Complement components and their activation products in pleural fluid

BACKGROUND: This study examined the influences of isoflurane versus halothane anesthesia on basal and agonist-stimulated nitric oxide in the cerebellum of intact rats. Nitric oxide was measured using the hemoglobin-trapping method in an in vivo microdialysis technique. This method uses the stoichiometric reaction of nitric oxide with oxyhemoglobin to produce methemoglobin and nitrate; the change in methemoglobin concentration is measured spectrophotometrically to estimate nitric oxide concentration. METHODS: Male Wistar rats were anesthetized with isoflurane (1.4%) or halothane (1.2%), mechanically ventilated and paralyzed (intravenous pancuronium, 1 mg/kg). Microdialysis probes were implanted into the cerebellum. Bovine oxyhemoglobin dissolved in artificial cerebrospinal fluid was pumped through the probe (2 microl/min) and assayed at 15-min intervals. The glutamatergic agonist, kainic acid (KA, 5 mg/kg, intraarterially), was used to stimulate nitric oxide production. NG-nitro L-arginine methyl ester (L-NAME, 40 mg/kg, intravenously) was used to inhibit nitric oxide synthase. RESULTS: Unstimulated cerebellar nitric oxide concentrations were stable and greater during anesthesia with isoflurane (532+/-31 nM; mean +/- SEM) than with halothane (303+/-23 nM). L-NAME pretreatment reduced nitric oxide concentrations during isoflurane, but not halothane, anesthesia. Infusion of KA increased nitric oxide in both groups; however, the increase in nitric oxide was significantly greater during isoflurane anesthesia. Pretreatment with L-NAME inhibited the response to KA in both groups. CONCLUSIONS: Nitric oxide production in the cerebellum, monitored by microdialysis, was greater during isoflurane anesthesia than during halothane anesthesia. Increased nitric oxide production during isoflurane anesthesia would be expected to impact central neuronal function and cerebral blood flow and vascular resistance.     

Seroprevalence of selected disease agents from free-ranging black bears in Florida

Streptococcus pneumoniae is a common cause of meningitis. Nitric oxide (NO) has been implicated in causing cerebral edema. Modulating NO production in cerebrospinal fluid (CSF) may have a role in the treatment of bacterial meningitis. Experimental S. pneumoniae meningitis was induced in a rabbit model to determine CSF parameters and NO concentrations. An electrochemical probe in the CSF throughout the 7-hour experiment monitored NO concentrations. The animals had S. pneumoniae (10(5)) injected intracisternally and incubated for 1 hour. Cerebrospinal fluid 200-300 microl was obtained by intracisternal puncture at zero, 2, 4, and 7 hours after drug administration to measure glucose, protein, and lactic acid by standard chemical methods. White blood cell count was measured by hemocytometry. Three groups of five animals were used-control (C), ceftriaxone (CTX), and ceftriaxone plus dexamethasone (CTX+D). Ceftriaxone concentrations in CSF were obtained by microdialysis and analyzed by high-performance liquid chromatography. Mean (+/- SEM) CSF white blood cell count was significantly higher at 2 hours in the C group than in the other two groups (C 7307 +/- 1302, CTX 605 +/- 345, CTX+D 730 +/- 43/mm3, p<0.002). Ceftriaxone induced a significant rise in protein at 4 hours compared with the other groups (C 364 +/- 107, CTX 1158 +/- 797, CTX+D 365 +/- 100 mg/dl, p<0.02). Cerebrospinal fluid lactic acid was significantly different at 4 and 7 hours between C and CTX+D groups (4-hr C 8.0 +/- 2.2, CTX+D 2.0 +/- 0.4 mmol/L, p<0.05; 7-hr C 10.2 +/- 2.4, CTX+D 2.8 +/- 0.8 mmol/L, p<0.01). Median NO concentrations were significantly elevated in the control group compared with the other two groups (C 11.7, CTX 6.8, CTX+D 6.5 micro, p<0.02 C vs CTX, p<0.01 C vs CTX+D). Average (+/- SEM) NO concentrations were significantly higher in the C group at 4 hours (18.1 +/- 0.4, CTX 5.8 +/- 1.8 microM, p<0.05; CTX+D 11.5 +/- 4.0 microM, p>0.05), whereas they did not rise significantly until 7 hours in the CTX group (CTX 18.7 +/- 0.7, C 8.9 +/- 0.4 microM, p=0.055; CTX+D 8.1 +/- 2.2 microM, p<0.05). These results indicate that ceftriaxone with or without dexamethasone significantly decreases lactic acid concentrations and white cell penetration into the CSF in an experimental model of S. pneumoniae meningitis. In addition, ceftriaxone induced a significant elevation in CSF protein. Median NO production in the CSF was significantly attenuated by ceftriaxone.     

