Nonylphenol,an environmental estrogen,affects voltage-gated K currents and L-type Ca currents in a non-monotonic manner in GH3 pituitary cells

We have investigated the characteristics of voltage-gated K⁺ channels and L-type Ca²⁺ channels in GH₃ rat pituitary cells and the effects of the xenoestrogen (XEs) nonylphenol (NP) on these ion channel currents. Our results have shown that the lower concentrations (10⁻¹⁵–10⁻¹⁴ M) of NP decreased the amplitudes of voltage-gated K⁺ currents (I_Kv) and activated L-type Ca²⁺ currents (I_Ca-L) by reducing half-activation membrane potentials of activation kinetics curves. However, the higher concentrations (10⁻¹⁰–10⁻⁹ M) of NP increased the amplitudes of I_Kv and inhibited I_Ca-L by reducing the peak values of I_Ca-L. Thus, NP affects I_Kv and I_Ca-L in an opposite and non-monotonic manner.     

Effect of arvanil (N-arachidonoyl-vanillyl-amine), a nonpungent anandamide-capsaicin hybrid,on ion currents in NG108-15 neuronal cells

The effects of arvanil (N-arachidonoyl-vanillyl-amine), a structural hybrid between capsaicin and anandamide, on ion currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. Arvanil (0.2-50 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Arvanil produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of arvanil-induced inhibition of I(Ca,L) was 2 microM. Arvanil (5 microM) could shift the steady-state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. No effect of arvanil (20 microM) on delayed rectifier K(+) current (I(K(DR))) was observed; however, capsaicin (20 microM), glyceryl nonivamide (20 microM) and capsinolol (20 microM) suppressed it significantly. Arvanil (20 microM) caused a slight reduction in the amplitude of erg (ether-à-go-go-related)-mediated K(+) current (I(K(erg))) without modifying the activation curve of this current, while capsaicin and glyceryl nonivamide were more effective in suppressing I(K(erg)). Under current-clamp configuration, arvanil decreased the firing frequency of action potentials. Arvanil-mediated inhibition of I(Ca,L) appeared to be independent of its binding to either vanilloid or cannabinoid receptors. The channel-blocking properties of arvanil may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.     

Inhibitory action of ICI-182,780, an estrogen receptor antagonist, on BK(Ca) channel activity in cultured endothelial cells of human coronary artery

ICI-182,780 is known to be a selective inhibitor of the intracellular estrogen receptors. The effect of ICI-182,780 on ion currents was studied in cultured endothelial cells of human coronary artery. In whole-cell current recordings, ICI-182,780 reversibly decreased the amplitude of K(+) outward currents. The decrease in outward current caused by ICI-182,780 could be counteracted by further application of magnolol or nordihydroguaiaretic acid, yet not by 17beta-estradiol. Under current-clamp condition, ICI-182,780 (3microM) depolarized the membrane potentials of the cells, and magnolol (10 microM) or nordihydroguaiaretic acid (10 microM) reversed ICI-182,780-induced depolarization. In inside-out patches, ICI-182,780 added to the bath did not alter single-channel conductance of large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels), but decreased their open probability. ICI-182,780 reduced channel activity in a concentration-dependent manner with an IC(50) value of 3 microM. After BK(Ca) channel activity was suppressed by 2-methoxyestradiol (3 microM), subsequent application of ICI-182,780 (3 microM) did not further reduce the channel activity. The application of ICI-182,780 shifted the activation curve of BK(Ca) channels to positive potentials. Its decrease in the open probability primarily involved a reduction in channel open duration. ICI-182,780 also suppressed the proliferation of these endothelial cells with an IC(50) value of 2 microM. However, in coronary smooth muscle cells, a bell-shaped concentration-response curve for the ICI-182,780 effect on BK(Ca) channel activity was observed. This study provides evidence that ICI-182,780 can inhibit BK(Ca) channels in vascular endothelial cells in a mechanism unlikely to be linked to its anti-estrogen activity. The inhibitory effects on these channels may partly contribute to the underlying mechanisms by which ICI-182,780 affects endothelial function.     

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells

The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.     

Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels

Background and purpose:

Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John''s Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels.

Experimental approach:

We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na⁺, I_A and I_K currents.

Key results:

Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I_A and delayed rectifier I_K potassium currents. In contrast, hypericin exerted no significant effect on both Na⁺ peak current and its decay kinetics.

Conclusions and implications:

Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I_A and I_K currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.     

