Mechanisms of action of isoflurane on contraction of rabbit conduit artery

Isoflurane may cause differential effects on different vascular beds of the same animal species. The mechanisms of this action have not been elucidated. Accordingly, we compared in rabbit aorta and femoral artery the effects of isoflurane (1-3.3%) in isolated rings (endothelium denuded) activated by norepinephrine, and isoflurane effects on Ca2+ fluxes from the sarcoplasmic reticulum in skinned strips. When < 30 nM norepinephrine was used to cause ring contraction, isoflurane increased the force of contraction in aortic rings, but decreased force in femoral arterial rings. At 30 nM norepinephrine stimulation, 3.3% isoflurane decreased the force and, in the presence of verapamil, isoflurane actually increased the force in both arterial types. In skinned strips of both arterial types, isoflurane present during Ca2+ uptake decreased the caffeine-induced tension transients, whereas isoflurane present during Ca2+ release enhanced the transients. Isoflurane potentiated the depression of the tension transients by ryanodine. Isoflurane directly caused contracture even in the absence of caffeine. Thus, isoflurane has similar cellular mechanisms of action in the aortic and femoral arterial smooth muscle: inhibiting Ca2+ influx through the sarcolemma, decreasing Ca2+ uptake by the sarcoplasmic reticulum, and enhancing caffeine-induced Ca2+ release from the sarcoplasmic reticulum.     

Leukotrienes in acetylcholine-induced contraction of esophageal circular smooth muscle in experimental esophagitis

BACKGROUND & AIMS: Phospholipase A2 (PLA2) participates in acetylcholine (ACh)-induced contraction of esophageal circular smooth muscle. Because PLA2, arachidonic acid, and its metabolites are involved in inflammatory responses, their role after induction of experimental esophagitis was examined. METHODS: Experiments were performed in esophageal smooth muscle cells (ESO) isolated by enzymatic digestion from the circular layer of normal and esophagitis animals. Content of peptidoleukotrienes (leukotriene [LT] C4, LTD4, and LTE4) was measured in esophageal circular muscle tissue. RESULTS: The cytosolic PLA2 antagonist trifluoromethyl ketone analogue of arachidonic acid inhibited ACh-induced contraction of normal and esophagitis ESO. Inhibition by secreted PLA2 antagonists AM5 and MJ33 was significantly greater in esophagitis ESO. The lipoxygenase inhibitor nordihydro-guaiaretic acid and the LTD4 antagonist ICI 198,615 inhibited ACh-induced contraction of esophagitis but not of normal ESO. Secreted PLA2 and LTD4 contracted normal ESO more than esophagitis ESO. However, in esophagitis, ESO contraction was increased by threshold diacylglycerol concentration. Resting levels of LTs were greater in esophagitis than in normal circular esophageal muscle and increased in response to ACh in esophagitis but not in normal esophageal muscle. CONCLUSIONS: Esophagitis shifts the signal transduction pathway activated by ACh. Esophagitis increased the contribution of secreted PLA2 and of LTs to ACh-induced contraction.     

Detection of platelets activated during acetylcholine-induced coronary vasospasm

Although platelet activation may play a role in coronary artery spasm, platelets activated following coronary vasospasm have not been clinically detected. We performed flow cytometric analysis of activation-dependent granular proteins, CD62P (P-selectin), CD63, PAC-1 (activated glycoprotein [GP] IIb/IIIa) and thrombospondin on the platelet plasma membrane in patients who exhibited acetylcholine-induced coronary vasospasm and compared findings with those in control patients without vasospasm. We simultaneously investigated the plasma levels of thrombin anti-thrombin III complex (TAT), plasmin alpha2-plasmin inhibitor complex (PIC), and thrombomodulin. In patients with vasospasm, the expression of CD62P, CD63 and PAC-1 on the platelet membrane surface increased in coronary sinus blood samples following coronary vasospasm, although the expression in aortic samples did not change. The TAT level also increased in the coronary sinus after vasospasm. Platelets might be activated by coronary vasospasm within the coronary circulation. The platelet activation process may be modulated by thrombin generation.     

