The effect of lead selection on traditional and heart rate-adjusted ST segment analysis in the detection of coronary artery disease during exercise testing

1. The present studies were designed to measure the affinity of UP 269-6, a newly developed angiotensin AT1 receptor antagonist, for vascular AT1 receptors from normotensive and hypertensive rats and to investigate in vitro, its effects on angiotensin II (AII)-induced hyperplasia and hypertrophy of vascular smooth muscle cells (VSMC). In addition the in vivo effects of UP 269-6 on neointimal proliferation in a carotid artery balloon injury in normotensive rats were also investigated. 2. UP 269-6 selectively inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors present on VSMC derived from normotensive Wistar rat and from SHR (Ki = 16.6 +/- 3.6 nM and 7.5 +/- 2.0 nM, respectively). In comparison, losartan and its metabolite, EXP 3174, inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors derived from both cell models with Ki values slightly lower (losartan) and higher (EXP 3174), respectively, than that of UP 269-6. 3. AII (1 microM) induced a weak and variable hyperplastic response (4 to 32% increase in cell number) in Wistar rat VSMC after 96 h. 4. AII (1 microM) induced a time-dependent increase in cell number in VSMC from SHR. UP 269-6 inhibited concentration-dependently this effect with an IC50 value of 159 +/- 58 nM. Losartan was clearly less potent and EXP 3174 showed nearly the same inhibitory potency, compared to UP 269-6. UP 269-6 (1 microM) inhibited nearly completely the action of AII. 5. AII (500 nM) caused maximal stimulation of protein synthesis in Wistar rat VSMC (117 +/- 36%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 28 +/- 6 nM, 3504 +/- 892 nM and 21 +/- 3 nM, respectively. 6. AII (50 nM) induced maximal stimulation of protein synthesis in SHR VSMC (237 +/- 67%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 16 +/- 3 nM, 282 +/- 122 nM and 3.3 +/- 1.0 nM, respectively. 7. UP 269-6 (75 mg kg-1 day-1) administered orally in the diet for 20 days induced a 38% reduction in neointimal area and a 36% reduction in neointima/media ratio associated with the intimal thickening induced by carotid artery balloon injury. 8. In conclusion, UP 269-6 was shown to be a potent antiproliferative agent both in vitro on AII-induced hyperplasia and hypertrophy of VSMC derived from normotensive and hypertensive rats, and in vivo upon intimal thickening induced by carotid artery balloon injury in the rat.     

Drug-induced vasodilation in an in vitro and in vivo study: the effects of nicardipine, papaverine, and lidocaine on the rabbit carotid artery

Extreme arterial vasoconstriction (vasospasm) is a common problem encountered in microvascular surgery. An ideal pharmacologic tool able to counteract ischemia during microsurgery should be easy to apply and exert its action both locally and distally in the microcirculation of the flap. We have compared in vitro and in vivo vascular properties of nicardipine, papaverine, and lidocaine in the rabbit carotid artery. In vitro, rings from the rabbit carotid artery (n = 7) were bathed in Krebs-Ringers solution and stretched progressively to an optimal tension of 3.7 to 4.2 g. The specimens were contracted with norepinephrine (1 microM), and a cumulative dose response curve was established. In vivo, microvascular anastomoses were performed bilaterally in the rabbit carotid artery in 35 animals using 9-0 nylon suture and standard microsurgical techniques. During and after the anastomoses, nicardipine (0.1, 0.01 mg topical, or 0.1 mg/hour IV), papaverine (30 mg/cc topical), and lidocaine (2% with and without epinephrine) were applied (blinded) at the anastomotic site in five rabbits each. Heparinized sodium chloride was used as topical irrigation for control and to clean the anastomosis. Blood flow changes were monitored continuously with the transonic Doppler for 30 minutes after the procedure. The systemic blood pressure was also monitored in a group of pilot experiments. A documented decrease in blood flow was noted in all animals after the microvascular anastomosis. Nicardipine and papaverine evoked a concentration-dependent relaxation to precontracted rings to norepinephrine. Nicardipine was greater than papaverine in inducing relaxation. Lidocaine demonstrated a biphasic response with low concentrations potentiating contraction. Systemic nicardipine and papaverine significantly increased the blood flow in the rabbit carotid artery. Topical application of nicardipine and lidocaine did not significantly alter the blood flow; however, the application of nicardipine demonstrates a trend toward increased flow. Lidocaine with epinephrine significantly decreased the blood flow. No drug was found to alter the blood pressure of the animals. Our results demonstrate that nicardipine and papaverine seem to be pharmacologic tools able to increase the blood flow in anastomotic arteries. In contrast, the use of 2% lidocaine as a spasmolytic agent should be re-evaluated, since this substance may act as a partial agonist.     

