Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

加替沙星-壳聚糖纳米粒胶联羊膜药膜的制备及体外实验研究

目的制备一种新型生物多功能复合材料——加替沙星．壳聚糖纳米粒胶联羊膜药膜,并观察其体外缓释性能及抑菌作用。方法采用离子胶联法制备加替沙星－壳聚糖纳米粒并检测其表征,然后将栽药纳米粒加入到纤维蛋白胶中,混匀后再与羊膜胶联制成加替沙星－壳聚糖纳米粒胶联羊膜药膜,光镜及扫描电镜下观察其形态学特征,高效液相色谱法测定其中加替沙星的浓度,微量肉汤稀释法检测其体外抑菌活性。结果加替沙星纳米粒的粒径为291±33nm,载药量为10．46％,包封率为61．32％。载有纳米药物的纤维蛋白胶与羊膜基底面粘合紧密,呈圆形的加替沙星纳米粒附着在胶原纤维的网状结构上。在体外,加替沙星－壳聚糖纳米粒胶联羊膜药膜可持续释药达15天,对金黄色葡萄球菌和铜绿假单胞菌的最低抑菌浓度分别为0．05μg／ml和0．42μg／ml。结论加替沙星－壳聚糖纳米粒胶联羊膜药膜在体外具有良好的缓释性能及抑茵作用。     

Influence de l'anaérobiose et de différentes tensions d'oxygène sur la libération d'histamine dans la réaction anaphylactiquein vitro

Summary The influence of anaerobiose and of varied oxygen tensions upon respiration and histamine release during anaphylactic reaction in guineapig lung slicesin vitro was studied.No histamine was liberated in the absence of oxygen. With increasing oxygen tensions, increasing histamine quantities were released; these quantities reached the control values when the atmosphere contained 10% oxygen.These results indicate that the mechanism of histamine release in the anaphylactic reaction is linked with the aerobic metabolism of the cell.

---

---

Boursicrs du Consclho Nacional de Pesquisas.     

Sulfhydryl oxidation induces calcium release from fragmented sarcoplasmic reticulum even in the presence of glutathione

Alcian blue and plumbagin induced transient Ca²⁺ release from fragmented sarcoplasmic reticulum. Dithiothreitol (DTT) and glutathione (GSH) partially blocked Ca²⁺ release induced by these oxidizing compounds. Pretreatment of alcian blue and plumbagin with DTT or GSH for more than 1 min was required to abolish the ability of the oxidizing compounds to release Ca²⁺. Mg²⁺ and ruthenium red completely blocked alcian blue-and plumbagin-induced Ca²⁺ release. These results suggest that oxidation of sulfhydryls on Ca²⁺ release channels induces Ca²⁺ release even in the presence of GSH in situ.     

Depression of spontaneous and ionophore-induced neurotransmitter release bySalmonella

Summary Exposure of frog neuromuscular junctions to heat-killed, lyophilizedSalmonella typhimurium (SR 11) produces an early increase in spontaneous transmitter release followed by depression of release and blockade of the obligatory release usually induced by ionophore X537A.This study was supported by the Office of Naval Research, Biophysics Section, NR 202-160, N00014-77-C-0630. X537A was a gift of Hoffman-LaRoche. I am greatly indebted to Dr R. McCallum for preparing the Salmonella material. J.A. Kuhn provided the technical assistance.     

Dynamic insulin secretion from perifused rat pancreatic islets

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 μM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 μM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K⁺ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED₅₀ = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED₅₀ = 89 nM) and DA (ED₅₀ = 2.2 μM). γ-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances. Received 16 January 1998; received after revision 6 May 1998; accepted 8 May 1998     

Flow-induced release of adenosine 5′-triphosphate from endothelial cells of the rat mesenteric arterial bed

Adenosine 5-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.     

Effect of a restricted diet on the in vitro glucose-induced insulin release of aging rats

To study the effect of a sudden loss of body weight on the -cell function of aging rats, basal and glucose-induced insulin secretion was measured in pancreatic islets obtained from young (2-month-old), adult (12-month-old) and aging (24-month-old) rats, either fed ad libitum or fed a restricted diet (50% caloric restriction). Basal insulin secretion was similar in islets of young, adult and older rats. Glucose stimulated insulin release was significantly reduced in aging rats as compared to young animals. Animals fed a restricted diet showed a prolonged and higher secretory rate during first phase release when compared to animals fed ad libitum.     