Nitric oxide production is increased in the paraventricular nucleus of the hypothalamus of male rats during non-contact penile erections and copulation

Male rats put in the presence of a receptive female rat that they can see, hear and smell, but cannot touch, show penile erection episodes. These non-contact erections occur concomitantly with an increase in nitric oxide production in the paraventricular nucleus of the hypothalamus, as detected by the increase in the NO2- and NO3- concentration in the paraventricular dialysate obtained from these males by in vivo microdialysis. NO2- concentration increased from 0.81+/-0.12 to 2.51+/-0.43 microM and that of NO3- from 4.50+/-0.73 to 8.31+/-2.3 microM. The NO2- increase was prevented by the nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (20 microg) given unilaterally in the paraventricular nucleus, which also prevented non-contact erections. In contrast, the nitric oxide scavenger haemoglobin (20 microg) prevented the NO2- increase, but not non-contact erections; while the guanylate cyclase inhibitor methylene blue (20 microg) was ineffective on either response. NO2-and NO3- concentration was also increased in the paraventricular dialysate of male rats during in copula penile erections, that is, when sexual activity was allowed with the receptive females. As found with non-contact erections, NG-nitro-L-arginine methylester prevented NO2- increase and impaired copulatory behaviour; haemoglobin prevented NO2- increase only; and methylene blue was ineffective on either response. The present results confirm that nitric oxide is a physiological mediator of penile erection at the level of the paraventricular nucleus of the hypothalamus.     

Prostaglandins reduce the contractile responses to noradrenaline and angiotensin II in canine femoral arteries after but not before chronic inhibition of nitric oxide synthesis

This study was designed to compare the contractile responses to graded concentrations of noradrenaline (1 nM-100 microM) and angiotensin II (0.1-100 nM) of femoral arteries isolated from normal control dogs and from dogs after long-term inhibition of nitric oxide (NO) by N(omega)-nitro-L-arginine (L-NNA; 20 mg/kg/day for 7 days). Maximal contraction to noradrenaline was similar in rings obtained from control and L-NNA-treated dogs. In the latter, however, sensitivity to noradrenaline was reduced compared with control rings, whether the endothelium was present [50% effective concentration (EC50) = 6.04 +/- 0.06 vs. 6.37 +/- 0.08; p < 0.01] or absent (EC50 = 6.00 +/- 0.11 vs. 6.45 +/- 0.05; p < 0.01). Indomethacin reversed this hyporesponsiveness to noradrenaline in arteries obtained from L-NNA-treated dogs but had no effect in rings isolated from control dogs. An almost complete inhibition of the contractile response to angiotensin II, also reversed by indomethacin, was observed in arteries taken from L-NNA-treated dogs both in the presence and in the absence of endothelium. These results suggest that the cyclooxygenase pathway might be upregulated in the smooth muscle cells of canine femoral arteries after long-term inhibition of NO synthesis and that relaxing prostanoids mediate the hypocontractile response of these arteries to both noradrenaline and angiotensin II.     

