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.     

Two types of P2x-purinoceptors in neurons of the guinea pig ileum submucous plexus

Effects of exogenous adenosine 5′-triphosphate (ATP) on dissociated guinea pig ileum submucous neurons were studied using a conventional whole-cell patch-clamp technique. With the holding potential of −50 mV, application of 50–1,000 μM ATP evoked an inward current (ATP-induced current) in most (90%) of the tested neurons (n-35). ATP-induced currents were observed regardless of whether or not guanosine 5′-triphosphate (GTP, 0.2 mM) and ATP (2 mM) were present in the intracellular solution, or GTP was replaced with equimolar concentration of guanosine 5′-O-3-thiotriphosphate (n-5). In 26 of 29 neurons studied, which responded to ATP, applications of 50–1,000 μM ATP induced slowly declining currents. ATP receptors did not appear to be completely desensitized during a long pulse (up to 4 min) of 200 μM ATP. Suramin (200 μM) accelerated an increase to peak of the current induced by 200 μM ATP without affecting the maximum response amplitude (n−4_. In about 10% of the neuronsn−3), 50 μM ATP evoked rapidly declining (about 1 sec) currents. Application of 100 μM α,β-Me-ATP to these neurons evoked similar responses. The above results suggest that submucous neurons express two specific subtypes of ionotropic P_2x-purinoceptors, which might be involved in distinct excitatory processes in these neurons.     

Modulation of Glutamate and Glycine Transporters by Niflumic,Flufenamic and Mefenamic Acids

Three fenamates—niflumic, flufenamic and mefenamic acids—were tested for effects on substrate-induced currents of glutamate and glycine transporters (EAAT1, EAAT2, GLYT1b and GLYT2a) expressed in Xenopus laevis oocytes. All fenamates inhibited EAAT1 currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 53 ± 4% (P < 0.001). EAAT2 currents were less sensitive, but 100 μM flufenamic acid inhibited the I _max by 34 ± 5% (P = 0.006). All fenamates inhibited GLYT1b currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 61 ± 1% (P < 0.001). At 100 μM, effects on the GLYT2a I _max were mixed: 13 ± 2% inhibition by flufenamic acid (P = 0.002), 30 ± 6% enhancement by niflumic acid (P = 0.002), and no effect by mefenamic acid. Minor effects on substrate affinity suggested non-competitive mechanisms. These data could contribute to the development of selective transport modulators.     

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.     

Characterization of TRPM8-like channels activated by the cooling agent icilin in the macrophage cell line RAW 264.7

Icilin is recognized as a chemical agonist of nociceptors and can activate TRPM8 channels. However, whether this agent has any effects on immune cells remains unknown. In this study, the effects of icilin on ion currents were investigated in RAW 264.7 murine macrophage-like cells. Icilin (1–100 μM) increased the amplitude of nonselective (NS) cation current (I _NS) in a concentration-dependent manner with an EC₅₀ value of 8.6 μM. LaCl₃ (100 μM) or capsazepine (30 μM) reversed icilin-induced I _NS; however, neither apamin (200 nM) nor iberiotoxin (200 nM) had any effects on it. In cell-attached configuration, when the electrode was filled with icilin (30 μM), a unique population of NS cation channels were activated with single-channel conductance of 158 pS. With the use of a long-lasting ramp pulse protocol, increasing icilin concentration produced a left shift in the activation curve of NS channels, with no change in the slope factor of the curve. The probability of channel opening enhanced by icilin was increased by either elevated extracellular Ca²⁺ or application of ionomycin (10 μM), while it was reduced by BAPTA-AM (10 μM). Icilin-stimulated activity is associated with an increase in mean open time and a reduction in mean closed time. Under current-clamp conditions, icilin caused membrane depolarization. Therefore, icilin interacts with the TRPM8-like channel to increase I _NS and depolarizes the membrane in these cells.     

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.     

Synergic Effects of β-Estradiol and Erythromycin on hERG Currents

The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties, the underlying mechanisms of the gender-based differences are not fully understood. In particular I _Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents were inhibited by β-estradiol maximally to 62% of control with an IC₅₀ of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin, which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced. Namely, hERG currents were inhibited maximally to 45.8% of control with an IC₅₀ of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher risk of drug-induced TDP in women than men.     

