg (n = 5) and 0.27 +/- 0.01 (n = 9), respectively; P < 0.001 vs. MCT]. Medial wall thickness of lung vessels in the MCT group was increased compared with C [31 +/- 1.5% (n = 9) vs. 13 +/- 0.66% (n = 9); P < 0.001], and MD partially prevented MCT-induced pulmonary vascular remodeling [22 +/- 1.2% (n = 11); P < 0.001 vs. MCT and C]. These results indicate that a defect in the availability of bioactive NO may play an important role in the pathogenesis of MCT-induced PH.

Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise.

This study determined the changes in NO production from the coronary circulation of the conscious dog during exercise. The role of endogenous NO as it relates to coronary flow, myocardial work, and metabolism was also studied. Mongrel dogs were chronically instrumented for measurements of coronary blood flow (CBF), ventricular and aortic pressure, and ventricular diameter, with catheters in the aorta and coronary sinus. Acute exercise (5 minutes at 3.6, 5.9, and 9.1 mph) was performed, and hemodynamic measurements and blood samples were taken at each exercise level. Nitro-L-arginine (NLA, 35 mg/kg IV) was given to block NO synthesis, and the exercise was repeated. Blood samples were analyzed for oxygen, plasma nitrate/nitrite (an index of NO), lactate, glucose, and free fatty acid (FFA) levels. Acute exercise caused significant elevations in NO production by the coronary circulation (46 +/- 23, 129 +/- 44, and 63 +/- 32 nmol/min at each speed respectively, P < .05). After NLA, there was no measurable NO production at rest or during exercise. Blockade of NO synthesis resulted in elevations in myocardial oxygen consumption and reductions in myocardial FFA consumption for comparable levels of CBF and cardiac work. The metabolic changes after NLA occurred in the absence of alterations in myocardial lactate or glucose consumptions. NO production by the coronary circulation is increased with exercise and blocked by NLA. The absence of NO in the coronary circulation during exercise does not affect levels of CBF, because it shifts the relationship between cardiac work and myocardial oxygen consumption, suggesting that endogenous NO modulates myocardial metabolism.

Preconditioning with ischemia or adenosine protects skeletal muscle from ischemic tissue reperfusion injury.

Prolonged tissue ischemia and subsequent reperfusion results in significant tissue injury due to the ischemic-reperfusion (IR) syndrome. Ischemic preconditioning (IPC) or adenosine (ADO) pretreatment are known to protect IR injury in cardiac muscle. Our aim was to determine whether IPC or ADO pretreatment attenuates and protects against ischemic tissue reperfusion injury in skeletal muscle. Rats were anesthetized and global hindlimb ischemia was induced by 60 min of suprarenal aortic clamping followed by 30 min of reperfusion period. The degree of skeletal muscle dysfunction was determined by decreases in maximum contractile force, and adenosine triphosphate (ATP) and creatine phosphate (CP) levels of extensor digitorum longus (EDL) muscle. The distal tendon of the EDL was attached to a force transducer for maximum isometric force measurement. Samples were taken from the EDL for measurement of ATP and CP levels. The following were protective protocols prior to the IR challenge: (1) four consecutive 5-min periods of ischemia separated by 5-min reperfusion periods (PC/I) or (2) i.v. adenosine infusion (350 microg/kg/min x 10 min, PC/A). Our data suggest that pretreatment with brief periods of ischemia or systemic ADO infusion attenuates ischemic tissue reperfusion injury in skeletal muscle. [Table: see text]

Pharmacodynamics of plasma nitrate/nitrite as an indication of nitric oxide formation in conscious dogs.

BACKGROUND: The present investigation was undertaken to better understand the production of nitric oxide (NO) in vivo as measured by alterations in plasma nitrite or nitrate in blood samples from studies in experimental animals or clinical studies in humans. METHODS AND RESULTS: Plasma samples were taken from the aorta, the coronary sinus, a peripheral vein in the leg (skeletal muscle), or the right ventricle (mixed venous) in chronically instrumented conscious dogs. Plasma nitrite was converted to NO gas in an argon environment by use of hydrochloric acid, and plasma nitrate was converted first to nitrite with nitrate reductase and then to NO gas with acid. Standard curves were constructed, and the amount of nitrite and nitrate in plasma was determined. The primary metabolite was nitrate, whereas nitrate was approximately 10% of the total and remained constant. In the resting dog, the only vascular bed with a positive arterial-venous nitrate difference, evidence for production of NO, was the heart. Nitrate infusion into quietly resting dogs resulted in increases in plasma nitrate up to 38 +/- 3.4 mmol/L, increases in systemic arterial pressure, and a marked diuresis. The plasma half-life was calculated as 3.8 hours. The volume of distribution was calculated as 0.215 L/kg, or equivalent to the extracellular volume. CONCLUSIONS: These studies indicate that nitrate is a reliable measure of NO metabolism in vivo but that because of the long half-life, nitrate will accumulate in plasma once it is produced. Because of the large volume of distribution (21% of body weight versus the 4% of body weight usually attributed to plasma volume, the compartment in which nitrate is measured), simple measures of plasma nitrate underestimate by a factor of 4 to 6 the actual production of nitrate or NO by the body. In disease states, such as heart failure, in which renal function and extracellular volume are altered, caution should be exercised when increases in nitrate in plasma as an index of NO formation are evaluated.

