P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods

Background. P-gp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Fluorescence techniques have been widely applied to measure the P-gp activity. In this paper, there is a comparison between the advantages of two fluorescence approaches of commonly available and affordable instruments: the microplate reader (MPR) and the flow cytometer to detect the P-gp efflux activity using calcein-AM.Results. The selectivity, sensibility, and reproducibility of the two methods have been defined. Our results showed that the MPR is more powerful for the detection of small inhibition, whereas the flow cytometry method is more reliable at higher concentrations of the inhibitors. We showed that to determine precisely the inhibition efficacy the flow cytometry is better; hence, to get the correctEmaxand EC50values, we cannot only rely on the MPR.Conclusion. Both techniques can potentially be used extensively in the pharmaceutical industry for high-throughput drug screening and in biology laboratories for academic research, monitoring the P-gp efflux in specific assays.

Metabolic amplification of insulin secretion by glucose is independent of β-cell microtubules

Glucose-induced insulin secretion (IS) by β-cells is controlled by two pathways. The triggering pathway involves ATP-sensitive potassium (KATP) channel-dependent depolarization, Ca2+ influx, and rise in the cytosolic Ca2+ concentration ([Ca2+]c), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [Ca2+]c. After exclusion of the contribution of actin microfilaments, we here tested whether amplification implicates microtubule-dependent granule mobilization. Mouse islets were treated with nocodazole or taxol, which completely depolymerized and polymerized tubulin. They were then perifused to measure [Ca2+]c and IS. Metabolic amplification was studied during imposed steady elevation of [Ca2+]c by tolbutamide or KCl or by comparing [Ca2+]c and IS responses to glucose and tolbutamide. Nocodazole did not alter [Ca2+]c or IS changes induced by the three secretagogues, whereas taxol caused a small inhibition of IS that is partly ascribed to a decrease in [Ca2+]c. When [Ca2+]c was elevated and controlled by KCl or tolbutamide, the amplifying action of glucose was unaffected by microtubule disruption or stabilization. Both phases of IS were larger in response to glucose than tolbutamide, although triggering [Ca2+]c was lower. This difference, due to amplification, persisted in nocodazole- or taxol-treated islets, even when IS was augmented fourfold by microfilament disruption with cytochalasin B or latrunculin B. In conclusion, metabolic amplification rapidly augments first and second phases of IS independently of insulin granule translocation along microtubules. We therefore extend our previous proposal that it does not implicate the cytoskeleton but corresponds to acceleration of the priming process conferring release competence to insulin granules.

In vitro testing of New Zealand manuka oil and Lema oil for inhibition of Erwinia amylovora

Current tools for the control of fire blight disease of apples caused by Erwinia amylovora have limitations including the increasing pressure by markets against the use of streptomycin Coast Manuka and Coast Lema Oil products have been previously shown to provide control against some bacterial fungal and yeast diseases Experiments were carried out to determine whether either of these products provided control against Erwinia amylovora Coast Lema Oil (05 1 2 3 4 w/v) inhibited E amylovora when added to a bacterial suspension Coast Manuka Oil (04 w/v) failed to inhibit E amylovora when added to the bacterial suspension It was also demonstrated that Coast Lema Oil (05 1 2 3 4 w/v) and Coast Manuka Oil (05 1 2 3 4 w/v) inhibited E amylovora replication when added to agar Filter paper discs soaked in Coast Lema Oil (2 3 4 w/v) caused small inhibition zones around the product when placed directly onto E amylovora Coast Manuka Oil (04 w/v) was unsuccessful in causing inhibition zones around the discs when placed directly onto E amylovora These initial results indicate that Lema oil has the potential to control fire blight in pipfruit trees

Phenotypes and genotypes of macrolide-resistant Streptococcus pyogenes isolated in Seoul, Korea

The mechanisms of resistance to macrolides in 51 erythromycin-resistant clinical isolates of Streptococcus pyogenes collected from 1997 through 2003 in Seoul, Korea were evaluated. They were characterized by their antimicrobial susceptibility, phenotype (using triple-disc and induction tests), resistance genotype, emm genotyping (M typing) and phylogenetic analysis. Erythromycin resistance was observed in 23 % of isolates. Inducible phenotype was the most common (iMLS, 51 %, 26 strains), followed by the constitutive phenotype (cMLS, 31 %, 16 strains) and the M phenotype (18 %, 9 strains). Eight of twenty-six iMLS isolates exhibited the iMLS-C phenotype. The remaining 18 isolates gave small inhibition zones (<12 mm) around all three discs, and mild blunting of the spiramycin and clindamycin zones of inhibition proximal to the erythromycin disc. They showed remarkable inducibility in erythromycin and clindamycin resistance. The MIC90 of erythromycin and clindamycin rose from 8 to >128 μg ml−1 and from 0.5 to >128 μg ml−1, respectively. Their resistance characteristics did not fit into any known iMLS subtype reported so far in the literature. So, it was named as an iMLS-D, new subtype. All of these iMLS-D strains harboured the erm(B) gene, demonstrated the emm12 genotype, except one, and formed a tight cluster in a phylogenetic tree, with 89.2 to 100 % sequence homology, suggesting that they are closely related. Nine of sixteen cMLS strains had the emm28 genotype, which had been reported to be associated with multiple drug resistance.