Action potential changes associated with the inhibitory effects on voltage-gated sodium current of hippocampal CA1 neurons by silver nanoparticles

Nano-sized materials are now being used in medicine, biotechnology, energy, and environmental technology. Although a wide and growing number of applications for nanomaterials exist, there are limited studies available on toxicity of nanoparticles for their human risk and environmental assessment. The aim of this study was to investigate the effects of silver nanoparticles (nano-Ag) on voltage-activated sodium currents in hippocampal CA1 neurons. Nano-Ag was tested at increasing concentrations (10⁻⁶, 5 × 10⁻⁶, 10⁻⁵ g/ml). The research results showed that only nano-Ag (10⁻⁵ g/ml) reduced the amplitude of voltage-gated sodium current (I_Na). The nano-Ag particles produced a hyperpolarizing shift in the activation–voltage curve of I_Na and also delayed the recovery of I_Na from inactivation. Action potential properties and the pattern of repetitive firing were examined using whole cell current-clamp recordings. Peak amplitude and overshoot of the evoked single action potential were decreased and half-width was increased in the present of the 10⁻⁵ g/ml nano-Ag solution, and the firing rate of repetitive firing had no change. The results suggest that nano-Ag may alter the action potential of hippocampal CA1 neurons by depressing voltage-gated sodium current.     

Evidence for aconitine-induced inhibition of delayed rectifier K(+) current in Jurkat T-lymphocytes

Aconitine (ACO) is a highly toxic diterpenoid alkaloid and known to exert the immunomodulatory action. However, whether it has any effects on ion currents in immune cells remains unknown. The effects of ACO and other related compounds on ion currents in Jurkat T-lymphocytes were investigated in this study. ACO suppressed the amplitude of delayed-rectifier K⁺ current (I_K(DR)) in a time- and concentration-dependent manner. Margatoxin (100 nM), a specific blocker of K_V1.3-encoded current, decreased the I_K(DR) amplitude in these cells and the ACO-induced inhibition of I_K(DR) was not reversed by 1-ethyl-2-benzimidazolinone (30 μM) or nicotine (10 μM). The IC₅₀ value for ACO-mediated inhibition of I_K(DR) was 5.6 μM. ACO accelerated the inactivation of I_K(DR) with no change in the activation rate of this current. Increasing the ACO concentration not only reduced the I_K(DR) amplitude, but also accelerated the inactivation time course of the current. With the aid of minimal binding scheme, the inhibitory action of ACO on I_K(DR) was estimated with a dissociation constant of 6.8 μM. ACO also shifted the inactivation curve of I_K(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for I_K(DR) was enhanced in the presence of ACO. In Jurkat cells incubated with amiloride (30 μM), the ACO-induced inhibition of I_K(DR) remained unaltered. In RAW 264.7 murine macrophages, ACO did not modify the kinetics of I_K(DR), although it suppressed I_K(DR) amplitude. Taken together, these effects can significantly contribute to its action on functional activity of immune cells if similar results are found in vivo.     

Cross talk of cAMP and flavone in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and Na+/K+/2Cl- cotransporter in renal epithelial A6 cells

We have reported that in renal epithelial A6 cells flavones stimulate the transepithelial Cl- secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and/or the Na+/K+/2Cl- cotransporter. On the other hand, it has been established that cAMP activates the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter. However, no information is available on the interaction between cAMP and flavones on stimulation of the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter. To clarify the interaction between cAMP and flavones, we studied the regulatory mechanism of the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter by flavones (apigenin, luteolin, kaempherol, and quercetin) under the basal and cAMP-stimulated conditions in renal epithelial A6 cells. Under the basal (cAMP-unstimulated) condition, these flavones stimulated the Cl- secretion by activating the Na+/K+/2Cl- cotransporter without any significant effects on the CFTR Cl- channel activity. On the other hand, these flavones diminished the activity of the cAMP-stimulated Na+/K+/2Cl- cotransporter without any significant effects on the CFTR Cl- channel activity. Interestingly, the level of the flavone-induced Cl- secretion under the basal condition was identical to that under the cAMP-stimulated condition. Based on these results, it is suggested that although both cAMP and flavones activate the Na+/K+/2Cl- cotransporter, these flavones have more powerful effects than cAMP on the Na+/K+/2Cl- cotransporter.     

H89, an inhibitor of protein kinase A (PKA), stimulates Na+ transport by translocating an epithelial Na+ channel (ENaC) in fetal rat alveolar type II epithelium

The present study was performed to clarify the effect of H89, an inhibitor of cAMP-activated protein kinase (protein kinase A; PKA), on Na(+) absorption in fetal rat alveolar type II epithelium. H89 stimulated the Na(+) absorption by increasing the open probability (Po) and number of a nonselective cation (NSC) channel composed of four alpha subunits of epithelial Na(+) channel (ENaC). Brefeldin A (BFA), an inhibitor of intracellular protein translocation, blocked the stimulatory action of H89 on the Na(+) absorption by interrupting the action of H89 on the Po and number of the NSC channel. H85, an inactive form of H89, showed an effect similar to H89, suggesting that H89 does not show its effect by inhibiting PKA, but acts on the channel depending the structure. These observations indicate that: (1) the H89 induced increase in number of the channel at the apical membrane is due to translocation of alpha subunit of ENaC to the apical membrane, (2) the elevation of Po of the channel is mediated through translocation of a protein activating alpha subunit of ENaC, and (3) the effect of H89 is dependent on its structure without any relation to PKA.     

Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling

Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system. AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound. Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9. In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines. Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr. The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective. Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively. During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8. However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation. Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells.