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.     

The combined effect of Cd2+ and ACh on action potentials of Nitellopsis obtusa cells

Interrelations between the action of acetylcholine (ACh) and cadmium ions (Cd²⁺) on bioelectrogenesis of Nitellopsis obtusa cells were investigated. We analyzed repetitively triggered action potentials (AP), their reproducibility, shape and dynamics of membrane potential after AP induction. ACh significantly increased membrane permeability only at high concentrations (1 mM and 5 mM). Repolarisation level of action potential after the first stimulus was much more positive in all cells treated with ACh as compared to the control. Differences of membrane potentials between points just before the first and the second stimuli were 23.4±.0 mV (control); 40.4±5.9 mV (1 mM ACh solution) and 57.7 ± 8.5 mV (5 mM ACh solution). Cd²⁺ at 20 μM concentration was examined as a possible inhibitor of acetylcholinesterase (AChE) in vivo. We found that cadmium strengthens depolarizing effect of acetylcholine after the first stimulus. The highest velocity of AP repolarization was reduced after ACh application and Cd²⁺strengthened this effect. There were no differences in dynamics of membrane potential after repetitively triggered action potentials in ACh or ACh and Cd²⁺ solutions. This shows that cadmium in small concentration acts as inhibitor of acetylcholinesterase.     

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca<Superscript>2+</Superscript>-channels and ryanodine receptors

L-type Ca²⁺-channel blockers, verapamil (5 μM) and nifedipine (10 μM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca²⁺-channel activator BAY K 8644 (1 μM) has a decreased mediator secretion level. Nifedipine (10 μM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 μM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 μM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 μM).     

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.     

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.     

Peculiarities of dopamine receptors on the membrane of multipolar spinal cord neurons of the brook lamprey <Emphasis Type="Italic">Lampetra planeri</Emphasis>

On isolated multipolar neurons of spinal cord of amniocoete (larva of the brook lamprey Lampetra planeri) by the patch-clamp method in configuration “the whole cell,” a modulating effect of dopamine on potential-activated Na⁺ currents was studied. Application of dopamine (10 μM) was shown to produce a complex action on the sodium current amplitude. In some cases a decrease of the amplitude, on average, by 13.5 ± 2.2% was found, while in others—an increase, on average, by 8.6 ± 6.1%. The modulation dopamine effect was not accompanied by any changes either of the threshold of the current appearance or of resistance of neuronal cell membranes. Pharmacological analysis with use of dopamine agonist has shown that the agonist of D1-receptors (−)-SKF-38393 (10 μM) decreases the Na+ current amplitude, whereas the agonist of D2-receptors (−)-quinpirole (10 μM) can produce in different cells both an increase, by 30.7 ± 17.0%, and a decrease, by 13.2 ± 3.1%, of the Na+ current amplitude. The obtained data indicate the existence of D1-and D2-receptors on the membrane of multipolar spinal neurons of the amniocoete (larva of the brook lamprey). Study of action of antagonists has shown that the antagonist of D1-receptors (+)-SCH-23390 (10 μM) does not affect action of the agonist of D1-receptors (−)-SKF-38393 (10 μM); the antagonist of D2-receptors (−)-sulpiride (10 μM) blocks completely effects both of the agonist of D1-receptors (−)-SKF-38393 (10 μM) and of the agonist of D2-receptors (−)-quinpirole (10 μM). The antagonist of D1-receptors (+)-SCH-23390 (10 μM) produced no effect on action of the agonist of D1-receptors (−)-SKF-38393 (10 μM). The obtained data indicate peculiarities of dopamine receptors of Cyclostomata as compared with those in mammals. Original Russian Text ? A. A. Bukinich, E. A. Tsvetkov, and N. P. Vesselkin, 2007, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2007, Vol. 43, No. 1, pp. 39–45.     

Two Types of Pharmacologically Distinct Ca2+ Currents with Voltage-dependent Similarities in Zona Fasciculata Cells Isolated from Bovine Adrenal Gland

Voltage-activated Ca²⁺ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca²⁺: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba²⁺, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: −40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15 and −34 mV, respectively. The sustained component was larger in Ba²⁺ than in Sr²⁺ and Ca²⁺. Ni²⁺ (20 μm) selectively blocked the transient component while Cd²⁺ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca²⁺ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells. Received: 12 July 1999/Revised: 5 October 1999     

