Partial inhibition of AT-EAE by an antibody to ICAM-1: clinico-histological and MRI studies

The aim of this study was to characterise the response to acute hypoxia in pulmonary artery rings isolated from rats exposed to chronic hypoxia for 2 weeks (CH) and following recovery in room air for 24 h (post hypoxic, PH). Large intrapulmonary artery (IPA) rings (internal diameter = 1.5 +/- 0.11 mm; n = 13) from CH and PH rats and age-matched controls were studied. These were precontracted with phenylephrine using standard organ bath procedures at an oxygen tension of 152 mmHg and subjected to an acute hypoxia stimulus (bubbling with 0% O2 giving Po2 = 7 mmHg or 2% O2 giving PO2 = 20 mmHg). Acute hypoxia-induced pulmonary vasoconstriction (HPV) consisted of a transient contraction, a relaxation and a sustained contraction over 30 min. Pulmonary vasoconstriction induced by 0% O2 was significantly reduced in IPA rings from the CH but not PH group compared with the response obtained from the control group. HPV induced by 2% O2 in IPA rings from CH and PH rats was not significantly different from that in control rats not subjected to chronic hypoxia. Mechanical removal of the endothelium or inhibition of nitric oxide (NO) synthase by L-NOARG (300 microM) reduced the contractile phases of HPV in IPA rings from control and CH rats. Carbachol-induced endothelium-dependent relaxation in phenylephrine precontracted IPA rings was significantly attenuated in the CH but not PH group. In conclusion, the present study demonstrates that HPV induced by 0% O2 in rat IPA rings was blunted in CH rats and restored following 24 h in room air, in parallel with changes in endothelium function.     

Protective effects of intravascular pressure and nitric oxide in ischemic lung injury

Cessation of blood flow during ischemia will decrease both distending and shear forces exerted on endothelium and may worsen ischemic lung injury by decreasing production of nitric oxide (NO), which influences vascular barrier function. We hypothesized that increased intravascular pressure (Piv) during ventilated ischemia might maintain NO production by increasing endothelial stretch or shear forces, thereby attenuating ischemic lung injury. Injury was assessed by measuring the filtration coefficient (Kf) and the osmotic reflection coefficient for albumin (sigmaalb) after 3 h of ventilated (95% O2-5% CO2; expiratory pressure 3 mmHg) ischemia. Lungs were flushed with physiological salt solution, and then Piv was adjusted to achieve High Piv (mean 6.7 +/- 0.4 mmHg, n = 15) or Low Piv (mean 0.83 +/- 0.4 mmHg, n = 10). NG-nitro-L-arginine methyl ester (L-NAME; 10(-5) M, n = 10), NG-nitro-D-arginine methyl ester (D-NAME; 10(-5) M, n = 11), or L-NAME (10(-5) M)+L-arginine (5 x 10(-4) M, n = 6) was added at the start of ischemia in three additional groups of lungs with High Piv. High Piv attenuated ischemic injury compared with Low Piv (sigmaalb 0.67 +/- 0.04 vs. 0. 35 +/- 0.04, P < 0.05). The protective effect of High Piv was abolished by L-NAME (sigmaalb 0.37 +/- 0.04, P < 0.05) but not by D-NAME (sigmaalb 0.63 +/- 0.07). The effects of L-NAME were overcome by an excess of L-arginine (sigmaalb 0.56 +/- 0.05, P < 0.05). Kf did not differ significantly among groups. These results suggest that Piv modulates ischemia-induced barrier dysfunction in the lung, and these effects may be mediated by NO.     

Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.     

Endothelium-dependent modulation of resistance vessel contraction: studies with NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine

1. The effect of NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NOARG) on noradrenaline (NA)-induced contractility and acetylcholine (ACh)-induced endothelium-dependent relaxation was studied in rat mesenteric resistance arteries. 2. Third order branches of mesenteric arteries were dissected and mounted on two forty micron wires in a Mulvany myograph. 3. Incubation with L-NAME and L-NOARG (10 microM) caused a time-dependent shift in the 50% response to NA (ED50) (0.01 microM-10 microM) but was not associated with an increase in the maximum contractile response. 4. L-NAME and L-NOARG (10 microM) caused a time-dependent inhibition of ACh (1 microM)-induced relaxation with a maximum effect after 120 min. 5. Following endothelium removal, incubation with either L-NAME or L-NOARG caused no significant shift in the ED50, although the residual relaxation response to ACh (1 microM) was further attenuated. 6. Incubation with the cyclo-oxygenase inhibitor, indomethacin, enhanced the relaxation to ACh and reduced the inhibitory effects of L-NAME and L-NOARG. 7. In conclusion, L-NAME and L-NOARG are potent inhibitors of acetylcholine-induced endothelium-dependent relaxation in mesenteric resistance arteries. The shift in ED50 associated with these inhibitors suggests a probable role for the endothelium in modulating the contractility of the resistance vasculature.     

