ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at –50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current. The EC₅₀ was 150.0±18.5 µM, while the Hill number was 1.6. The current was selectively activated by ATP and was not blocked by P2 purinoreceptor antagonist suramin (50–300 µM).,-Methylene-ATP (100–200 µM) and,-methylene-ATP (100–200µM), which are P2-purinoreceptor agonists, as well as adenosine (100–300 µM), exerted no effects. Reactive blue 2, if applied up to 4 min, enhanced ATP-induced current, while its longer application partially suppressed this current. In most submucous neurons, acetylcholine (ACh) likewise activated an inward cationic current. The amplitude of ACh-induced current was lower if ACh was applied during a long-lasting application of ATP than if ACh only was applied. Hexamethonium (50 µM), d-tubocurarine (20–40 µM), and trimethaphan (30 µM) completely and reversibly blocked ACh-induced currents, regardless of the presence of ATP, and did not affect ATP-induced currents. The results suggest that ATP-induced currents in submucous neurons are due to activation of a unique type of P2 purinoreceptors, which function in connection with nicotinic ACh receptors.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 100–110, March–June, 1996.     

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose production in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Guanosine 5′-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5′-diphosphate (GDP)-l-fucose. In this study, improvement of GDP-l-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-l-fucose. The effects of overexpression of inosine 5′-monophosphate (IMP) dehydrogenase, guanosine 5′-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine–inosine kinase (Gsk) on GDP-l-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-l-fucose production. Maximum GDP-l-fucose concentration of 305.5 ± 5.3 mg l⁻¹ was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes. Such an enhancement of GDP-l-fucose production could be due to the increase in the intracellular level of GMP.     

Induction of Long-Term Depression of Synaptic Transmission in a Co-Culture of DRG and Spinal Dorsal Horn Neurons of Rats

In a co-culture of dissociated neurons of lumbar dorsal root ganglia (DRG) and spinal dorsal horn (DH) neurons of newborn rats, we examined peculiarities of induction of long-term depression (LTD) of synaptic transmission through synapses formed by primary afferents on DH neurons. Induction of LTD was provided by low-frequency (5 sec⁻¹) microstimulation of single DRG neurons. Ion currents were simultaneously recorded in pre- and post-synaptic cells using a dual whole-cell path-clamp technique. Parameters of evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) initiated in DH neurons by intracellular stimulation of DRG neurons were analyzed. Monosynaptic eEPSC mediated by activation of AMPA receptors demonstrated no sensitivity to blockers of NMDA and kainate receptors (20 μM D_L-AP5 and 10 μM SIM 2081, respectively), but were entirely blocked upon applications of 10 μM DNQX. Monosynaptic glycinergic eIPSCs found in some of the DH neurons were blocked by 1 μM strychnine and were insensitive to 10 μM bicuculline and blockers of glutamatergic neurotransmission, D_L-AP5 and DNQX. Long-lasting (360 sec) low-frequency stimulation of DRG neurons did not affect the amplitude of glycineinduced eIPSCs in DH neurons. At the same time, such stimulation of DRG neurons evoked a drop in the amplitude of AMPA-activated eEPSCs in DH neurons to 41.6 ± 2.5%, on average, as compared with the analogous index in the control. This effect lasted at least 20 min after stimulation. Long-term depression of glutamatergic transmission in DH neurons was observed at the holding potential of −70 mV and did not change after applications of 10 μM bicuculline and 1 μM strychnine. The LTD intensity depended on the duration of low-frequency stimulation of primary afferent neurons. Sequential stimulation of DRG neurons lasting 120, 160, 200, and 240 sec resulted in decreases in the eEPSC amplitude in DH neurons to 85.6 ± 3.9, 62.7 ± 4.3, 51.8 ± 3.5, and 41.6 ±2.5% with respect to control values. Our findings show that use-dependent induction of homosynaptic LTD of glutamatergic transmission is possible at the level of a separate pair of synaptically connected DRG and DH neurons under co-culturing conditions. Such LTD of glutamatergic synaptic transmission mostly mediated by activation of AMPA receptors depends on the duration of activation of a presynaptic DRG neuron and does not need depolarization of a postsynaptic DH neuron.     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