The ETA antagonist CI-1020 inhibits hypoxic pulmonary vasoconstriction in small isolated rat pulmonary arteries

Hypoxic pulmonary vasoconstriction (HPV) of rat pulmonary arteries in vitro occurs in four phases. Initial vasodilation (phase 1), is followed by transient contraction (phase 2), further vasodilation (phase 3) and finally a second sustained contraction (phase 4). We have investigated the role of ET-1 in HPV using the ETA receptor antagonist CI-1020. Small rat pulmonary arteries (SPA, n=32, diameter=454+/-22 microM) were mounted in a wire myograph. Two contractions to 80 microM KCl ensured response reproducibility and relaxation to 10 microM acetyl choline following constriction with 100 microM prostaglandin F2alpha (PGF2alpha) to indicate endothelial integrity. A control hypoxic response was produced following priming with 5 microM PGF2alpha. Vessels (n=8) were then exposed to either vehicle or CI-1020 (1, 10 or 100 microM) for 30 min in the dark before re-exposure to PGF2alpha and hypoxia. Responses were standardized as a percentage of contraction to 80 mM KCl. Vehicle caused an increase in phase 2 of HPV of +2.51+/-4.20% (expressed as difference between pre- and post-drug values). CI-1020 (1, 10 and 100 microM) caused a significant reduction in phase 2 of HPV of -9. 76+/-1.40%, -9.23+/-2.30% and -7.96+/-1.70%, respectively (P<0.05). These results suggest that phase 2 of HPV in rat SPA is attributed, in part, to the action of ET-1 at the ETA receptor.     

Effects of the novel antimigraine agent, frovatriptan, on coronary and cardiac function in dogs

The effects of frovatriptan (VML 251/SB-209509) on coronary artery function were investigated in isolated coronary arteries from beagle dogs. Low concentrations of frovatriptan produced contraction with -logEC50 7.55 +/- 0.08 (n = 11). The maximal observed contraction attained was 56 +/- 7% of the control 5-hydroxytryptamine (5-HT; 10 microM) response. At high concentrations of frovatriptan (>6 microM), reversal of sumatriptan (10 microM)-induced contractions was noted. In arteries precontracted with the thromboxane mimetic, U46619, frovatriptan produced a bell-shaped concentration-response relation with a maximal response at 600 nM. Concentrations of frovatriptan >2 microM produced marked reversal of tone, with full relaxation of precontracted tissues at 200 microM. In anesthetized, open-chest mongrel dogs, intravenous (n = 5) or intracoronary (n = 5) artery administration of frovatriptan (0.0001-1 mg/kg) had no consistent effect on left ventricular end-diastolic pressure, left end-systolic pressure, cardiac contractility, aortic blood flow, systemic peripheral resistance, coronary blood flow, coronary vascular resistance, mean arterial blood pressure, or heart rate when compared with vehicle (n = 3). Intravenous sumatriptan produced minor effects on blood pressure and heart rate. Intracoronary artery administration of sumatriptan (0.0003 mg/kg) produced an increase in systemic peripheral resistance to 120.5 +/- 8.2% compared with vehicle (97.8 +/- 5.4%; p < 0.05). This dose of sumatriptan also produced a significant increase in coronary blood flow and decrease in coronary vascular resistance. Intravenous administration of sumatriptan produced a dose-related reduction in left ventricular diastolic pressure with a reduction to 58.3 +/- 8.3% and 41.7 +/- 25% of control values observed at 0.3 and 1 mg/kg, respectively; however, administration of sumatriptan by an intracoronary route had no effect. In a model of myocardial infarction, comparable doses of sumatriptan (1.0 mg/kg) or frovatriptan (0.1 mg/kg), in terms of their effect on carotid vascular resistance, had no significant effect on infarct size. Frovatriptan had no effect on coronary blood flow after reperfusion; however, sumatriptan produced a significant reduction in coronary blood flow for < or =3 h. These studies show that frovatriptan has the capability of relaxing coronary arteries in vitro, has no overall effect on cardiac function at rest with no effect on coronary hemodynamics after myocardial infarction, and has a profile superior to that of sumatriptan.     

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.     