Neurochemical mechanisms responsible for depotentiation of synaptic transmission

It was established in experiments on murine hippocampal slices that low-frequency (1 sec⁻¹, 15 min) stimulation of the Schaffer collaterals applied 45 to 60 min after their high-frequency repetitive stimulation (60 sec⁻¹, 0.5 sec) results, in 2/3 of the slices, in reduction of the amplitude of population EPSP recorded from pyramidal neurons of theCA1 area, almost to its level before high-frequency stimulation. Depotentiation was practically completely prevented by application of a non-competitive blocker of NMDA glutamate receptors (GR), ketamine (100 μM), was weakened by a blocker of voltage-dependent L-type Ca²⁺ channels, nifedipine (10 μM), and remained significant after a competitive blocker of the AMPA/kainate receptors, CNQX (10 μM), had been applied to the slices. Depotentiation was significantly reduced by 10 μM of a calmodulin inhibitor, trifluoroperazine, by an increase in the intracellular cAMP concentration caused by activation of A2-adenosine receptors and D5-dopamine receptors, but was resistant to the action of 50 μM of a protein kinase C (PKC) inhibitor, polymixin B. Nootropic compounds possessing anti-amnestic activity enhanced the depotentiation. It is suggested that depotentiation is due to an increase in the intracellular Ca²⁺ concentration, activation of protein phosphatases, and dephosphorylation of pre- and post-synaptic substrates involved in the expression of long-term post-tetanic potentiation of synaptic transmission, which result from cooperative activation of NMDA GR and metabotropic GR.     

Comparative study of effects of cadmium cations in free and chelated forms on activity of glutathione S-transferase, growth, and endocytosis in culture of the infusoriumTetrahymena pyriformis

Effects of cadmium cations in free (Cd²⁺) and chelated with EDTA (Cd²⁺-EDTA) forms were studied on growth, endocytosis, and activity of glutathione S-transferase (GT) in the free-living infusoriaTetrahymena pyriformis. It is shown that the cytotoxicity of Cd²⁺ in the free form at a concentration of 10 μM is much higher than of the Cd²⁺-EDTA complex at the equimolar concentration. Even at a low concentration (2 μM), Cd²⁺ produces an inhibition of the growth rate and endocytosis in theT. pyriformis culture, while the Cd²⁺-EDTA complex suppresses these functions insignificantly. Cd²⁺ in the free form at concentrations of 10 and 100 μM reduced activity of glutathione S-transferase by 39 and 61%. The chelated Cd²⁺-EDTA complex at these concentrations inhibited the GT activity by 5 and 55%, respectively.     

Kinetic Analysis of the Calcium- and Cadmium-Induced Development of Nonspecific Permeability of the Mitochondrial Inner Membrane

We studied the Са²⁺- and Cd²⁺-induced development of the nonspecific permeability of the mitochondrial inner membrane in preparations obtained from rat liver tissue, which is accompanied by swelling of these organelles and intensification of light dispersion of their suspension. Addition of 5 to 100 μM Са²⁺ or 1 to 50 μM Сd²⁺ to the medium caused swelling of the mitochondria. With increase in concentrations of Са²⁺ and Cd²⁺, the latency of the effect decreased, and the rate of swelling of these organelles increased. Upon isolated action of Са²⁺, the intensity of the process (amplitude of changes) did not depend significantly on the concentration of the above ions, while upon isolated action of Cd²⁺, it was the maximum at the concentration of 1 mM and noticeably decreased with increase in the concentration. The dependence of the rate of Са²⁺- and Cd²⁺-induced swelling of the mitochondria on the concentration of these ions was described by power and sigmoid functions, respectively. The calculated maximum rate and the constant of 50% saturation of these processes were equal to 0.609 and 1.084 extinction units/min⋅mg protein and 19.85 and 7.28 μM for Са²⁺- and Cd²⁺-induced swelling of the mitochondria, respectively. Cyclosporine A (10 μM) suppressed completely the Са²⁺-induced swelling of the mitochondria and decreased only partly the Cd²⁺-induced swelling. Dithiothreitol (1 mM) inhibited completely the latter effect but did not influence significantly the Са²⁺-stimulated process. Therefore, the distinctions between the kinetics of Са²⁺- and Cd²⁺-induced swelling of the mitochondria, as well as the different sensitivity of these processes to cyclosporine A and dithiothreitol, prove that the mechanisms underlying interactions between the cations of the above metals and the inner mitochondrial membrane in the course of the development of nonspecific permeability of these organelles are dissimilar. *Deceased     

Protective Effect of Retinal Ischemia by Blockers of Voltage-dependent Calcium Channels and Intracellular Calcium Stores