Release of labile cyclo-oxygenase products of arachidonic acid from kidney by endotoxin

Summary The possible release of prostaglandin (PG)-like substances was studied in isolated perfused kidneys from intact and from intrarenal endotoxin (Lipopolysaccharide-LPS)-injected rabbits, using the venous outflow superflow superfuse assay organ technique. Injection of LPS into the renal artery of an LPS-pretreated kidney caused a release of thromboxane A₂ (TXA₂) and prostacyclin (PGI₂)-like materials into the venous effluent as verified by the responses of the specific assay organs. No detectable release of these substances was found in the venous outflow of LPS-injected intact kidney. The possible role of labile cyclo-oxygenase products of arachidonic acid in the Shwartzman reaction is discussed.The authors are indepted to Upjohn, Klamazoo (USA) for the generous gift of PGI₂ and to Squibb, New Jersey (USA) for SQ 80338.     

Somatostatin reduces the release of colony-stimulating activity (CSA) from PHA-activated mouse spleen lymphocytes

Summary PHA-activated lymphocytes release colony-stimulating activity (CSA) for macrophage-granulocyte precursor cells (colony forming units, CFU_C) in the culture medium. Somatostatin, known to interfer with ribosomal protein synthesis, was demonstrated to reduce the release of CSA from PHA-treated mouse spleen lymphocytes.The technical assistance of Ulrike Wallner and Paulette Bais is thankfully acknowledged.     

Impaired amylase release from the parotid gland of rats treated with reserpine

Summary Using an automated system for the analysis of amylase, the release of this enzyme was compared in superfused parotid gland segments from control and reserpine treated rats. Stimulant-evoked amylase release was delayed and of smaller magnitude in the glands of the treated animals and a reduction of the transmembrane K⁺ gradient caused a smaller and short lasting reduction in Ach-evoked release of amylase in the glands from these animals.This work was supported by grants from the Cystic Fibrosis Research Trust (U.K.), Cystic Fibrosis Foundation (USA) and University of Missouri.     

Macrophage origin of platelet activating factor]

Platelet-activating factor (P.A.F.) is a mediator of anaphylaxis released from human and Rabbit basophils which causes aggregation of platelets and release of their vasoactive amines. We have induced the release of P.A.F. from Rat peritoneal cells (P.C.) with ionophore A 23187. After fractionation of P.C. on 5-15% Ficoll gradients, P.A.F. was obtained from macrophage-rich but not from mastocyte-rich fractions and from adherent cells but not from non adherent cells. These data suggest an important new function for the macrophage: aggregation of platelets and release of their vasoactive amines and others mediators of inflammation.     

Mitochondrial and cytosolic glutathione after depletion by phorone in isolated hepatocytes

Summary The glutathione content of cytosol and mitochondria of isolated hepatocytes was depleted by addition of a low concentration of phorone (0.5 mM) by 75% and 40% respectively. Different rates of replenishment indicate metabolic separation of cytosolic and mitochondrial glutathione pools. The release from hepatocytes occurred at a rate of about 8 nmol/g wet weight/min, both in controls and after phorone depletion.This work was supported by the Ministerium für Wissenschaft und Forschung. Nordrhein-Westfalen, and by Deutsche Forschungsgemeinschaft.     

Chronic sensory denervation reduces thrombin-stimulated endothelin release from aortic endothelial cells

The long-term (trophic) influence of perivascular nerves on the endothelium was investigated by measuring changes in thrombin-stimulated release of the potent vasoconstrictor, endothelin, after selective chronic denervation. Rat pups were treated with either guanethidine or capsaicin to destroy sympathetic or sensory nerves, respectively. The abdominal aortas from the rats at three months of age (5 pooled per experiment) were incubated with 4U thrombin/ml in medium for 24 h at 37°C, and the amount of endothelin released from the preparation determined by immunoassay. After neonatal sensory denervation there was a significant reduction in the thrombinstimulated release of endothelin compared to the controls (0.012±0.012 (4) compared to 0.063±0.012 (6), pmol/cm²/24 h, p<0.02). There was no change in endothelin release after sympathetic denervation. In summary, sensory nerves play a trophic role in the expression of endothelin in endothelial cells of the intima.     

