Ring test assessment of the mKir2.1 growth based assay in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using parametric models and model-free fits

Inward rectifying K⁺ (Kir) channels are a subfamily of the potassium channel superfamily. They mediate potassium influx into the cells, a process responding to the polarization state, a variety of intracellular messengers and specific auxiliary proteins, thereby they are involved in important physiological processes such as the pacemaker activity in the heart, insulin release, and potassium uptake in glial cells. The Saccharomyces cerevisiae mKir2.1 in vitro assay was subjected to a ring test assessment. Compound-associated mKir2.1 modulating effects were detected by growth determination of functionally complemented S. cerevisiae cells in a 96-well format within 15 h. Dose–response diagrams and EC₅₀ value calculations were determined by parametric model and model-free fits using cubic spline interpolation. These characteristics were evaluated by statistical methods to determine reproducibility among working groups. Nonparametric bootstrap simulations of the variability of the data revealed that EC₅₀ values of the mKir2.1 indicator strain were well-matched (81–92 μM), enabling unambiguous quantitative statements about inhibitory effects and no significant influence of the different laboratory conditions. Limitations of the assay include compounds/samples that are either insoluble under the conditions of the test or strongly cytotoxic to yeast. Thus, the described test is a sensitive and reliable tool that can be used in different laboratories and is applicable in drug discovery and development as simple and reliable prescreening procedure.     

Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells

Interaction of Li+ with the voltage-dependent Na+ channel has been analyzed in neuroblastoma X glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage-dependent Na+ channels. Veratridine and aconitine increased the uptake of Li+ to the same degree (EC50 30 microM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine-stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 micrograms/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage-dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.     

Ca-dependent modulation of human glycine receptors expressed in cultured cell lines

Glycine receptors (GlyRs) provide the main inhibitory neurotransmission in spinal cord and brainstem synapses of vertebrates. Fucile et al. (2000) discovered that elevation of intracellular Ca2+ caused rapid potentiation of GlyRs. This modulation develops in less than 100 ms. It is characterized by an increase in GlyR apparent affinity for glycine. It has been suggested that the phenomenon of Ca-induced potentiation involves an unknown Ca2+-binding protein (CaBP). Using the yeast two-hybrid system, screening of human brain cDNA library against the cytoplasmic loop of human alpha 1 subunit (GlyRhl) allowed us to identify five new interactors. One of them belongs to a family of Ca-binding proteins. We analyzed effect of "short" forms of this protein (CaBP-S) on functional properties of GlyRhl expressed in HEK-293 and CHO cells. Using whole-cell recordings and rapid agonist application we constructed concentration dependencies of glycine-induced currents. This analysis revealed statistical differences in EC50s between control cells (expressing only GlyRhl) and those expressing CaBP-S. In HEK-293 cells recorded under conditions of low intracellular Ca concentration (BAPTA 20 mM in the recording pipette), EC50 for glycine in control cells and expressing GlyRhl + CaBP-S were, correspondently, 68+/-49 microM (n = 29) and 409 +/-421 microM (n = 60). In CHO cells EC50 were 54+/-43 microM (n = 25) and 123 +/-104 microM (n = 28). These differences were statistically not significant at recording with intracellular solution containing high Ca concentration (50 microM). In this case EC50 were correspondently 35+/-28 microM (n = 7) and 64 +/-38 microM (n = 7). These results suggest that CaBP-S causes decrease of GlyR sensitivity to agonist through interaction with cytoplasmic domain of GlyR.     

Cadmium uptake and toxicity via voltage-sensitive calcium channels

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.     

Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel

The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.     

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.     

Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.     

Arachidonyl dopamine as a ligand for the vanilloid receptor VR1 of the rat

The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.     

Characterization of a Ca2+ uniporter from Bacillus subtilis by partial purification and reconstitution into phospholipid vesicles

Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent Km for Ca2+ of about 250 microM. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5.28-5.33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent Km for Ca2+ in the reconstituted system was about 260 microM. The specific activity was increased about 50-fold by purification with isoelectric focusing.     

Growth factors activate the bumetanide-sensitive Na+/K+/Cl-cotransport in hamster fibroblasts

alpha-Thrombin, a potent mitogen for the hamster fibroblast cell line CCL 39, stimulates by approximately 3-fold 86Rb+ uptake in a mutant lacking the Na+/H+ antiport activity (PS 120). The major component of this stimulated 86Rb+ (K+) uptake is a bumetanide-sensitive flux (IC50 = 0.4 microM), which accounts for 50% of total K+ uptake in Go-arrested cells and is approximately 4-fold stimulated by maximal thrombin concentrations (EC50 = 5 X 10(-4) units/ml). This bumetanide-sensitive 86Rb+ uptake represents a Na+/K+/Cl- cotransport, as indicated by its dependence on extracellular Na+ and Cl- and by the existence in PS 120 cells of a bumetanide-sensitive K+-dependent 22Na+ uptake. The stimulation reaches its maximum within 2 min, is reduced at acidic intracellular pH values (half-maximal at pHi = 6.8), and can also be induced, to a lesser extent, by EGF and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the effects of which are nearly additive. In contrast, preincubation with 12-O-tetradecanoylphorbol 13-acetate results in inhibition of thrombin- and EGF-induced stimulations, suggesting that activated protein kinase C might exert a feedback inhibitory control. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport is separated from the activation of the Na+/H+ antiport. However, activation of this cotransporter does not seem to play a major role in the mitogenic signaling pathway since its complete inhibition with bumetanide reduces only by 25-30% reinitiation of DNA synthesis.     

Development of recombinant cell line co-expressing mutated Nav1.5, Kir2.1, and hERG for the safety assay of drug candidates

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 μM, 19 nM, 17 nM, 74 nM, and 3 μM, respectively, whereas 10 μM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.     

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa + pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.     

Altered expression, polymerisation and cellular distribution of alpha-/beta-tubulins and apoptosis-like cell death in arsenite resistant Leishmania donovani promastigotes

Studies in mammalian systems have shown specific affinity of arsenite for tubulin proteins. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which is a 13-fold increase in resistance compared to the wild type. Data presented in this study shows decreased expression of alpha- and beta-tubulin in wild type L. donovani promastigotes on exposure to NaAsO2 from 0.0016 to 5.0 microM (IC50 in the wild type strain) in a dose-dependent manner. alpha- and beta-tubulins in the resistant strain show decreased expression levels only at 65.0 microM NaAsO2 (IC50 in the resistant strain). Treatment with respective IC50 concentrations of NaAsO2 caused alterations in tubulin polymerisation dynamics and deregulated the cellular distribution of the microtubules in wild type and resistant strains. The NaAsO2-induced cell death exhibited characteristics of apoptosis-like DNA laddering and fragmentation in both the affected wild type and resistant cells. However, poly(ADP-ribose)polymerase cleavage was evident in the wild type strain but not in the resistant strain.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.     

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.