Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach

The role that nitric oxide, an endothelium-derived relaxing factor, may play in the regulation of gastric mucosal defence was investigated by assessing the potential protective actions of this factor against the damage caused by ethanol in an ex vivo chamber preparation of the rat stomach. Topical application of glyceryl trinitrate and sodium nitroprusside, which have been shown to release nitric oxide, markedly reduced the area of 70% ethanol-induced hemorrhagic damage. Topical application of a 0.01% solution of authentic nitric oxide also significantly reduced the severity of mucosal damage. Pretreatment with indomethacin precluded the involvement of endogenous prostaglandins in the protective effects of these agents. The protective effects of NO were transient, since a delay of 5 minutes between NO administration and ethanol administration resulted in a complete loss of the protective activity. The protection against ethanol afforded by 10 micrograms/ml nitroprusside could be completely reversed by intravenous infusion of either 1% methylene blue or 1 mM hemoglobin, both of which inhibit vasodilation induced by nitric oxide. Intravenous infusion of 1% methylene blue significantly increased the susceptibility of the mucosa to damage induced by topical 20% ethanol. These results indicate that ethanol-induced gastric damage can be significantly reduced by nitric oxide. The mechanisms underlying the protective actions of nitric oxide are unclear, but may be related to its vasodilator or anti-aggregatory properties.     

Endothelium-derived relaxing factor contributes to the regulation of endothelial permeability.

To determine whether endothelium-derived relaxing factor (EDRF) contributes to the regulation of endothelial permeability, the transendothelial flux of 14C-sucrose, a marker for the paracellular pathway across endothelial monolayers (Oliver, J. Cell. Physiol. 145:536-548, 1990), was examined in monolayers of bovine aortic endothelial cells grown on collagen-coated filters. The permeability coefficient of 14C-sucrose was significantly decreased by 10(-3) M 8-Bromoguanosine 3',5'-cyclic monophosphate or by 5 x 10(-6) M glyceryl trinitrate, an activator of soluble guanylate cyclase. Depletion of L-arginine from endothelial monolayers increased 14C-sucrose permeability from 3.21 +/- 0.59 to 3.88 +/- 0.50 x 10(-5) cm.sec-1 (mean +/- SEM; n = 6; P < 0.05). The acute administration of 5 x 10(-4) M L-arginine to monolayers depleted of this amino acid decreased 14C-sucrose permeability from 2.91 +/- 0.27 to 2.52 +/- 0.26 x 10(-5) cm.sec-1 (n = 11; P < 0.05). 14C-sucrose permeability was increased by 10(-7) M bradykinin and this effect was enhanced by the presence of each one of the following compounds: 10(-5) M methylene blue, 4 x 10(-6) M oxyhemoglobin, 5 x 10(-4) M NG-methyl-L-arginine or 5 x 10(-4) M N omega-nitro-L-arginine. These results suggest that EDRF contributes to the sealing of the endothelial monolayer and that EDRF released by bradykinin acts as a feedback inhibitor attenuating the increase in endothelial permeability induced by this peptide. Because endothelial cells have the ability to contract and relax and possess guanylate cyclase responsive to nitric oxide, our results suggest that EDRF decreases 14C-sucrose permeability by relaxing endothelial cells, thereby narrowing the width of endothelial junctions.     

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10⁻⁷ M: SHR +37 ± 2 [SE] vs. WKY +32 ± 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K⁺ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.     

Arterial remodeling in response to increased blood flow using a constituent-based model

Previous theoretical models of arterial remodeling in response to changes in blood flow were based on the assumption that material properties of the arterial wall remain unchanged during the remodeling process. According to experimental findings, however, remodeling due to increased flow is accompanied by alteration in the structural properties of elastin, which results in a decrease in its effective elastic stiffness. To account for these effects, we propose a predictive model of arterial remodeling hypothesizing that the variation in mechanical properties of elastin is initiated and driven by the deviation of the intimal shear stress from its baseline value. Geometrical remodeling restores the wall stress distribution as it was under normal flow conditions. A constrained mixture approach is followed. Artery is modeled as a thick-walled cylindrical tube made of non-linear, elastic, anisotropic and incompressible material. Data for a rabbit thoracic aorta have been employed. At the final adapted state, the model predicts a non-monotonic dependence of arterial compliance on the magnitude of flow. This result is in agreement with available experimental data in the literature.     