Lovastatin maintains nitric oxide--but not EDHF-mediated endothelium-dependent relaxation in the hypercholesterolemic rabbit carotid artery

The endothelium contributes to the regulation of vascular tone by producing nitric oxide (NO) and the endothelium-derived hyperpolarising factor (EDHF). In hypercholesterolemia, endothelium-dependent relaxation is impaired but can be restored by treatment with lovastatin (LOVAS). We investigated the effects of LOVAS on NO and EDHF-mediated relaxation. Rabbits were fed 1% cholesterol diet for 4 weeks and 0.5%) cholesterol for the following 12 weeks (CHOL-group). The LOVAS group additionally received 10 mg of lovastatin over the last 12-week period. Experiments were performed in carotid artery rings. Relaxant responses to acetylcholine (ACh) were recorded in the presence of indomethacin. Nitro-L-arginine (NOARG, 100 microM) and potassium chloride (KCl, 35 mM) were used to differentiate between NO- and EDHF-mediated relaxations. Cholesterol impaired ACh-induced relaxations and this effect was prevented by LOVAS (control 100+/-1%, CHOL 81+/-6%, LOVAS 98+/-1%). In the presence of NOARG, relaxations to ACh were not different between the LOVAS and CHOL groups (control 78+/-4%, CHOL 64+/-6%, LOVAS 64+/-5%). When KCl was used, ACh-induced relaxations were similar in the LOVAS and control group (control 75+/-5%, CHOL 49+/-6%, LOVAS 76+/-2%). In arteries treated with NOARG and KCl together, no relaxations were observed. Relaxations of arteries from the control group were not affected by 18 h preincubation with lovastatin (10 microM). Lovastatin selectively maintains nitric oxide-mediated endothelium-dependent relaxation in hypercholesterolemic rabbit carotid arteries.     

Perindopril treatment prevents the loss of endothelial nitric oxide function and development of neo-intima in rabbits

An atheroma-like neo-intima was produced by positioning a flexible collar around the common carotid arteries of normocholesterolaemic rabbits. Vessel segments taken from the mid-region of the collared and control region of the same artery were studied 7 days after surgery. Placebo rabbits were provided ab libitum with regular tap water, and treated animals were supplied with water containing perindopril (0.3 mg/kg/day) for 14 days. Perindopril treatment reduced plasma angiotensin converting enzyme (ACE) activity by 88%, but did not significantly alter arterial blood pressure or heart rate. In control rings from placebo rabbits perindoprilat in vitro (0.1-1.0 microM) reduced the sensitivity to angiotension I up to 20-fold but did not affect that of angiotensin II. In placebo rabbits, the collared arterial segments were approximately five-fold more sensitive to the vasoconstrictor action of 5-HT (P < 0.05) than the corresponding control segments. Perindopril treatment did not prevent the supersensitivity of the collared vessels to 5-HT. Development of the lesion in placebo or perindopril-treated rabbits did not alter the vascular sensitivity to either angiotensin I (10(-9)-10(-5)M) or angiotensin II (10(-10)-10(-6)M). The vasorelaxant action of sodium nitroprusside was similar in collared and control rings, whereas the maximum endothelium-dependent vasorelaxant response to acetylcholine was reduced from 68 +/- 5% in control rings, to 44 +/- 8% (mean +/- S.E.M., n = 9, P < 0.05) in collared rings of placebo-treated rabbits. In the perindopril-treated animals, this impairment of relaxation was restored in collared vessels and was no longer significantly different from the control sections. In contrast, perindoprilat in vitro (1.0 microM) did not alter the vasorelaxant response to acetylcholine in control or collared rings in a separate series of placebo rabbits. Morphologically, vessel segments taken from the centre of the collared artery of all placebo rabbits showed a thickened intima filled with cells that had the appearance of synthetic-state smooth muscle. The intimal/medial cross-sectional area ratio was reduced from 0.11 +/- 0.02 (n = 10) in placebo rabbits to 0.05 +/- 0.01 (n = 9) in perindopril-treated rabbits, whereas cross-sectional area of media of the collared vessels was similar in the two groups. Thus ACE may have important roles in the initiation and progression of atheroma-like lesions. Inhibition of ACE with perindopril reduces intimal thickening and restores the defective vasodilatation induced by the endothelial-dependent vasodilator, acetylcholine.     