Uptake of amino acids by the aquatic resurrection plant Chamaegigas intrepidus and its implication for N nutrition

Chamaegigas intrepidus is a poikilohydric aquatic plant that lives in rock pools on granitic outcrops in Central Namibia. The pools are filled intermittently during the summer rains, and the plants may pass through up 20 rehydration/dehydration cycles during a single wet season. Rehydrated plants also have to cope with substantial diurnal fluctuations in the pH and extreme nutrient deficiency. Ammonium concentrations are normally around 30 μM. Additional nitrogen sources are amino acids. Total free amino acids are up to 15 μM with glycine and serine as the predominant amino acids. Experiments on uptake of radiolabelled amino acids into roots of C. intrepidus showed high␣affinity (K _M= 16 μM) and low-affinity (K _M= 159 μM) uptake systems. The K _M of the high-affinity system is well in accordance with the free amino acid concentration found in the water of the pools. We conclude that amino acids, predominantly glycine and serine, can be utilised by C. intrepidus in its natural habitat. Since glycine uptake showed a strong reduction at pH 10, nitrogen uptake from glycine or serine should occur mainly in the morning when the pH of the pool water is slightly acid. Further experiments with ¹⁵N-labelled ammonium in combination with non-labelled glycine demonstrated high ¹⁵N values in plant tissues. Under experimental conditions C. intrepidus preferred ammonium as a nitrogen source. The implication of amino acids for nitrogen nutrition of C. intrepidus may depend on the relation of inorganic and organic nitrogen available in the pool water and the preferential utilisation of one or the other nitrogen source may change during the day corresponding with pH changes in the water. Received: 28 January 1998 / Accepted: 24 July 1998     

Effects of mibefradil on synaptic transmission in the hippocampus and on voltage-dependent currents in isolated hippocampal and thalamic neurons of the rat

The effects of a novel anti-hypertensive drug, mibefradil, on voltage-dependent currents in isolated thalamic and hippocampal neurons, as well as on synaptic transmission in the hippocampus have been studied. Mibefradil exerted a potent inhibitory action on low-threshold calcium currents in thalamic neurons (IC₅₀=160 nM). In higher concentrations (1–20 μM), this drug blocked not only low-threshold calcium current but also voltage-dependent sodium and delayed potassium currents in pyramidal hippocampal neurons. The amplitude of population action potentials in hippocampal slices decreased by 55% in the presence of 20μM mibefradil. All of the effects of mibefradil were almost completely reversible. In our experiments, the sensitivity of low-threshold calcium channels in thalamic neurons to mibefradil was higher than that observed on other objects. The ability of mibefradil to block not only calcium currents but also other types of voltage-dependent ion conductances in hippocampal neurons may be considered an essential factor that determines the specificity of the pharmacological profile of this drug.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (I_gaba and I_Mus), respectively in the Mg²⁺-free external solution containing 1 μmol/L glycine at a holding potential (V _H ) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of I_gaba; (ii) when the neurons were incubated in a Ca²⁺-free bath or pre-loaded with a membrane-permeable Ca²⁺ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on I_GAba disappeared. Cd²⁺ (10 μmol/L) or La³⁺ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of I_gaba by NMDA application; (iii) the suppression of I_GAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca²⁺-dependent and CaMKII is involved in the process of the Ca²⁺-dependent inhibition.     

cAMP-dependent regulation of Ca2+ channels expressed inXenopus oocytes

Rat forebrain- and heart-derived mRNA were used to express Ca²⁺ channels inXenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca²⁺ channel currents (I _Ba, 40 mM Ba²⁺ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directedI _Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by 70% by 1 μM of ω-conotoxin GVIA, whileI _Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directedI _Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac”I _Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM), as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted no effect on the basal level ofI _Ba, suggesting that the expressed Ca²⁺ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration (2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA intoXenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca²⁺ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific, since N-type Ca²⁺ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of functional significance of phosphorylation.     

Memantine and the amino-alkyl-cyclohexane MRZ 2/579 are moderate affinity uncompetitive NMDA receptor antagonists –in vitro characterisation