Autosomal dominant hypertension and brachydactyly in a Turkish kindred resembles essential hypertension

1. The aim of this study was to investigate, by use of spectral analysis, (1) the blood pressure (BP) variability changes in the conscious rat during blockade of nitric oxide (NO) synthesis by the L-arginine analogue NG-nitro-L-arginine methyl ester (L-NAME); (2) the involvement of the renin-angiotensin system in these modifications, by use of the angiotensin II AT1-receptor antagonist losartan. 2. Blockade of NO synthesis was achieved by infusion for 1 h of a low-dose (10 micrograms kg-1 min-1, i.v., n = 10) and high-dose (100 micrograms kg-1 min-1, i.v., n = 10) of L-NAME. The same treatment was applied in two further groups (2 x n = 10) after a bolus dose of losartan (10 mg kg-1, i.v.). 3. Thirty minutes after the start of the infusion of low-dose L-NAME, systolic BP (SBP) increased (+10 +/- 3 mmHg, P < 0.01), with the effect being more pronounced 5 min after the end of L-NAME administration (+20 +/- 4 mmHg, P < 0.001). With high-dose L-NAME, SBP increased immediately (5 min: +8 +/- 2 mmHg, P < 0.05) and reached a maximum after 40 min (+53 +/- 4 mmHg, P < 0.001); a bradycardia was observed (60 min: -44 +/- 13 beats min-1, P < 0.01). 4. Low-dose L-NAME increased the low-frequency component (LF: 0.02-0.2 Hz) of SBP variability (50 min: 6.7 +/- 1.7 mmHg2 vs 3.4 +/- 0.5 mmHg2, P < 0.05), whereas the high dose of L-NAME not only increased the LF component (40 min: 11.7 +/- 2 mmHg2 vs 2.7 +/- 0.5 mmHg2, P < 0.001) but also decreased the mind frequency (MF: 0.2-0.6 Hz) component (60 min: 1.14 +/- 0.3 mmHg2 vs 1.7 +/- 0.1 mmHg2, P < 0.05) of SBP. 5. Losartan did not modify BP levels but had a tachycardic effect (+45 beats min-1). Moreover, losartan increased MF oscillations of SBP (4.26 +/- 0.49 mmHg2 vs 2.43 +/- 0.25 mmHg2, P < 0.001), prevented the BP rise provoked by the low-dose of L-NAME and delayed the BP rise provoked by the high-dose of L-NAME. Losartan also prevented the amplification of the LF oscillations of SBP induced by L-NAME; the decrease of the MF oscillations of SBP induced by L-NAME was reinforced after losartan. 6. We conclude that the renin-angiotensin system is involved in the increase in variability of SBP in the LF range which resulted from the withdrawal of the vasodilating influence of NO. We propose that NO may counterbalance LF oscillations provoked by the activity of the renin-angiotensin system.     

