Biomedical subjects

S N Wu

Publications and source records attributed to S N Wu.

At least 19 recordsLinked to original sources

PubMed2001

Functional coupling of voltage-dependent L-type Ca2+ current to Ca2+-activated K+ current in pituitary GH3 cells.

Ca2+-activated K+ currents (I(K(Ca)) can contribute to action potential repolarization and after-hyperpolarization in GH3 cells. In this study, we examined how the activation of I(K(Ca) at the cellular level could be functionally coupled to Ca2+ influx through L-type Ca2+ channels. A 30-msec Ca2+ influx step to 0 mV was found to exhibit substantial contribution of Ca2+ influx through the activation of I(Ca,L) to the activation of I(K(Ca)). A bell-shaped relationship between the conditioning potentials and the integrated I(K(Ca)) was observed, suggesting that the magnitude of integrated I(Ca,L) correlates well with that of integrated I(K(Ca)) in the same cell. A linear relationship of integrated I(Ca,L) and integrated I(K(Ca)) was found with a coupling ratio of 69+/-7. The value of the coupling ratio was unaffected by the presence of Bay K 8644 or nimodipine, although these compounds could effectively affect the amplitudes of both I(K(Ca)) and I(Ca,L). However, tetrandrine could decrease the coupling ratio. Paxilline or intracellular Ca2+ buffer with EGTA decreased the coupling ratio, while apamin had no effect on it. Interestingly, phorbol 12-myristate 13-acetate also reduced the coupling ratio significantly, whereas thapsigargin increased this value. Thus, the present study indicates that the activation of I(K(Ca)) during brief Ca2+ influx, which is inhibited by paxilline, is coupled to Ca2+ influx primarily through the L-type channels. The selective modulation of I(K(Ca)) by second messengers or Ca2+ release from internal stores may affect the coupling efficiency and hence cellular excitability.

Alkaloids↗

PubMed2001

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells.

2-Methoxyestradiol, an endogenous metabolite of 17beta-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3-30 microm) reversibly suppressed the amplitude of K+ outward currents. The IC50 value of the 2-methoxyestradiol-induced decrease in outward current was 3 microm. Evans blue (30 microm) or niflumic acid (30 microm), but not diazoxide (30 microm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 microm) to the bath did not modify the single-channel conductance of large-conductance Ca2+-activated K+ (BKCa) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 microm) produced a shift in the activation curve of BKCa channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BKCa channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17beta-estradiol (10 microm) nor estriol (10 microm) affected BKCa channel activity, whereas 2-hydroxyestradiol (10 microm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 microm) caused membrane depolarization and Evans blue (30 microm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BKCa channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells.

2-Methoxyestradiol↗

PubMed2001

Vinpocetine-induced stimulation of calcium-activated potassium currents in rat pituitary GH3 cells.

The effects of vinpocetine, an inhibitor of cyclic GMP phosphodiesterase, on ionic currents were examined in rat pituitary GH3 lactotrophs with the aid of the patch-clamp technique. In GH3 cells bathed in normal Tyrode's solution, vinpocetine (10 microM) reversibly increased the amplitude of Ca2+-activated K+ current (I(K)Ca) with an EC50 value of 4 microM. When the recording pipettes were filled with 10 mM EGTA, vinpocetine also stimulated I(K)Ca. In the cell-attached configuration, application of vinpocetine to the bath increased the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels. In excised membrane patches, application of vinpocetine (10 microM) to the bath did not change the single-channel conductance of BK(Ca) channels; however, it did increase channel activity. In the inside-out configuration, neither 8-bromo cyclic GMP nor YC-1 applied intracellularly affected BK(Ca) channel activity. The vinpocetine-induced change in the kinetic behavior of BK(Ca) channels was due to an increase in mean open time and a decrease in mean closed time. Vinpocetine (10 microM) caused a leftward shift in the midpoint for the voltage-dependent opening. Under the current-clamp mode, vinpocetine (10 microM) decreased the firing rate of spontaneous action potentials induced by thyrotropin-releasing hormone (10 microM) in GH3 cells. In pheochromocytoma PC12 cells, vinpocetine (10 microM) applied intracellularly also enhanced the activity of BK(Ca) channels without altering single-channel conductance. Thus, the present study suggests that vinpocetine-mediated stimulation of I(K)Ca may result from the direct activation of BK(Ca) channels and indirectly from elevated cytosolic Ca2+.