Ischemic preconditioning attenuates postischemic coronary artery endothelial dysfunction in a model of minimally invasive direct coronary artery bypass grafting

OBJECTIVE: Unmodified reperfusion without cardioplegia in minimally invasive direct coronary artery bypass grafting procedures causes endothelial dysfunction that may predispose to polymorphonuclear neutrophil-mediated myocardial injury. This study tested the hypothesis that ischemic preconditioning in a minimally invasive direct coronary artery bypass grafting model attenuates postischemic endothelial dysfunction in coronary vessels. METHODS: In anesthetized dogs, the left anterior descending coronary artery was occluded for 30 minutes and reperfused for 3 hours without ischemic preconditioning (no-ischemic preconditioning; n = 7); in 7 dogs, the left anterior descending occlusion was preceded by 5 minutes occlusion followed by 5 minutes of reperfusion. Relaxation responses to stimulators of nitric oxide synthase were used to evaluate endothelial function in arteries from the ischemic-reperfused (left anterior descending) and nonischemic (left circumflex coronary artery) zones. RESULTS: Stimulated endothelial-dependent relaxation of epicardial left anterior descending artery to incremental concentrations of acetylcholine in the no-ischemic preconditioning animals was shifted to the right, and maximal relaxation was attenuated compared with the nonischemic left circumflex coronary artery (117% +/- 4% vs 138% +/- 5%). In contrast, acetylcholine-induced maximal relaxation was comparable in the left anterior descending artery versus left circumflex coronary artery in the ischemic preconditioning group (130% +/- 6% vs 135% +/- 5%). In 150- to 200- microm left anterior descending microvessels, 50% relaxation occurred with a lower concentration (log[M]) of acetylcholine in ischemic preconditioning versus no-ischemic preconditioning (-8.0 +/- 0.4 vs -7.0 +/- 0.1) with no group differences in smooth muscle relaxation to sodium nitroprusside, suggesting endothelial-specific damage. Adherence of fluorescent labeled polymorphonuclear neutrophils to epicardial coronary artery endothelium, used as an index of basal (unstimulated) anti-polymorphonuclear neutrophil function, was significantly attenuated by ischemic preconditioning versus no-ischemic preconditioning (293 +/- 25 polymorphonuclear neutrophils/mm2 vs 528 +/- 29 polymorphonuclear neutrophils/mm2). CONCLUSION: In this minimally invasive direct coronary artery bypass grafting model, both agonist-stimulated and basal postischemic endothelial dysfunction were attenuated by ischemic preconditioning.     

Hypoxic contraction of isolated canine coronary artery. Mediation by potassium-dependent exocytosis of norepinephrine

Effects of hypoxia on arterial tone, efflux of potassium, and efflux of norepinephrine were monitored for isolated canine coronary arteries labeled with radioactive potassium (42K) and norepinephrine (3HNE). Hypoxia elicited transient relaxation and subsequent sustained contraction accompanied by marked increases in the effluxes of 42K and 3HNE. After sympathetic nerve injury with 6-hydroxydopamine or cold storage, arteries responded to hypoxia with sustained relaxation. Sustained relaxation occurred also after pretreatment with L-propranolol, but not with D-propranolol or phentolamine. Inhibition of hypoxic contraction by L-propranolol did not alter 42K or 3HNE efflux. Colchicine, an inhibitor of the exocytosis of NE, suppressed hypoxic 3HNE efflux and contraction, but not 42K efflux. Proadifen inhibited 42K and 3HNE efflux as well as contraction. During proadifen-inhibited 42K efflux, exogenous K+ augmented overflow of 3HNE, indicating that proadifen relaxed the hypoxic artery primarily by inhibiting K+-dependent exocytosis of NE. The ratio of NE to dopamine beta-hydroxylase activity was similar in effluents from oxygenated arteries exposed to elevated K+ concentrations and in effluents from hypoxic arteries. Thus, hypoxia evoked exocytotic release of norepinephrine which promoted contraction by a beta-adrenergic mechanism.     

The surgical treatment of heart valve defects combined with multiple coronary artery involvement

The authors analyze 144 surgeries in patients with single and multiple valvular defects combined with multiple involvement of the coronary arteries; multiple (3-4) coronary artery shunting was carried out in 21 patients, the mortality being 19.4%. Recovery of the blood stream in all involved coronary arteries and correction of defective valves were found conducive to reduction of incidence of myocardial infarction and acute heart failure. Retrograde blood cardioplegia to a considerable measure shortened the duration of aorta clamping and improved myocardial protection. Such methods and improvement of surgical technique helped us reduce the mortality rate by 2-3 times.     