A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) by either carbachol (pEC50 [negative logarithm of the EC50] = 6.19 +/- 0.01, Rmax [maximum response] = 93.2 +/- 0.4%; n = 14) or calcium ionophore A23187 (pEC50 = 6.46 +/- 0.02, Rmax = 83.6 +/- 3.6%; n = 8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC50 = 6.31 +/- 0.06, Rmax = 94.7 +/- 4.6%; n = 10; with L-NAME: pEC50 = 6.33 +/- 0.04, Rmax = 93.4 +/- 6.0%; n = 4). 2. The selective cannabinoid receptor antagonist, SR 141716A (1 microM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 microM) greatly reduced the Rmax values for EDHF-mediated relaxations to carbachol (control, 93.2 +/- 0.4%; SR 141716A, 10.7 +/- 2.5%; n = 5; P < 0.001) and A23187 (control, 84.8 +/- 2.1%; SR 141716A, 3.5 +/- 2.3%; n = 6; P < 0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting Rmax. 3. Precontraction with 60 mM KCl significantly reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 68.8 +/- 5.6% versus 17.8 +/- 7.1%), A23187 (control 71.4 +/- 6.1% versus 3.9 +/- 0.45%) and anandamide (control 71.1 +/- 7.0% versus 5.2 +/- 3.6%). Similar effects were seen in the presence of 25 mM K+. Incubation of vessels with pertussis toxin (PTX; 400 ng ml-1, 2 h) also reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 63.5 +/- 7.5% versus 9.0 +/- 3.2%), A23187 (control 77.0 +/- 5.8% versus 16.2 +/- 7.1%) and anandamide (control 89.8 +/- 2.2% versus 17.6 +/- 8.7%). 4. Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 microM) significantly potentiated (P < 0.01), to a similar extent (approximately 2 fold), relaxation to A23187 (pEC50: control, 6.45 +/- 0.04; PMSF, 6.74 +/- 0.10; n = 4) and anandamide (pEC50: control, 6.31 +/- 0.02; PMSF, 6.61 +/- 0.08; n = 8). PMSF also potentiated carbachol responses both in the presence (pEC50: control, 6.25 +/- 0.01; PMSF, 7.00 +/- 0.01; n = 4; P < 0.01) and absence (pEC50: control, 6.41 +/- 0.04; PMSF, 6.88 +/- 0.04; n = 4; P < 0.001) of L-NAME. Responses to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC50: control, 7.51 +/- 0.06; PMSF, 8.00 +/- 0.05, n = 4, P < 0.001). 5. EDHF-mediated relaxation to carbachol was significantly attenuated by the K+ channel blocker tetraethylammonium (TEA; 1 mM) (pEC50: control, 6.19 +/- 0.01; TEA, 5.61 +/- 0.01; n = 6; P < 0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to A23187 (pEC50: control, 6.47 +/- 0.04; TEA, 6.41 +/- 0.02, n = 4) or on anandamide (pEC50: control, 6.28 +/- 0.06; TEA, 6.09 +/- 0.02; n = 5). TEA (10 mM) significantly (P < 0.01) reduced the Rmax for anandamide (control, 94.3 +/- 4.0%; 10 mM TEA, 60.7 +/- 4.4%; n = 5) but had no effect on the Rmax to carbachol or A23187. 6. BaCl2 (100 microM), considered to be selective for blockade of inward rectifier K+ channels, had no significant effect on relaxations to carbachol or A23187, but caused a small shift in the anandamide concentration-response curve (pEC50: control, 6.39 +/- 0.01; Ba2+, 6.20 +/- 0.01; n = 4; P < 0.01). BaCl2 (1 mM; which causes non-selective block of K+ channels) significantly (P < 0.01) attenuated relaxations to all three agents (pEC50 values: carbachol, 5.65 +/- 0.02; A23187, 5.84 +/- 0.04; anandamide, 5.95 +/- 0.02; n = 4 for each). 7. Apamin (1mu M), a selective blocker of small conductance, Ca2+-activated, K+ channels (SKCa), 4-aminopyridine (1mM), a blocker of delayed rectifier, voltage-dependent, K+ channels (Kv), and ciclazindol (10mu M), an inhibitor of Kv and adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on A23187 or anandamide responses. 8. Glibenclamide (10mu M), a KATP inhibitor and charybdotoxin (100 or 300nM), a blocker of several K+ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50nM), an inhibitor of large conductance, Ca2+-activated, K+ channels (BKCa), had no significant effect on the relaxation responses, either alone or in combination with apamin (1muM). Also, a combination of apamin (1muM) with either glibenclamide (10muM) or 4-aminopyridine (1mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to A23187 or anandamide. 9. A combination of apamin (1muM) with charybdotoxin (100nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to A23187. Apamin (1muM) and charybdotoxin (300nM) together consistently inhibited the response to A23187, while apamin (1muM) and ciclazindol (10muM) together inhibited relaxations to both carbachol and A23187. None of these toxin combinations had any significant effect on relaxation to anandamide. 10. It was concluded that the differential sensitivity to K+ channel blockers of EDHF-mediated responses to carbachol and A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX-sensitive G-proteins to activation of K+ conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K+ channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.     

Age-related differences and roles of endothelial nitric oxide and prostanoids in angiotensin II responses of isolated, perfused mesenteric arteries and veins of rats

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.     

