Hydrogen Peroxide-Dependent Uptake of Iodine by Marine Flavobacteriaceae Bacterium Strain C-21

The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I⁻). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, ¹²⁵I⁻. In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.     

Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L.

The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g⁻¹ DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l⁻¹ produced a 5-fold enhancement of OA accumulation (0.37 mg g⁻¹ DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l⁻¹ for 96 h or with pectin at 2 mg l⁻¹ for 48 h produced identical cellular levels of OA (0.22 mg g⁻¹ DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g⁻¹ DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.     

Biotechnological potential of Chlorella vulgaris for accumulation of Cu and Ni from single and binary metal solutions

This study provides information on the mechanism(s) of Cu and Ni ion biosorption by C. vulgaris, distinguishing adsorption from intracellular accumulation under various conditions. Surface adsorption was found to contribute maximally (>70%) to total Cu/Ni ion accumulation by the test alga (total accumulation efficiencies were 60 and 53 g metal ion mg protein^–1, respectively for Cu and Ni). Maximum intracellular uptake was reported at a pH range of 6.5–7.5, whereas adsorption reached its maximum at pH 3.5 for Cu, and pH 3.5 and 6.5 in the case of Ni. 35 °C was found to be the best temperature for maximum adsorption, whereas intracellular uptake was highest at 25 °C. Though exponentially grown C. vulgaris registered maximum metal ion uptake, adsorption maxima reached the highest values in the declining phase of the culture. Heat-killed and air-dried C. vulgaris accumulated Cu and Ni at about 80% of the values for viable samples, whereas formaldehyde-treated and immobilized biomasses depicted better accumulating potential than the control cells. Na, K, Mn and Zn caused competitive inhibition, whereas for Ca a mixed-type inhibition was evident. Thus, the present study suggests that the general concept that cations inhibit metal ion accumulation by competing with them for the same binding sites on the cell surface is not absolutely valid. As these results also demonstrate that a large amount of the bound metal (>70%) is in the adsorbed fraction, it is advantageous in the sense that it could be recovered by a suitable desorbing agent, especially in case of precious metals and the biomass could be exploited for repeated use in reactors.     

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l^–1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.     

Rate of in vivo verapamil exchange within the hypothalamus of the cat as examined by push-pull perfusion

To investigate the characteristics of the uptake within hypothalamic tissue of the Ca²⁺-channel blocker, verapamil, push-pull canulae were implanted bilaterally above the anterior hypothalamic-preoptic area (AH/POA) and posterior hypothalamus (PH) of the cat. The functional reactivity of these two anatomical regions was verified in the unrestrained cat, prior to a push-pull perfusion, by a microinjection of either 5–7 g norepinephrine (NE) into AH/POA, or by perfusion of 50 mM Ca²⁺ within the PH, both of which induce a transient decline in the cat's core temperature. Verapamil was perfused in a concentration of 0.4, 2.0 or 4.0 g/l for successive 10 and 20 min intervals within these NE and Ca²⁺-sensitive sites. A quantitative analysis of verapamil in each sample of perfusate was performed double-blind by HPLC-spectrophotometric detection. The results showed that the percent recovery of verapamil after the 10 min interval was always less than that after the next 20 min period of perfusion. These recovery values were independent of the site of perfusion and the concentration of verapamil. However, the mean uptake of verapamil into tissue after 10 min was significantly greater than that after the 20 min period for all concentrations tested. These results demonstrate that the hypothalamus has a time-dependent characteristic to incorporate a Ca²⁺-channel blocker into the parenchyma. Once the point of tissue saturation is reached, a steady-state level of verapamil uptake is established.     

Characterization of Desmodesmus pleiomorphus isolated from a heavy metal-contaminated site: biosorption of zinc

Microalgae have been proven efficient biological vectors for heavy metal uptake. In order to further study their biosorption potential, a strain of Desmodesmus pleiomorphus (L) was isolated from a strongly contaminated industrial site in Portugal. Under different initial Zn²⁺ concentrations, metal removal by that strain reached a maximum of 360 mg Zn/g biomass after 7 days, at 30 mg Zn/l, after an initial rapid phase of uptake. Comparative studies were carried out using a strain of the same microalgal species that is commercially available (ACOI 561): when exposed to 30 mg Zn/l, it could remove only 81.8 mg Zn/g biomass. Biosorption experiments using inactivated biomass of the isolated strain reached a maximum Zn²⁺ uptake of 103.7 mg/g. Metal removal at various initial pH values was studied as well; higher removal was obtained at pH 5.0. The microalga strain L, isolated from the contaminated site, exhibited a much higher removal capacity than the commercial strain, and the living biomass yielded higher levels of metal removal than its inactivated form.     

