14C]-2-deoxyglucose uptake studies in Leydig cells

The present study examines several aspects of [14C]-2-deoxyglucose uptake by rat Leydig cells: the characteristics of uptake by Leydig cells and type of inhibition by cytochalasin B, the effect of other drugs on [14C]-2-deoxyglucose uptake, the specificity of the glucose transporter for other substrates and the effect of various hormones. The apparent Km and V for [14C]-2-deoxyglucose were 0.4 mM and 0.17 mumol/min/10(6) cells, respectively. The inhibition of [14C]-2-deoxyglucose by cytochalasin B was competitive in nature, with an apparent Ki of 0.28 microM. Both phloretin and phlorizin (1.0-50 microM) inhibited [14C]-2-deoxyglucose uptake in a dose-dependent manner. Although D-glucose inhibited [14C]-2-deoxyglucose uptake by Leydig cells, L-glucose was ineffective, reflecting the stereospecificity of the glucose transporter for the former substrate. Various hormones, including: insulin, LH, 17 beta-estradiol or growth hormone which have been reported to stimulate glucose uptake in cells from other tissues, had no effect on [14C]-2-deoxyglucose uptake in Leydig cells under the current conditions. It remains to be determined how glucose uptake in Leydig cells is regulated.     

Downregulation of protein kinase C and insulin-stimulated 2-deoxyglucose uptake in rat adipocytes by phorbol esters, glucose, and insulin

Phorbol esters translocatively activate and subsequently downregulate protein kinase C and insulin-stimulated glucose uptake in rat adipocytes. This study examined the possibility that other translocative activators of protein kinase C in rat adipocytes, e.g., insulin and glucose, provoke similar downregulating effects. Pretreatment of rat adipocytes for 20-24 h with phorbol esters, 3 nM insulin, 20 mM glucose, or 3 nM insulin plus 20 mM glucose resulted in concomitant decreases in protein kinase C and insulin-stimulated (or phorbol ester-stimulated) [3H]-2-deoxyglucose uptake. Downregulating effects of glucose on protein kinase C and insulin-stimulated [3H]-2-deoxyglucose uptake were also evident within 30 min in adipocytes freshly incubated in medium containing 5-20 mM, rather than 0, glucose. These findings confirm that protein kinase C is required during insulin-stimulated glucose uptake and raise the possibility that downregulation of protein kinase C by continued translocative activation of the enzyme may contribute (along with other factors) to impaired responsiveness of the glucose transport system after prolonged insulin and/or glucose treatment.     

Modulation of insulin action on 2-deoxyglucose uptake by chloroquine in chick embryo fibroblasts

Blazar et al. recently found that chloroquine therapy decreased intravenous insulin requirements in a case of extreme insulin resistance. However, no relationship has been shown to exist between insulin degradation and the stimulation of glucose uptake. In this study we investigate the action of insulin on glucose uptake by the ability of this hormone to stimulate 2-deoxyglucose. The effect on alpha-aminoisobutyrate uptake, which is known to be insulin sensitive, is also investigated. Cell-associated 125I-labeled insulin and trichloroacetic acid-soluble and -precipitable substances were measured in parallel. Chloroquine increased insulin-stimulated uptake of 2-deoxyglucose and alpha-aminoisobutyrate. Three hours were required for this effect to appear, and it did not depend on DNA synthesis. Chloroquine also increased cell-associated insulin and slightly decreased the percentage of trichloroacetic acid-soluble products. Methylamine affected neither nutrient uptake processes nor insulin binding and degradation; however, it did abolish the effect of chloroquine on these parameters. These data suggest that in chick embryo fibroblasts a relationship may exist between the increase in undegraded cell-associated insulin and the ability of the hormone to stimulate sugar and amino acid uptake.     

Cellular and regional localization of pentobarbital-2-14C by radioautography of selected areas of mouse brain at loss and return of withdrawal response.

Regional and cellular distribution of pentobarbital-14C in mouse brain was determined by frozen-section radioautographic methods. The mice were studied at the times of loss (WRL) and return (WRR) of withdrawal response following a single intravenous dose of either 40 or 50 mg/kg body weight. At WRL, grey matter areas had higher concentrations of pentobarbital-2-14C than white matter. At WRR grey matter concentrations were not altered, but white matter areas were now similar to the grey. At WRL pentobarbital concentration was 55 per cent higher in large pyramidal cells in the parietal cortex than in surrounding neuropil. At WRL hippocampal pyramidal cell bodies (stratum pyramidalis) and glial cells in corpus callosum had pentobarbital levels similar to that of surrounding neuropil. Levels in the neuropil of these three areas were higher at WRR than at WRL. Lipid-rich compartments had higher pentobarbital concentrations at WRR than at WRL. The results suggest that return of consciousness after pentobarbital anesthesia is associated with intracerebral redistribution of pentobarbital even while there is continuing uptake into brain. (Key words: Brain, pentobarbital uptake; hypnotics, barbiturates, pentobarbital; pharmacokinetics, pentobarbital uptake.).     