8%-9% and 12%-13% hypoxic gas induced free radicals generation in rat's left and right myocardium

Alpha-phenyl-tert-butylnitrone (PBN) was administered intravenously to capture free radicals of rat's myocardium. Rats were exposed to hypoxic gas (8%-9% O2 in N2) for 15 min. The ESR (electron spin resonance) signal intensity of PBN-spin adduct in the left myocardium increased significantly as compared with the normoxia group (n = 5, P < 0.05), but in the right myocardium there was no significant changes between hypoxia and normoxia. Rats exposed to hypoxic gas (12%-13% O2 in N2) were divided into four groups: I (hypoxia for 15 min), II (hypoxia for 60 min), III (hypoxia for 30 min/normoxia for 15 min/hypoxia for 30 min) and IV (injected MPEG- SOD intravenously before hypoxia for 60 min). The ESR signal intensity of PBN-spin adduct of left and right myocardium in group II increased significantly as compared with normoxia group (n = 5, P < 0.01), but the ESR signal intensity of group I didn't show obvious change as compared with normoxia group (n = 5, P > 0.05). In the right myocardium of group III the ESR signal intensity of PBN-spin adduct decreased significantly as compared with group II (n = 5, P < 0.05) and in the left myocardium did not decrease evidently. In the left myocardium of group IV the ESR signal intensity of PBN-spin adduct decreased evidently as compared with group II (n = 5, P < 0.05) and that in the right myocardium did not decrease evidently. When the rats were exposed to 8%-9% hypoxic gas for 15 min and 12%-13% hypoxic gas for 60 min, the SOD (superoxide dismutase, EC 1.11.1.9) activity of myocardium decreased and the content of MDA (malondialdehyde) increased significantly (n = 8, P < 0.05 or P < 0.01). The above results suggested that one way of myocardium free radical gereration may be relevant to decrease of SOD activity. The generation of free radicals pertained chiefly to superoxide free radical in the left myocardium and the membrane structure of myocardium cells might have been damaged largely during hypoxia.     

Oxygen modulation of guanylate cyclase-mediated retinal pericyte relaxations with 3-morpholino-sydnonimine and atrial natriuretic peptide

PURPOSE: This study explores at which level of the guanylate cyclase pathway oxygen modulates retinal pericyte relaxation induced by nitric oxide (NO). METHODS: Bovine retinal microvascular pericytes were grown on silicone. On silicone, pericyte contractile tone induces wrinkles. Drug-induced relaxation was quantified as a reduced number of wrinkles after exposure to 3-morpholino-sydnonimine (SIN-1) or atrial natriuretic peptide (ANP) in the absence or in the presence of either 0.3 microM methylene blue (MB), a guanylate cyclase inhibitor, or 10 microM hemoglobin, a NO scavenger; and under 100% oxygen (hyperoxia), ambient air (normoxia), or 100% nitrogen (hypoxia). RESULTS: Pericytes were relaxed with SIN-1 and ANP in a concentration-dependent manner (EC50: 0.1 microM and 0.01 microM, respectively). Relaxations induced by SIN-1 or ANP were inhibited (P < 0.001) by MB, whereas hemoglobin inhibited only SIN-1 relaxations (P < 0.001). Relaxations induced by SIN-1, but not by ANP were increased (P < 0.001) under hypoxia and decreased (P = 0.002) under hyperoxia. CONCLUSIONS: SIN-1 and ANP relax pericytes through the activation of guanylate cyclase (inhibited by MB), but only SIN-1 through an extracellular release of NO (inhibited by hemoglobin). That oxygen only modulates pericyte relaxations induced by SIN-1 (NO-mediated) but not those induced by ANP suggests that an interaction between oxygen and NO might participate in the capillary network's blood-flow modulation according to local tissue oxygen tension.     

Single-stage repair of aortic arch obstruction and associated intracardiac defects with pulmonary homograft patch aortoplasty

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.     

Preservation of hypoxic pulmonary vasoconstriction during sevoflurane and desflurane anesthesia compared to the conscious state in chronically instrumented dogs

