Agonist-induced tension determines vascular reactivity during anoxia and reoxygenation.

To determine the influence of pre-existing pharmacologically-induced tension on vascular reactivity during anoxia and reoxygenation, rat aortic rings were contracted with norepinephrine, epinephrine, endothelin or KCl to 1, 2 or 4 g of tension. The rings were then exposed to anoxia (95% N2) for 10 min followed by reoxygenation (95% O2). The degree of anoxia-mediated contraction varied with the magnitude of tension before anoxia and resembled the length-tension relationship in myocardial fibers. The optimal agonist-induced tension for maximal anoxic contraction was approximately 1 to 2 g. This relationship between tension and anoxic contraction was observed in all but KCl-contracted rings. The agonist- as well as KCl-contracted rings showed normal relaxant response to acetylcholine, suggesting that a decrease in endothelium-derived relaxing factor (EDRF) alone cannot be the basis of anoxic contraction and release of endothelium-derived constricting factors (EDCFs) may relate to anoxic contraction in agonist-preconstricted rings. The relationship between the magnitude of agonist-induced tension and the extent of anoxia-mediated contraction may relate to the ability of endothelium to release EDRF and EDCFs as well as to the degree of phosphorylation in vascular smooth muscle cells. The reoxygenation-mediated contraction was noted to progressively increase in all experiments regardless of the pharmacologic agent used. This increase in reoxygenation-mediated contraction correlated with pre-existing pharmacologic tension, and may relate to calcium influx and restoration of ATP and other mediators in the vascular tissues during reoxygenation.     

Prostacyclin (PGI2) mediates hypoxic relaxation of bovine coronary arterial strips

Bovine coronary arterial strips (BCA) exhibiting spontaneous tone, relax in response to a decrease in the PO₂ of the batching medium. Experiments were performed to determine if prostaglandins (PGs) mediate the oxygen-induced changes in tension. BCA were equilibrated in Krebs-bicarbonate solution at 37 °C gassed with 95% O₂, 5% CO₂ and tension was measured isometrically. When the PO₂ of the bathing medium was decreased, BCA exhibited reversible reductions in tension. Switching from 95% O₂, 5% CO₂ to 95% N₂, 5% CO₂ (anoxia) elicited an initial relaxation followed by a contraction. In contrast, a change to 5% O₂, 5% CO₂, 90% N₂ (hypoxia) was followed by a sustained relaxation. Re-introduction of O₂ to anoxic strips produced a biphasic response: relaxation followed by contraction. Indomethacin or eicosatetraynoic acid (EYA) increased tone and inhibited the relaxation produced by anoxia or hypoxia. Indomethacin or EYA did not inhibit the relaxation of anoxic strips during re-introduction of O₂, but did inhibit the contraction partially. Relaxation of arterial strips to arachidonic acid (AA) was similar to relaxation to prostacyclin (PGI₂). Anoxia limited the relaxation to AA but not to PGI₂. We conclude that PG synthesis contributes to the basal tone and the hypoxia-induced relaxation of CBA. In addition, hypoxia, unless severe, does not prevent the conversion of AA to PGI₁.     

Salvia miltiorrhiza attenuates the changes in contraction and intracellular calcium induced by anoxia and reoxygenation in rat cardiomyocytes

The aim of the present study is to investigate the effect of Salvia miltiorrhiza (SM) on contraction and the intracellular calcium of isolated ventricular myocytes during normoxia or anoxia and reoxygenation using a video tracking system and spectrofluorometry. Cardiac ventricular myocytes were isolated enzymatically by collagenase and exposed to 5 min of anoxia followed by 10 min of reoxygenation. SM (1-9 g/L) depressed both contraction and the [Ca(2+)](i) transient in a dose-dependent manner. SM did not affect the diastolic calcium level and the sarcolemmal Ca(2+) channel of myocytes but decreased the caffeine-induced calcium release. During anoxia, the +/-dL/dtmax, amplitudes of contraction (dL) of cell contraction and [Ca(2+)](i) transients were decreased, while the diastolic calcium level was increased. None of the parameters returned to the pre-anoxia level during reoxygenaton. However, SM (3 g/L) did attenuate the changes in cell contraction and intracellular calcium induced by anoxia and reoxygenation. It is concluded that SM has different effects on normoxic and anoxic cardiomyocytes. The SM-induced reduction of changes in contraction and intracellular calcium induced by anoxia/reoxygenation indicates that SM may be beneficial for cardiac tissue in recovery of mechanical function and intracellular calcium homeostasis.     