Role of known risk factors in explaining the difference in the risk of coronary heart disease between eastern and southwestern Finland

BACKGROUND: Estradiol (E2) has a potent antioxidant effect on low density lipoproteins (LDL) in vitro and in vivo, which could be important in explaining the cardioprotective effect of hormone replacement therapy (HRT) in post menopausal women. Estriol (E3), on the other hand, is a weak estrogen with low metabolic effects on different tissues, and at present no cardioprotective effect has been attributed to this steroid. AIM: To study the antioxidant effect of E3 on LDL and to compare it with the potent antioxidant action exhibited by E2. SUBJECTS AND METHODS: After LDL was isolated by ultra centrifugation from plasma of 12 healthy untreated post menopausal women, it was divided into aliquots containing 0.5 mg of LDL protein. Estriol and E2 in doses of 0, 1, 5, 15 and 50 microM were incubated with different aliquots of LDL. CuSO4 15 microM was added to each aliquot to induce an oxidative stress. The aliquots were then incubated during 4 hours at 37 degrees C. Malonaldehyde (MDA) was measured as a marker of LDL oxidation, and expressed as nM/mg protein. RESULTS: (mean +/- SD): Estriol induced a dose-dependent decrease in MDA concentration (baseline 62.8 +/- 21.7; 1 microM: 61.5 +/- 23.0; 5 microM: 52.9 +/- 20.3; 15 microM 43.5 +/- 20.1 and 50 microM: 31.0 +/- 17.6 nM/mg protein; F = 92.4; p < 0.0001), reaching a mean decrease of 50.7% at the highest dose tested. Estradiol has a similar dose-dependent decrease in MDA concentration (F = 60.2; p < 0.0001), revealing a more potent effect than E3 (p < 0.05), with a mean decrease of 67.4% at the highest dose tested. CONCLUSIONS: Our results demonstrate that estriol shows an important antioxidant action of LDL in vitro, although its effect is less potent than estradiol. These results raise the possibility that estriol could have a cardioprotective effect in post menopausal women, possibility that has not been yet demonstrated.     

Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils

1. The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2. A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 microM) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 microM) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.68 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectively. 3. A role for cyclic GMP as a mediator of chemotaxis was supported by the finding that the guanylyl cyclase inhibitor LY 83583 (100 microM) completely inhibited chemotaxis and suppressed the maximal response; EC50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 microM); EC50 for fMLP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823, respectively. 4. The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 microM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/10(6) cells with and without DPG, respectively. 5. Although the NOS inhibitors L-NMMA and L-canavanine (500 microM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36.15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response from 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O2-/10(6) cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6. A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 microM) and Rp-8-pCPT-cGMPS (100 microM) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS. 7. The phosphatase inhibitor DPG (500 microM) inhibited SAG; EC50 for fMLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respectively. 8. The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 microM) > GEA 5024 (IC50 = 18.44 +/- 0.43 microM) > SIN-1 (IC50 > 1000 microM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most effective (SIN-1 (EC50 = 37.62 +/- 0.9 microM) > GEA 3162 (EC50 = 39.7 +/- 0.53 microM) > GEA 5024 (EC50 = 89.86 +/- 1.62 microM)). 9. In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for these agents in neutrophil activation. However, the increases in cyclic GMP and NO induced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as observed with NO releasing compounds, inhibit chemotaxis.     

Effects of ischemia, preconditioning, and adenosine deaminase inhibition on interstitial adenosine levels and infarct size