BACKGROUND: The authors' objective was to assess the extent to which sevoflurane and desflurane anesthesia alter the magnitude of hypoxic pulmonary vasoconstriction compared with the response measured in the same animal in the conscious state. METHODS: Left pulmonary vascular pressure-flow plots were generated in seven chronically instrumented dogs by continuously measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure-left atrial pressure) and left pulmonary blood flow during gradual (approximately 1 min) inflation of a hydraulic occluder implanted around the right main pulmonary artery. Pressure-flow plots were generated during normoxia and hypoxia on separate days in the conscious state, during sevoflurane (approximately 3.5% end-tidal), and during desflurane (approximately 10.5% end-tidal) anesthesia. Values are mean+/-SEM. RESULTS: In the conscious state, administration of the hypoxic gas mixture by conical face mask decreased (P < 0.01) systemic arterial PO2 from 94+/-2 mmHg to 50+/-1 mmHg and caused a leftward shift (P < 0.01) in the pressure-flow relationship, indicating pulmonary vasoconstriction. The magnitude of hypoxic pulmonary vasoconstriction in the conscious state was flow-dependent (P < 0.01). Neither anesthetic had an effect on the baseline pressure-flow relationship during normoxia. The magnitude of hypoxic pulmonary vasoconstriction during sevoflurane and desflurane was also flow-dependent (P < 0.01). Moreover, at any given value of flow the magnitude of hypoxic pulmonary vasoconstriction was similar during sevoflurane and desflurane compared with the conscious state. CONCLUSION: These results indicate that hypoxic pulmonary vasoconstriction is preserved during sevoflurane and desflurane anesthesia compared with the conscious state. Thus, inhibition of hypoxic pulmonary vasoconstriction is not a general characteristic of inhalational anesthetics. The flow-dependent nature of the response should be considered when assessing the effects of physiologic or pharmacologic interventions on the magnitude of hypoxic pulmonary vasoconstriction.     

Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation of rat isolated mesenteric artery

1. The NO donor 3-morpholino-sydnonimine (SIN-1; 0.01-10 microM) evoked concentration-dependent relaxation of rat isolated mesenteric arteries pre-constricted with phenylephrine (1-3 microM). The relaxation to SIN-1 was not significantly different between endothelium-intact or denuded arterial segments or segments in which basal nitric oxide (NO) synthesis was inhibited (n = 8; P > 0.05). In contrast, the membrane permeable analogue of guanosine 3':5'-cyclic monophosphate (cyclic GMP), 8-Br-cyclic GMP (0.01-1 mM), was much less effective in relaxing intact than denuded arterial segments or intact arterial segments pre-incubated with NO synthase blockers (n = 4; P < 0.01). 2. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min) alone, did not alter SIN-1-evoked relaxation in any tissues (n = 5; P > 0.05). However, in parallel experiments, ODQ almost completely inhibited both basal and SIN-1-stimulated production of cyclic GMP in both the presence and absence of NO synthase blockers (n = 6; P < 0.01) indicating that full relaxation to SIN-1 can be achieved in the absence of an increase in cyclic GMP. 3. Exposure of endothelium-intact arterial segments to the potassium channel blocker charybdotoxin (50 nM; 10 min), significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 90% (n = 5; P < 0.01). In contrast, in arterial segments in which either the endothelial cell layer had been removed or basal NO synthesis inhibited, relaxation to SIN-1 was not reduced in the presence of charybdotoxin (n = 6; P > 0.05). However, in the presence of NO synthase blockers and L-arginine (300 microM) together, charybdotoxin did significantly inhibit SIN-1-evoked relaxation to a similar extent as intact tissues (maximum response induced by around 80%; n = 4; P < 0.01). 4. Pre-incubation with apamin (30 nM; 10 min) or glibenclamide (10 microM; 10 min) did not alter SIN-1-evoked relaxation of phenylephrine-induced tone in any tissues (n = 4 and n = 6, respectively; P > 0.05). However, in the presence of either ODQ and apamin, or ODQ and glibenclamide, SIN-1-evoked relaxation was significantly attenuated in intact arterial segments and segments in which NO synthesis was blocked. 5. Exposure of intact arterial segments to charybdotoxin and apamin, in the presence of NO synthase blockers, also significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 80% (n = 4; P < 0.01). 6. Addition of superoxide dismutase (SOD; 30 u ml-1), potentiated relaxations to SIN-1 in all tissues, but did not alter the effects of charybdotoxin and ODQ and SIN-1-evoked relaxation. 7. These data show that although relaxation to the NO-donor SIN-1 is not significantly different between endothelium-intact and denuded arterial segments, the mechanisms which mediate SIN-1-evoked relaxation in the rat isolated mesenteric artery appear to be modulated by the basal release of endothelium-derived NO. In the presence of an intact endothelial cell layer, the major mechanism for SIN-1-evoked relaxation appears to be the activation of charybdotoxin-sensitive potassium channels. In contrast, when basal NO synthesis is inhibited, SIN-1 appears to cause full relaxation by both the activation of a charybdotoxin-sensitive pathway and the stimulation of soluble guanylyl cyclase.     