Cytotoxic and non-cytotoxic exometabolites of the cyanobacterium Nostoc insulare

The isolation, identification and quantification of exometabolites from culture media of the cyanobacterium Nostoc insulare are described. Besides the known exometabolite 4,4′-dihydroxybiphenyl (I), two more compounds, the β-carboline 9H-pyrido(3,4-b)indole (norharmane, II) and N,N′-(4,5-dimethyl-1,2-phenylene)bis-acetamide (III), were discovered. Concentrations of all three compounds in media and biomass of five 250 L cultures of N. insulare were determined. Culture medium values for I ranged between 200 and 1,250 μg L⁻¹ (1.1–6.7 μmol L⁻¹), for III between 115 and 390 μg L⁻¹ (0.5–1.8 μmol L⁻¹), whereas concentrations of II were conspicuously lower (2–16 μg L⁻¹ or 0.014–0.094 μmol L⁻¹). Amounts of III per volume of culture medium were about tenfold higher than in the biomass contained in an equal culture volume. This difference is an indication for an active excretion of III. Amounts of I and II in biomass and media were of no significant difference. In the neutral red uptake assay, I and II were found to be toxic against eukaryotic cells as follows: I was of considerable cytotoxicity in concentrations from 1,000 to 10 mg L⁻¹ and of lower cytotoxicity (causing a 27 % decrease of cell viability) in a concentration of 1,000 μg L⁻¹, whereas II was merely of considerable cytotoxicity in concentrations from 1,000 to 100 mg L⁻¹. Because of the cytotoxicity of I and because of the many known pharmacological effects of II there is a possibility that a certain amount of risk to humans and livestock comes from cultures or even from biomass- free culture media of N. insulare. The natural function of the examined exometabolites is discussed.     

Characterization of the Mn2+-stimulated (di)adenosine polyphosphate hydrolase encoded by the Deinococcus radiodurans DR2356 nudix gene

The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6–9.0 and showed a strong preference for Mn²⁺ as activating cation. Mg²⁺ ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn²⁺. K _m and k _cat values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 μM and 11.4, 28.6 and 12.0 s⁻¹, respectively, while for GTP they were 20.3 μM and 1.8 s⁻¹, respectively. The K _m for adenosine 5′-pentaphosphate was <1 μM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn²⁺ accumulates.     

Modulating effect of dopamine on amplitude of GABA-produced chemocontrolled currents in multipolar spinal cord neurons of ammocaete

By using the patch-clamp method in the whole cell configuration, modulating effect of dopamine on GABA-activated currents has been studied on isolated multipolar spinal cord neurons of the ammocaete (larva of the lamprey Lampetra planeri). At application of dopamine (5 μM), there was observed in some cases a decrease of the GABA-activated current, on average, by 33.3 ± 8.7% (n = 8, p < 0.01), in other cases—an increase of the amplitude, on average, by 37.3 ± 11.8% (n = 5, p < 0.01). Concentration of GABA amounted to 2 mM. Study of action of agonists of D1- and D2-receptors on amplitude of chemocontrolled currents has shown that agonist of D1-receptors (+)-SKF-38393 (5 μM) decreases the GABA-activated current amplitude, on average, by 63.1 ± 11.7% (n = 8, p s< 0.01); the agonist of D2-receptors (−)-quinpirole (5 μM) produces in various cells the dopamine-like effects: an increase of the GABA-activated current amplitude, on average, by 61.0 ± 13.8% (n = 8, p < 0.01) and a decrease of amplitude, on average, by 55.7 ± 2.0% (n = 6, p < 0.01). It has been shown that antagonist of D2-receptors sulpiride (5 μM) does not block effects produced by dopamine. The dopamine effects were partially blocked by antagonist of D1-receptors (+)-SCH-23390 (5 μM): a decrease of the GABA-activated amplitude current amounted, on average, to 11.7 ± 1.8% (n = 7, p < 0.01), while an increase of amplitude—8.3 ± 2.0% (n = 5, p < 0.01). At the same time, effects of agonist of D1-receptors quinpirole (5 μM) were partially blocked by antagonist of D1-receptors (+)-SCH-23390: a decrease of the GABA-activated current amplitude amounted, on average, to 9.2 ± 3.4% (n = 6, p < 0.01) and an increase of amplitude—6.3 ± 1.8% (n = 10, p < 0.01). The obtained data indicate differences of mechanisms of the receptor-mediated effect of agonists of dopamine receptors on GABA-activated and potential-activated currents of multipolar neurons of the ammocaete spinal cord.     

Ion currents induced by ATP and angiotensin II in cultured follicular cells of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at −60 mV revealed that ATP (20–250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at −60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known F_in and S_in-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using wholecell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at −60 mV. The net Ang II response, mediated by the activation of the AT₁ receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes.     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

Depletion of adenosine triphosphate inDesulfovibrio by oxyanions of group VI elements

Oxyanions of elements from group VI of the periodic table, i.e., analogs of SO₄ ²⁻, destroyed adenosine 5′-triphosphate (ATP) in cells of sulfate-respiring bacteria (Desulfovibrio spp.), probably via the ATP sulfurylase reaction. The approximate order of effectiveness was CrO₄ ²⁻> MoO₄ ²⁻=WO₄ ²⁻>SeO₄ ²⁻. Cultures of aerobically grown or nitrate-respiring bacteria were less susceptible and with fermentatively grownEscherichia coli the oxyanions even appeared to stimulate ATP levels. The selective depletion of ATP in sulfate-respiring bacteria might provide the basis for a rapid and simple assay of their biomass in mixed cultures or environmental samples.     