High-affinity Neurotensin Receptor is Involved in Phosphoinositide Turnover Increase by Inhibition of Sodium Pump in Neonatal Rat Brain

Phosphoinositide (PI) metabolism is enhanced in neonatal brain by activation of neurotransmitter receptors and by inhibition of the sodium pump with ouabain or endogenous inhibitor termed endobain E. Peptide neurotensin inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity, an effect blocked by SR 48692, a selective antagonist for high-affinity neurotensin receptor (NTS1). The purpose of this study was to evaluate potential participation of NTS1 receptor on PI hydrolysis enhancement by sodium pump inhibition. Cerebral cortex miniprisms from neonatal Wistar rats were preloaded with [³H]myoinositol in buffer during 60 min and further preincubated for 0 min or 30 min in the absence or presence of SR 48692. Then, ouabain or endobain E were added and incubation proceeded during 20 or 60 min. Reaction was stopped with chloroform/methanol and [³H]inositol-phosphates (IPs) accumulation was quantified in the water phase. After 60-min incubation with ouabain, IPs accumulation values reached roughly 500% or 860% in comparison with basal values (100%), if the preincubation was omitted or lasted 30 min, respectively. Values were reduced 50% in the presence of SR 48692. In 20-min incubation experiments, IPs accumulation by ouabain versus basal was 300% or 410% if preincubation was 0 min or 30 min, respectively, an effect blocked 23% or 32% with SR 48692. PI hydrolysis enhancement by endobain E was similarly blocked by SR 48692, being this effect higher when sample incubation with the endogenous inhibitor lasted 60 min versus 20 min. Present results indicate that PI hydrolysis increase by sodium pump inhibition with ouabain or endobain E is partially diminished by SR 48692. It is therefore suggested that NTS1 receptor may be involved in cell signaling system mediated by PI turnover.     

d-Xylose absorption test: A tool for the assessment of the effect of anticoccidials on the intestinal absorptive capacity of broilers during experimental coccidiosis

Coccidiosis in chickens causes intestinal mucosal lesions and disrupts its integrity leading to a disturbance in absorption of dietary components. The d-xylose absorption test is a sensitive tool of measuring the absorption capacity of the intestine in diseased chickens. In an experiment on broilers, the influence of different anticoccidials on the intestinal absorption capacity of the birds challenged with experimental coccidiosis was evaluated, using the d-xylose absorption test. The experiment had 5 groups of 10 Ross male broiler chickens (24-days-old) as follows: Group 1— negative control received no Eimeria oocystes, Group 2—positive control challenged with mixed Eimeria oocystes, Group 3—positive control dosed with an attenuated oral coccidiosis vaccine, Group 4—positive control dosed with 25 ppm toltrazuril in drinking water and Group 5—positive control received 66 ppm salinomycin sodium, in the diet. The d-xylose absorption test was carried out 5 days after the coccidial infection. Results showed that coccidiosis highly reduced the plasma d-xylose peak level of Group 2 when compared with Group 1 (31 mg/dl at 90 min versus 50 mg/dl at 30–60 min after the d-xylose administration, respectively). The concentration of d-xylose followed cubic (P<0.001, r² = 0.886) and quadratic (P<0.001, r² = 0.686) correlations with time in Group 1 and 2, respectively. Anticoccidials enhanced the uptake of d-xylose in the infected birds. The plasma d-xylose reached to its peak in Group 3, 4, and 5 (38.9, 50 and 47.0 mg/dl, respectively) at 60–90 min after the d-xylose administration and had quadratic functions with time (r² = 0.802, 0.883 and 0.860, respectively, P<0.001). The d-xylose absorption test was a sensitive test for evaluating the influence of anticoccidials on the absorption capacity of intestinal mucosae during coccidiosis in broiler chickens.     

One-step radiosynthesis and initial evaluation of a small molecule PET tracer for PD-L1 imaging