Endothelium-dependent relaxation in response to ethanol in the porcine isolated pulmonary artery

Many drugs cannot be dissolved in distilled water and so other solvents such as ethanol, dimethylsulphoxide and methanol are used. Because very little is known about the direct effects of these three solvents on the cardiovascular system, we have examined their effects on isolated pulmonary and coronary arteries from the pig. Increasing concentrations of ethanol, dimethylsulphoxide and methanol induced relaxation in porcine pulmonary (at 1.2% v/v, 59.9+/-9.0% (n =9), 55.9+/-9.0% (n =6) and 12.3+/-6.4% (n = 8), respectively, of U46619-induced tone) and coronary arteries (at 1.2% v/v, 69.9+/-7.1% (n = 10), 78.9+/-6.1% (n = 7) and 12.9+/-8.2% (n = 6) respectively, of U46619-induced tone). In the pulmonary arteries the relaxation in response to ethanol was found to be endothelium-dependent whereas the responses to dimethylsulphoxide and methanol were unaffected by removal of the endothelium. In the coronary arteries the relaxation to all three solvents was independent of the presence of the endothelium. Comparison of the sensitivity of the tissues to the solvents showed that ethanol and dimethylsulphoxide produced comparative responses in both the pulmonary and coronary arteries, whereas methanol was much less potent. The endothelium-dependent response to ethanol in the porcine pulmonary artery (maximum response, Emax, 67.1+/-9.3% of U46619-induced tone, n = 7) was attenuated by the cyclooxygenase inhibitor, flurbiprofen (Emax 31.9 +/- 12.0%, n=7), the nitric oxide synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester; Emax 23.5+/-10.2%, n = 7)) and the combination of both inhibitors (Emax 18.3+/-7.8%, n = 7). The residual relaxatory response to ethanol was abolished, and converted into a contractile response, both by removal of the endothelium (at 1.7% v/v ethanol 27.3+/-11.5% of U46619-induced tone, n=7) and by the addition of a low concentration of KC1 (49.9-/+10.3%, n=6), suggesting the release of a non-prostanoid, non-nitric oxide factor from the endothelium. This response, however, was not attenuated by the cannabinoid receptor-antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide HCL; 52.5-/+4.3% relaxation, n =8), suggesting that the factor released in this preparation by ethanol is not a cannabinoid. The results of this study indicate that many solvents commonly used in pharmacological experiments have pronounced vasoactive properties. Methanol might be the vehicle of choice, because it was the least active solvent, whereas high concentrations of ethanol might influence vascular function at both the level of the smooth muscle and the endothelium, with the action on the endothelium involving the release of endothelium-derived relaxing factors.     

Role of nitric oxide in vascular hyper-responsiveness to norepinephrine in hypertensive Dahl rats

OBJECTIVE: To determine whether the abnormal vascular responses observed in salt-sensitive hypertension are caused by an impairment in vascular nitric oxide function. DESIGN: Isometric tension was measured in aortic rings isolated from Dahl salt-sensitive and salt-resistant rats fed a regular-salt (0.4% NaCl) or a high-salt (8% NaCl) diet, with and without inhibition of endogenous nitric oxide synthesis. METHODS AND RESULTS: Systolic arterial pressure, measured weekly by the tail-cuff method, increased markedly in DS rats with a high-salt diet but did not increase in the other groups. In aortic rings, norepinephrine evoked dose-dependent contractions which were significantly increased in rings from DS rats with a high-salt diet Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, increased the norepinephrine-induced contraction in all groups and abolished differences in contractile responses between high-salt DS rats and the other groups. Acetylcholine induced endothelium-dependent relaxation, which was significantly depressed in high-salt DS rats. L-NAME attenuated the acetylcholine-induced relaxation in all groups and abolished the difference in relaxation response between high-salt DS rats and the other groups. Sodium nitroprusside-induced relaxation was significantly depressed in high-salt DS rats. CONCLUSIONS: Vascular hypercontractile responses to norepinephrine in DS hypertensive rats can, in part, be explained by an impairment in endothelial nitric oxide production.     

