Preparation and in vivo evaluation of PEGylated spherulite formulations

Spherulites are multilamellar vesicles obtained by shearing a lamellar phase of lipids and surfactants. They consist of concentric bilayers of amphiphiles alternating with layers of aqueous medium in which hydrophilic drugs can be sequestered with high yield. To be useful for drug targeting applications, spherulites should be small and long circulating. The objectives of this work were threefold. First, the spherulite size was optimized to obtain a mean diameter of less than 300 nm. Second, the vesicle composition was adjusted to minimize in vitro leakage of internal content. Third, the spherulites were coated with 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy poly(ethylene glycol)] (DSPE-PEG) to impart them with a long half-life. Then, the PEGylated spherulites (Phospholipon 90G/Solutol HS15/cholesterol/DSPE-PEG 2000 or 5000) were loaded with 1-beta-d-arabinofuranosylcytosine (ara-C) and injected intravenously to rats. They were compared to uncoated spherulites and to an ara-C solution. The surface-modified vesicles exhibited long circulation times with areas under the blood concentration vs. time curve exceeding by 3.1- to 6.9-fold that of uncoated spherulites. Similarly, blood levels of ara-C encapsulated in PEGylated vesicles were higher than those of the controls, but they did not parallel the carrier pharmacokinetics. Two hours post-injection, most of the drug was cleared from the systemic circulation, reflecting rapid leakage of ara-C from the vesicles.     

Preparation and in vivo evaluation of PEGylated spherulite formulations

Spherulites are multilamellar vesicles obtained by shearing a lamellar phase of lipids and surfactants. They consist of concentric bilayers of amphiphiles alternating with layers of aqueous medium in which hydrophilic drugs can be sequestered with high yield. To be useful for drug targeting applications, spherulites should be small and long circulating. The objectives of this work were threefold. First, the spherulite size was optimized to obtain a mean diameter of less than 300 nm. Second, the vesicle composition was adjusted to minimize in vitro leakage of internal content. Third, the spherulites were coated with 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy poly(ethylene glycol)] (DSPE-PEG) to impart them with a long half-life. Then, the PEGylated spherulites (Phospholipon 90G/Solutol HS15/cholesterol/DSPE-PEG 2000 or 5000) were loaded with 1-β-d-arabinofuranosylcytosine (ara-C) and injected intravenously to rats. They were compared to uncoated spherulites and to an ara-C solution. The surface-modified vesicles exhibited long circulation times with areas under the blood concentration vs. time curve exceeding by 3.1- to 6.9-fold that of uncoated spherulites. Similarly, blood levels of ara-C encapsulated in PEGylated vesicles were higher than those of the controls, but they did not parallel the carrier pharmacokinetics. Two hours post-injection, most of the drug was cleared from the systemic circulation, reflecting rapid leakage of ara-C from the vesicles.     

Chelation therapy and vanadium: effect on reproductive organs in rats

Present investigation was planned to evaluate the therapeutic effectiveness of chelating agents against vanadium intoxication on blood and reproductive organs of rats. Male and female albino rats were injected vanadyl sulphate (7.5 mg/kg, po, for 21 days, 5 days in a week). Chelating agents tiron (T) alone and in combination with lipoic acid (LA), vitamin E (vit E) and selenium (Se) were given for 2 days/week. With the administration of vanadyl sulphate to rats fructose level in seminal vesicles was significantly (P< or =0.05) declined. The activities of alkaline phosphatase and adenosine triphosphatase were also decreased, whereas glycogen content and acid phosphatase activity increased in testis, seminal vesicles, ovaries and uterus after toxicant exposure. Significant changes in serum transaminases, serum alkaline phosphatase and lactate dehydrogenase were recouped by chelation therapy. Lipid peroxidation, reduced glutathione level and triglycerides levels altered significantly after exposure to vanadium in rats. The ultrastructural damage in spermatogenic stages in treated animals showed recovery pattern after therapy. Co-treatment with antioxidants restored these activities. The most effective combination was tiron + selenium followed by tiron + vitamin E, and tiron + lipoic acid.     