Cardiac desensitization to adenosine analogues after prolonged R-PIA infusion in vivo.

To determine the effects of chronic in vivo stimulation of adenosine receptors, R-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), a selective A1 receptor agonist, was administered to rats as a continuous 7-day infusion (200 nmol/h). Inotropic and chronotropic responses of isolated atria to adenosine receptor agonists were markedly desensitized compared with the responses of atria from age-matched control animals. Carbachol's negative chronotropic effect was also attenuated, indicating a heterologous mode of desensitization. Antagonist radioligand binding assays indicated a 52% reduction in A1 adenosine receptor maximum binding, and competition binding assays revealed a significant loss of G protein-coupled high-affinity A1 receptors in atria from R-PIA-treated rats. Inhibitory G proteins (Gi) were significantly reduced, as quantified by immunoblot analysis, with no change in the amount of stimulatory G proteins. Ventricular membranes from R-PIA rats showed loss of Gi and uncoupling of A1 receptors, without a significant change in A1 receptor density. Thus chronic R-PIA infusion desensitized rat atrial muscle to the effects of adenosine receptor agonists via several regulatory adaptations, including downregulation of A1 adenosine receptors, uncoupling of A1 receptors from their associated G proteins, and loss of Gi proteins.

Propensity to form conditioned taste aversions augments anorexia in obese (ob/ob) mice with B16 melanoma.

Ob/ob mice (OB) with B16 melanoma become anorectic, but lean mice (LN) do not. Present studies suggest that this difference reflects a greater bent for OB to form conditioned taste aversions (CTA). In Exp 1, healthy OB formed stronger CTAs than LN to a saccharin taste paired with lithium chloride (LiCl, 3 mEq/kg ip). In Exp 2, the OB-LN difference of Exp 1 was decreased by giving naltrexone (10 mg/kg sc) before LiCl, which suggested opiate involvement. Exp 3 showed that OB tumor anorexia vanishes if foods dissociated from tumor growth are given: OB fed a constant diet became anorectic 16 days after B16 inoculation; giving a new diet on Day 16 delayed anorexia onset for 8 days; a second new diet on Day 32 abolished anorexia for 24 hr. LN with tumors ate all diets at nontumor control levels. OB survived melanoma longer than LN regardless of diet, but OB fed a varied diet died first; thus, anorexia may enhance OB survival.

Differential sensitization of cardiac actions of adenosine in rats after chronic theophylline treatment.

To determine the effect of chronic adenosine receptor blockade on atrial responsiveness, we administered theophylline to rats in their drinking water (0.6 mg/ml) for 2 wk. Inotropic and chronotropic responses to the adenosine receptor agonists N6-cyclopentyladenosine (CPA) and 5'-(N-ethylcarboxamido)-adenosine (NECA) were then measured in isolated atria from treated and control animals. The indirect (antiadrenergic) actions of CPA and NECA on force and rate, measured during beta-adrenergic receptor stimulation by isoproterenol, were markedly sensitized (2- to 10-fold reductions in the agonist concentration needed to obtain a half-maximal response) after theophylline. The sensitization was homologous because inotropic and chronotropic responses to carbachol were not affected by theophylline. The direct negative inotropic and chronotropic actions of CPA and NECA, measured without concomitant beta-adrenergic stimulation, were not sensitized after theophylline. The number of atrial A1-receptors, measured by antagonist radioligand binding (maximum specific binding at saturation), was increased by 22% in theophylline-treated rats [66.2 +/- 3.4 vs. 54.3 +/- 1.9 (control) fmol/mg protein, P < 0.05]. Competition binding indicated that the fraction of coupled (high-affinity) receptors was unchanged. The number of ventricular A1-receptors was increased to a similar extent without any change in coupling. Thus chronic dietary theophylline upregulated cardiac A1-adenosine receptors without changing coupling state or affinity and sensitized rat atria to the indirect, antiadrenergic, inhibitory inotropic and chronotropic actions of adenosine receptor agonists.