Comparison of the effect of etomidate and desflurane on brain tissue gases and pH during prolonged middle cerebral artery occlusion

BACKGROUND: The authors compared the effects of etomidate and desflurane on brain tissue oxygen pressure (PO2), carbon dioxide pressure (PCO2), and pH in patients who had middle cerebral artery occlusion for > 15 min. METHODS: After a craniotomy, a probe that measures PO2, PCO2, and pH was inserted into cortical tissue at risk for ischemia during middle cerebral artery occlusion. A burst suppression pattern of the electroencephalogram was induced with etomidate (n = 6) or 9% end-tidal desflurane (n = 6) started before middle cerebral artery occlusion. Mean blood pressure was supported with phenylephrine to 90-95 mmHg. RESULTS: During baseline conditions, tissue PO2, PCO2, and pH were similar between the two groups (PO2 = 15 mmHg, PCO2 = 60 mmHg, pH = 7.1). During administration of etomidate before middle cerebral artery occlusion, tissue PO2 decreased in five of six patients without a change in PCO2 or pH. During administration of 9% desflurane, tissue PO2 and pH increased before middle cerebral artery clipping. Middle cerebral artery occlusion for an average of 33 min with etomidate and 37 min with desflurane produced a decrease in pH with etomidate (7.09 to 6.63, P < 0.05) but not with desflurane (7.12 to 7.15). CONCLUSION: These results suggest that tissue hypoxia and acidosis are often observed during etomidate treatment and middle cerebral artery occlusion. Treatment with desflurane significantly increases tissue PO2 alone and attenuates acidotic changes to prolonged middle cerebral artery occlusion.     

Influences of morphine on the ventilatory response to isocapnic hypoxia

BACKGROUND: The ventilatory response to hypoxia is composed of the stimulatory activity from peripheral chemoreceptors and a depressant effect from within the central nervous system. Morphine induces respiratory depression by affecting the peripheral and central carbon dioxide chemoreflex loops. There are only few reports on its effect on the hypoxic response. Thus the authors assessed the effect of morphine on the isocapnic ventilatory response to hypoxia in eight cats anesthetized with alpha-chloralose-urethan and on the ventilatory carbon dioxide sensitivities of the central and peripheral chemoreflex loops. METHODS: The steady-state ventilatory responses to six levels of end-tidal oxygen tension (PO2) ranging from 375 to 45 mmHg were measured at constant end-tidal carbon dioxide tension (P[ET]CO2, 41 mmHg) before and after intravenous administration of morphine hydrochloride (0.15 mg/kg). Each oxygen response was fitted to an exponential function characterized by the hypoxic sensitivity and a shape parameter. The hypercapnic ventilatory responses, determined before and after administration of morphine hydrochloride, were separated into a slow central and a fast peripheral component characterized by a carbon dioxide sensitivity and a single offset B (apneic threshold). RESULTS: At constant P(ET)CO2, morphine decreased ventilation during hyperoxia from 1,260 +/- 140 ml/min to 530 +/- 110 ml/ min (P < 0.01). The hypoxic sensitivity and shape parameter did not differ from control. The ventilatory response to carbon dioxide was displaced to higher P(ET)CO2 levels, and the apneic threshold increased by 6 mmHg (P < 0.01). The central and peripheral carbon dioxide sensitivities decreased by about 30% (P < 0.01). Their ratio (peripheral carbon dioxide sensitivity:central carbon dioxide sensitivity) did not differ for the treatments (control = 0.165 +/- 0.105; morphine = 0.161 +/- 0.084). CONCLUSIONS: Morphine depresses ventilation at hyperoxia but does not depress the steady-state increase in ventilation due to hypoxia. The authors speculate that morphine reduces the central depressant effect of hypoxia and the peripheral carbon dioxide sensitivity at hyperoxia.     