Uptake kinetics of14C L-leucine and14C L- and14C D-methionine in rat brain and incorporation into protein

Because the amino acids¹¹C L-leucine,¹¹C L-methionine, and¹¹C D-methionine are used for examinations of brain tumors with positron emission tomography (PET), the uptake of the corresponding¹⁴C substances and their incorporation into protein was studied in the rat brain. The uptake of all three substances from the plasma, across the bloodbrain barrier, and into the brain took place quite quickly; return to the plasma seemed negligible. Incorporation into protein took place much more slowly.     

Insulin response in individual tissues of control and gold thioglucose-obese mice in vivo with [1-14C]2-deoxyglucose

The dose-response characteristics of several glucose-utilizing tissues (brain, heart, white adipose tissue, brown adipose tissue, and quadriceps muscle) to a single injection of insulin have been compared in control mice and mice made obese with a single injection of gold thioglucose (GTG). Tissue content of [1-14C]2-deoxyglucose 6-phosphate and blood disappearance rate of [1-14C]2-deoxyglucose (2-DG) were measured at nine different insulin doses and used to calculate rates of 2-DG uptake and phosphorylation in tissues from control and obese mice. The insulin sensitivity of tissues reflected in the ED50 of insulin response varied widely, and brown adipose tissue was the most insulin-sensitive tissue studied. In GTG-obese mice, heart, quadriceps, and brown adipose tissue were insulin resistant (demonstrated by increased ED50), whereas in white adipose tissue, 2-DG phosphorylation was more sensitive to insulin. Brain 2-DG phosphorylation was insulin independent in control and obese animals. The largest decrease in insulin sensitivity in GTG-obese mice was observed in brown adipose tissue. The loss of diet-induced thermogenesis in brown adipose tissue as a result of the hypothalamic lesion in GTG-obese mice could be a major cause of insulin resistance in brown adipose tissue. Because brown adipose tissue can make a major contribution to whole-body glucose utilization, insulin resistance in this tissue may have a significant effect on whole-animal glucose homeostasis in GTG-obese mice.     

Effect of peroperative hypertension on 125I-albumin uptake by vein graft media

Aortocoronary bypass, in use for over 20 years, is followed by lesions in 80% of vein grafts after 10 years. In 8 patients (mean age 60, SD = 7.8) before cardiopulmonary bypass, segments of saphenous vein were harvested, and following hydraulic distension (760 mmHg x 10 min), stripped of adventitia, de-endothelialized, cannulated, immersed in oxygenated Krebs solution pH 7.4 37 deg C, and pressurized (100 mmHg) with 4% 125I-albumin-Evans blue for two hours. The samples were then frozen, and serially sectioned in the plane of the lumenal surface. The radioactivity of the 20 microns-thick sections was then determined, and expressed as a tissue: labelled solution concentration ratio (cpm unit volume wet tissue/cpm unit volume labelled solution). Distended portions had a higher mean transmural concentration ratio, as compared with the undistended portion; the ratio distended/undistended was 1.23, SD = 0.17, n = 8, p less than 0.01 (paired t test). This suggests alteration in the transport properties of vein interstitium by hypertension. Random histologic examination of native veins revealed no structural damage apart from age-related dystrophy of elastic fibres and intimal and medial fibrosis in grossly normal veins. These results suggest that the quality of the vein used, combined with hypertension during its preparation, may be factors in the genesis of postoperative graft failure.     

金黄散药膏用于碘海醇静脉外渗的效果研究

目的评价金黄散药膏外敷干预碘海醇静脉外渗损伤的效果。方法选取低渗性含碘造影剂碘海醇以16只新西兰大白兔为研究对象,模拟造影剂静脉外渗损伤构建动物模型。将大白兔随机分为金黄散湿敷实验组和注射用水湿敷对照组,观察金黄散治疗造影剂静脉外渗损伤的疗效。结果湿敷后24h及48h,实验组局部肿胀面积显著小于对照组(均P<0.01);病理检测显示充血及炎细胞浸润程度显著低于对照组(均P<0.01)。结论金黄散湿敷能有效促进造影剂静脉外渗造成组织肿胀的消退,改善组织充血程度及炎细胞浸润。     