Antiplatelet effect of FK633, a platelet glycoprotein IIb/IIIa antagonist, on thrombus formation and vascular patency after thrombolysis in the injured hamster carotid artery

The antithrombotic and restenosis-preventing effects of FK633, an inhibitor of platelet aggregation via binding to the glycoprotein (GP) IIb/IIIa receptor, were studied, IC50 value of FK633 against platelet aggregation ex vivo induced by 2.5 microM adenosine diphosphate (ADP) was 5.4 x 10(-7) M as determined using hamster platelet rich plasma. The inhibitory effect was also investigated in vivo on thrombus formation at the carotid arterial wall injured by a modified catheter. As a control, the left carotid artery was injured and the time required to develop a thrombotic occlusion (3.9 +/- 1.1 min, mean +/- S.E.M., n = 18) was determined. Then, the right carotid artery of the same animal was injured while a continuous intravenous (i.v.) infusion of FK633 was administered at doses of 0 (saline), 0.1, 0.3 or 1.0 mg/kg/h. The time to occlusion was dose-dependently prolonged. In a separate experiment, 10% of the total tPA dose (0.52 mg/kg) was injected into the injured artery as a bolus and the remaining was infused i.v. at a constant rate for 30 min. When FK633 (0.3 or 1.0 mg/kg/h) was infused together with tPA, late patency of the reperfused artery was much improved as compared with that of treatment with tPA alone. Bleeding time, measured at the end of the tPA infusion, was markedly prolonged when the higher dose of FK633 (1.0 mg/kg/h) was coadministered, however coadministration of the lower dose of FK633 (0.3 mg/kg/h) was almost without prolongation on the bleeding time, despite a significant effect on the vascular patency after thrombolysis. Next, neointima formation was evaluated 2 weeks after the vascular injury. When FK633 (0.3 mg/kg/h) was continuously infused i.v. by an implanted osmotic pump for 3, 7 or 14 days after the vascular injury, the neointimal area formation was significantly suppressed in the treatment groups for 7 or 14 days. These findings suggest that FK633 inhibits platelet activation in the injured artery and improves vascular patency after thrombolysis with tPA with a concomitant suppression of neointima formation.     

Effect of the antioxidant Nicanartine on the proliferative and inflammatory response after experimental balloon angioplasty

BACKGROUND: Antioxidant treatment seems to reduce the development of restenosis after percutaneous transluminal angioplasty. In this study, the effect of Nicanartine, a new antioxidant drug with both antiproliferative and lipid-lowering properties, on the proliferative and inflammatory response after balloon angioplasty was investigated in a rabbit model of restenosis. METHODS: To induce pre-interventional plaques in the common carotid artery of 48 New Zealand White rabbits, electrostimulation was carried out for 28 days. After a break of 7 days, balloon angioplasty was performed in 36 animals, of which 18 received Nicanartine at a dose of 120 mg/kg body weight; the other 18 served as a control group. The vessels were excised by day 7 and 28 after balloon angioplasty and examined for intimal plaque size, macrophage content and proliferative activity. Bromodeoxyuridine labeling was used to determine proliferating cells in the dilated segment; macrophages were detected using the RAM-11 antibody. RESULTS: In the Nicanartine-treated group, immunohistological quantification 7 days after intervention showed a statistically significant (P< 0.05) reduction of both cells undergoing DNA synthesis (1.6+/-1.4% versus 3.7+/-2.2%) and intimal macrophages (0.7+/-1.2% versus 1.3+/-0.6%). Twenty-eight days after balloon angioplasty, proliferative activity in both groups was decreased to a level comparable to the non-dilated control groups. A clear trend towards smaller plaques could be seen in the Nicanartine group (0.146+/-0.077 mm2 versus 0.255+/-0.174 mm2). Total cholesterol levels did not differ significantly between the groups. CONCLUSION: Under treatment with Nicanartine a clear reduction in the proliferative and inflammatory response after balloon angioplasty was observed. Antioxidant treatment, especially with compounds having antiproliferative and lipid-lowering properties, appears to be an effective secondary preventive strategy after interventional treatment in patients with coronary artery disease.     