Animals↗

PubMed2001

Quantitative analysis of end-tidal carbon dioxide during mechanical and spontaneous ventilation in infants and young children.

Capnography provides a substitute for monitoring of arterial carbon dioxide tension (PCO(2)). We performed a prospective study to evaluate a new application of capnography, using quantitative curve analysis in the pediatric ICU. Twenty-five infants and children admitted to the pediatric ICU after cardiovascular surgery for congenital heart diseases were included in the study. Capnographic curves were recorded during 3 phases of mechanical and spontaneous ventilation: phase 1, immediate postoperative period; phase 3, preextubation period; and phase 2, period between phases 1 and 3. Each recording included 17 sec of capnographic tracings from consecutive spontaneous and/or ventilator-driven breaths. Quantitative curve analysis was made to define parameters including peak value of exhaled PCO(2) (P), mean rate of rise of PCO(2) (R), and area under each capnographic curve (A). Qualitative inspection of the wave contour showed no obvious difference in phase 3 during spontaneous and mechanically assisted ventilator breaths. However, an obvious difference existed between spontaneous and mechanically assisted breaths in phase 2. For each parameter (P, R, and A), there was a significant difference in phases 2 and 3 from spontaneous breaths. However, there was no significant difference in phases 2 and 3 from ventilator-assisted breaths. We further calculated the ratio of parameters of spontaneous breaths (S) and ventilator-assisted breaths (V) in phase 2 and phase 3. The ratio of S/V for P, R, and A showed significant differences between phase 2 and phase 3. We conclude that quantitative analysis of exhaled end-tidal PCO(2) curves revealed significant changes of specific parameters during the transition from the ventilator-dependent state to the spontaneously breathing ventilator-independent state. This new approach provides a new way to estimate respiratory status in infants and children receiving ventilator therapy. Through quantitative capnographic curve analysis, if P, R, and A from spontaneous breaths approached those of ventilator-assisted breaths, patients have resumed reasonable pulmonary mechanics, and extubation may then be considered.

Capnography↗

PubMed2001

Characterization of action potential waveform-evoked L-type calcium currents in pituitary GH3 cells.

The response of the L-type Ca2+ current (ICa,L) in pituitary GH3 cells to variations in the action potential (AP) waveform was examined using the whole-cell configuration of the patch-clamp technique. ICa,L evoked during an AP waveform exhibited an early and a late component. The early component occurred on the rising phase of the AP; the late component coincided with the falling phase. Prolonging the falling phase of the AP increased the Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced. Prolonging the peak voltage of the AP waveform, however, increased the amplitude of the late component. ICa,L inactivated during a train of AP waveforms. When Ba2+ was used as the charge carrier, current inactivation during a train of APs decreased. Likewise, ICa,L evoked by the AP templates with irregular bursting pattern was inactivated. When the repetitive firing of APs with depolarizing potentials was replayed to cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory. After application of thyrotropin releasing hormone (TRH; 10 microM), ICa,L in response to rectangular pulses was increased and the current/voltage relation shifted slightly to more negative values. TRH (10 microM), thapsigargin (10 microM) or cyclopiazonic acid (30 microM) enhanced the late component of the AP-evoked ICa,L. TRH also attenuated the inactivation of ICa,L during a train of APs. These results indicate that in pituitary GH3 cells, the time course and kinetics of ICa,L during the AP waveforms is distinct from that evoked by rectangular voltage clamp. Changes in the shape and firing pattern of APs in GH3 cells can modulate Ca2+ influx through L-type Ca2+ channels. Ca2+ release from internal stores may affect the magnitude of AP-evoked ICa,L in these cells.

Action Potentials↗

PubMed2001

Rutaecarpine-induced block of delayed rectifier K+ current in NG108-15 neuronal cells.