嘌呤拟似物对豚鼠胃窦环行肌自发性收缩及电活动的影响

为探讨非肾上腺素能非胆碱能神经递质对胃窦环行肌功能的调节作用,在离体胃平滑肌上观察了嘌呤拟似物对胃窦环行肌自发性收缩活动和电活动的影响。电活动用传统的细胞内微电极记录,并和收缩活动同步描记于多道生理记录仪。结果表明,嘌呤能P2Y受体激动剂,三磷酸腺苷(ATP)和2-methylthio ATP(2-MeSATP)均增强胃窦平滑肌的收缩活动,但不影响电活动,而且ATP和2-MeSATP对胃平滑肌收缩活动的增强作用可被嘌呤能P2Y受体阻断剂,reactive blue-2和苏拉明(suramin)所阻断。用100μmol／L α,β-MeATP引起的脱敏感使P2X受体被阻断,ATP增强胃窦平滑肌收缩活动的效应不受影响。嘌呤能P2X受体激动剂,α,β-MeATP明显抑制胃窦环行肌自发性收缩活动,同时使膜电位明显超极化。ATP对胃窦平滑肌的收缩作用不被L型钙通道阻断剂尼卡地平(nicardipine)阻断,但细胞外用无钙液灌流时这种效应则完全被阻断。用前列腺素合成抑制剂消炎痛预先处理20min后,ATP和2-MeSATP仍能增强胃窦平滑肌的自发性收缩活动。以上结果提示：(1)ATP和2-MeSATP通过嘌呤能P2Y受体增强胃窦平滑肌的自发性收缩活动,而α,β-MeATP或β,γ-MeATP通过嘌呤能P2X受体使膜电位超极化,引起胃窦平滑肌的舒张;(2)ATP和2-MeSATP增强胃窦平滑肌自发性收缩活动的效应依赖于细胞外钙,但钙离子进入细胞的途径并不是电压依赖性钙通道;(3)ATP和2-MeSATP增强胃窦平滑肌自发性收缩活动的效应不通过前列腺素介导。     

Induction of 72-kDa Inducible Heat Shock Protein (HSP72) in Cultured Rat Astrocytes After Energy Depletion

Abstract: Protein synthesis is important in the readaptive processes for cultured astrocytes after hypoxia and subsequent reoxygenation. We have identified 72-kDa inducible heat shock protein (HSP72) as a major stress protein in reoxygenated astrocytes. To assess the mechanism for reoxygenation-mediated induction of HSP72, a reporter gene that consists of a human HSP promoter fused to the luciferase gene was transfected into cultured astrocytes. Analysis of cellular energy nucleotides showed an increase of the ADP/ATP ratio after reoxygenation, which synchronized with activation of the HSP promoter. Activation of the HSP promoter was also observed after an addition of iodoacetic acid to hypoxic astrocytes, which reached the maximum when the ADP/ATP ratio reached 50%, but further decline in the energy profile caused inactivation of this promoter. Inhibition of protein synthesis after reoxygenation resulted in temporary restoration of the energy profile and suppression of the DNA binding activity of the heat shock factor. Addition of quercetin greatly decreased the [³H]leucine incorporation in the polysome fraction without any effect on the mature mRNA formation. These data suggest that the energy depletion in reoxygenation triggers induction of HSP72 after reoxygenation, which may act as a pivotal mediator in the stress response of reoxygenated astrocytes by facilitating protein synthesis.     