In order to examine the relationship between local adenosine concentrations before, during, and after ischemia and the extent of ischemic myocardial damage, measurements of interstitial fluid (ISF) nucleosides were made using microdialysis probes implanted in the ischemic region of isoflurane anesthetized Micropigs undergoing 60' coronary artery occlusion (CAO) and 3 h of reperfusion (REP). Nucleoside concentrations in the dialysate collected from the microdialysis probes were used as an index of ISF levels. Dialysate nucleoside concentrations (ADO, inosine and hypoxanthine), myocardial infarct size, and myocardial blood flow (MBF) were determined in control animals (n = 6), animals preconditioned with a single 10' cycle of CAO and REP (PC, n = 6), and those treated with the adenosine deaminase inhibitor pentostatin (n = 6, 0.2 mg/Kg i.v. 30' prior to CAO). The brief PC occlusion resulted in a transient but significant increase in dialysate ADO (6.7 +/- 1.8 microM vs. 0.67 +/- 0.1 microM at baseline). Pentostatin administration had no significant effect on either dialysate nucleosides or MBF at baseline. During the 60' CAO, dialysate ADO increased in control animals. In PC animals, however, dialysate ADO during CAO was lower than control. Pretreatment with pentostatin resulted in a six-fold augmentation in dialysate ADO during the 60 min CAO when compared to the control values (110.62 +/- 30.2 microM vs. 16.31 +/- 2.1 microM at 60 min of ischemia). Pentostatin also resulted in a significant reduction in the accumulation of inosine and hypoxanthine, indicating inhibition of adenosine deaminase activity. There were no significant differences in MBF between groups at any time point. Following 3 h REP, infarct size was 35.4 +/- 5.5%, 8.1 +/- 1.5% and 8.3 +/- 1.8% of the region at risk in control, PC, and pentostatin groups, respectively. These data suggest that marked increase in ISF ADO during CAO, may be as effective in reducing INF as a modest increase in ISF ADO prior to prolonged CAO.     

Increased resting and exercise-induced oxidative stress in young IDDM men

The effects of local application of the 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide (CPBG), and i.p. administration of ethanol on the extracellular levels of dopamine (DA) in the ventral tegmental area (VTA) were studied using in vivo microdialysis. Adult female Wistar rats were implanted with microdialysis probes in the VTA at least 24 h before each experiment. Stable extracellular levels of DA (101 +/- 9 fmol/20 min) were established before initiating the experiments. Application of 10-250 microM CPBG through the microdialysis probe dose-dependently enhanced the extracellular concentrations of DA but did not alter the levels of either 3,4-dihydroxyphenylacetic acid or homovanillic acid in the dialysate. The effects of CPBG were reversible and dependent upon Ca2+. Co-perfusion with the 5-HT3 receptor antagonist, 3-tropanyl-indole-3-carboxylate (ICS 205-930), inhibited the effects of CPBG on enhancing extracellular DA levels. The i.p. administration of 2 g/kg ethanol significantly (p < 0.005) enhanced the levels of DA to 150% of baseline values; this ethanol-induced increase was prevented by local perfusion with 100 microM ICS 205-930. These results suggest that 5-HT3 receptors in the VTA are involved in regulating the somatodendritic release of DA and in mediating the stimulatory effects of ethanol on this neuronal system.     

AT2-antagonist sensitive potentiation of angiotensin II-induced vasoconstrictions by blockade of nitric oxide synthesis in rat renal vasculature

1. Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II-induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT-R) antagonists on Ang II-induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single-pass circuit. 2. In the absence of NO blockade, the AT1-R antagonist L-158809 (500 nM) antagonized the Ang II-induced vasoconstrictions, while the AT2-R antagonist PD-123319 (500 nM) had no effect. 3. Perfusing kidneys in the presence of either NO synthase inhibitors, L-NAME (100 microM) or L-NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY-83583 (10 microM), significantly increased both molar pD2 (from 9.40+/-0.25 to 10.36+/-0.11) and Emax values (from 24.9+/-3.1 to 79.9+/-4.9 mmHg) of the concentration-response curve for Ang II-induced vasoconstriction. 4. In the presence of L-NAME, 500 nM L158809 abolished the Ang II-induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD-123319 reversed the left shift of the concentration-response curve for Ang II (molar pD2 value 9.72+/-0.13) leaving Emax value unaffected (91.3+/-7.6 mmHg). 5. In the presence of L-NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration-dependent manner by L-158809 (0.05-500 nM). By contrast, PD-123319 (0.5-500 nM) did not affect the 10 nM Ang II-induced vasoconstriction and concentration-dependently decreased the 0.1 nM Ang II-induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6. Similar to PD-123319, during NO blockade the AT2-R antagonist CGP-42112A at 5 nM decreased by 50% the 0.1 nM Ang II-induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II-induced vasoconstriction. 7. In conclusion, the renal Ang II-induced vasoconstriction, which is antagonized only by AT1-R antagonist in the presence of endogenous NO, becomes sensitive to both AT1- and AT2-R antagonists during NO synthesis inhibition. While AT1-R antagonist inhibited both L-NAME-potentiated and -augmented components of Ang II-induced vasoconstriction, AT2-R antagonists inhibited only the L-NAME-potentiated component.     