Bisphosphonates stimulate an endogenous nonselective cation channel in Xenopus oocytes: potential mechanism of action

The mechanisms of action of bisphosphonates (BPs) have been poorly determined. Besides their actions on osteoclasts, these agents exhibit gastrointestinal complications. They have also recently been described as affecting various preparations that express an epithelial Na+ channel (ENaC). To understand the effects of BP on ion channels and the ENaC in particular, we used the Xenopus oocyte expression system. Alendronate, and similarly risedronate, two aminobisphosphonates, caused a large stimulation of an endogenous nonselective cation conductance (NSCC). This stimulation averaged 63 ± 12 μS ( n = 18) 60 min after the addition of 2 mM alendronate. The effects on the endogenous NSCC were blocked by extracellular acidification to pH 6.4. On the other hand, alendronate caused a small inhibition of ENaC conductance at pH 7.4 and 6.4, but the effects at pH 6.4 were more readily observed in the absence of changes of the endogenous conductance. The effects on membrane capacitance were also markedly different, with a clear decrease at pH 6.4 and no consistent changes at pH 7.4. The effects on the endogenous channel were further augmented by genistein and were inhibited by a tyrosine phosphatase inhibitor, indicating the involvement of the tyrosine kinase pathway. Stimulation of NSCC with BP is expected to cause membrane depolarization and may explain, in part, its mechanisms of action in inhibiting osteoclasts.

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects of reducing Ca2+ influx (Cain) on K+ currents ( I K) in myocytes from rat small mesenteric arteries by 1) adding external Cd2+ or 2) lowering external Ca2+ to 0.2 mM. When measured from a holding potential (HP) of −20 mV ( I K20), decreasing Cain decreased I K at voltages where it was active (>0 mV). When measured from a HP of −60 mV ( I K60), decreasing Cain increased I K at voltages between −30 and +20 mV but decreased I K at voltages above +40 mV. Difference currents (Δ I K) were determined by digital subtraction of currents recorded under control conditions from those obtained when Cain was decreased. At test voltages up to 0 mV, Δ I K60 exhibited kinetics similar to control I K60, with rapid activation to a peak followed by slow inactivation. At 0 mV, peak Δ I K60 averaged 75 ± 13 pA ( n = 8) with Cd2+ and 120 ± 20 pA ( n = 9) with low Ca2+ concentration. At test voltages from 0 to +60 mV, Δ I K60 always had an early positive peak phase, but its apparent “inactivation” increased with voltage and its steady value became negative above +20 mV. At +60 mV, the initial peak Δ I K60 averaged 115 ± 18 pA with Cd2+ and 187 ± 34 pA with low Ca2+. With 10 mM pipette BAPTA, Cd2+ produced a small inhibition of I K20 but still increased I K60between −30 and +10 mV. In Ca2+-free external solution, Cd2+ only decreased both I K20 and I K60. In the presence of iberiotoxin (100 nM) to inhibit Ca2+-activated K+ channels (KCa), Cd2+ increased I K60 at all voltages positive to −30 mV while BAY K 8644 (1 μM) decreased I K60. These results suggest that Cain, through L-type Ca2+ channels and perhaps other pathways, increases KCa(i.e., I K20) and decreases voltage-dependent K+currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin

The release of glucokinase from digitonin-permeabilized hepatocytes shows different characteristics with respect to ionic strength and [MgCl2] from the release of other cytoplasmic enzymes. Release of glucokinase is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) and is inhibited by increasing concentration of KCl [concn. giving half-maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosphoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydrogenase is independent of ionic strength, but shows a small inhibition by MgCl2 (20%, versus > 80% for glucokinase). Lactate dehydrogenase release increases with increasing ionic strength [concn. giving half-maximal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of glucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with which the hepatocytes have been preincubated. In hepatocytes pre-cultured with 5 mM glucose the release of glucokinase was much slower than that of other cytoplasmic enzymes measured. However, preincubation with glucose (10-30 mM) or fructose (50 microM-1 mM) markedly increased glucokinase release. This suggests that, in cells maintained in 5 mM glucose, glucokinase is present predominantly in a bound state and this binding is dependent on the presence of Mg2+. The enzyme can be released or translocated from its bound state by an increase in [glucose] (A50 15 mM) or by fructose (A50 50 microM). The effects of glucose and fructose were rapid (t1/2 5 min) and reversible, and were potentiated by insulin and counteracted by glucagon. They were inhibited by cyanide, but not by cytochalasin D, phalloidin or colchicine. Mannose had a glucose-like effect (A50 approximately 15 mM), whereas galactose, 3-O-methyl-D-glucose and 2-deoxyglucose were ineffective. When hepatocytes were incubated with [2-3H, U-14C]glucose, the incorporation of 3H/14C label into glycogen correlated with the extent of glucokinase release. Since 2-3H is lost during conversion of glucose 6-phosphate into fructose 6-phosphate, substrate-induced translocation of glucokinase from a Mg(2+)-dependent binding site to an alternative site might favour the partitioning of glucose 6-phosphate towards glycogen, as opposed to phosphoglucoisomerase.

Effects of carbonic anhydrase inhibitors on basolateral base transport of rabbit proximal straight tubule

The effect of carbonic anhydrase inhibitors on basolateral base transport was examined in rabbit proximal straight tubules (S2 segment) perfused in vitro by double-barreled pH microelectrodes. Bath HCO3- reduction or Na+ removal induced an initial basolateral voltage (Vbl) depolarization followed by a late-phase depolarization. Administration of 1 mM acetazolamide (ACTZ) to the bath fluid caused a small inhibition of the initial depolarization, and a larger inhibition of the late phase depolarization. Bath HCO3- reduction decreased intracellular pH (pHi), and this pHi decrease was attenuated by ACTZ. Bath Na+ removal also decreased pHi, and this pHi decrease was completely blocked by ACTZ. However, a slow pHi decrease was observed in response to bath Na+ removal when the bath fluid contained ACTZ and the luminal fluid contained 4 mM amiloride. The addition of ACTZ or ethoxyzolamide to the bath caused a rapid Vbl hyperpolarization and pHi increase. These results demonstrate that carbonic anhydrase inhibitors inhibit basolateral Na(HCO3)n transport in intact rabbit proximal tubule cells (S2). The data suggest that at least one of the base species transported by the transporter is HCO3-, and cytosolic carbonic anhydrase is important in converting intracellular OH- to HCO3-.

Short-term effects of corticosterone treatment on muscle protein synthesis in relation to the response to feeding

1. Rates of protein synthesis in liver and muscle of 100 g male rats were measured in vivo at 1 h or 4 h after injection of 2.5 mg of corticosterone and compared with those from animals given carrier medium alone. 2. In post-absorptive rats, corticosterone for 1 h had no effect on either muscle or liver protein synthesis. After 4 h there was a decrease in both tissues, but this was only statistically significant in muscle. 3. In fed rats, rates of protein synthesis were higher than those in post-absorptive animals, but the effects of corticosterone injection were similar. 4. Re-feeding of post-absorptive rats led to an increase in muscle protein synthesis after 1 h and 4 h. At 1 h this increase was not inhibited when plasma corticosterone concentrations were maintained high by injection of the hormone immediately before feeding commenced, but at 4 h there was a small inhibition. 5. It is concluded that the action of corticosterone in depressing muscle protein synthesis is time-dependent and requires longer than 1 h to develop. The failure of the hormone to alter the response to re-feeding for 1 h in post-absorptive rats suggest that corticosteroids are not important mediators of the acute stimulation of muscle protein synthesis by food intake.

Functional dedifferentiation of parathyroid cells results both from a defective regulation and action of cytoplasmic Ca2+

Monolayer culture of bovine parathyroid cells for 24 hours resulted in a right-shift of the dose-effect relationships for Ca2+-inhibition of parathyroid hormone (PTH) release and the dependence of the cytoplasmic Ca2+ concentration (Ca2+) on extracellular Ca2+ as well as in a less suppressible hormone release. After 4 days of culture, hormone secretion was almost non-suppressible and Cai2+ increased poorly in response to a rise in extracelluiar Ca2+. Ionomycin, a Ca2+ ionophore, raised Cai2+, but there was only a small inhibition of PTH release and the correlation between Cai2+ and secretion was weak. A deteriorated Cai2+ regulation and a decreased inhibitory action of cytoplasmic Ca2+ on PTH release were also found in ceils from human parathyroid adenomas. Functional dedifferentiation of the parathyroid cell thus results from both defective regulation and action of cytoplasmic Ca2+.