Effects of hypoxia,simulated ischemia and reoxygenation on the contractile function of human atrial trabeculae

Hypoxia, ischemia and reoxygenation cause contractile dysfunction which will be characterized by the time course of isometric contraction of human atrial trabeculae. Post-rest potentiation (PRP) and postextrastimulatory potentiation (PEP) were elicited to obtain indirect information about the role of the sarcoplasmic reticulum (SR) in excitation-contraction coupling. As lipid peroxidation could cause SR dysfunction, thiobarbituric acid reactive substances (TBARS) were measured. After 30 min of hypoxia (H) or simulated ischemia (H combined with acidosis-SI), contractile force decreased to 15% and 6%, respectively, of control (p <- 0.05), whereas the normalized rate of both contraction and relaxation increased. In group H, rapid reoxygenation produced a recovery of contractile force to about 60%. After post-hypoxic reoxygenation the TBARS concentration was increased. In group SI, rapid reoxygenation and a rather gradual correction of acidosis produced complete recovery of contractile force. PRP and PEP were maintained during H and SI. Particularly post-ischemic reoxygenation caused a marked depression of PRP and partly of PEP. Thus, alteration of SR Ca²⁺ handling occurs predominantly during reoxygenation rather than during H or SI, probably associated with the damaging effect of increased oxygen radicals. The depression of potentiation occured along with delayed relaxation, temporary increased resting force, mechanical alternans, and spontaneous activity which are further characteristics for SR dysfunction. Thus, for a possibly beneficial effect of low pH during SI combined with its gradual correction during reoxygenation on the recovery of contractile function, developed force should not be the only index.     

Underlying mechanism of hypoxic preconditioning decreasing apoptosis induced by anoxia in cultured hippocampal neurons

It is known that hypoxic preconditioning (HP, a brief period of sublethal hypoxia) provides neuroprotection against subsequent severe anoxia, but the mechanisms of this increased tolerance have not been fully elucidated. A hypoxic preconditioning model was established by exposing a 4-day hippocampal culture to 1% O(2) for 20 min/day for 8 days. The preconditioning significantly decreased the number of apoptotic neurons at reoxygenation 24 h after 4 h of severe anoxia (0% O(2)). Further study demonstrated that the degradation of mitochondrial membrane potential (MMP) was greatly inhibited and the expression of B-cell lymphoma protein-2 (Bcl-2) was increased considerably after severe anoxia in the HP groups. These results indicate that the increased anoxic tolerance, which is induced by HP in cultured hippocampal cells, may be correlated with Bcl-2 overexpression and enhanced stability of MMP, which ultimately reduces apoptosis 24 h after reoxygenation.     

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.     

Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture

The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.     

枸杞多糖对肾性高血压大鼠离体血管反应性的影响及其机制

本工作利用两肾一夹肾性高血压大鼠模型,观察枸杞多糖对高血压大鼠血压的影响以及离体主动脉环丙皮细胞在调节血管张力中的功能改变,探讨ＬＢＰ对高血压发生发展的影响及其机制。结果表明,ＬＢＰ可防止２Ｋ１Ｃ大鼠高血压的形成。离体主动脉环灌流表明ＲＨ组对苯肾上腺素的收缩反应明显高于对照组,去除内皮后组间差异消失．     

缺氧对离体猫心乳头肌力学的影响

本文研究缺氧对离体猫心乳头肌力学的影响,以探讨缺血性心肌损伤中缺氧这一因素所起的作用。结果表明:①-dT/dt max/Tmax(舒张指标之一)在缺氧 lmin 时明显降低(P<0.05),而所有收缩指标无变化。②缺氧4min 时,-dT/dt max 降低16.7%(P<0.01),而 dT/dt max 在缺氧7min 时才降低16.6%(P<0.05)。③在缺氧4min 时,所有舒张指标已发生显著的变化,而所有收缩指标在缺氧7min 时才有明显改变。缺氧对离体猫心乳头肌的收缩过程和舒张过程都有影响,但是舒张过程的变化较之收缩过程出现更早,且较明显。     

Effect of the antioxidant ionol on energy metabolic indices and heart function during acute hypoxia and subsequent reoxygenation

The effect of preliminary administration of antioxidant ionol on the heart energy metabolism and contractile function was estimated in hypoxic hypoxia and subsequent reoxygenation. The protective effect of ionol on the energy metabolism in hypoxia was shown to occur mainly at the level of glycolysis. In reoxygenation, the protective effect of ionol manifested at the level of creatine kinase system to provide a rapid restoration of the CP synthesis rate. This shift correlated with the velocity of restoration of the developed pressure and the velocities of contraction and relaxation. On the whole the data obtained correspond to the notion that creatine kinase system and ATP play an important role in the depression and subsequent restoration on the heart contractile function in acute hypoxia and reoxygenation and ionol provides more effective performance of this system and correspondingly more rapid restoration of the contractile function in reoxygenation.     