The endothelium-dependent response of human hepatic artery after preservation with the UW solution

The University of Wisconsin's (UW) solution has been used commonly for current liver transplantation. However, its effect on the vascular endothelium remains unclear. Experiments were designed to study the effects. Human hepatic arteries harvested from patients with hepatocellular carcinoma undergoing liver resection were preserved in 4 degree C physiological solution (group 1, the content showed on the text) and UW solution (group 2) for 1 hr. Segments of preserved and control (group 3) hepatic arteries were suspended in organ chamber to measure the isometric force. The relaxations to acetylcholine (ACH) and adenosine diphosphate in segments of hepatic artery with endothelium were significantly greater than those segments without endothelium. The maximal relaxation to ACH in arterial segments with endothelium of group 2 was significantly different from those of group 1 and 3 (group 1 to group 3, 82 +/- 2%, 57 +/- 6%, and 83 +/- 4% of the initial tension contracted by neoepinephrine (3 X 10-7 mole/l, P < 0.05). The maximal relaxation to adenosine diphosphate was similar to the response to ACH. Perfusate hypoxia (oxygen tension 30 +/- 5 mmHG) caused endothelium-dependent contraction of the arterial segments (group 1 to group 3, 233 +/- 32%, 276 +/- 35%, and 251 +/- 40% of the initial tension, P < 0.05). Endothelium-independent relaxation and contraction of human hepatic artery to sodium nitroprusside and norepinephrine were not altered by UW solution. In summary, the impaired endothelium-dependent relaxation by UW solution and prominent endothelium-dependent contraction to hypoxia of human hepatic artery would favor vasospasm and thrombus formation after liver transplantation.     

Chloroaluminum sulfonated phthalocyanine partitioning in normal and intimal hyperplastic artery in the rat. Implications for photodynamic therapy

Photodynamic therapy, the light activation of photosensitizers into cytotoxic mediators, has been a successful treatment for experimental intimal hyperplasia (IH). To understand the basis of the photosensitizer chloroaluminum sulfonated phthalocyanine (CASPc)-mediated photoinhibition of intimal hyperplasia in the rat common carotid artery model, we studied photosensitizer partitioning in hyperplastic as compared to normal arterial tissue. Serum clearance of CASPc is exponential with, a half-life of 300 minutes. Laser-induced fluorescence and spectrofluorimetric analyses of artery tissue demonstrated an approximately 60% lower uptake and retention of CASPc by normal arterial tissue as compared to arteries with IH; the differences become more pronounced at 24 h. Fluorescent microscopy of arterial tissue demonstrated increased uptake of the CASPc by the artery with IH. However, by 24 h it is primarily the IH tissue that has retained the CASPc, with clearance of the dye from the media of normal or hyperplastic arteries. These data demonstrate that IH, like neoplastic tissue, has an increased accumulation of CASPc compared to normal artery. The preferential partitioning into hyperplastic tissue has implications for therapeutic targeting of this cellular population with photodynamic therapy.     

IL-10-mediated suppression of TNF-alpha production is independent of its ability to inhibit NF kappa B activity

It is hypothesized that carotid body chemosensory activity is coupled to neurosecretion. The purpose of this study was to examine whether there was a correspondence between carotid body tissue dopamine (DA) levels and neuronal discharge (ND) measured from the carotid sinus nerve of perfused cat carotid bodies and to characterize interaction between CO2 and O2 in these responses. ND and tissue DA were measured after changing from normoxic, normocapnic control bicarbonate buffer (PO2 >120 Torr, PCO2 25-30 Torr, pH approximately 7.4) to normoxic hypercapnia (PCO2 55-57 Torr, pH 7.1-7.2) or to hypoxic solutions (PO2 30-35 Torr) with normocapnia (PCO2 25-30 Torr, pH approximately 7.4) or hypocapnia (PCO2 10-15 Torr, pH 7.6-7.8). Similar temporal changes for ND and tissue DA were found for all of the stimuli, although there was a much different proportional relationship for normoxic hypercapnia. Both ND and DA increased above baseline values during flow interruption and normocapnic hypoxia, and both decreased below baseline values during hypoxic hypocapnia. In contrast, normoxic hypercapnia caused an initial increase in ND, from a baseline of 175 +/- 12 (SE) to a peak of 593 +/- 20 impulses/s within 4.6 +/- 0.9 s, followed by adaptation, whereas ND declined to 423 +/- 20 impulses/s after 1 min. Tissue DA initially increased from a baseline of 17.9 +/- 1.2 microM to a peak of 23.2 +/- 1.2 microM within 3.0 +/- 0.7 s, then declined to 2.6 +/- 1.0 microM. The substantial decrease in tissue DA during normoxic hypercapnia was not consistent with the parallel changes in DA with ND that were observed for hypoxic stimuli.     