On the characterization of pH-sensitive liposome/polymer complexes

A randomly alkylated copolymer of N-isopropylacrylamide, methacrylic acid and N-vinyl-2-pyrrolidone was characterized with regard to its pH- and temperature-triggered conformational change. It was then complexed to liposomes to produce pH-responsive vesicles. Light scattering and differential scanning calorimetry experiments performed at neutral pH revealed that the polymer underwent coil-to-globule phase transition over a wide range of temperatures. At 37 degrees C and pH 7.4, although the polymer was water-soluble, Fourier transform infrared spectroscopy analysis showed that it was partly dehydrated. At acidic pH, the decrease in the lower critical solution temperature was accompanied by an increase in cooperativity degree of the phase transition. Complexation of copolymer to liposomes did not substantially influence its phase transition. The liposome/copolymer complexes were stable at neutral pH but rapidly released their contents under acidic conditions. The copolymer slightly increased liposome circulation time following intravenous administration to rats. The addition of poly(ethylene glycol) to the formulation had a detrimental effect on pH-sensitivity but enhanced substantially the circulation time.     

Effect of cisplatin on H+ transport by H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane

The effect of the potent anticancer drug cisplatin, cis-diamminedichloroplatinum (II) (CDDP), on H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane was examined. To measure H+ transport by vacuolar H+ -ATPase in renal brush-border membrane vesicles, we employed a detergent-dilution procedure, which can reorientate the catalytic domain of H+ -ATPase from an inward-facing configuration to outward-facing one. ATP-driven H+ pump activity decreased markedly in brush-border membrane prepared from rats two days after CDDP administration (5 mg/kg, i.p.). In addition, N-ethylmaleimide and bafilomycin A1 (inhibitors of vacuolar H+ -ATPase)-sensitive ATPase activity also decreased in these rats. The decrease in ATP-driven H+ pump activity was observed even at day 7 after the administration of CDDP. Suppression of ATP-driven H+ pump activity was also observed when brush-border membrane vesicles prepared from normal rats were pretreated with CDDP in vitro. In contrast with H+ -ATPase, the activity of Na+/H+ exchanger, which was determined by measuring acridine orange fluorescence quenching, was not affected by the administration of CDDP. These results provide new insights into CDDP-induced renal tubular dysfunctions, especially such as proximal tubular acidosis and proteinuria.     

Processing and secretion in the neurohypophysis. Stability of isolated secretory vesicles and role of internal pH.

A possible role of low pH in secretory vesicles for processing and secretion in the neurohypophysis was investigated. Subcellular fractionation of guinea-pig neural lobes revealed that a proton present in the membranes from this tissue could not be ascribed to secretory vesicles. However, a proton pump was found in coated microvesicles. Secretory vesicles isolated from rats and guinea pigs were stable under conditions known to lyse secretory vesicles from the adrenal medulla owing to the generation of a proton gradient. These results suggest that the internal pH of secretory vesicles from the neurohypophysis is closer to neutral than is the pH in chromaffin secretory vesicles. Processing of a neurophysin-glycopeptide intermediate from the biosynthesis of vasopressin in intact secretory vesicles incubated in vitro was activated by the addition of NH4Cl, known to increase the intravesicular pH. This activation of neurohormone processing was also apparent in isolated nerve endings incubated in the presence of NH4Cl, suggesting that NH4Cl can also be used to increase the intravesicular pH in intact nerve endings. However, NH4Cl did not affect the secretion of neurohormones, indicating that a low intravesicular pH is not important for exocytosis in the neurohypophysis. Our results indicate that a low pH generated during processing by mechanisms other than ATP-dependent proton transport may inhibit the processing enzymes, thereby preventing extensive breakdown of neurohormone precursors.     

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.     