Oxidized low-density lipoprotein antagonizes the activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor but does not interfere with its biosynthesis.

Oxidized low-density lipoprotein (LDLox) is a molecule with strong atherogenic properties. In a concentration dependent fashion, LDLox antagonized the activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor (EDRF), which was produced in vitro by incubation of a partially purified EDRF-forming enzyme in the presence of L-arginine, Ca2+ and NADPH. The inhibitory effect of LDLox was potentiated by preincubation of the soluble guanylate cyclase with LDLox, but not when the EDRF-forming enzyme was pretreated with LDLox. As LDLox did not diminish the calmodulin-dependent conversion of L-arginine into L-citrulline by the EDRF-forming enzyme it would appear that EDRF-biosynthesis was not affected by LDLox. It is suggested that the impaired relaxant response of atherosclerotic blood vessels to endothelium-dependent vasodilators was not due to a reduced formation of EDRF but due to a diminished responsiveness of soluble guanylate cyclase.     

Peptidoleukotrienes induce an endothelium-dependent relaxation of guinea pig main pulmonary artery and thoracic aorta

The purpose of our investigation was to assess the role of the endothelium in the vasomotor effects of leukotrienes. Norepinephrine-preconstricted rings isolated from guinea pig main pulmonary artery and thoracic aorta responded to LTC4 and LTD4 with a concentration-dependent relaxation. In endothelium-denuded rings, both LTC4 and LTD4 caused a concentration-dependent contraction. The LTD4 receptor antagonist ICI 198,615 inhibited both LTC4- and LTD4-induced relaxation and contraction. Inhibition of gamma-glutamyl transpeptidase with AT-125 prevented the effects of LTC4, but not those of LTD4. The relaxant effect of LTD4 was not modified by indomethacin, but was abolished by methylene blue. We conclude that: 1) LTD4 induces a receptor-mediated endothelium-dependent relaxation of cavian pulmonary artery and aorta; 2) the vasorelaxant effect of LTC4 requires its conversion to LTD4; 3) the vasorelaxant effect of LTD4 is unrelated to PGI2 release, and is probably due to the release of an "EDRF"; 4) the removal of the endothelium reveals a direct receptor-mediated vasoconstricting effect of leukotrienes.     

EDRF inhibition attenuates the increase in pulmonary blood flow due to oxygen ventilation in fetal lambs.

At birth, pulmonary vasodilation occurs during rhythmic distension of the lungs and oxygenation. Inhibition of prostaglandin synthesis prevents pulmonary vasodilation during rhythmic distension of the lungs but not during oxygenation. Because endothelium-derived relaxing factor (EDRF) modulates pulmonary vascular tone at birth, at rest, and during hypoxia in older animals, we hypothesized that EDRF may modulate pulmonary vascular tone during oxygenation in fetal lambs. We studied the responses to N omega-nitro-L-arginine, a competitive inhibitor of EDRF synthesis, in nine near-term fetal lambs and to drug vehicle in six of these lambs and the subsequent responses to in utero ventilation with 95% O2 in these fetal lambs. In all fetal lambs, prostaglandin synthesis was prevented by meclofenamate. N omega-nitro-L-arginine increased pulmonary and systemic arterial pressures by 28% (P < 0.05) and 31% (P < 0.05), respectively, and decreased pulmonary blood flow by 83% (P < 0.05). In the controls, ventilation with 95% O2 increased pulmonary blood flow by 1,050% (P = 0.05) without changing pressures, thereby decreasing pulmonary vascular resistance by 88% (P = 0.05). During N omega-nitro-L-arginine infusion, ventilation with 95% O2 increased pulmonary blood flow by 162% (P = 0.05) and decreased pulmonary vascular resistance by 74% (P = 0.05). This suggests that EDRF may play an important role in modulating resting pulmonary vascular tone in fetal lambs and in the vasodilatory response to ventilation with O2 in utero.     

Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells

Endothelial cells are critical targets in both hypoxia-and reoxygenation-mediated lung injury. Reactive O₂ species (ROS) have been implicated in the pathogenesis of hypoxic and reoxygenation lung injury, and xanthine dehydrogenase/oxidase (XDH/XO) is a major generator of the ROS. Porcine pulmonary artery endothelial cells (PAEC) have no detectable XDH/XO. This study was undertaken to examine (1) ROS production by hypoxic porcine PAEC and their mitochondria and (2) ROS production and injury in reoxygenated PAEC lacking XDH/XO activity. Intracellular H₂O₂ generation and extracellular H₂O₂ and O/₂ release were measured after exposure to normoxia (room air-5% CO₂), hypoxia (0% O₂ -95% N-5% CO₂), or hypoxia followed by normoxia or hyperoxia (95% O₂-5% CO₂). Exposure to hypoxia results in significant reductions in intracellular H₂ O₂ formation and extracellular release of H₂ O₂ and O₂ by PAEC and mitochondria. The reductions occur with as little as a 2 h exposure and progress with continued exposure. During reoxygenation, cytotoxicity was not observed, and the production of ROS by PAEC and their mitochondria never exceeded levels observed in normoxic cells. The absence of XDH/XO may prevent porcine PAEC from developing injury and increased ROS production during reoxygenation. © 1995 Wiley-Liss, Inc.     

Possible role of nitric oxide and arachidonic acid pathways in hypoxia-induced contraction of rabbit coronary artery rings

In isolated coronary arteries, hypoxia induces an increase in tone by releasing an unidentified endothelium-derived contracting factor (EDCF). Isometric force was measured in an isolated rabbit coronary artery ring at 37 degrees C in control and high K+ (40 mM) pre-contracted conditions. Hypoxia (15 mmHg pO2) induced by equilibrating the perfusate with nitrogen. Hypoxia did not affect the resting tone but induced an endothelium-dependent contraction on pre-contracted rings. Inhibitors of nitric oxide (NO) were tested, L-NAME (10(-4) M) totally and L-NMMA (10(-4) M) partially convert the hypoxic contraction to an hypoxic relaxation. The addition of L-arginine (10(-4) or 10(-3) M) did not restore the response. Methylene blue (10( -5) M) and ODQ (1 H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one, 10(-5) M), both inhibitors of guanylate cyclase, also changed the hypoxic contraction into a hypoxic relaxation. Catalase (1200 U/ml), which decomposes hydrogen peroxide (H2O2), and superoxide dismutase (150 U/ml, SOD), a free radical scavenger, did not change the hypoxic response but quinacrine (50 microM), an inhibitor of phospholipase A2, significantly decreased it. Inhibitors of arachidonic acid metabolism (indomethacin, diethylcarbamazine, miconazole) however did not affect the hypoxic response. We conclude that in K+ pre-contracted rabbit coronary artery rings, hypoxia induces a contraction which is nitric oxide and arachidonic acid dependent.     

Endothelium-dependent transfer of ethanol tolerance in the aorta

Recent studies have suggested that the endothelium-dependent tolerance to the direct vasoconstrictor effect of ethanol in the rat aorta is mediated by endothelium-derived relaxing factor (EDRF). This hypothesis was tested directly by employing a sandwich technique which has been used to demonstrate the release and action of EDRF. These experiments measured the ethanol-induced contraction of a spirally-cut aortic strip without endothelium obtained from an ethanol naive control rat. The response of the spiral strip was measured before and after it was sandwiched with a longitudinally-cut aortic strip with or without endothelium obtained from either control or ethanol tolerant rats. There was no significant difference in the ethanol-induced contraction of the spiral strip after beginning sandwiched with a longitudinal strip with or without endothelium obtained from a control rat or with a longitudinal strip without endothelium from a tolerant rat. In contrast, when a longitudinal strip with endothelium from a tolerant rat was sandwiched with the spiral strip the ethanol-induced contraction was significantly reduced. This effect was inhibited by methylene blue but not by indomethacin. Further, the magnitude of the carbachol-induced relaxation of the sandwiched preparation was significantly greater when the longitudinal strip with endothelium was obtained from a tolerant rat than from control rat. These data demonstrate the involvement of EDRF in the endothelium-dependent tolerance to ethanol in the rat aorta.     