In the present study, the neuroprotective effect of blockers of voltage-dependent calcium channels (VDCC) and intracellular calcium stores on retinal ischemic damage induced by oxygen deprivation-low glucose insult (ODLG) was investigated. Retinal damage induced by ODLG was dependent on the calcium concentration in the perfusion medium. When incubated in medium containing 2.4 mM CaCl₂, cell death in ischemic retinal slices treated with blockers of VDCC, ω-conotoxin GVIA (1.0 μM), ω-conotoxin MVIIC (100 nM) and nifedipine (1.0 μM), was reduced to 62 ± 2.3, 46 ± 4.3 and 47 ± 3.9%, respectively. In the presence of blockers of intracellular calcium stores, dantrolene (100 μM) and 2-APB (100 μM), the cell death was reduced to 46 ± 3.2 and 55 ± 2.9%, respectively. Tetrodotoxin (1.0 μM), reducing the extent of the membrane depolarization reduces the magnitude of calcium influx trough VDCC causing a reduction of the cell death to 55 ± 4.3. Lactate dehydrogenase content of untreated ischemic retinal slices was reduced by 37% and treatment of ischemic slices with BAPTA-AM (100 μM) or 2-APB (100 μM) abolished the leakage of LDH. Dantrolene (100 μM) and nifedipine (1.0 μM) partially blocked the induced reduction on the LDH content of retinal ischemic slices. Histological analysis of retinal ischemic slices showed 40% reduction of ganglion cells that was prevented by BAPTA-AM or dantrolene. 2-APB partially blocked this reduction whilst nifedipine had no effect, p > 0.95. Conclusion Blockers of VDCC and intracellular calcium-sensitive receptors exert neuroprotective effect on retinal ischemia.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

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.     

Different characteristics of roots in the cadmium-tolerance and Cd-binding complex formation between mono- and dicotyledonous plants

Effects of Cd²⁺ on growth and Cd-binding complex formation in roots were examined with various seedlings of mono- and dicotyledonous plants. Maize, oat, barley and rice exhibited the greater tolerance to Cd²⁺ (100 μM) than did azuki bean, cucumber, lettuce, pea, radish, sesame and tomato (10–30 μM). Azuki bean was the most sensitive to Cd²⁺ (<10 μM). Under these Cd-treatments, cereal roots accumulated Cd²⁺ in the cytoplasmic fractions and transported Cd²⁺ into the same fractions of shoot tissues, to larger extents than did dicotyledonous roots. Cereal roots synthesized a Cd-binding complex containing phytochelatins in the cytoplasmic fractions, depending upon Cd²⁺ concentrations applied (30–100 μM). Such a complex was not detected from the same fractions of dicotyledonous roots treated with Cd²⁺. These results suggest that the Cd-binding complex formation has an important role in the tolerance of cereal roots against Cd²⁺.     

Lhcb2 gene expression analysis in two ecotypes of Sedum alfredii subjected to Zn/Cd treatments with functional analysis of SaLhcb2 isolated from a Zn/Cd hyperaccumulator

The Lhcb2 gene from hyperaccumulator Sedum alfredii was up-regulated more than three-fold while the non-hyperaccumulator accumulated one or two-fold higher amount of the mRNA than control plants under different concentrations of Cd²⁺ for 24 h. Lhcb2 expression was up-regulated more than five-fold in a non-hyperaccumulator S. alfredii when exposed to 2 μM Cd²⁺ or 50 μM Zn²⁺ for 8 d and the hyperaccumulator had over two-fold more mRNA abundance than the control plants. Over-expression of SaLhcb2 increased the shoot biomass by 14–41% and the root biomass by 21–57% without Cd²⁺ treatment. Four transgenic tobacco lines (L5, L7, L10 and L11) possessed higher shoot biomass than WT plants with Cd²⁺. Four transgenic lines (L7, L8, L10 and L11) accumulated 6–35% higher Cd²⁺ amounts in shoots than the wild type plants.     

Characteristics of Single, Large-Conductance Calcium-Dependent Potassium Channels (BKCa) from Smooth Muscle Cells Isolated from the Rabbit Mesenteric Artery

Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca²⁺-dependent channels. In excised patches with symmetrical (140 mm) K⁺ solutions, these channels had an average slope conductance of 235 ± 3 pS, and reversed in direction at −6.1 ± 0.4 mV. The channel showed K⁺ selectivity and its open probability (P _o ) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased P _o , whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca²⁺] on P _o was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 μm Ca²⁺, V _1/2 was 77.1 ± 5.3 (n= 18), 71.2 ± 4.8 (n= 16), 47.3 ± 10.1 (n= 11) and −14.9 ± 10.1 mV (n= 6), respectively. Values of k obtained in 1 and 10 μm [Ca²⁺] were significantly larger than that observed in 0.1 μm [Ca²⁺]. With 30 μm NS 1619 (a BK_Ca channel activator), V _1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a K _d of 59 mm. In summary, BK_Ca channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V _1/2 and k. These findings suggest the possibility that different types (isoforms) of BK_Ca channels may exist in this vascular tissue. Received: 22 December 1997/Revised: 27 March 1998     

State-Dependent Block of Na<Superscript>+</Superscript> Channels by Articaine Via the Local Anesthetic Receptor