Summary. There is general agreement that moderate affinity uncompetitive NMDA receptor antagonists combine good efficacy and tolerability in animal models of disturbances in glutamatergic transmission. There are several theories on which properties are important for this profile including 1, rapid access to the channel at the start of pathological overactivity 2, rapid, voltage-dependent relief of blockade during physiological synaptic activation and 3, partial untrapping. Merz has developed a series of novel uncompetitive NMDA receptor antagonists based on the cyclohexane structure. In cultured hippocampal neurones MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane) shows similar blocking kinetics to memantine (K_on 10.7 ^* 10⁴ M⁻¹ sec⁻¹, K_off 0.20 sec⁻¹ at −70 mV) and binds at the same depth in the NMDA receptor channel (δ = 0.8). The potency of MRZ 2/579 assessed as Kd = K_off/K_on = 1.87 μM agrees well with the IC₅₀ of 1.29 μM against steady-state currents in cultured hippocampal neurones (at −70 mV) and with the Ki in [³H]-MK-801 binding of 0.65 μM. MRZ 2/579 protected cultured cortical neurones against glutamate toxicity with an IC₅₀ of 2.16 μM and was also effective in protecting hippocampal slices against hypoxia / hypoglycaemia-induced reduction of fEPSP amplitude in CA1 with an EC₅₀ of 7.01 μM. MRZ 2/579 has similar potency and bio-availability to memantine in vivo assessed using microdialysis, microiontophoresis and MES-induced seizures. Initial characterization in animal models provides strong support for the assump-tion that MRZ 2/579 could be a useful therapeutic in morphine/alcohol dependence, inhibition of morphine tolerance, chronic pain and as a neuroprotective agent. Received August 31, 1999 Accepted September 20, 1999     

In vitro study of the tocolytic effect of oroxylin A from Scutellaria baicalensis root

Scutellariae Radix is one of the well-known tocolytic Chinese herbs. Oroxylin A is isolated from the root of Scutellaria baicalensis. The main syndrome of preterm birth is caused by uterus contractions from excitatory factors. Administration of tocolytic agents is a strategy to prevent the occurrence of preterm births. The aim of this study was to investigate the effects of oroxylin A on contractions of uterine strips isolated from non-pregnant female Wistar rats (250~350 g). Contractions of the uterus were induced with acetylcholine (Ach) (1 μM), PGF_2α (0.1 μM), oxytocin (10^-3 U/ml), KCl (56.3 mM), tetraethylammonium (TEA; 1 and 10 mM), 4-aminopyridine (4-AP; 5 mM), glipizide (30 μM), a nitric oxide synthase (NOS) inhibitor (LNNA; 10^-3M), a β-receptor blocker (propranolol; 10 μM), and a cyclooxygenase inhibitor (indomethacin; 60 μM). The inhibitory effects of the amplitude and frequency of spontaneous contractions by oroxylin A were antagonized with Ach (IC₅₀ 22.85 μM), PGF_2α (IC₅₀27.28 μM), oxytocin (IC₅₀ 12.34 μM), TEA; 1 and 10 mM (IC₅₀ 52.73 and 76.43 μM), 4-AP (IC₅₀ 67.16 μM), and glipizide (IC₅₀27.53 μM), but oroxylin A was not influenced by Ca²⁺-free medium, LNNA, propranolol, or indomethacin. Otherwise, oroxylin A-mediated relaxation of the rat uterus might occur through opening of uterine calcium-dependent potassium channels or adenosine triphosphate potassium channel activation. This suggests that oroxylin A is the tocolytic principle constituent of Scutellariae Radix, and oroxylin A may provide a lead compound for new tocolytic drug development in the future.     

Role of Prostanoid Production and Receptors in the Regulation of Retinal Endogenous Amino Acid Neurotransmitters by 8-Isoprostaglandin E<Subscript>2</Subscript>, Ex Vivo

The role of enzymes and receptors of the prostanoid pathway in the inhibitory effect of 8-isoprostaglandin E₂ (8-isoPGE₂) on endogenous amino acid neurotransmitter levels was examined, ex vivo. Freshly isolated bovine eyeballs were injected intravitreally with IsoPs, incubated in Krebs buffer for 30 min and retina prepared for HPLC-ECD detection of amino acids. 8-isoPGE₂ attenuated retinal glutamate and its metabolite, glutamine and glycine in a concentration-dependent manner. The non-selective cyclooxygenase (COX)-inhibitor, flurbiprofen, COX-2 selective inhibitor, NS-398 and thromboxane (Tx) synthase inhibitor, furegrelate had no effect on both basal amino acid levels and the inhibitory effects of 8-isoPGE₂ (1–100 μM) on the retinal amino acids. Whereas the TP-receptor antagonist SQ-29548(10 μM) exhibited no effect, SC-19220(EP₁; 30 μM), AH-6809(EP_1–3; 30 μM) and AH-23848(EP₄; 30 μM) reversed the inhibitory effects of 8-isoPGE₂ (0.01–100 μM) on glutamate, glutamine and glycine levels. We conclude that prostanoid EP-receptors regulate the inhibitory effect of 8-isoPGE₂ on basal levels of endogenous amino acids in bovine retina, ex vivo.     