Stress-induced increase in noradrenaline release in the rat hypothalamus assessed by intracranial microdialysis

Summary The hypothalamic microdialysis of conscious rats was used to investigate the effects of immobilization stress (20 min) on extracellular noradrenaline(NA) levels. The stress significantly increased NA levels relative to basal efflux by 106% and this elevation continued for 40 min after release from stress.     

The lack of an effect of cholinergic agonists on anterior pituitary prolactin production in vitro

Summary The addition of dopamine to anterior pituitary incubations resulted in a marked decrease (88% for³H prolactin and 69% for RIA prolactin) in prolactin release. Incubation with the cholinergic agonists carbacol, arecoline and nicotine resulted in no significant change in prolactin secretion.Supported in part by NSF Research grant No. BMS 74-17332.The authors appreciate receiving as a gift from the National Institute of Arthritis, Metabolic and Digestive Diseases, the rat prolactin used for iodination (RP-I₂) and standards (RP-1).     

Stress-induced increase in noradrenaline release in the rat hypothalamus assessed by intracranial microdialysis

The hypothalamic microdialysis of conscious rats was used to investigate the effects of immobilization stress (20 min) on extracellular noradrenaline (NA) levels. The stress significantly increased NA levels relative to basal efflux by 106% and this elevation continued for 40 min after release from stress.     

Peroxovanadate inhibits Ca2+ release from mitochondria

Mitochondria contain a specific Ca²⁺ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed. NAD⁺ hydrolysis and therefore Ca²⁺ release is possible when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxovanadate inhibits the specific Ca²⁺ release pathway. In mitochondria, peroxovanadate causes a complete loss of reduced glutathione, which is not accompanied by formation of glutathione disulfide, and a partial loss of protein thiols. In model reactions, peroxovanadate oxidizes reduced glutathione predominantly to the sulfonate derivative, but does not react with glutathione disulfide. When the vicinal thiols relevant for Ca²⁺ release are cross-linked, Ca²⁺ release is no longer inhibited by peroxovanadate. Conversely, pretreatment of mitochondria with peroxovanadate makes them insensitive to compounds promoting the disulfide state. These results suggest that peroxovanadate inhibits the prooxidant-induced Ca²⁺ release from mitochondria by (i) depleting mitochondria of reduced glutathione and (ii) oxidizing the vicinal thiols relevant for Ca²⁺ release to a state higher than disulfide, presumably the sulfonate state. The findings provide further insight into the regulation of Ca²⁺ release from intact mitochondria, and may be relevant for a better understanding of the action of peroxovanadate in cells, where the compound can be insulin mimetic. Received 28 March 2002; received after revision 8 May 2002; accepted 15 May 2002     

Inhibition of TSH-stimulated thyroid hormone release and potentiation of TRH-stimulated TSH release by indomethacin in perifusion systems of rat thyroids and pituitaries

Summary Using indomethacin (Ind), a prostaglandin, synthesis inhibitor, in vivo experiments in rats and in vitro experiments with perifusion systems of rat thyroids and pituitaries were conducted. After 35 days of intragastric infusion of Ind, serum TSH levels were markedly increased, the thyroid was swollen and, as a consequence, T₃ and T₄ levels were normal. The T₃ release from perifused rat thyroids under continuous stimulation with 10 mU/ml TSH was inhibited significantly (p<0.01) by 1.0×10^–6 M Ind. On the other hand, the TSH release from perifused rat pituitaries under TRH stimulation was enhanced conspicuously by Ind. It was concluded that Ind decelerated thyroid hormone release from the thyroid and accelerated TSH release from the pituitary in perifusion systems.     

Differences in the stimulation by calcium ionophore of juvenile hormone III release from corpora allata of solitarious and gregariousLocusta migratoria

Summary Incubation of the calcium ionophore A23187 resulted in an increase in the median rate of juvenile hormone III release by corpora allata (CA) of both gregarious and solitarious adultLocusta migratoria females at 3, 5 and 8 days after fledging. At all 3 datapoints, the enhancement of release rates was highly significant for CA from gregarious females but not significant for CA from solitarious females.