Peroxynitrite: a biologically significant oxidant

A prolongation of the intracellular acidosis after myocardial ischemia can protect the myocardium against reperfusion injury. In isolated hearts, this was achieved by prolongation of the extracellular acidosis. The aim of this study was to investigate whether regional reperfusion with acidotic blood after coronary artery occlusion can reduce infarct size and improve myocardial function in vivo. Anesthetized open-chest dogs were instrumented for measurement of regional myocardial function, assessed by sonomicrometry as systolic wall thickening (sWT). Infarct size was determined by triphenyltetrazolium staining after 3 h of reperfusion. The left anterior descending coronary artery (LAD) was perfused through a bypass from the left carotid artery. The animals underwent 1 h of LAD occlusion and subsequent bypass-reperfusion with normal blood (control, n = 6) or blood equilibrated to pH = 6.8 by using 0.1 mM HCl during the first 30 min of reperfusion (HCl, n = 5). Regional collateral blood flow (RCBF) at 30-min occlusion was measured by using colored microspheres. There was no difference in recovery of sWT in the LAD-perfused area between the two groups at the end of the experiments [-2.8+/-1.2% (HCl) vs. -4.4+/-2.5% (control); mean +/- SEM; p = NS]. RCBF was comparable in both groups. Infarct size (percentage of area at risk) was reduced in the treatment group (12.8+/-2.8%) compared with the control group (26.2+/-4.8%; p < 0.05). These results indicate that reperfusion injury after coronary artery occlusion can be reduced by a prolonged local extracellular acidosis in vivo.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Locally applied antisense oligonucleotide to proliferating cell nuclear antigen inhibits intimal thickening in experimental vein grafts

This study examines the effect of antisense oligonucleotide to proliferating cell nuclear antigen (PCNA) on the formation of vein graft intimal hyperplasia in vivo, using localized administration. Twenty-four New Zealand white rabbits had a right carotid interposition bypass graft using the external jugular vein and were sacrificed on the 28th postoperative day. To determine the effect of PCNA on the development of intimal hyperplasia, 6 animals had their grafts coated with a pluronic gel containing 18 base antisense oligonucleotide to PCNA (1 mg/ml), 6 received a pluronic gel containing an 18 base nonsense oligonucleotide (1 mg/ml), and 12 animals were controls (6 with and 6 without pluronic gel). These grafts were harvested for morphology and videomorphometry. There was no change in the intimal thickness between the control and gel-treated groups. (70 +/- 4 microm versus 72 +/- 4 microm; mean +/- s.e.m.; p = ns). The presence of nonsense oligonucleotide had no further effect. Antisense PCNA produced a 26% decrease in intimal thickness to 50 +/- 4 microm in the treated vein grafts (p < 0.03) without a change in medial thickness. This study shows that a local single application of antisense oligonucleotide to PCNA will reduce the intimal hyperplasia in experimental vein grafts over 28 days.     

Influence of applied tension and nitric oxide on responses to endothelins in rat pulmonary resistance arteries: effect of chronic hypoxia