Quantitative microdialysis of ethanol in rat striatum

We have applied a steady-state theory of microdialysis to characterize the diffusion of ethanol through a microdialysis membrane and through rat striatum. Quantitative characterization required measurement of in vitro and in vivo extraction fractions for ethanol and determination of the clearance of ethanol from brain tissue during steady-state perfusion through a microdialysis probe. Extraction fraction of ethanol was determined in vitro by perfusing a known concentration of ethanol through probes immersed in water at 37 degrees C with stirring. The in vitro extraction fraction yielded a probe permeability value of 0.046 +/- 0.004 cm/min that is comparable with an estimate from published measurements for similar dialysis membranes. The in vivo extraction fraction was determined for probes placed in the striatum. Clearance of ethanol and a brain slice concentration profile of ethanol were determined by measurement of the amount of ethanol remaining in the brain tissue during steady-state perfusion of the probe. Steady state was achieved within 10 min after beginning the ethanol perfusion in vivo, and the extraction fraction was not altered by sedation of the rat with pentobarbital. The tissue concentration profile was symmetrical around the probe track, and ethanol was detected 1 mm from the probe. The experimental clearance rate constant value obtained for ethanol (2.0 +/- 0.3 min(-1)) was higher than that expected for removal solely by loss to the blood. The tissue diffusivity for ethanol, Dt, derived from the experimental measurements was 1.2 +/- 0.2 x 10(-5) cm2/sec. This value is greater than expected for interstitial diffusion, suggesting a substantial contribution by transcellular diffusion of ethanol as well. The predicted tissue concentration profile had a higher peak value and did not extend into the tissue (0.5 mm) as much as the experimental profile (1 mm), although there was reasonable agreement between experiment and theory. Our quantitative characterization of the microdialysis behavior of ethanol in brain provides a framework for interpretation of brain microdialysis experiments using ethanol by supplying, inter alia, a means for estimating the ethanol concentration achieved in the tissue volume being sampled by the probe.     

Microdialysis probe for transcutaneous monitoring of ethanol and glucose in humans

A heated (42 degrees C) microdialysis probe and its application for continuous transcutaneous sampling of ethanol and glucose through cellophane-stripped forearm skin are described. Ethanol and glucose concentration in the dialysate were measured on-line with continuous-flow analysis and compared with blood values in human volunteers after ethanol consumption (n = 4) and oral glucose testing (n = 5), respectively. For ethanol and glucose, the dialysate and blood concentrations were linearly related in each subject (r > or = 0.91, P < 0.005), although the dialysate-to-blood ratio varied among subjects. The recovery in vivo was 22.4 +/- 22.7 and 4.7 +/- 2.3% (SD) of the recovery in vitro for ethanol and glucose, respectively. The dialysate glucose concentration was independent of blood flow. When the probe temperature was increased from 32 to 42 degrees C, the dialysate-to-blood glucose ratio increased, with 2.4 +/- 1.4%/degrees C (SD) in fasting subjects (n = 4), which was similar to an increase of 2.1 +/- 0.045%/degree C in dialysate-to-medium ratio in vitro. The present approach for transcutaneous sampling may possibly be used for other substances of low molecular weight as well.     