金黄散药膏用于碘海醇静脉外渗的效果研究

目的评价金黄散药膏外敷干预碘海醇静脉外渗损伤的效果。方法选取低渗性含碘造影剂碘海醇以16只新西兰大白兔为研究对象,模拟造影剂静脉外渗损伤构建动物模型。将大白兔随机分为金黄散湿敷实验组和注射用水湿敷对照组,观察金黄散治疗造影剂静脉外渗损伤的疗效。结果湿敷后24h及48h,实验组局部肿胀面积显著小于对照组（均P〈0.01）;病理检测显示充血及炎细胞浸润程度显著低于对照组（均P〈0.01）。结论金黄散湿敷能有效促进造影剂静脉外渗造成组织肿胀的消退,改善组织充血程度及炎细胞浸润。     

Effect of N-acetylsphingosine (C2) and the ceramidase inhibitor (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol on the regulation of Sertoli cell function.

In the present study, a possible role of ceramide in the regulation of Sertoli cell function was investigated. Intracellular ceramide levels were increased by adding N-acetylsphingosine (C2) or by inhibiting ceramidase with (1 S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP). Cultured Sertoli cells were stimulated for 3 days with different doses of C2, MAPP, and their corresponding inactive analogs. The effect of these drugs was evaluated along four well-known Sertoli cell parameters: lactate and transferrin secretion, gamma-glutamyl transpeptidase (gamma-GTP) activity, and estradiol production. C2 and MAPP increased lactate production and decreased transferrin secretion. The inactive analogs did not produce any effect. In FSH (follicle-stimulating hormone)-stimulated cultures, C2 and MAPP produced a further increment in lactate production and decreased FSH-stimulated transferrin secretion. No effect was observed under basal or FSH-stimulated gamma-GTP activity, and both treatments decreased estradiol production in response to FSH. Results obtained in dbcAMP (dibutyryladenosine 3':5'-cyclic monophosphate)-stimulated cultures suggest that the observed effects of ceramide on transferrin secretion are secondary to a decrease in cAMP production, whereas the effects of ceramide on lactate and estradiol productions are post-cAMP formation regulatory events. In summary, our results show that ceramide can regulate Sertoli cell function. Similar to what has been observed for other signaling molecules, ceramide can interact with the FSH-dependent pathway, but the precise steps involved in this interaction are still unknown.     

The uptake of ISO-flurane by the foetal lamb in utero: Effect on regional blood flow

Because isoflurane has recently been approved for clinical use in anaesthesia, we have studied the effect of this agent in the foetus using the pregnant ewe as an animal model. Eight pregnant ewes of 120–125 days gestation were surgically prepared with indwelling catheters and tracke-ostonty. Prior to anaesthesia, labelled microspheres were injected into the foetal circulation to determine cardiac output and regional blood flows to all organs. The ewes were anaesthetized with a constant inspired concentration of 2 .0 per cent isoflurane in oxygen. Blood samples were drawn to construct an uptake curve for both mother and foetus. At 60 and 96 minutes of anaesthesia, microspheres were injected into the foetal circulation to measure changes in organ blood flow from the control period. Isoflurane crossed the placenta and appeared in the foetal circulation within two minutes. By 96 minutes, maternal and foetal arterial levels were 116.3 ± 9.9 and 99.3 ± 9.1 mEqIL (0.98 vol% and 0.75 vol%). There were no significant changes in foetal blood pressure or pulserate but foetal pH decreased significantly from 7,39 ± 0.02 to 7.26 ± 0.2 (mean ± SEM) and base excess decreased from -1.1 ± 1.5 to -6.2 ± 0.7. Foetal cardiac index decreased from 390.8 ± 26 9ml-kg^-1 min^-1 to 292.0 ± 13.8 after 96 minutes of anaesthesia. There were no significant changes in any of the maternal cardiovascular or acid-base parameters. In the foetal lamb, isoflurane anaesthesia produces foetal acidosis and decreases foetal cardiac index after 96 minutes of anaesthesia.     