1. The effect of basal tension (transmural tensions 235 +/- 29 mg wt (low tension: equivalent to approximately 16 mmHg) and 305 +/- 34 mg wt (high tension: equivalent to 35 mmHg)) on rat pulmonary resistance artery responses to endothelin-1 (ET-1) and the selective ET(B)-receptor agonist sarafotoxin S6c (S6c) were studied. The effects of nitric oxide synthase inhibition with N(omega)-nitro-L-arginine methylester (L-NAME, 100 microM) on ET receptor-induced responses, as well as vasodilator responses to acetylcholine (ACh) and S6c, were also investigated. Changes with development of pulmonary hypertension, induced by two weeks of chronic hypoxia, were determined. 2. Control rat preparations showed greatest sensitivity for ET-1 when put under low tension (pEC50: 8.1 +/- 0.1) compared with at the higher tension (pEC50: 7.7 +/- 0.1) and there were significant increases in maximum contractile responses to S6c (approximately 80%) and noradrenaline (approximately 60%) when put under high tension. 3. In control pulmonary resistance arteries, both ET-1 and S6c produced potent vasoconstrictor responses. S6c was 12 fold more potent than ET-1 in vessels set at low tension (S6c pEC50: 9.2 +/- 0.1) and 200 fold more potent than ET-1 when the vessels were set at high tension (S6c pEC50: 9.0 +/- 0.1). Chronic hypoxia did not change the potencies of ET-1 and S6c but did significantly increase the maximum contractile response to ET-1 by 60% (at low tension) and 130% (at high tension). 4. In control rat vessels, L-NAME itself caused small increases in vascular tone (5-8 mg wt tension) in 33-56% of vessels. In the chronic hypoxic rats, in vessels set at high tension, L-NAME-induced tone was evident in 88% of vessels and had increased to 26.9 +/- 6.6 mg wt tension. Vasodilatation to sodium nitroprusside, in non-preconstricted vessels, was small in control rat vessels (2-6 mg wt tension) but increased significantly to 22.5 +/- 8.0 mg wt tension in chronic hypoxic vessels set at the higher tensions. Together, these results indicate an increase in endogenous tone in the vessels from the chronic hypoxic rats which is normally attenuated by nitric oxide production. 5. L-NAME increased the sensitivity to S6c 10 fold (low tension) and 6 fold (high tension) only in chronic hypoxic rat pulmonary resistance arteries. It had no effect on responses to ET-1 in any vessel studied. 6. Vasodilatation of pre-contracted vessels by ACh was markedly greater in the pulmonary resistance arteries from the chronic hypoxic rats (pIC50: 7.12 +/- 0.19, maximum: 72.1 +/- 0.2.0%) compared to their age-matched controls (pIC50: 5.77 +/- 0.15, maximum: 28.2 +/- 2.0%). There was also a 2.5 fold increase in maximum vasodilatation induced by ACh. 7. These results demonstrate that control rat preparations showed greatest sensitivity for ET-1 when set at the lower tension, equivalent to the pressure expected in vivo (approximately 16 mmHg). Pulmonary hypertension due to chronic hypoxia potentiated the maximum response to ET-1. Pulmonary resistance arteries from control animals exhibited little endogenous tone, but exposure to chronic hypoxia increased endogenous inherent tone which is normally attenuated by nitric oxide. Endogenous nitric oxide production may increase in pulmonary resistance arteries from chronic hypoxic rats and attenuate contractile responses to ET(B2) receptor stimulation. Relaxation to ACh was increased in pulmonary resistance arteries from chronic hypoxic rats.     

Effects of halothane and isoflurane on carbon monoxide-induced relaxations in the rat aorta

BACKGROUND: Halothane and isoflurane previously were reported to attenuate endothelium-derived relaxing factor/nitric oxide-mediated vasodilation and cyclic guanosine monophosphate (cGMP) formation in isolated rat aortic rings. Carbon monoxide has many chemical and physiologic similarities to nitric oxide. This study was designed to investigate the effects of halothane and isoflurane on carbon monoxide-induced relaxations and cGMP formation in the isolated rat aorta. METHODS: Isometric tension was recorded continuously from endothelium denuded rat aortic rings suspended in Krebs-filled organ baths. Rings precontracted with submaximal concentrations of norepinephrine were exposed to cumulative concentrations of carbon monoxide (26-176 microM). This procedure was repeated three times, with anesthetics delivered 10 min before the second procedure. Carbon monoxide responses of rings contracted with the same concentration of norepinephrine (10(-6) M and 2 x 10(-6) M) used in the anesthetic-exposed preparations also were examined. The concentrations of cGMP were determined in denuded rings using radioimmunoassay. The rings were treated with carbon monoxide (176 microM, 30 s) alone, or carbon monoxide after a 10-min incubation with halothane (0.34 mM or 0.72 mM). To determine whether the sequence of anesthetic delivery influenced results, vascular rings pretreated with halothane were compared with nonpretreated rings. RESULTS: Carbon monoxide (26-176 microM) caused a dose-dependent reduction of norepinephrine-induced tension, with a maximal relaxation of 1.51 +/- 0.07 g (85 +/- 7% of norepinephrine-induced contraction). Halothane (0.34 mM and 0.72 mM) significantly attenuated the carbon monoxide-induced relaxations, but only the highest concentration of isoflurane (0.53 mM) significantly attenuated the carbon monoxide-induced relaxations. Carbon monoxide (176 microM) significantly increased cGMP content (+88.1 +/- 7.1%) and preincubation of the aortic rings with halothane (0.34 mM and 0.72 mM) inhibited this increase (-70.7 +/- 6.8% and -108.1 +/- 10.6%, respectively). When aortic rings and carbon monoxide were added simultaneously to Krebs solution equilibrated with halothane (0.72 mM), no inhibition of cGMP formation occurred. CONCLUSION: Carbon monoxide-induced endothelium-independent relaxations of rat aortic rings were decreased by clinically relevant concentrations of halothane and isoflurane. The carbon monoxide-induced elevations of cGMP were attenuated by halothane only when the anesthetic was incubated with aortic rings before carbon monoxide treatment. The possible clinical significance of the actions of the anesthetics on this endogenous vasodilator is yet to be determined.     