Cyclooxygenase-pathway participates in the regulation of regional cerebral blood flow in response to neuronal activation under normo- and hypercapnia

The present study was designed to investigate whether cyclooxygenase products are involved in the regulation of the regional cerebral blood flow, evoked by somatosensory activation (evoked rCBF) under normo- and hypercapnia. Indomethacin (IMC) was used as cyclooxygenase inhibitor. It was applied intravenously (i.v., 10 mg/kg/h) in two experimental protocols-before hypercapnia (i) and after hypercapnia (ii). Somatosensory activation was induced by electrical hind paw stimulation (5 Hz frequency, 5 s duration, 1.5 mA). The evoked rCBF-response was measured in alpha -chloralose anesthetized rats using laser-Doppler flowmetry. IMC abolished completely the effect of hypercapnia on the baseline level of CBF. The drug reduced significantly evoked rCBF-response also. The inhibitory effect of IMC on evoked rCBF-response is better expressed under normocapnia (approximately 70%) than that under hypercapnia (approximately 40%). After IMC application, the normalized evoked rCBF curves peaked earlier as compared to that before its application (P<0.05), although the rise time of 0.5 s was nearly constant regardless of stimulus frequency. In conclusion, the results suggest a participation of IMC-sensitive and cyclooxygenase-dependent mechanisms in the regulation of evoked rCBF, induced by somatosensory stimulation.     

Contributions of endothelium-derived relaxing factors to control of hindlimb blood flow in the mouse in vivo

We determined the contributions of various endothelium-derived relaxing factors to control of basal vascular tone and endothelium-dependent vasodilation in the mouse hindlimb in vivo. Under anesthesia, catheters were placed in a carotid artery, jugular vein, and femoral artery (for local hindlimb circulation injections). Hindlimb blood flow (HBF) was measured by transit-time ultrasound flowmetry. N(omega)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg plus 10 mg x kg(-1) x h(-1)), to block nitric oxide (NO) production, altered basal hemodynamics, increasing mean arterial pressure (30 +/- 3%) and reducing HBF (-30 +/- 12%). Basal hemodynamics were not significantly altered by indomethacin (10 mg x kg(-1) x h(-1)), charybdotoxin (ChTx, 3 x 10(-8) mol/l), apamin (2.5 x 10(-7) mol/l), or ChTx plus apamin (to block endothelium-derived hyperpolarizing factor; EDHF). Hyperemic responses to local injection of acetylcholine (2.4 microg/kg) were reproducible in vehicle-treated mice and were not significantly attenuated by L-NAME alone, indomethacin alone, L-NAME plus indomethacin with or without co-infusion of diethlyamine NONOate to restore resting NO levels, ChTx alone, or apamin alone. Hyperemic responses evoked by acetylcholine were reduced by 29 +/- 11% after combined treatment with apamin plus charybdotoxin, and the remainder was virtually abolished by additional treatment with L-NAME but not indomethacin. None of the treatments altered the hyperemic response to sodium nitroprusside (5 microg/kg). We conclude that endothelium-dependent vasodilation in the mouse hindlimb in vivo is mediated by both NO and EDHF. EDHF can fully compensate for the loss of NO, but this cannot be explained by tonic inhibition of EDHF by NO. Control of basal vasodilator tone in the mouse hindlimb is dominated by NO.     

Frataxin participates to the hypoxia-induced response in tumors

Defective expression of frataxin is responsible for the degenerative disease Friedreich''s ataxia. Frataxin is a protein required for cell survival since complete knockout is lethal. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. We therefore sought to investigate the pathways through which frataxin enhances stress resistance in tumor cells. We found that frataxin expression is upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, frataxin upregulation in response to hypoxia is dependent on hypoxia-inducible factors expression and modulates the activation of the tumor-suppressor p53. Importantly, we show for the first time that frataxin is in fact increased in human tumors in vivo. These results show that frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could have a critical role in tumor cell survival and/or progression.     

Effects of L-glutamic acid on acid secretion and mucosal blood flow in the rat stomach

The effect of intravenous administration of L-glutamic acid (L-Glu) on gastric acid secretion and gastric mucosal blood flow (GMBF) in anesthetized rats were investigated. Infusion with synthetic L-Glu alone had no effect on spontaneous acid secretion. However, L-Glu reduced histamine- (2 mg/kg/hr) or oxotremorine- (1 microg/kg/hr) stimulated acid secretion, whereas L-Glu had no effect on acid secretion induced by pentagastrin (8 microg/kg/hr). Furthermore, this inhibitory effect of L-Glu on histamine- or oxotremorine-stimulated acid secretion was blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist. The effect of L-Glu on gastric mucosal microcirculation in the anesthetized rats was evaluated by using Laser Doppler Flowmetry (LDF). The results showed that L-Glu did not significantly reduce both mucosal and serosal blood flow in stomach. No significant modulatory effect on histamine- or oxotremorine-stimulated increase in GMBF was noted after infusion with L-Glu. It is concluded that L-glutamic acid is capable of the modulating of gastric acid secretion via ionotropic non-NMDA receptors, but do not affect on GMBF. However, L-glutamic acid showed no effect on acid secretion by itself.     