Corneal endothelial involvement in pseudoexfoliation syndrome

Information on corneal manifestation in pseudoexfoliation syndrome is restricted to specular microscopic studies. We investigated morphologic changes of the posterior cornea of two corneal buttons with Fuchs' endothelial dystrophy obtained at penetrating keratoplasty and of one enucleated glaucomatous eye from three patients with ocular pseudoexfoliation syndrome. By transmission electron microscopy, large clumps of typical pseudoexfoliation material were found adhering to the corneal endothelium and masses of pseudoexfoliation material were incorporated into the posterior Descemet's membrane. In the affected areas, the endothelial layer appeared irregular and discontinuous, with loosely adherent, degenerating cells producing pseudoexfoliation fibers and fibroblastic cells spreading to cover denuded Descemet's membrane. The present findings indicate that the pseudoexfoliation material is initially formed by degenerative endothelial cells and that it becomes subsequently buried by overgrowing cells. The corneal endothelial involvement may potentiate complications of cataract surgery and open angle glaucoma in patients with pseudoexfoliation syndrome.     

A new model for occluding the left coronary artery in the rabbit without thoracotomy

A small, nonmagnetic coil was fluoroscopically guided into the left circumflex coronary artery (LCXA) at the midventricular level via the left femoral artery in seven rabbits. Coronary artery occlusion was confirmed by intracoronary contrast injection, electrocardiographic changes, akinesis of the posterolateral wall distal to the occluded area, and histopathological staining of the myocardium. This new model of a closed-chest coronary artery occlusion in the rabbit may be suitable for acute ischemic myocardium studies that do not require reperfusion.     

Possible involvement of proteases in the regulation of spermatogenesis

Proteolytic enzymes, which are synthesized and secreted by cells of the seminiferous tubule of the testis, have important functions in spermatogenesis. We performed metabolic studies using small peptide hormones as a substrate to investigate the activity of proteases in cultured Sertoli cells of the rat. High-performance liquid chromatographic analysis of the cell culture supernatants showed cleavage of met- and leu-enkephalin, substance P, and bradykinin. No peptidolysis was observed for the cyclic peptide oxytocin. The hormone cleavage pattern and the use of specific protease inhibitors in peptide degradation experiments demonstrated activities of several proteases in Sertoli cells. These are mainly metalloproteinases including neutral metalloendopeptidases, angiotensin-converting enzyme and aminopeptidases. In addition, activities of serine and aspartic proteases were detected. Only marginal proteolytic activities were observed in Sertoli cell conditioned supernatants, indicating that the investigated proteases are mainly located on Sertoli cell membranes. The peptide hormones used in this study have been found to play a potential role in the endocrine, paracrine or autocrine regulation of testicular cells. The membrane-associated proteases reported here may therefore be involved in the metabolism and inactivation of these peptides.     

Possible involvement of neuropeptidergic sensory nerves in alopecia areata

Alopecia areata (AA) is a dermatosis involving the sudden occurrence of bald patches on the scalp. Although the aetiology is unknown, experimental data indicate that cutaneous microcirculation plays an important role. The skin is richly innervated by neuropeptidergic sensory nerves that help regulate microvascular circulation. This study shows a reduction of cutaneous levels of substance P and calcitonin gene-related peptide (CGRP) but not of vasoactive intestinal polypeptide in scalp biopsies from patients with AA. Laser-Doppler flowmetry was used to study microcirculation of the scalp. Results indicate that patients with AA have lower basal blood flow and greater vasodilatation following intradermal CGRP injection than control subjects. A vascular hyper-reactivity to vasodilatatory substances such as neuropeptides, probably because of the lack of these substances, is hypothesized.     

Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation

The contractile effect of norepinephrine (NE) on isolated rabbit bronchial artery rings (150-300 microns in diameter) and the role of alpha 1- and alpha 2-adrenoceptors (AR) on smooth muscle and endothelium were studied. In intact arteries, NE increased tension in a dose-dependent manner, and the sensitivity for NE was further increased in the absence of endothelium. In intact but not in endothelium-denuded arteries, the response to NE was increased in the presence of both indomethacin (Indo; cyclooxygenase inhibitor) and NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], indicating that two endothelium-derived factors, NO and a prostanoid, modulate the NE-induced contraction. The alpha 1-AR antagonist prazosin shifted the NE dose-response curve to the right, and phenylephrine (alpha 1-AR agonist) induced a dose-dependent contraction that was potentiated by L-NAME or removal of the endothelium. The sensitivity to NE was increased slightly by the alpha 2-AR antagonists yohimbine and idazoxan, and this effect was abolished by Indo or removal of the endothelium. Similarly, contractions induced by UK-14304 (alpha 2-AR agonist) were potentiated by Indo or removal of the endothelium. These results suggest that NE-induced contraction is mediated through activation of alpha 1- and alpha 2-ARs on both smooth muscle and endothelium. Activation of the alpha 1- and alpha 2-ARs on the smooth muscle causes contraction, whereas activation of the endothelial alpha 1- and alpha 2-ARs induces relaxation through release of NO (alpha 1-ARs) and a prostanoid (alpha 2-ARs).     

Developmental changes in the effect of acidosis on contraction, intracellular pH, and calcium in the rabbit mesenteric small artery

The purpose of the present study was to determine developmental changes in the effect of respiratory acidosis on vascular smooth muscle contraction. Vessel diameter, intracellular pH (pHi), and calcium concentration ([Ca]i) were measured in a cannulated preparation of the small mesenteric artery of newborn and adult rabbits. In the artery precontracted by high KCl, acidosis caused a vasorelaxation both in the newborn and the adult; the vasorelaxation was greater in the newborn than in the adult. The fura-2 fluorescence ratio, an indicator of [Ca]i, decreased transiently during acidosis and the decrease was similar in the two age groups. In the artery precontracted by norepinephrine, acidosis caused a transient vasoconstriction in the adult and a vasorelaxation in the newborn. In these vessels, the fura-2 fluorescence ratio increased transiently during acidosis; the increase was similar in the two groups. Upon induction of acidosis, pHi fell rapidly in the artery precontracted by norepinephrine or high KCl, and the depression of pHi was similar in the two groups. In the skinned smooth muscle preparation, a tension-[Ca] relationship curve at pH 7.1 was not significantly different from that at pH 6.8 in the adult. In the newborn, the tension-[Ca] curve at pH 6.8 was shifted to the right, compared with that at pH 7.1. These data suggest that the vasorelaxant effect of respiratory acidosis in the premature vessel is greater than in the adult. The greater vasorelaxation in the newborn cannot be explained by the age-related difference in pHi or [Ca]i during acidosis. The greater sensitivity of myofibrils to low pHi in the newborn may, at least in part, be responsible for the greater vasorelaxation in this age group.     

L-2-Oxothiazolidine-4-carboxylic acid reverses endothelial dysfunction in patients with coronary artery disease

The effective action of endothelium-derived nitric oxide (EDNO) is impaired in patients with atherosclerosis. This impairment has been attributed in part to increased vascular oxidative stress. EDNO action is improved by administration of ascorbic acid, a water-soluble antioxidant. Ascorbic acid is a potent free-radical scavenger in plasma, and also regulates intracellular redox state in part by sparing cellular glutathione. We specifically investigated the role of intracellular redox state in EDNO action by examining the effect of L-2-oxo-4-thiazolidine carboxylate (OTC) on EDNO-dependent, flow-mediated dilation in a randomized double-blind placebo-controlled study of patients with angiographically proven coronary artery disease. OTC augments intracellular glutathione by providing substrate cysteine for glutathione synthesis. Brachial artery flow-mediated dilation was examined with high-resolution ultrasound before and after oral administration of 4.5 g of OTC or placebo in 48 subjects with angiographically documented coronary artery disease. Placebo treatment produced no change in flow-mediated dilation (7.0+/-3.9% vs. 7.2+/-3.7%), whereas OTC treatment was associated with a significant improvement in flow-mediated dilation (6.6+/-4.4% vs. 11.0+/-6.3%; P = 0.005). OTC had no effect on arterial dilation to nitroglycerin, systemic blood pressure, heart rate, or reactive hyperemia. These data suggest that augmenting cellular glutathione levels improves EDNO action in human atherosclerosis. Cellular redox state may be an important regulator of EDNO action, and is a potential target for therapy in patients with coronary artery disease.     