The effects of rutaecarpine on ionic currents of NG108-15 neuronal cells were investigated in this study. Rutaecarpine (2-100 microM) suppressed the amplitude of delayed rectifier K+ current (I(K(DR))) in a concentration-dependent manner. The IC50 value for rutaecarpine-induced inhibition of I(K(DR)) was 11 microM. I(K(DR)) present in these cells is sensitive to the inhibition by quinidine and dendrotoxin, yet not by E-4031. The presence of rutaecarpine enhanced the rate and extent of I(K(DR)) inactivation, although it had no effect on the initial activation phase of I(K(DR)). Recovery from block by rutaecarpine (5 microM) was fitted by a single exponential with a value of 2.87 s. Crossover of tail currents in the presence of rutaecarpine was also observed. Cell-attached single-channel recordings revealed that rutaecarpine decreased channel activity, but it did not alter single-channel amplitude. With the aid of the binding scheme, a quantitative description of the rutaecarpine actions on I(K(DR)) was provided. However, rutaecarpine (20 microM) had no effect on L-type Ca2+ current. Under current-clamp configuration, rutaecarpine prolonged action potential duration in NG108-15 cells. These results show that rutaecarpine is a blocker of the K(DR) channel. The increase in action potential duration induced by rutaecarpine can be explained mainly by its blocking actions on I(K(DR)).

Action Potentials↗

PubMed2001

Ceramide inhibits the inwardly rectifying potassium current in GH(3) lactotrophs.

The effects of ceramide on ion currents in rat pituitary GH(3) cells were investigated. Hyperpolarization-elicited K(+) currents present in GH(3) cells were studied to determine the effect of ceramide and other related compounds on the inwardly rectifying K(+) current (I(K(IR))). Ceramide (C(2)-ceramide) suppressed the amplitude of I(K(IR)) in a concentration-dependent manner, with an IC(50) value of 5 microM. Ceramide caused a rightward shift in the midpoint for the activation curve of I(K(IR)). Pretreatment with PD-98059 (30 microM) or U-0126 (30 microM) did not prevent ceramide-mediated inhibition of I(K(IR)). However, the magnitude of ceramide-induced inhibition of I(K(IR)) was attenuated in GH(3) cells preincubated with dithiothreitol (10 microM). TNF alpha (100 ng/g) also suppressed I(K(IR)). In the inside-out configuration, application of ceramide (30 microM) to the bath slightly suppressed the activity of large conductance Ca(2+)-activated K(+) channels. Under the current clamp mode, ceramide (10 microM) increased the firing of action potentials. Cells that exhibited an irregular firing pattern were converted to those displaying a regular firing pattern after application of ceramide (10 microM). Ceramide also suppressed I(K(IR)) in neuroblastoma IMR-32 cells. Therefore, ceramide can produce a depressant effect on I(K(IR)). The blockade of this current by ceramide may affect cell function.

Action Potentials↗

PubMed2001

Fenamates stimulate BKCa channel osteoblast-like MG-63 cells activity in the human.

BACKGROUND: The fenamates, a family of nonsteroidal anti-inflammatory drugs that are derivatives of N-phenylanthranilic acid, are the inhibitors of cyclo-oxygenase. The ionic mechanism of actions of these compounds in osteoblasts is not well understood. METHODS: The effects of the fenamates on ionic currents were investigated in a human osteoblast-like cell line (MG-63) with the aid of the whole-cell and inside-out configurations of the patch-clamp technique. RESULTS: In MG-63 cells, niflumic acid and meclofenamic acid increased K+ outward currents (IK). The niflumic acid-stimulated IK was reversed by subsequent application of iberiotoxin or paxilline, yet not by that of glibenclamide or apamin. In the inside-out configuration, niflumic acid (30 micromol/L) added to the bath did not modify single-channel conductance but increased the activity of large-conductance Ca2+-activated K+ (BKCa) channels. The EC50 values for niflumic acid- and meclofenamic acid-induced channel activity were 22 and 24 micromol/L, respectively. Niflumic acid (30 micromol/L) and meclofenamic acid (30 micromol/L) shifted the activation curve of BKCa channels to less positive membrane potentials. Membrane stretch potentiated niflumic acid-stimulated channel activity. The rank order of potency for the activation of BKCa channels in these cells was niflumic acid = meclofenamic acid > tolfenamic acid > flufenamic acid > nimesulide. Evans blue and nordihydroguaiaretic acid increased channel activity; however, indomethacin, piroxicam, and NS-398 had no effect on it. CONCLUSIONS: The fenamates can stimulate BKCa channel activity in a manner that seems to be independent of the action of these drugs on the prostaglandin pathway. The activation of the BKCa channel may hyperpolarize the osteoblast, thereby modulating osteoblastic function.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2000

Characterization of ATP-sensitive potassium channels functionally expressed in pituitary GH3 cells.

ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 microM) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mM) or sodium cyanide (300 microM) increased KATP channel activity, while nicorandil (100 microM) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 microM. The activation of phospholipase A2 caused by mellitin (1 microM) was found to enhance KATP channel activity and further application of aristolochic acid (30 microM) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4'-dithiodipyridine (100 microM) also induced KATP channel activity. Diazoxide, mellitin and 4,4'-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 microM) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that KATP channels similar to those seen in pancreatic beta cells are functionally expressed in GH3 cells. In addition to the presence of Ca(2+)-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential.

2,4-Dinitrophenol↗

PubMed2000

The mechanism of actions of 3-(5'-(hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) on Ca(2+)-activated K(+) currents in GH(3) lactotrophs.

The effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an activator of soluble guanylyl cyclase, on ionic currents have been assessed in rat pituitary GH(3) lactotrophs. In GH(3) cells bathed in normal Tyrode's solution, YC-1 (1 microM) reversibly suppressed the amplitude of the Ca(2+)-activated K(+) current (I(K(Ca))). YC-1 at a concentration above 10 microM produced a biphasic response in the amplitude of I(K(Ca)), i.e., an initial decrease followed by a sustained increase. When the pipette solutions were filled with high EGTA (10 mM), the YC-1-induced stimulatory effect on I(K(Ca)) was abolished. Over a similar concentration range, YC-1 also effectively inhibited the voltage-dependent K(+) current (I(K(V))) in GH(3) cells. The IC(50) value required for the inhibition of I(K(V)) by YC-1 was 1 microM. Unlike YC-1, 8-bromo cGMP did not inhibit I(K(Ca)). However, YC-1 (10 microM) did not affect the amplitude of L-type Ca(2+) current. In the cell-attached configuration, application of YC-1 (10 microM) to the bath did not change the single-channel conductance of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels; however, it did increase the opening probability of BK(Ca) channels. In contrast, in the outside-out configuration, YC-1 (10 microM) significantly suppressed the opening probability of BK(Ca) channels. The present study shows dual effects of YC-1 on I(K(Ca)) in GH(3) cells. The YC-1-mediated stimulation of I(K(Ca)) may result from elevated cytosolic Ca(2+), whereas the inhibition of I(K(Ca)) and I(K(V)) by YC-1 appears to be direct and independent of the activation of soluble guanylyl cyclase. Caution thus needs to be used in attributing the YC-1-mediated response to the activation of soluble guanylyl cyclase.

Animals↗

PubMed2000

Actions of epoxyeicosatrienoic acid on large-conductance Ca(2+)-activated K(+) channels in pituitary GH(3) cells.

Epoxyeicosatrienoic acids (EETs), a family of cytochrome P450 epoxygenase metabolites of arachidonic acid, are believed to have an autocrine or paracrine role in the regulation of neurons or neuroendocrine cells. The effects of 14,15-EET on ionic currents were investigated in rat pituitary GH(3) cells. In the whole-cell configuration, 14,15-EET (3 microM) reversibly increased the amplitude of the Ca(2+)-activated K(+) current (I(K(Ca))). The 14, 15-EET-induced increase in I(K(Ca)) was unaffected in the presence of 10 microM thyrotropin-releasing hormone externally or 10 microM inositol trisphosphate in the recording pipette. In cells preincubated with pertussis toxin or herbimycin A, the 14, 15-EET-induced increase in I(K(Ca)) was also not changed. In the inside-out configuration, 14,15-EET applied intracellularly did not change single-channel conductance, but did increase the opening probability of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. 14,15-EET (3 microM) shifted the activation curve of BK(Ca) channels to less positive membrane potential by approximately 15 mV. The change in the kinetic behavior of BK(Ca) channels caused by 14,15-EET is explained by a lengthening of open and a shortening of closed times. 14,15-EET increased the activity of BK(Ca) channels in a concentration-dependent manner with an EC(50) value of 1 microM. However, 14,15-EET did not affect the Ca(2+) sensitivity of BK(Ca) channels. The present study indicates that 14,15-EET is an opener of BK(Ca) channels in GH(3) cells and that the stimulatory effect of 14, 15-EET on these channels may, at least in part, contribute to the underlying cellular mechanisms by which EETs affect neuronal or neuroendocrine function.