The alpha adrenoceptors on endothelial cells

Endothelial cells release a powerful factor (endothelium-derived relaxing factor [EDRF]) that relaxes smooth muscle cells in response to some vasodilating agents such as acetylcholine. Contraction curves to norepinephrine (NE) in greyhound, mongrel dog, and pig coronary artery rings were studied in vitro in the presence of propranolol. Removal of endothelium increased the sensitivity and maximum contraction in response to NE. In other experiments pig coronary rings were precontracted with a thromboxane mimetic U 46619 in the presence of propranolol. NE relaxed these arteries only if endothelium was present. Methoxamine was without effect but the relaxation response to NE was antagonized by phentolamine, idazoxan, and yohimbine, which suggests that there are alpha 2 adrenoceptors on endothelial cells that mediate the release of EDRF. Greyhound and mongrel dog large coronary arteries relaxed to NE only if prazosin was present, which suggests that alpha 1-adrenoceptor stimulation on the vascular smooth muscle can override the relaxation response to EDRF. Comparison of NE responses in carotid, mesenteric, renal, and femoral large arteries of the pig, greyhound, and mongrel dog indicate the nonuniformity of distribution of alpha 2 adrenoceptors on endothelium and alpha 1 and alpha 2 adrenoceptors on vascular smooth muscle. The integrity of the endothelium must now be considered in interpreting the vascular responses to alpha-adrenoceptor agonists.     

Epr Spectroscopic Studies of Detection of a Carbon-Centered Free Radical During Acetylcholine-Induced and Endothelium-Dependent Relaxation of Guinea Pig Pulmonary Artery

Vascular smooth muscle relaxation by several vasodilators, including acetylcholine (Ach) and ATP, depends on the presence of intact endothelium. Ach is thought to activate muscarinic receptors on endothelium to release an endothelium-derived relaxing factor (EDRF) which brings about relaxation of smooth muscle. In order to assess the role of free radicals in the endothelium-dependent relaxation of blood vessel, we have studied the effect of a spin-trapping agent, phenyl t-butyl nitrone (PBN). on Ach-, ATP-, and sodium nitroprusside-induced relaxation of guinea pig pulmonary artery. Arterial strips were mounted in a 5-ml organ bath containing Krebs solution equilibrated with 95% O₂ and 5% CO₂ at 37°C. After increasing vascular tone by a synthetic prostaglandin endoperoxide analog (50 ng/ml), the strips relaxed dose-dependently in response to Ach (5 × 10^-8M), ATP (1.5 × 10^-6M) or sodium nitroprusside (6 × 10^-9 M). Removal of the endothelium abolished the relaxation by Ach or ATP, but did not affect the relaxation by sodium nitroprusside. PBN inhibited Ach-induced relaxation of pulmonary artery dose-dependently, but had no effect on relaxations by ATP or sodium nitroprusside. PBN did not block radioligand binding to muscarinic cholinergic membrane receptors on both chick embryonic heart and guinea pig pulmonary artery endothelial cells indicating that it does not block the muscarinic receptors. Spin trapping in combination with electron paramagnetic resonance (EPR) spectral analysis revealed a carbon-centered radical with hyperfine splitting constants of a_N = 16.0 G and a_β^H: = 3.85 G in the lipid extracts of pulmonary artery (0.2-0.4g) incubated with PBN (14mM) and Ach (3 × 10^-6M) for 20min. No signal was detected when endothelium was removed. Our data suggest that the endothelium-dependent relaxation of pulmonary artery by Ach is associated with the generation of a free-radical and can be prevented by a spin-trapping agent. ATP, however, relaxes the arterial smooth muscle by a different mechanism.