Programmed cell death protein-ligand 1 (PD-L1) is a crucial biomarker in immunotherapy and its expression level plays a key role in the guidance of anti-PD-L1 therapy. It had been reported that PD-L1 was quantified by noninvasive imaging with more developed radiotracers. In our study, a novel [¹⁸F]fluoride labeled small molecule inhibitor, [¹⁸F]LN was designed for positron emission tomography (PET) imaging in both PD-L1 transfected (A375-hPD-L1) and non-transfected (A375) melanoma-bearing mice. LN showed the specificity (IC₅₀ = 50.39 ± 2.65 nM) to PD-L1 confirmed by competitive combination and cell flow cytometry (FACS) analysis. The radiotracer [¹⁸F]LN was obtained via ¹⁸F-¹⁹F isotope exchange from precursor LN. After radiosynthesis, [¹⁸F]LN was achieved with a high radiochemical purity (RCP) above 95% and got a favorable molar activity of 36.34 ± 5.73 GBq/μmol. [¹⁸F]LN displayed the moderate affinity (K_d = 65.27 ± 3.47 nM) to PD-L1 by specific binding assay. And it showed 1.3-fold higher uptake in A375-hPD-L1 cells than that in A375 cells. PET imaging revealed that [¹⁸F]LN could enter into PD-L1 expressing tumor site and visualize the outline of tumor. And tumor uptake (1.96 ± 0.27 %ID/g) reached the maximum at 15 min in the positive group, showed 2.2-fold higher than the negative (0.89 ± 0.31 %ID/g) or the blocked (1.07 ± 0.26 %ID/g) groups. Meanwhile, biodistribution could slightly distinguish the positive from the negative. The results indicated [¹⁸F]LN would become an efficient tool for evaluating PD-L1 expression with further optimization.     

Efficient Zn and Pb uptake by filamentous fungus Paecilomyces marquandii with engagement of metal hydrocarbonates precipitation

Zinc and lead biosorption by living non-growing filamentous fungus Paecilomyces marquandii was examined for its potential application in heavy metals elimination from contaminated areas. Metal uptake by the studied fungus was pH dependent and reached the level of 308 mg of Zn²⁺ g⁻¹ and 505 mg of Pb²⁺ g⁻¹ at pH of 7.5 caused by microprecipitation in slightly alkaline environment. All other metal studies were cultivated with unregulated pH yielding the maximum of 186.2 mg of Zn²⁺ g⁻¹ and 305.8 mg of Pb²⁺ g⁻¹. Interestingly, zinc binding by mycelium increased intensively after 15 h of incubation, whereas the lead concentration in biomass extended gradually and proportionally to the initial concentration and the time of contact. The study showed that thermal pretreatment of mycelium led to a decline in metal uptake, especially in the case of zinc. The mycelium slightly digested by the cell wall lytic enzyme complex, could adsorb lead twice as well after 2 h of exposure whereas zinc loading did not differ from the metal uptake by mycelia without any digestion procedure. The release of potassium ions from the mycelium, concomitant with lead uptake was observed suggesting ion exchange participation in lead binding. Energy-dispersive X-ray analysis, X-ray diffraction and FTIR spectroscopy revealed the presence of both metals hydrocarbonates on the mycelium surface. Additionally, the contribution of carboxyl and amide groups, originating from the mycelium, in metal binding was confirmed by FTIR analysis.The obtained results suggest that the effective metals uptake by P. marquandii was due to a combined mechanism with a dominant role of metabolism dependent microprecipitation.     

Pollution from mining in South Greenland: uptake and release of Pb by blue mussels (Mytilus edulis L.) documented by transplantation experiments

Long-term impact of former mining activities on the marine sub-Arctic ecosystem in the Ivittuut area, Arsuk Fjord, South Greenland, was studied by transplantation experiments with the blue mussel Mytilus edulis. Measurements of metal concentration in mussels were conducted using atomic absorption spectrometry (flame AAS) and graphite furnace atomic absorption spectrometry (graphite furnace AAS). Uptake and release of Pb were documented to be slow processes. For mussels transplanted from the pristine Kugnait Bay to the polluted mining site at Ivittuut, a continuous accumulation throughout the experiments was found. Linear uptake rates of 5.86, 6.94 and 11.62 μg Pb month⁻¹ for small, medium and large mussels were found for a 6-week experiment, whereas exponential uptake rates of 0.26, 0.20 and 0.28 month⁻¹ were found for a 9-month experiment. It is estimated that the transplanted mussels will reach the same level as the resident population 12–16 months following transplantation. Mussels transplanted from the polluted mining site at Ivittuut to the pristine Kugnait Bay depurated only 7–21% of their original Pb content, and the release was within the first 10 days following transplantation, after which the Pb content remained constant.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria. IV. Relationship to the energy state of the mitochondria

1. The energy-dependent accumulation of iron by isolated rat liver mitochondria, respiring on endogenous substrates, is strongly dependent on the efficiency of energy coupling in the respiratory chain as measured by respiratory control with ADP and the endogenous energy dissipation. The accumulation reached a saturation level at respiratory control with ADP values (with succinate as the substrate) of approx. 4.0.2. In the presence of exogenous substrate, the energy-dependent accumulation of iron was markedly reduced, primarily due to binding of iron as carboxylate complexes having less favourable dissociation constants than the iron(III)-sucrose complex(es).3. The effect of added ATP was at least 2-fold, i.e. that of providing energy and that of chelating iron. When the mitochondria respired on endogenous substrate, the energy-dependent accumulation of iron increased at low concentrations of ATP, whereas higher concentrations (> 50 μM) gradually inhibited the uptake.4. Energization of the mitochondria by the generation of an artificial K⁺ gradient across the inner membrane with valinomycin in a K⁺-free medium increased the energy-dependent accumulation of iron.     