异丙酚对大鼠中枢多巴胺再摄取的抑制作用

目的　观察异丙酚麻醉的大鼠中枢神经系统纹状体中多巴胺转运蛋白的变化,了解异丙酚麻醉时中枢多巴胺增加的调控机制。方法（１）正常ＳＤ大鼠２４只随机分为４组,分别腹腔注射异丙酚 ５０ｍｇ／ｋｇ、７５ｍｇ／ｋｇ、１００ｍｇ／ｋｇ和同容积 １０％脂肪乳剂作对照,腹腔注射后 １０分钟动物断头处死,取脑组织做脑连续切片,进行放射自显影研究;（２）ＳＤ大鼠６只,断头处死迅速取脑组织做脑连续切片,用~（１２５）Ｉ－β－ＣＴＴ为配基做放射受体结合体外抑制实验。结果　腹腔注射１００ｍｇ／ｋｇ异丙酚能明显抑制~（１２５） Ｉ－β－ＣＩＴ与纹状体中多巴胺转运蛋白结合（Ｐ＜０．０５）。体外抑制实验随异丙酚浓度增加,与脑组织多巴胺转运蛋白结合的~（１２５） Ｉ－β－ＣＩＴ的放射活性明显下降。结论异丙酚致中枢多巴胺增加与异丙酚抑制多巴胺转运蛋白对突触中多巴胺的再摄取有关。     

微管功能变化对C6胶质瘤细胞葡萄糖摄取的影响

目的 观察微管功能变化对C6胶质瘤细胞葡萄糖摄取的影响。方法 用影响微管功能的药物长春花碱(抑制微管聚合)、紫杉醇(促进微管聚合)与C6胶质瘤细胞在37℃下孵育3h后，再用含125μmol／L未标记2—脱氧葡萄糖(2—deoxy—D-glucose，2—DOG)和37kBq[^3H]2-DOG的磷酸盐缓冲溶液(PBS)1ml室温下孵育5min。用液闪计数分析仪检测C6细胞葡萄糖摄取。GLUT1cDNA转染C6胶质瘤细胞后再与紫杉醇作用，观察转染后C6细胞的紫杉醇刺激葡萄糖摄取。结果 长春花碱抑制C6细胞葡萄糖摄取24％-36％。紫杉醇刺激葡萄糖摄取增加16％-23％；紫杉醇刺激GLUT1转染C6胶质瘤细胞的2—DOG摄取增加约35％。结论 微管功能变化与C6细胞葡萄糖摄取及葡萄糖转运体介导的葡萄糖转运过程密切相关。     

Effect of estrogen and androgen administration on alpha-difluoromethylornithine-enhanced putrescine uptake by the rat prostate

Radiolabeled putrescine is a potential imaging agent for carcinoma of the prostate. The intrinsically high uptake of putrescine into the rat prostate and prostatic carcinoma can be further stimulated by pretreatment of the animals with alpha-difluoromethylornithine (DFMO) uptake, a polyamine synthesis inhibitor. Pharmacological castration of the animals followed by androgen stimulation and DFMO pretreatment achieved a 30:1 prostate/muscle ratio of 14C-putrescine uptake in elderly rats.     

7-N -(2-([2-(Gamma-L-glutamylamino)-ethyl]-dithio)-ethyl)-mitomycin C (KW-2149) is more active than mitomycin C on chemonaive and drug-resistant urothelial carcinoma cells

This in vitro study aimed to investigate the cytotoxic activity of 7-N-(2-([2-(gamma-l-glutamylamino)ethyl]dithio)ethyl)-mitomycin C (KW-2149) versus mitomycin C (MMC) against cell lines from human transitional cell carcinoma (TCC). Direct cytotoxicity of the two drugs was measured employing a colorimetric cytotoxicity assay on chemonaive and chemoresistant cancer cell populations. The results revealed that all cell lines (n= 19) were significantly more inhibited by treatment (2 h, 96 h) with KW-2149 than by MMC (P < 0.03–0.001). pH 6.0 decreased the stronger activity of KW-2149 (P < 0.013–0.004). Creatinine ≥10 mmol/l and nitrosourea ≥100 mg/l also inhibited the activity of KW-2149 significantly. Tumor cells with relative drug-resistance against MMC (RT112-MMC: 55-fold) exerted minor cross-resistance to KW-2149 (fourfold). In conclusion, the present in vitro data suggest KW-2149 to be a superior drug for intravesical therapy of patients with primary or recurrent superficial bladder carcinoma. Since pH and concentrations of creatinine and nitrosourea influence the activity of KW-2149, patients are supposed to profit from neutralizing the urinary pH and enhanced diureses. Received: 5 February 1998 / Accepted: 15 April 1998     

Electroencephalography (EEG) after introduction of water soluble contrast media into cerebrospinal fluid (CSF)

Summary Thirty-six patients undergoing Dimer X or Amipaque ventriculography or myelography underwent electroencephalographic investigations. In most cases EEG alterations were found. They ranged from general slowing to seizure potentials. These findings and their causes are discussed.