Germline mutations of the BRCA1 and BRCA2 genes in a breast and ovarian cancer patient

Nitric oxide (NO) has been implicated as a modulator of the vascular effects of angiotensin II (ANG II) in the kidney. We used a NO-sensitive microelectrode to study the effect of ANG II on NO release, and to determine the effect of selective inhibition of the ANG II subtype I receptor (AT1) with losartan (LOS) and candesartan (CAN). NO release from isolated and perfused renal resistance arteries was measured with a porphyrin-electroplated, carbon fiber. The vessels were microdissected from isolated perfused rat kidneys and perfused at constant flow and pressure in vitro. The NO-electrode was placed inside the glass collection cannula to measure vessel effluent NO concentration. ANG II stimulated NO release in a dose-dependent fashion: 0.1 nM, 10 nM and 1000 nM ANG II increased NO-oxidation current by 85+/-18 pA (n = 11), 148+/-22 pA (n = 11), and 193+/-29 pA (n = 11), respectively. These currents correspond to changes in effluent NO concentration of 3.4+/-0.5 nM, 6.1+/-1.1 nM, and 8.2+/-1.3 nM, respectively. Neither LOS (1 muM) nor CAN (1 nM) significantly affected basal NO production, but both AT1-receptor blockers markedly blunted NO release in response to ANG II (10 nM): 77+/-6% inhibition with LOS (n = 8) and 63+/-9% with CAN (n = 8). These results are the first to demonstrate that ANG II stimulates NO release in isolated renal resistance arteries, and that ANG II-induced NO release is blunted by simultaneous AT1-receptor blockade. Our findings suggest that endothelium-dependent modulation of ANG II-induced vasoconstriction in renal resistance arteries is mediated, at least in part, by AT1-receptor-dependent NO release.     

Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma

Oxidative stress contributes to airway inflammation and exhaled hydrogen peroxide (H2O2) and nitric oxide (NO) are elevated in asthmatic patients. We determined the concentrations of expired H2O2 and NO in 116 asthmatic (72 stable steroid-naive, 30 stable steroid-treated, and 14 severe steroid-treated unstable patients) and in 35 healthy subjects, and studied the relation between exhaled H2O2, NO, FEV1, airway responsiveness, and eosinophils in induced sputum. Both exhaled H2O2 and NO levels were elevated in steroid-naive asthmatic patients compared with normal subjects (0.72 +/- 0.06 versus 0.27 +/- 0.04 microM and 29 +/- 1.9 versus 6.5 +/- 0. 32 ppb, respectively; p < 0.001) and were reduced in stable steroid-treated patients (0.43 +/- 0.08 microM, p < 0.05, and 9.9 +/- 0.97 ppb, p < 0.001). In unstable steroid-treated asthmatics, however, H2O2 levels were increased, but exhaled NO levels were low (0.78 +/- 0.16 microM and 6.7 +/- 1.0 ppb, respectively). There was a correlation between expired H2O2, sputum eosinophils and airway hyperresponsiveness (methacholine PC20). Exhaled NO also correlated with sputum eosinophils, but not with airway hyperresponsiveness. Our findings indicate that measurement of expired H2O2 and NO in asthmatic patients provides complementary data for monitoring of disease activity.     

Potency and kinetics of nitric oxide-mediated vascular smooth muscle relaxation determined with flash photolysis of ruthenium nitrosyl chlorides

Flash photolysis of thermally stable, photolabile 'caged' precursors permits rapid and precise changes of ligand concentration at their site of action. This approach was used to determine the concentration-dependence and time course of NO-mediated relaxation of aortic smooth muscle, by use of two photolabile NO donors, trichloronitrosylruthenium (Ru(NO)Cl3) and dipotassium pentachloronitrosylruthenate (K2Ru(NO)Cl5). At concentrations up to 500 microM, both compounds were non-toxic before photolysis, and produced non-toxic by-products on photolysis. Photolytic release of NO produced relaxations of intact and endothelium-denuded aortic rings precontracted with noradrenaline (0.1-0.5 microM), with an EC50 for NO-mediated relaxations of 10.5 nM and 13 nM, respectively. NO-mediated relaxations were reversibly blocked by 1 microM oxyhaemoglobin. The time course of NO-mediated relaxation comprised a delay of 3-7 s, followed by a sigmoidal decline in tension with peak rates that were strongly dependent on NO concentration.     