血清反应因子参与RhoA诱导的人血管内皮细胞肌动蛋白骨架重构

为进一步阐明RhoA调控人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)肌动蛋白骨架重构的分子机制,用逆转录病毒感染并筛选出稳定表达持续活化型RhoA(Q63LRhoA)和主导抑制型RhoA(T19NRhoA)的HUVECs。应用免疫组化和Western blot方法分析去血清前后HUVECs血清反应因子(serum response factor,SRF)的表达及定位,Rhodamine-Phalloidine染色观察F-actin动态变化。结果显示,Q63LRhoA组细胞核中SRF表达增加,F-actin重排形成大量应力纤维;T19NRhoA组中SRF表达较弱,F-actin无明显改变,无应力纤维形成。去血清后,正常HUVECs(对照组)和感染细胞中SRF的表达均显著增加,但其亚细胞定位明显不同。对照组去血清培养3d,SRF主要定位在细胞核,去血清培养5d,SRF出核转位入细胞浆。Q63LRhoA组SRF发生核滞留,不随去血清培养时间延长发生出核转位现象。T19NRhoA组SRF的表达主要定位于细胞核周。对照组去血清培养3d,F-actin表达增加,同时形成大量应力纤维,去血清培养5d,细胞F-actin表达下调,应力纤维解聚。Q63LRhoA组F-actin重构持续发生并形成大量应力纤维,但不随去血清培养时间延长发生明显解聚。而T19NRhoA组F-actin表达不随去血清时间延长而增加。上述结果提示,RhoA介导HUVECs F-actin的重构与SRF的核转位现象密切相关。     

NaCl induced ornithine decarboxylase and DNA synthesis in rat stomach mucosa

A sensitive method is described that detects an alteration in the structure of tRNA that is caused by cadmium but not by magnesium or zinc ions. The chromatographic system, RPC-5, separates Drosophila tyrosyl-tRNA into two fractions. These two isoacceptors differ by a single position in the anticodon where either a guanosine or queuine resides. Cadmium ions apparently interact with the tRNA and prevent the chromatographic separation. This is the first instance where cadmium is shown to cause a selective change in nucleic acid structure. The RPC-5 system seems to be uniquely useful in detecting such a change.     

Endothelium-derived hyperpolarizing factor in coronary microcirculation: responses to arachidonic acid

In coronary resistance vessels, endothelium-derived hyperpolarizing factor (EDHF) plays an important role in endothelium-dependent vasodilation. EDHF has been proposed to be formed through cytochrome P-450 monooxygenase metabolism of arachidonic acid (AA). Our hypothesis was that AA-induced coronary microvascular dilation is mediated in part through a cytochrome P-450 pathway. The canine coronary microcirculation was studied in vivo (beating heart preparation) and in vitro (isolated microvessels). Nitric oxide synthase (NOS) (N(omega)-nitro-L-arginine, 100 microM) and cyclooxygenase (indomethacin, 10 microM) or cytochrome P-450 (clotrimazole, 2 microM) inhibition did not alter AA-induced dilation. However, when a Ca(2+)-activated K(+) channel channel or cytochrome P-450 antagonist was used in combination with NOS and cyclooxygenase inhibitors, AA-induced dilation was attenuated. We also show a negative feedback by NO on NOS-cyclooxygenase-resistant AA-induced dilation. We conclude that AA-induced dilation is attenuated by cytochrome P-450 inhibitors, but only when combined with inhibitors of cyclooxygenase and NOS. Therefore, redundant pathways appear to mediate the AA response in the canine coronary microcirculation.     

Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis

This study evaluated the effects of 25% ethanol, a mild irritant, on endogenous prostanoid synthesis in the rat stomach before and after exposure to oral 100% ethanol. Rats received water or 25% ethanol orally. After 15 min, a portion of each group was sacrificed and the remaining animals treated with 100% ethanol prior to sacrifice one minute later. Microsomal membrane fractions were prepared from the glandular gastric mucosa in all groups and incubated with 14C arachidonic acid in the presence of cofactors. Endogenous mucosal prostanoid synthesis was analyzed by radiochromatography and results correlated with the presence or absence of gastric injury macroscopically. Prostanoids measured included PGI2, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane A2. Additional experiments were performed in like manner to those just described with the exception that indomethacin (5 mg/kg intraperitoneally) pretreatment was rendered. Stomachs exposed to water or 25% ethanol alone demonstrated a modest and equivalent level of synthesis of all prostanoids measured. Exposure to 100% ethanol (with and without mild irritant pretreatment) significantly increased prostanoid synthesis (especially PGI2, PGF2 alpha, and PGE2) compared with stomachs exposed to water or 25% ethanol alone; only mild irritant treated mucosa was protected from injury by 100% ethanol. Indomethacin pretreatment reversed the increased prostanoid synthesis in mucosa exposed to 100% ethanol, with or without mild irritant pretreatment, and partially reversed the protective effect of 25% ethanol. Other experiments using tissue slices in which perturbations in mucosal levels of prostanoids were measured by radioimmunoassay under identical experimental conditions exhibited similar results. These data dispute the notion that adaptive cytoprotection is mediated by increased endogenous prostanoid synthesis. The partial reversal of this process by indomethacin was most likely secondary to some other action of this agent, such as a reduction in gastric blood flow, rather than direct effects on prostanoid synthesis.