8,11,14-Eicosatrienoic Acid↗

PubMed2000

Evidence for the stimulatory effect of resveratrol on Ca(2+)-activated K+ current in vascular endothelial cells.

OBJECTIVE: Resveratrol, a natural phytoalexin compound, is present in grapes and wine, and it can produce vasorelaxation. However, little is known of its mechanisms of action on ionic currents in endothelial cells. METHODS: The effect of resveratrol on Ca(2+)-activated K+ currents in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein was investigated with the aid of the patch-clamp technique. RESULTS: In the whole-cell configuration, resveratrol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by resveratrol was greatly inhibited by iberiotoxin (200 nM) or paxilline (1 microM), but not by glibenclamide (10 microM), tamoxifen (10 microM), or beta-bungarotoxin (200 nM). Thus, this outward current is believed to be Ca(2+)-activated K+ current (I K(Ca)). In the inside-out configuration, bath application of resveratrol (30 microM) caused no change in the single-channel conductance, but increased the activity of large-conductance Ca(2+)-activated K+ (BKCa) channels. Resveratrol enhanced the channel activity in a concentration-dependent manner. The EC50 value for resveratrol-induced channel activity was 20 microM. The resveratrol-stimulated increase in the channel activity was independent of internal Ca2+. Resveratrol (30 microM) also shifted the activation curve of BKCa channels to less positive membrane potentials. The change in the kinetic behavior of BKCa channels caused by resveratrol in these cells in due to an increase in mean open time and a decrease in mean closed time. In a pancreatic islet endothelial cell line (MS1), resveratrol (30 microM) also increased the activity of intermediate-conductance KCa channels. CONCLUSIONS: These results provide evidence that in addition to the presence of antioxidative activity, resveratrol can also stimulate KCa channels in endothelial cells. The direct stimulation of these KCa channels by resveratrol may be responsible for its effect on the functional activities of endothelial cells.

Cell Line↗

PubMed2000

Characterization of inhibition by risperidone of the inwardly rectifying K(+) current in pituitary GH(3) cells.

The effects of risperidone on ionic currents in rat pituitary GH(3) cells were investigated with the aid of the patch-clamp technique. Hyperpolarization-activated K(+) currents in GH(3) cells bathed in high-K(+) Ca(2+)-free solution were studied to determine the effect of risperidone and other related compounds on the inwardly rectifying K(+) current (I(K(IR))). Risperidone (0.1-10 microM) suppressed the amplitude of I(K(IR)) in a concentration-dependent manner. The IC(50) value for the risperidone-induced inhibition of I(K(IR)) was 1 microM. Risperidone (3 microM) was found to slow the rate of activation. An increase in current deactivation by the presence of risperidone was also observed. Haloperidol (10 microM) and thioridazine (10 microM) inhibited the amplitude of I(K(IR)) effectively, and clozapine slightly suppressed it; however, metoclopramide (10 microM) had no effect on it. Risperidone (10 microM) had no effect on voltage-dependent K(+) and L-type Ca(2+) currents. However, in the inside-out configuration, risperidone (10 microM) did not alter the single-channel conductance, but reduced the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Under the current-clamp mode, risperidone (3 microM) depolarized the membrane potential and increased the firing rate. With the aid of the spectral analysis, cells that exhibited an irregular firing pattern were also converted to those displaying a regular firing pattern after addition of risperidone (3 microM). The present study provides evidence that risperidone, in addition to the blockade of dopamine receptors, can produce a depressant effect on I(K(IR)) and BK(Ca) channels, and implies that the blockade of these ionic currents by risperidone may affect membrane excitability and prolactin secretion in GH(3) cells.