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.     

GR144053, a fibrinogen receptor antagonist, enhances the suppression of neointima formation by losartan, an angiotensin II receptor antagonist, in the injured carotid artery of hamster

1. The present study investigated the inhibitory effect of losartan, a type 1 angiotensin II (AT1) antagonist, and of combined treatment with losartan and GR144053, a fibrinogen receptor (GPIIb/IIIa) antagonist, on neointima formation subsequent to vascular injury in the hamster carotid artery. Vascular injury was achieved by a roughened-tip 2F catheter and the neointimal area was measured up to 2 weeks inducing the injury. 2. Compared to non-treated hamsters (intimal area (IA/internal elastic laminal area (IELA) ratio = 60.3 +/- 5.9%, n = 12), losartan dissolved in drinking water (1, 3 and 10 mg kg-1 per day, n = 8 each) reduced neointimal area dose-dependently, a significant decrease (IA/IELA = 39.7 +/- 5.6%) being attained with the highest dose when it was administered from 1 day before injury. However, neointima formation was not prevented even with the highest dose of losartan when the administration was started after injury. 3. When the administration of GR144053 (1.0 mg kg-1 per hour) via an implanted osmotic pump was started 30 min before the injury and continued for the next 2 weeks, no suppression of neointima formation was observed, although platelet aggregation evoked ex vivo by adenosine diphosphate (ADP) at the end of treatment period was efficiently inhibited. 4. In separate experiments in which 5-bromo-2-deoxy-Uridine (BrdU) was used to test smooth muscle cell (SMC) proliferation 1 and 7 days after injury, the ratio of SMC proliferation in the injured area was only slightly decreased by losartan when its administration was started after the injury, despite the marked reduction of SMC proliferation when treatment was started before the injury. Treatment with GR144053 as indicated above also significantly decreased the SMC proliferating index 1 day after the injury. 5. To examine the potential benefit of the coadministration of the GPIIb/IIIa antagonist with the AT1 receptor antagonist, GR144053 (1.0 mg kg-1 per hour) was combined with post-injury treatment with losartan (10 mg kg-1 per day). This markedly reduced the proliferation of SMCs and significantly decreased the neointimal area (IA/IELA = 31.2 +/- 4.6%) measured 2 weeks following the catheterization. 6. According to the results of a time-dependent study in which GR144053 was given in combination with post injury treatment with losartan for 1, 3, 7 or 14 days, neointima formation could be reduced by treatment with GR144053 for just 7 days. 7. In conclusion, GR144053, a fibrinogen receptor antagonist, enhanced the inhibitory effect of losartan, an AT1 receptor antagonist, on neointima formation in the damaged carotid artery of hamsters.     

Mirror, mirror on the wall, who''s the thinnest one of all? Effects of self-awareness on consumption of full-fat, reduced-fat, and no-fat products

The aim of this study was to determine whether adenosine receptor blockade before ischemia would enhance the degree of stunning and induce a sustained decrease in glucose uptake after reperfusion. METHODS: Stunning was induced in 14 anesthetized swine by partially occluding the left anterior descending artery (LAD) for 20 min (> 80% flow reduction). Seven animals were pretreated with the nonspecific adenosine receptor blocker 8-phenyltheophylline (8-PT; 5 mg/kg), which decreased reactive hyperemia by an average of 38%. Myocardial glucose uptake was assessed 1 hr following reperfusion with PET and the glucose analog 18F-fluorodeoxyglucose (FDG). RESULTS: Before ischemia, systolic shortening in the LAD region was 15% +/- 6% in the control group and 16% +/- 4% in the 8-PT group and in both groups was reduced to - 1% +/- 2% during ischemia. After reperfusion, systolic shortening was 7% +/- 3% in the control group and 2% +/- 3% in the 8-PT group (p < 0.05). Myocardial oxygen consumption before ischemia was 4.58 +/- 3.03 micromol/min/g in the control group and 4.44 +/- 1.83 micromol/min/g in the 8-PT group (ns) and neither were different after reperfusion. In the postischemic LAD region, myocardial glucose uptake was 0.18 +/- 0.15 micromol/min/g in the control group and was similar to that of the 8-PT group (0.17 +/- 0.08 micromol/min/g; ns). CONCLUSION: The nonspecific adenosine blocker 8-PT enhanced the degree of stunning when given before ischemia but did not induce a sustained effect on myocardial glucose uptake after reperfusion.