In Vitro evaluation of the cytotoxic and anti-proliferative properties of resveratrol and several of its analogs

Resveratrol (RES), a component of red wine, possesses anti-inflammatory properties. The studies described in the present work were aimed at evaluating the potential for RES and related stilbene analogs (piceatannol, PIC; pterostilbene, TPS; trans-stilbene, TS; and trans-stilbene oxide, TSO) to exhibit toxicity towards RAW 264.7 mouse macrophages. The effect of TS, TSO, RES and TPS on RAW 264.7 macrophage viability was determined by two standard methods: (a) the MTT assay and (b) the trypan blue dye exclusion test. Whereas macrophages were more sensitive to PIC (LC_{50 trypan} ∼ 1.3 μM) and to TPS (LC_{50 trypan} ∼ 4.0 μM and LC_{50 MTT} ∼ 8.3 μM) than to RES (LC_{50 trypan} ∼ 8.9 μM and LC_{50 MTT} ∼ 29.0 μM), they were relatively resistant to TSO (LC_{50 trypan} ∼ 61.0 μM and LC_{50 MTT} > 100 μM) and to TS (LC_{50 trypan} ≥ 5.0 μM and LC_{50 MTT} ≥ 5.0 μM). The ability of selected stilbenes (RES, TPS and PIC) to exhibit growth inhibitory effects was also examined. Although RES and TPS were observed to inhibit cell proliferation in macrophages (IC₅₀ ≤ 25 μM), these cells were resistant to growth inhibition by PIC (IC₅₀ ≥ 50 μM). The data obtained in the present analysis demonstrate that substituted stilbene compounds such as RES have the capacity to exhibit cytotoxic and anti-proliferative activities in macrophages.     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

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.     

Role of cAMP, cGMP, and protein kinase C in regulation of calcium current through the L-type calcium channels in the electroexcitable membrane of smooth muscle cells

Regulation of calcium current through L-type calcium channels (I _Ca,L) of the guinea pigtaenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced theI _Ca,L amplitude in a dose-dependent manner. Dose-dependent suppression ofI _Ca,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects onI _Ca,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reducedI _Ca,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase inI _Ca,L. 8-Bromo-cAMP (1 μM) increasedI _Ca,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS onI _Ca,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pigtaenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase theI _Ca,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increasesI _Ca,L (probably through activation of PKA). PKG apparently mediatesI _Ca,L drops evoked by cAMP at high concentrations and by cGMP.     

Hypocotyl derived in vitro regeneration of pumpkin ash (<Emphasis Type="Italic">Fraxinus profunda</Emphasis>)

Pumpkin ash (Fraxinus profunda (Bush) Bush) is at risk for extirpation by an exotic insect, the emerald ash borer (EAB). Pumpkin ash is limited to wetland areas of the Eastern United States, and has been listed as an endangered species because of EAB activity. Pumpkin ash provides many benefits to the ecosystem, and its wood is used in the manufacturing industry. In vitro regeneration provides an integral tool for the mass propagation and genetic transformation of pumpkin ash to combat EAB. Therefore, a plant regeneration protocol was developed for pumpkin ash. Aseptically extracted hypocotyls formed adventitious shoots following 4 weeks on Murashige and Skoog (MS) medium supplemented with 0–22.2 μM 6-benzyladenine (BA) and 0–6.8 μM thidiazuron (TDZ) then transferred for an additional 4 weeks on MS medium with Gamborg B5 vitamins plus 0.2 g L⁻¹ glycine (B5G) containing 6.7 μM BA, 1 μM indole-3-butryic acid (IBA), and 0.29 μM gibberellic acid (GA₃). As adventitious shoots developed, these were transferred to a MSB5G medium with 13.3 μM BA, 1 μM IBA, and 0.29 μM GA₃ for shoot elongation. Elongated shoots were successfully micropropagated using MSB5 medium with 10 μM BA and 10 μM TDZ. Adventitious root formation was as high as 94% using woody plant medium supplemented with 4.9 μM IBA with shoots cultured for 10 days in the dark followed by culture under a 16-h photoperiod. Acclimatization to the greenhouse was successful and normal plant growth was observed. This protocol will provide a means for genetic transformation for EAB resistance and mass propagation for conservation.     

A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.