Tonic immobility in chickens: Possible involvement of monoamines.

In 4 experiments with a total of 259 Production Red chickens, it was found that tonic immobility was affected by a variety of drugs that act on monoaminergic systems. Compounds that enhanced the duration of tonic immobility were dextro-LSD, 2-bromo-dextro-lysergic acid (BOL-148), pargyline, and iproniazid; no effect was found when Ss were given 5-hydroxytryptophan, para-chlorophenylalanine, or atropine. Injections of serotonin depressed response duration. A suggestive parallel was noted between the results of the present study and those of previous work reporting drug-induced suppression of raphe electrical activity. The data implicate monoamines, especially serotonin, in the mediation of behavioral activation and suppression. (48 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Comparison of coronary endothelial dynamics with electrocardiographic and left ventricular contractile responses to stress in the absence of coronary artery disease

Coronary artery endothelial dysfunction has been proposed as a cause of myocardial ischemia and symptoms in patients with angina-like chest pain despite normal coronary angiograms, especially those with ischemic-appearing ST-segment depression during exercise (syndrome X). We measured coronary vasomotor responses to acetylcholine (3 to 300 microg/min) in 42 patients (27 women and 15 men) with effort chest pain and normal coronary angiograms who also had normal electrocardiograms and echocardiograms at rest. All patients underwent treadmill exercise testing and measurement of systolic wall thickening responses to dobutamine (40 microg/kg/min) during transesophageal echocardiography. There were no differences in the acetylcholine-stimulated epicardial coronary diameter (+5+/-13% vs +1+/-13%, p=0.386) and flow (+179+/-90% vs +169+/-96%, p=0.756), or in the systolic wall thickening responses (+134+/-65% vs +118+/-57%, p=0.445) from baseline values in the 12 syndrome X patients compared with the 30 patients with negative exercise test results. In patients in the lowest quartile of coronary flow responses to acetylcholine, dobutamine increased systolic wall thickening by 121+/-73%; 3 had ischemic-appearing ST-segment depression during this stress. This contractile response to dobutamine was no different than the increase in systolic wall thickening (129+/-48%, p=0.777) in patients in the highest quartile of coronary flow responses, 3 of whom also had ischemic-appearing ST-segment depression during this stress. Thus, coronary endothelial dysfunction in the absence of coronary artery disease does not account for ischemic-appearing ST-segment depression in patients with chest pain despite normal coronary angiograms. Further, coronary endothelial dysfunction is not associated with myocardial contractile responses to stress consistent with myocardial ischemia.     

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.     

Possible involvement of DNA methylation in 5-azacytidine-induced neuronal cell apoptosis

Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052-1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in microM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in microM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders.     

Difference in acetylcholine-induced nitric oxide release of arterial and venous grafts in patients after coronary bypass operations

OBJECT: In this study the authors tested the hypothesis that hemorrhagic hypotension and high intracranial pressure induce an increase in cerebrovascular resistance that is caused by sympathetic compensatory mechanisms and can be modified by alpha-adrenergic blockade. METHODS: Continuous measurements of cerebral blood flow were obtained using laser Doppler microprobes placed in the cerebral cortex in anesthetized pigs during induced hemorrhagic hypotension and high cerebrospinal fluid pressure. Eight pigs received 2 mg/kg phentolamine in 10 ml saline, and 13 pigs served as control animals. During high intracranial pressure occurring after blood loss, cerebral perfusion pressure (CPP) (p < 0.01) and cerebral blood flow (p < 0.01) decreased in both groups. Cerebrovascular resistance increased (p < 0.05) in the control group and decreased (p < 0.005) in the phentolamine-treated group. The cerebrovascular resistance was significantly lower in the phentolamine-treated group (p < 0.05) than in the control group. Cerebrovascular resistance increased at lower CPPs in the control group (linear correlation, r = 0.39, p < 0.01) and decreased with decreasing CPP in the phentolamine-treated group (linear correlation, r = 0.76, p < 0.001). CONCLUSIONS: This study shows that the deleterious effects on cerebral hemodynamics induced by blood loss in combination with high intracranial pressure are inhibited by alpha-adrenergic blockade. This suggests that these responses are caused by alpha-adrenergically mediated cerebral vasoconstriction.