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1.
A. A. Bukinich 《Journal of Evolutionary Biochemistry and Physiology》2010,46(1):61-68
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. 相似文献
2.
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 P2x-purinoceptors, which might be involved in distinct excitatory processes in these neurons. 相似文献
3.
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. 相似文献
4.
A. A. Bukinich E. A. Tsvetkov N. P. Vesselkin 《Journal of Evolutionary Biochemistry and Physiology》2007,43(1):43-50
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. 相似文献
5.
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. 相似文献
6.
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 DL-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, DL-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. 相似文献
7.
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. 相似文献
8.
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 相似文献
9.
Yu. V. Kirichok Ya. A. Boychuk M. V. Kopanitsa T. Sh. Tsintsadze N. A. Lozovaya O. A. Krishtal 《Neurophysiology》2000,32(1):12-16
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. 相似文献
10.
中国科学 《中国科学:生命科学英文版》2000,43(6):655-662
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 (Igaba and IMus), 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 Igaba; (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 IGAba 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 Igaba by NMDA application; (iii) the suppression of IGAba 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. 相似文献
11.
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. 相似文献
12.
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 (Kon 10.7 * 104 M−1 sec−1, Koff 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 = Koff/Kon = 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 相似文献
13.
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), PGF2α (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), PGF2α (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. 相似文献
14.
Min Zhao Christopher J. Destache Sunny E. Ohia Catherine A. Opere 《Neurochemical research》2009,34(12):2170-2180
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(EP1–3; 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. 相似文献
15.
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 (LC50 trypan ∼ 1.3 μM) and to TPS (LC50 trypan ∼ 4.0 μM and LC50 MTT ∼ 8.3 μM) than to RES (LC50 trypan ∼ 8.9 μM and LC50 MTT ∼ 29.0 μM), they were relatively resistant to TSO (LC50 trypan ∼ 61.0 μM and LC50 MTT > 100 μM) and to TS (LC50 trypan ≥ 5.0 μM and LC50 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. 相似文献
16.
The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster 总被引:2,自引:0,他引:2
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. 相似文献
17.
Montiel-Herrera M Zaske AM García-Colunga J Martínez-Torres A Miledi R 《Molecules and cells》2011,32(5):397-404
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 Fin and Sin-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. 相似文献
18.
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. 相似文献
19.
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. 相似文献
20.
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. 相似文献