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1. The functional role of the nitric oxide (NO)/guanosine 3':5'-cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2. Rat isolated hearts were perfused at constant pressure with Krebs-Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min(-1) for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3. At baseline, heart rate was 287+/-12 beats min(-1), coronary flow was 12.8+/-0.6 ml min(-1), left ventricular developed pressure (LVDevP) was 105+/-4 mmHg and left ventricular end-diastolic pressure 4.6+/-0.2 mmHg in vehicle-treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05). 4. In normoxic perfused hearts, 1 microM N(G)-nitro-L-arginine (L-NOARG) significantly reduced coronary flow from 13.5+/-0.2 to 12.1+/-0.1 ml min(-1) (10%) and LVDevP from 97+/-1 to 92+/-1 mmHg (5%; P<0.05, n=5). 5. Ischaemic contracture was 46+/-2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 microM) but reduced to approximately 30 mmHg (approximately 25%) at higher concentrations (100 or 1000 microM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 microM to 26+/-3 mmHg (23%), but increased it to 63+/-4 mmHg (59%) at 1000 microM (n=6). Dobutamine (10 microM) exacerbated ischaemic contracture (81+/-3 mmHg; n = 7) and the cyclic GMP analogue Sp-8-(4-p-chlorophenylthio)-3',5'-monophosphorothioate (Sp-8-pCPT-cGMPS; 10 microM) blocked this effect (63+/-11 mmHg; P<0.05 vs dobutamine alone, n=5). 6. At the end of reperfusion, LVDevP was 58+/-5 mmHg, i.e. 55% of pre-ischaemic value in control hearts, significantly increased to approximately 80% by high concentrations of nitroprusside (100 or 1000 microM) or L-NOARG at 1 microM, while a high concentration of L-NOARG (1000 microM) reduced LVDevP to approximately 35% (P<0.05 vs control; n=6). 7. Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 microM had no sustained effect, but increased cyclic GMP approximately 6 fold at 1000 microM; L-NOARG (1 or 1000 microM) was without effect (n=6). Nitroprusside (1 or 1000 microM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6). 8. In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.     

Anandamide amidase inhibition enhances anandamide-stimulated nitric oxide release in invertebrate neural tissues

Anandamide, an endogenous cannabinoid signaling molecule, in a concentration dependent manner, initiates the release of nitric oxide (NO) from leech and mussel ganglia. SR 141716A, a cannabinoid antagonist, blocks the anandamide stimulated release of NO from these tissues. Methyl arachidonyl fluorophosphonate (MAFP), a specific anandamide amidase inhibitor, when administered to either ganglia with anandamide (10-6 M) did not increase the peak level of NO release but did significantly extend NO release from 12 to 18 min (P<0.05). Lower levels of anandamide (10-8 and 10-7 M) do not stimulate the release of significant amounts of NO from these tissues. However, in the presence of MAFP (2.5 nM), the lower anandamide concentrations were able to release significant peak amounts of NO. In mussel neural tissues, the peak NO release increased from 2.2+/-1.3 nM to 8.6+/-2.1 nM. Taken together, the results indirectly demonstrate the presence of anandamide amidase in these tissues, suggesting that the enzyme may serve as an endogenous regulator of anandamide action.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Spontaneous and evoked inhibitory junction potentials in the circular muscle layer of mouse colon

Intracellular microelectrodes were used to record spontaneous and evoked inhibitory junction potentials (IJPs) from the circular muscle layer of an in vitro preparation of whole mouse colon. Membrane potential recordings were made from cells of the mid to distal region of colon at 36 +/- 1 degrees C in a modified Krebs' solution that contained atropine (1 microM) and nifedipine (1-2 microM). Spontaneously occurring hyperpolarisations of irregular amplitude and frequency (range: up to 20 mV and 2 Hz) were recorded that were resistant to N(G)-nitro-L-arginine (NOLA, 100 microM), but were abolished by tetrodotoxin (TTX, 1.6 microM) or apamin (250 nM). These were considered to be spontaneous IJPs as a consequence of activity in inhibitory motor neurons. Single electrical stimuli (0.6 ms, 15 V), elicited a fast IJP, whose time course could be superimposed on spontaneous IJPs of similar amplitude. The amplitude of evoked IJPs was not depressed by NOLA (100 microM). However, in NOLA (100 microM), further addition of apamin (250 nM) significantly depressed the amplitudes of the evoked IJPs by 44%. NOLA- and apamin-resistant evoked IJPs were abolished by TTX (1.6 microM). It is suggested, that in the circular muscle layer of mouse colon, NO does not mediate the fast hyperpolarisations associated with spontaneous or evoked IJPs. Apamin abolished spontaneous IJPs, but electrical stimuli evoked an IJP with apamin-sensitive and resistant components both of which were non-nitrergic in origin.     

In situ assessment of the role of the beta 1-, beta 2- and beta 3-adrenoceptors in the control of lipolysis and nutritive blood flow in human subcutaneous adipose tissue

1. The involvement of beta 1-, beta 2- and beta 3-adrenoceptors in the control of lipolysis and nutritive blood flow was investigated in abdominal subcutaneous adipose tissue of healthy young adults by use of an in situ microdialysis technique. 2. Dialysis probes were infused either with isoprenaline (non-selective beta-adrenoceptor agonist), CGP 12,177 (selective beta 3-adrenoceptor agonist having beta 1-/beta 2-antagonist properties), dobutamine (selective beta 1-adrenoceptor agonist) or terbutaline (selective beta 2-adrenoceptor agonist). The recovery of each probe used for perfusion was calculated by an in vivo calibration method. The local blood flow was estimated through the measurement of the escape of ethanol infused simultaneously with the drugs included in the probe. 3. Isoprenaline infusion at 0.01 microM had a weak effect while higher concentrations of isoprenaline (0.1 and 1 microM) caused a rapid, sustained and concentration-dependent increase of glycerol outflow; the maximum increase was 306 +/- 34% with 1 microM. Isoprenaline also increased the nutritive blood flow in adipose tissue; a significant effect appeared at 0.1 microM isoprenaline and was greater at 1 microM. 4. CGP 12,177 (10 and 100 microM) increased the glycerol concentration in the dialysate (128 +/- 8 and 149 +/- 12%, respectively) and nutritive blood flow. Terbutaline and dobutamine (100 microM) both provoked rapid and similar increases in glycerol outflow (252 +/- 18 and 249 +/- 18%, respectively). Both, terbutaline and dobutamine increased nutritive blood flow. 5. It is concluded that beta 1- and beta 2-adrenoceptor subtypes are both mainly involved in the mobilization of lipids and in the control of nutritive blood flow. beta 3-Adrenoceptors play a weaker role in the control of lipolysis and nutritive blood flow in human subcutaneous abdominal adipose tissue.     

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BK(Ca)) in smooth muscle cells isolated from the rat mesenteric artery

1. To assess the action of nitric oxide (NO) and NO-donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2. Inside-out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212+/-5 pS (n = 12) in symmetrical (140 mM) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK(Ca). 3. In five out of six inside-out patches, NO (45 or 67 microM) produced an increase in BK(Ca) activity. In inside-out patches, BK(Ca) activity was also enhanced in some patches with 100 or 200 microM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 microM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4. In inside-out patches, 100 microM SIN-1 failed to increase BK(Ca) activity (in all 4 patches tested), while at a higher (500 microM) concentration SIN-1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside-out patches increased (P < 0.05) BK(Ca) activity in two patches. 5. In the majority of cells (6 out of 7), application of NO (45 or 67 microM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 microM). With SIN-1, the increase in current was blocked with the BK(Ca) channel blocker, iberiotoxin (50 nM). 6. With conventional whole-cell voltage clamp, the increase in the outward K+ current evoked with SIN-1 (50-300 microM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+/-10.2% was recorded at 40 mV. 7. In cell-attached patches, large conductance voltage-dependent K+ channels were stimulated by SIN-1 (100 microM) applied to the cell (n = 5 patches). 8. These data indicate that NO and its donors can directly stimulate BK(Ca) activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK(Ca) channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.