Nifedipine-sensitive low voltage-activated calcium current in neurons of the rat laterodorsal thalamic nucleus

The kinetic and pharmacological properties of low voltage-activated (LVA) Ca²⁺ channels were studied in neurons of the laterodorsal (LD) thalamic nucleus in brain slices from 12-day-old rats. A homogeneous population of LVA Ca²⁺ channels was found in the tested neurons. LVA Ca²⁺ current evoked by a step depolarization from a holding potential more negative than −70 mV was found to be sensitive to nifedipine (K _d=2.6 (M). This current gained its maximum at −55 mV and demonstrated fast monoexponential decay with the time constant of 32.3±4.0 msec (n=15). Lanthanum (1 μM) effectively blocked LVA Ca²⁺ current, while nickel (25 μM) did not affect this current. It is concluded that the channels that, according to their pharmacological properties, provide the studied LVA Ca²⁺ current cannot be regarded as T-type Ca²⁺ channels and belong to some other type of LVA Ca²⁺ channels.     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997     

Use of 5′-[<Emphasis Type="Italic">p</Emphasis>-(Fluorosulfonyl)benzoyl] Guanosine as an Affinity Probe for the Guanine Nucleotide-Binding Site of Transducin

Transducin (T) mediates vision in retinal rods by transmitting light signals detected by rhodopsin to a cGMP phosphodiesterase. The flow of information relies on a subunit association/dissociation cycle of T regulated by a guanine nucleotide exchange/hydrolysis reaction. 5′-[p-(Fluorosulfonyl)benzoyl] guanosine (FSBG) was synthesized and examined here as an affinity label for the guanine nucleotide binding site of T. Although the relative binding affinity of FSBG to T was much lower than for GTP and β,γ-imido-guanosine 5′-triphosphate (GMPPNP), the incorporation of FSBG to T inhibited its light-dependent [³H] GMPPNP binding activity in a concentration dependent manner. Additionally, GDP, GTP and GTP analogs hindered the binding of [³H] FSBG to T. These results demonstrated that FSBG could be used to specifically modify the active site of T. In addition, FSBG was not capable of dissociating T from T:photoactivated rhodopsin complexes, suggesting that in this case FSBG is acting as a GDP analog.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Effects of cadmium stress on seed germination,seedling growth and antioxidative enzymes in <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> plants infected with a <Emphasis Type="Italic">Neotyphodium</Emphasis> endophyte

The effects of cadmium (Cd) on germination, and antioxidative enzyme activity (AEA) involving superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase, and on amounts of malondialdehyde and proline present within Achnatherum inebrians, were determined for specimens infected (E+) vs. non-infected (E−) by Neotyphodium gansuense, and cultivated in the presence of various concentrations of CdCl₂ (0, 50, 100, 200 and 300 μmol/l). Under high Cd concentrations (100, 200 and 300 μM), E+ (vs. E−) specimens exhibited a higher germination rate and index, and higher values for shoot length, root length and dry biomass, but there was no significant difference (P > 0.05) under low Cd concentrations (0 and 50 μM). AEA and the proline content increased, but malondialdehyde content declined in the E+ (vs. E−) specimens under high Cd concentrations (100, 200 and 300 μM). There was no significant difference (P > 0.05) under low Cd concentrations (0 and 50 μM). Endophyte infection was concluded to be of benefit to the germination and anti-oxidative mechanisms within A. inebrians under plant exposures to high CdCl₂ concentrations.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

An ionotropic GABA receptor in cultured mushroom body Kenyon cells of the honeybee and its modulation by intracellular calcium

GABAergic inhibitory transmission is very abundant within the insect brain. We, therefore, studied the functional properties of the ionotropic GABA receptor of honeybee mushroom body Kenyon cells in vitro. GABA applications elicit rapidly activating and desensitizing currents, which are concentration-dependent between 10 and 500 μM. The mean peak amplitude induced by 500 μM GABA at a holding potential of −110 mV is −1.55 ± 0.23 nA (SEM, n = 29). The GABA-induced current is mediated by Cl⁻ ions because (1) the reversal potential of the GABA-induced current of −40.6 mV is very close to the calculated Nernst potential of chloride (−44.8 mV). (2) With equimolar chloride concentrations the reversal potential shifted to about 0 mV. GABA or muscimol are equally efficient channel agonists, whereas CACA is a partial agonist. Picrotoxin or philanthotoxin (100 μM) completely and reversibly block the GABA-induced current, bicuculline (100 μM) has no effect. Elevating the intracellular Ca²⁺ concentration increases the GABA current amplitude. This modualtory effect is blocked by the kinase blocker K 252a, but not by blockers of CaMkinaseII (KN-93), PKC (bisindolylmaleimide) or PKA (KT 5720). We conclude that Kenyon cells express functional GABA receptors whose properties support an inhibitory role of GABAergic transmission.     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999