老年大鼠血管α1肾上腺素受体及其亚型的改变

本工作用离体与整体实验方法,研究了老年(18月龄)与年轻(3月龄)大鼠血管中 α_1肾上腺素受体储备和 α_1(?)与 α_(1b)亚型比值的差别。在离体实验中用机械方法分离血管,用 Krebs 溶液灌流,在灌流液中加入 α_2和 β肾上腺素受体拮抗剂,然后用去甲肾上腺素(NE)激动 α_1受体。结果显示;老年大鼠主动脉、肾动脉和肠系膜动脉由去甲肾上腺素(NE)引起的最大收缩反应与年轻大鼠无显著差别,但浓度-效应曲线显著右移,功能性解离常数 K_A 值不变,而 K_A 与EC_(50)的比值减小。此外,在老年大鼠主动脉和肠系膜动脉血管,选择性 α_(1b)亚型拮抗剂 CEC对 NE 引起的缩血管效应的阻断作用显著减弱,硝苯吡啶(选择性阻断 α_(1a) 亚型的效应)对 NE 缩血管效应的阻断作用显著增强。整体实验显示老年大鼠硝苯吡啶的降血压作用比年轻大鼠增强,在用硝苯吡啶的基础上给予苯肾上腺素升血压作用减弱。上述结果提示:与年轻大鼠相比较,老年大鼠 α_(1-) 肾上腺素受体储备减少,α_(1a) 亚型相对 α_(1b)亚型的比率增高。     

Effect of glutathione depletion on urinary acidification in the rat

Glutathione (GSH) depletion by diethyl maleate (DEM) administration and its rapid repletion were associated with the development of a moderate acidosis in the rat. The acidosis observed after DEM treatment could be a consequence of an impairment of lactate metabolism. GSH-depleted rats also showed an increased urine pH and a higher bicarbonate fractional excretion compared with control rats. Renal bicarbonate excretion was magnified when blood bicarbonate levels were normalized by means of a bicarbonate infusion in GSH-depleted rats; however, the amount of bicarbonate excreted in the urine was a very small fraction (less than 5%) of the calculated filtered load. GSH-depleted rats failed to elevate the relation urine minus blood (U-B) pCO2 as compared with control rats when they were subjected to a high bicarbonate load to the distal portions of the nephron. All these data were consistent with a distal renal tubular acidosis due to GSH depletion which could participate in the maintenance of the systemic acidosis, although it is unlikely that it is the primary cause of the acidosis.     

Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 X 10(-5) M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in Vmax of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50-70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.     

In vitro and in vivo evaluation of proximal tubular acidification in aging rats

The normal aging process is accompanied by a progressive deterioration of renal function. We studied the kinetics of proximal tubular acidification of young (3 mo) and aging (22 mo) rats using in vivo and in vitro techniques. Blood acid-base parameters were similar in both groups. The maximum velocity of the Na(+)/H(+) exchange (NHE) in brush-border membrane vesicles (BBMV) showed a 72% decrease in aging compared with young rats, whereas the Michaelis constant remained unchanged. The NHE3 isoform of the Na(+)/H(+) exchanger was detected in BBMV by Western blot in both groups, and a decrease of 90% in the abundance was observed in aging rats. Micropuncture experiments with simultaneous luminal and peritubular perfusion with phosphate Ringer and continuous measurement of intratubular pH showed an acidification rate constant 34% smaller in aging compared with young rats. Proton flux was 48% lower in aging than in young rats. The present results suggest that proximal tubular acidification is impaired with aging.     