The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres

1. The actions of nitric oxide (NO) have been investigated in an endotoxin-evoked ocular inflammatory model in the rabbit, with particular emphasis on the relationship between NO, sensory nerves (C-fibres) and the C-fibre neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP). 2. Endotoxin, injected intravitreally, evoked inflammatory responses, i.e. conjunctival hyperaemia, miosis and protein extravasation, reflected by the aqueous flare response (AFR). In control rabbits, the maximum AFR was 66.5 +/- 9.5 (arbitrary units). Pretreatment with the NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NAME, 200 mg kg-1) given by intravenous injection, inhibited the endotoxin-evoked responses; the AFR was 16.5 +/- 1.9 (n = 8, P < 0.001) and the conjunctival hyperaemia was abolished. 3. Endotoxin-evoked ocular inflammation is associated with the release of CGRP and PACAP from C-fibres. In the eyes challenged with endotoxin, the concentrations of PACAP-27, -38 and CGRP in the aqueous humour were 58.2 +/- 10.9, 54.4 +/- 12.4 and 5526 +/- 519 (pmoll'), respectively. L-NAME inhibited the release of PACAP-27, -38 and CGRP; the concentrations were 14.3 +/- 2.5, 13.5 +/- 2.5 and 510 +/- 67 (pmoll-1), respectively (n = 8, P < 0.01 or 0.001). 4. Intravitreal injection of 0.3 nmol CGRP induced conjunctival hyperaemia and AFR; the maximum AFR was 140.2 +/- 11.4. L-NAME suppressed the response induced by CGRP; the AFR was 23.4 +/- 5.5 (n = 8, P < 0.001). L-NAME abolished the conjunctival hyperaemia induced by PACAP-27 and -38 (0.3 nmol) and reduced the AFR. 5. The inflammatory cells that infiltrated the uvea, cornea and aqueous humour in large numbers in response to intravitreal injection of endotoxin were found to express inducible NOS. L-NAME prevented the appearance of such cells. 6. Our findings suggest that NO plays an important role in the endotoxin-evoked ocular inflammation in the rabbit: NO activates C-fibres causing release of C-fibre neuropeptides into the aqueous humour. In addition, NO mediates scme of the ocular effects of CGRP and PACAP, since L-NAME suppressed the AFR induced by these peptides.     

The excitatory response of the adrenal sympathetic nerve to severe hypoxia decreases in aged rats

The internal mammary artery (IMA) has become the conduct of choice for coronary artery bypass grafting. However, the IMA graft can exhibit vasoconstriction during the perioperative period. Experiments were designed to determine the role of cyclooxygenase products in human IMA during hypoxia. Rings of IMA, with and without endothelium, were suspended in organ baths containing physiologic salt solution. Rings were contracted with norepinephrine and then exposed to hypoxia for 15 minutes. In segments with endothelium, hypoxia induced a transient relaxation followed by contraction. This contraction was associated with a significantly increased production of thromboxane B2, the stable metabolite of thromboxane A2 (n = 10; from 120.7 +/- 3.5 pg/mg wet tissue before hypoxia to 175.8 +/- 5.2 pg/mg during hypoxia; p < 0.05). This hypoxic contraction could be attenuated by indomethacin. However, thromboxane B2 could not be detected in samples from organ baths containing IMA segments without endothelium before or during hypoxia. This study demonstrated that endothelium of human IMA grafts releases thromboxane A2 basally and that production is augmented by hypoxia, which acts to constrict the underlying vascular smooth muscle, increase vascular tone, and promote ischemic events such as vasospasm and thrombosis, particularly in hypoxemic patients.     

Hyporeactivity of rat diaphragmatic arterioles after exposure to hypoxia in vivo. Role of the endothelium

The effect of prior in vivo hypoxia on the in vitro responses to changes in transmural pressure, alpha-adrenoceptor activation, and depolarization with KCl were evaluated in first-order diaphragmatic arterioles. Rats (n = 14 per group) were exposed to normoxia (controls) or to hypoxia (inspired O2 concentration = 10%) for 12 or 48 h. The arteriolar pressure-diameter relationships were recorded over a pressure range from 10 to 200 mm Hg. In separate groups of arterioles (n = 12 per group), the diaphragmatic arteriolar responses to phenylephrine (10(-8) to 10(-5 M) or KCl (10 to 100 mM) were determined after exposure to either room air or hypoxia for 48 h. In half of the arterioles studied, the endothelium was removed. After 12 h of hypoxia, the pressure-diameter relationship was normal in endothelialized arterioles but was shifted upward in de-endothelialized vessels (p < 0.05). After 48 h of hypoxia, the constrictor response to increasing transmural pressure was severely suppressed in all arterioles. The intraluminal diameters during activation with phenylephrine and KCl were larger in arterioles from rats exposed to hypoxia (103 +/- 8 and 81 +/- 7 microns, respectively) than in control arterioles (41 +/- 5 and 54 +/- 6 microns, respectively; p < 0.05 for differences). During maximum phenylephrine- and KCl-induced constriction in de-endothelialized arterioles, diameters averaged 125 +/- 8 and 105 +/- 8 microns, respectively, for arterioles from hypoxic rats and 32 +/- 6 and 40 +/- 5 microns, respectively, for arterioles from control vessels. Exposure to hypoxia results in impairment of diaphragmatic arteriolar smooth muscle reactivity and reversal of the normal inhibitory influence of the endothelium on diaphragmatic arteriolar tone.