Activation of human T-lymphocytes

Summary Stereological data of phytohaemagglutinin (PHA)-activated human T-lymphocytes were recorded at intervals (12 to 72 h) together with biochemical (isotope-uptake, lymphotoxin-release) and morphological measurements. About 98 % of the cells were activated 12 h after PHA-stimulation. The activation phase lasted less than 48 h, i.e., cells entering the activation phase within 12 h were at their activation maximum by 48 h. The activated cell increased in size. The nuclear/cytoplasmic-ratio decreased. Most of the cytoplasmic organelles developed in phase with the increase of cytoplasmic volume. After 48 h, mitotic figures were frequently seen. Due to the increasing number of secondary, activated daughter cells, parameters of most cytoplasmic components declined between 48 and 72 h. Structural changes in the nucleus preceded the ³H-leucine uptake, which had not reached its maximum after 72 h of incubation. The ³H-leucine uptake started as early as 12 h after culture initiation, and its increase was proportional to the increasing polyribosome density. No maximum uptake was reached up to 72 h, but the development of structural components related to this uptake was at its maximum at the end of the activation phase (48 h). The formation of bound ribosomes occurred subsequent to the enlargement of the surface of the rough endoplasmic reticulum. Initial polysome formation occurred at the expense of existing free ribosomes.     

A cell permeable peptide analog as a potential-specific PET imaging probe for prostate cancer detection

Non-invasive detection of prostate cancer or metastases still remains a challenge in the field of molecular imaging. In our recent work of screening arginine- or lysine-rich peptides for intracellular delivery of a therapeutic agent into prostate cancer cells, an arginine-rich cell permeable peptide (NH₂GR₁₁) was found with an unexpectedly preferential uptake in prostate cancer cell lines. The goal of this work was to develop this peptide as a positron emission tomography (PET) imaging probe for specific detection of distant prostate cancer metastases. The optimal length of arginine-rich peptides was evaluated by the cell uptake efficiency of three fluorescein isothiocyanate (FITC)-tagged oligoarginines (NHGR₉, NHGR₁₁, and NHGR₁₃) in four human prostate cell lines (LNCaP, PZ-HPV-7, DU145, and PC3). Of the three oligoarginines, NH₂GR₁₁ showed the highest cell uptake and internalization efficiency with its subcellular localization in cytosol. The biodistribution of FITC-NHGR₉, FITC-NHGR₁₁, and FITC-NHGR₁₃ performed in control nude mice displayed the unique preferential accumulation of FITC-NHGR₁₁ in the prostate tissue. Further in vivo evaluation of FITC-NHGR₁₁ in PC3 tumor-bearing nude mice revealed elevated uptake of this peptide in tumors as compared to other organs. In vivo pharmacokinetics evaluated with ⁶⁴Cu-labeled NH₂GR₁₁ showed that the peptide was rapidly cleared from the blood (t _1/2 = 10.7 min) and its elimination half-life was 17.2 h. The PET imaging specificity of ⁶⁴Cu-labled NH₂GR₁₁ was demonstrated for the detection of prostate cancer in a comparative imaging experiment using two different human cancer xenograft models.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.     

A simple method for cryopreservation of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> callus

Two-years-old Ginkgo biloba cell culture initiated from cotyledonary explants was cryopreserved by a simple desiccation method. Preliminary incubation of callus clumps on MS preculture medium supplemented with 100 g l⁻¹ sucrose and 2 mg l⁻¹ ABA for 7 and 14 days resulted in accumulation of endogenous soluble sugars and was essential for cell culture post-cryopreservation survival. The optimal time for the preculture on sucrose-and-ABA containing medium was found to be 14 days. The sufficient desiccation duration was determined as 150 min. FCM profiles of calli maintained for 2 years remained stable and were not affected by cryopreservation.     