表皮葡萄球菌对大鼠变应原敏感性的影响

目的 筛选具有较好抑制金黄色葡萄球菌效果的表皮葡萄球菌,研究通过鼻腔进行表皮葡萄球菌调节观察大鼠对变应原敏感性的效果。方法 由鼻腔拭子稀释液分离筛选得到的9株表皮葡萄球菌（Staphylococcus epidermidis）,使用上清液抑制法选取抑制金黄色葡萄球菌效果最好的表皮葡萄球菌做体内实验。将Wistar大鼠分为阴性组、模型组、表葡调节组。模型组、表葡调节组使用OVA建立呼吸道炎症模型,使用表皮葡萄球菌DMSA16001进行滴鼻调节2周,同时用PBS进行滴鼻对照。统计抓鼻和喷嚏数,用ELISA进行肺灌洗液（BALF）和外周血IgE的检测。结果 （1）不同株的表皮葡萄球菌均对金黄色葡萄球菌有抑制作用,表皮葡萄球菌DMSA16001对金黄色葡萄球菌的抑制率最高。（2）与模型组相比,表葡调节组的抓鼻数显著下降（t=2.173,P=0.049）,喷嚏数有下降趋势,表葡调节组的肺灌洗液和外周血血清的IgE水平均呈现下降趋势。结论 对金黄色葡萄球菌有抑制作用的表皮葡萄球菌能使OVA造模的哮喘大鼠对变应原敏感性降低。     

Administration of microparticles from blood of the lipopolysaccharide-treated rats serves to induce pathologic changes of acute respiratory distress syndrome

This study was conducted to investigate the effect of intratracheal and intravenous administration of microparticles (MPs) on developing acute respiratory distress syndrome (ARDS). The blood MPs from lipopolysaccharide-treated rats were collected and examined by transmission electron microscopy (TEM). Cellular source of the MPs was identified by fluorescent-labeled antibodies after the circulating MPs were delivered to naïve rats. Levels of myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 productions in bronchoalveolar lavage fluid (BALF) and plasma were determined 24 h after the rats received intratracheal and intravenous administration of the MPs. Histopathologic examination of lungs was performed by light microscope. A TEM image of MPs showed spherical particles at a variable diameter from 0.1 to 0.5 µm. Endothelial- and leukocyte-derived vesicles were abundant in the investigated samples. Treatment with MPs may lead to significant increases in MPO, TNF-α, IL-1β, and IL-10 productions in BALF and plasma of the rats (all P < 0.001). Morphological observation indicated that alveolar structures were destroyed with a large amount of neutrophil infiltration in the lungs of the MP-treated rats. Perivascular and/or intra-alveolar hemorrhage were serious and hyaline membrane formed in the alveoli. Intratracheal and intravenous approaches to delivery of the circulating MPs to naïve recipient rats may induce ARDS. This presents an inducer of the onset of ARDS and provides potential therapeutic targets for attenuating lung injury.     

Clathrin beta-light chain of rat liver coated vesicles is phosphorylated in vitro and in vivo

Clathrin beta-light chain of rat liver coated vesicles is phosphorylated in vitro in the presence of poly(L-lysine) by an endogenous protein kinase which appears to be similar to casein kinase II. Clathrin beta-light chain is also phosphorylated in vivo. After injection of [32P]phosphate into rats and preparation of purified coated vesicles in the presence of phosphatase inhibitors, electrophoretic analysis showed the presence of several labeled polypeptides including clathrin beta-light chain. A polypeptide of 50 kDa, which may correspond to the major polypeptide phosphorylated in vitro of coated vesicles, is also labeled in vivo.     

Pneumadin in the ventral prostate and seminal vesicles of estradiol-treated rats: RIA and immunocytochemical studies