Articaine is widely used as a local anesthetic (LA) in dentistry, but little is known regarding its blocking actions on Na⁺ channels. We therefore examined the state-dependent block of articaine first in rat skeletal muscle rNav1.4 Na⁺ channels expressed in Hek293t cells. Articaine exhibited a weak block of resting rNav1.4 Na⁺ channels at −140 mV with a 50% inhibitory concentration (IC₅₀) of 378 ± 26 μM (n = 5). The affinity was higher for inactivated Na⁺ channels measured at −70 mV with an IC₅₀ value of 40.6 ± 2.7 μM (n = 5). The open-channel block by articaine was measured using inactivation-deficient rNav1.4 Na⁺ channels with an IC₅₀ value of 15.8 ± 1.5 μM (n = 5). Receptor mapping demonstrated that articaine interacted strongly with a D4S6 phenylalanine residue, which is known to form a part of the LA receptor. Thus the block of rNav1.4 Na⁺ channels by articaine is via the conserved LA receptor in a highly state-dependent manner, with a ranking order of open (23.9×) > inactivated (9.3×) > resting (1×) state. Finally, the open-channel block by articaine was likewise measured in inactivation-deficient hNav1.7 and rNav1.8 Na⁺ channels, with IC₅₀ values of 8.8 ± 0.1 and 22.0 ± 0.5 μM, respectively (n = 5), indicating that the high-affinity open-channel block by articaine is indeed preserved in neuronal Na⁺ channel isoforms.     

The Activity of the Antioxidative System in Cadmium-Treated Arabidopsis thaliana

Changes in the content of reactive oxygen species (ROS) and activity of the antioxidant system were measured in leaves of Arabidopsis thaliana (L.) Heynh exposed to Cd²⁺. Mature plants growing in the nutrient solution were treated with Cd²⁺ at different concentrations (0, 5, 25, 50, 100 μM). An increase of content in leaves was observed at 5, 25 and 50 μM Cd²⁺. A strong accumulation of H₂O₂ was found only at the lowest Cd²⁺ concentration. The content of OH^*. was high at 50 and 100 μM Cd²⁺. Superoxide dismutase (SOD) activity was always higher in Cd²⁺-treated plants than in control. Catalase (CAT) activity decreased with increasing Cd²⁺ concentration in the nutrient solution. Guaiacol peroxidase (POX) activity was particularly high at the lowest and highest Cd²⁺ concentrations and ascorbate peroxidase (APX) activity additionally at 50 μM Cd²⁺. Enhanced activity of monodehydroascorbate reductase (MDHAR) and strong reduction in ascorbate (AA) content were observed at 25 μM Cd²⁺. Glutathione reductase (GR) activity was always higher than in control but decreased as Cd²⁺ concentration increased. However, it was accompanied by gradual content increase of SH-groups. This revised version was published online in July 2006 with corrections to the Cover Date.     

Baclofen and Adenosine Inhibition of Synaptic Transmission at CA3-CA1 Synapses Display Differential Sensitivity to K<Superscript>+</Superscript> Channel Blockade

The metabotropic GABA_B and adenosine A₁ receptors mediate presynaptic inhibition through regulation of voltage-dependent Ca²⁺ channels, whereas K⁺ channel regulation is believed to have no role at the CA3-CA1 synapse. We show here that the inhibitory effect of baclofen (20 μM) and adenosine (300 μM) on field EPSPs are differentially sensitive to Cs⁺ (3.5 mM) and Ba²⁺ (200 μM), but not 4-aminopyridine (100 μM). Barium had no effect on paired-pulse facilitation (PPF) in itself, but gave significant reduction (14 ± 5%) when applied in the presence of baclofen, but not adenosine, suggesting that the effect is presynaptic and selective on the GABA_B receptor-mediated response. The effect of Ba²⁺ on PPF was not mimicked by tertiapin (30 nM), indicating that the underlying mechanism does not involve GIRK channels. Barium did not affect PPF in slices from young rats (P7–P8), suggesting developmental regulation. The above effects of Ba²⁺ on adult tissue were reproduced when measuring evoked whole-cell EPSCs from CA1 pyramidal neurons: PPF was reduced by 22 ± 3% in the presence of baclofen and unaltered in adenosine. In contrast, Ba²⁺ caused no significant change in frequency or amplitude of miniature EPSCs. The Ba²⁺-induced reduction of PPF was antagonized by LY341495, suggesting metabotropic glutamate receptor involvement. We propose that these novel effects of Ba²⁺ and Cs⁺ are exerted through blockade of inwardly rectifying K⁺ channels in glial cells, which are functionally interacting with the GABA_B receptor-dependent glutamate release that generates heterosynaptic depression.     

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3–0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with E _m,7 of +171 ± 10, +62 ± 10 and –45 ± 13 mV (561–575 nm), and +268 ± 6, +137 ± 10 and –43 ± 12 mV (551–540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (E _m,7≥ +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c ₂ class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60–70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 μM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 μM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c. Received: 8 June 1998 / Accepted: 25 August 1998