Denitrification capacity in response to increasing nitrate loads and decreasing organic carbon contents in injected leachate of a simulated landfill reactor

Ex situ nitrification followed by denitrification inside the landfill has been recommended to remove ammonia from leachate. The effects of increasing nitrate load and decreasing organic carbon content in the injected leachate on the denitrifying capacity of municipal solid waste (MSW) were investigated. Results showed that MSW possesses a high denitrification capacity. Nitrate reduction could be initiated within 48 h after the first addition of nitrate. Nitrate reduction rate increased with the increasing nitrate loading concentration. When the nitrate loading concentration was increased to 850 mg L^?1, nitrate reduction rate reached up to 35 mg L^?1 h^?1. Nitrite accumulation could be found after the addition of nitrate in each test. However, the maximum nitrite accumulation efficiency declined with increased nitrate load. Organic carbon played an important role in the reduction of nitrate, and both endogenous and exogenous organic materials could act as electron donors.     

Bioaccumulation of cobalt in silkworm (Bombyx mori L.) in relation to mulberry, soil and wastewater metal concentrations

The present study was planned to evaluate Co(II) toxicity in silkworm population. The soil was irrigated using synthetic wastewater to determine the effects of pH and initial cobalt concentration in its bioaccumulation in silkworm (Bombyx mori L.) food chain. The amount of cobalt in wastewater, soil, mulberry and silkworm was determined by atomic absorption spectrophotometer (AAS) analysis. The obtained results clearly indicate that silkworm can be used as template to indicate local cobalt pollution as its body length, body weight and mortality rate was found to be strongly related to cobalt concentration. Higher the cobalt amount in mulberry leaves more the toxicity to silkworm population. At 400 mg/L Co concentration and pH 4 there was maximum deposition of Co in the soil from the synthetic effluent. However, in plants and silk worm the accumulation of Co was maximum at pH 4.5 at an initial Co concentration of 400 mg/L in the synthetic effluent. The maximum cobalt found in wastewater, soil, mulberry and silkworm was 400 ± 0.01, 273.5 ± 0.04, 42.85 ± 0.01, 36.62 ± 0.22 mg/kg, respectively.     

Calcium-independent inhibition of glucose transport in PC-12 and L6 cells by calcium channel antagonists

The goal of these studies was todetermine whether different calcium channel antagonists affect glucosetransport in a neuronal cell line. Rat pheochromocytoma (PC-12) cellswere treated with L-, T-, and N-type calcium channel antagonists beforemeasurement of accumulation of 2-[³H]deoxyglucose(2-[³H]DG). The L-type channel antagonistsnimodipine, nifedipine, verapamil, and diltiazem all inhibited glucosetransport in a dose-dependent manner (2-150 µM) withnimodipine being the most potent and diltiazem only moderatelyinhibiting transport. T- and N-type channel antagonists had no effecton transport. The L-type channel agonist l-BAY K 8644 alsoinhibited uptake of 2-[³H]DG. The ability of these drugsto inhibit glucose transport was significantly diminished by thepresence of unlabeled 2-DG in the uptake medium. Some experiments wereperformed in the presence of EDTA (4 mM) or in uptake buffer withoutcalcium. The absence of calcium in the uptake medium had no effect oninhibition of glucose transport by nimodipine or verapamil. To examinethe effects of these drugs on a cell model of a peripheral tissue, westudied rat L6 muscle cells. The drugs inhibited glucose transport in L6 myoblasts in a dose-dependent manner that was independent of calciumin the uptake medium. These studies suggest that the calcium channelantagonists inhibit glucose transport in cells through mechanisms otherthan the antagonism of calcium channels, perhaps by acting directly onglucose transporters.

     

Quantitation of fluid phase uptake in paramecium. 1. Kinetics in the cells blocked in phagocytic activity

Uptake of horseradish peroxidase (HRP) was quantified in the unicellular eukaryote Paramecium aurelia. About 80% of the total HRP accumulated within 20 min entered the cells via a fluid phase pathway as demonstrated measuring the HRP internalization in the presence of yeast mannan. The rate of HRP accumulation was concentration-dependent and was found to be linear over the range of 50–500 μg HRP per ml of the extracellular medium. During the first 10 min of exposure to HRP, the mannan-uninhibitable uptake was found to reach 1.2–1.65 ng HRP per mg protein (depending on the concentration of the marker in the medium), which corresponds to 0.68 fl per cell/min. Accumulation of HRP reached a plateau within the next 10–15 min and its intracellular uptake was 2–2.55 ng HRP per mg protein. When the phagocytic activity of the cells was blocked with 1-propranolol, the amount of cell-accumulated HRP was 1.8–2.1-fold higher than in the control and the rate of the marker uptake within the first 10 min of incubation reached 1.114 fl per cell/min.