Pneumadin (PNM) is a decapeptide (the rat peptide: Tyr-Gly-Glu-Pro-Lys-Leu-Asp-Ala-Gly-Val-NH2) isolated from mammalian lungs. Human and rat PNM differ only by substitution of one amino acid--Tyr/Ala. PNM evokes an antidiuretic effect via a potent stimulation of arginine-vasopressin (AVP) release. By means of recently established, highly specific RIA method, high concentration of PNM had been found in the rat ventral prostate. Castration resulted in a profound drop in PNM concentration, an effect prevented by testosterone replacement. The present studies were aimed at investigating the effect of prolonged estradiol administration on PNM concentration, content and localization in the prostate and seminal vesicles of the rat. Depo estradiol (estradiolum valerianicum) administration to adult male rats resulted in a notable atrophy of ventral prostate and seminal vesicles. During the entire experiment (till day 30 after administration), PNM concentration in ventral prostate was similar to that seen in intact animals, while peptide content per gland was markedly lowered. PNM immunostaining was observed in prostate epithelium of estradiol-treated rats and its localization resembled that observed in intact animals. Nearly 40 times lower PNM concentration than in ventral prostate was found in seminal vesicles. In contrast to prostate, on days 20 and 30 of estradiol treatment PNM concentration in seminal vesicles was higher than in intact rats. However, due to profound seminal vesicle atrophy, PNM content per entire gland was notably lowered in estradiol-injected rats. By immunocytochemistry, PNM-immunoreactive substances were not found in seminal vesicles of either intact or estradiol-administered rats. High PNM concentration in the rat prostate suggests its important role in the function of the gland.     

TAK-599, a novel N-phosphono type prodrug of anti-MRSA cephalosporin T-91825: synthesis,physicochemical and pharmacological properties

Crystalline 1 (TAK-599) is a novel N-phosphono prodrug of anti-methicillin-resistant Staphylococcus aureus (MRSA) cephalosporin 2a (T-91825) that has high affinity for penicillin-binding protein (PBP) 2' (IC(50); 0.90 microg/mL) and shows potent in vitro anti-MRSA activity (MIC against MRSA N133; 1.56 microg/mL), comparable to that of vancomycin (1.56 microg/mL). Although 2a had insufficient water solubility (2.3 mg/mL) for parenteral administration, 1 showed excellent water solubility (>100 mg/mL, pH 7) as well as good chemical stability in the solid state and solution. In pharmacokinetic studies, when 1 was administered intravenously to rats and monkeys, it was rapidly converted into 2a in the blood. These results show that 1 (TAK-599) is a highly promising parenteral cephalosporin targeted for MRSA infection.     

Combined strategies for liposome characterization during in vitro digestion

Three types of pyranine (HPTS)-containing liposomes were prepared by high-pressure homogenization under optimized conditions. At 37°C, they were 1) fluid-state vesicles made from soybean phosphatidylcholine (SPC), 2) gel-state liposomes made from hydrogenated SPC (HSPC), and 3) solid-disordered membranes obtained from HSPC and cholesterol (HSPC-Chol). These liposome formulations were characterized before, during, and after in vitro digestion, which involved the presence of pH gradients, enzymes, and bile salts. Mean sizes and size distributions of the vesicles were determined by DLS; ³¹P-NMR (nuclear magnetic resonance) was used to quantify lyso-PC forms; internal pH was monitored throughout digestion with two different fluorescent pH probes; and changes in bilayer permeability and HPTS encapsulation were determined by size-exclusion chromatography and fluorimetry. Differential scanning calorimetry analysis was also performed in order to study the effect of digestion on HSPC vesicles. SPC liposomes were physically stable during digestion; they presented 8% lyso-forms and an HPTS encapsulation around 85% after in vitro digestion. However, they were extremely permeable to ions, so that the internal pH immediately equilibrated with the bulk pH. HSPC liposomes were the most affected by the digestive process. Even though they were chemically stable, as inferred from the low lyso-PC content, very important changes in their size distribution were observed. A final 50% HPTS leakage was quantified after in vitro digestion. Nevertheless, they were the least permeable to protons under pH gradients. HSPC-Chol vesicles presented intermediate permeability to protons, having their internal pH decreased from approximately 6.8 to 4.6 after 1 hour of incubation at pH 2. This was the most chemically stable formulation and showed the highest encapsulation, even after in vitro digestion. Therefore, HSPC-Chol liposomes would be the most adequate choice for the design of lipid products for oral administration.     

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter.

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.