Action Potentials↗

PubMed2000

Differential regulation of Ca2+ influx by fMLP and PAF in human neutrophils: possible involvement of store-operated Ca2+ channel.

Calcium (Ca2+) influx into human polymorphonuclear cells (PMNs) in response to N-formyl-Met-Leu-Phe (fMLP) and platelet-activating factor (PAF) stimulation was studied. Whole blood was taken by venous puncture from healthy human volunteers. PMNs were isolated, diluted, and incubated with 2 microM fura-2 AM. The cytosolic free calcium concentration, [Ca2+]i, in human neutrophils was determined by microfluorometry. We found that the net area under the fMLP- or PAF-induced [Ca2+]i rise curve in Ca2+-free medium decreased to 75% or 30% of the area under the curve in Ca2+ medium. Treatment of PMNs with phorbol myristate acetate (PMA), a protein kinase C activator, completely abolished the intracellular Ca2+ level stimulated by PAF, but not the intracellular Ca2+ level stimulated by fMLP. Treatment of PMNs with PAF did not abolish the intracellular Ca2+ level elevation stimulated by fMLP. In addition, treatment of PMNs with fMLP did not abolish intracellular Ca2+ level elevation stimulated by PAF. Loperamide, a positive modulator for store-operated calcium (SOC) channels, elicited an increase in intracellular calcium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3',5'-cyclic monophosphate, N2,2'-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF- or fMLP-induced PMNs intracellular Ca2+ fluorescences was well preserved, but the slope and the peak height of fluorescence curves declined compared with the curves without db-cGMP. In conclusion, we found that PAF and fMLP regulate the Ca2+ influx of PMNs in different ways. Most of the PAF-induced [Ca2+]i rise resulted from Ca2+ influx, and most of the fMLP-induced [Ca2+]i elevation resulted from intracellular stores release. The initial mobilization of intracellular Ca2+ stores in PAF-stimulated signals is mediated by protein kinase C (PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are present and important in the fMLP- or PAF-induced PMNs Ca2+ influx. There was no apparent cross-regulation between PAF- and fMLP-stimulated intracellular Ca2+ influx.

Calcium↗

PubMed2000

Characterization of tetrandrine-induced inhibition of large-conductance calcium-activated potassium channels in a human endothelial cell line (HUV-EC-C).

The effects of tetrandrine, a blocker of voltage-dependent Ca(2+) channels, on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In whole-cell configuration, tetrandrine (0.5-50 microM) reversibly decreased the amplitude of K(+) outward currents. The IC(50) value of tetrandrine-induced decrease in outward current was 5 microM. The K(+) outward current in response to depolarizing voltage pulses was also inhibited by iberiotoxin (200 nM), yet not by glibenclamide (10 microM) or apamin (200 nM). The reduced amplitude of outward current by tetrandrine can be reversed by the further addition of Evans' blue (30 microM) or niflumic acid (30 microM). Thus, the tetrandrine-sensitive component of outward current is believed to be Ca(2+)-activated K(+) current. Pretreatment with thapsigargin (1 microM) or sodium nitroprusside (10 microM) for 5 h did not prevent tetrandrine-mediated inhibition of outward current. In outside-out configuration, bath application of tetrandrine (5 microM) did not change the single-channel conductance but significantly reduced the opening probability of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. The tetrandrine-mediated decrease in the channel activity was independent on internal Ca(2+) concentration. Tetrandrine (5 microM) can also shift the activation curve of BK(Ca) channels to more positive potentials by approximately 20 mV. The change in the kinetic behavior of BK(Ca) channels caused by tetrandrine is due to a decrease in mean open time and an increase in mean closed time. The present study provides substantial evidence that tetrandrine is capable of suppressing the activity of BK(Ca) channels in endothelial cells. The direct inhibition of these channels by tetrandrine should contribute to its effect on the functional activities of endothelial cells.

Alkaloids↗

PubMed2000

Stimulatory effects of delta-hexachlorocyclohexane on Ca(2+)-activated K(+) currents in GH(3) lactotrophs.

delta-Hexachlorocyclohexane (delta-HCH), a lipophilic neurodepressant agent, has been shown to inhibit neurotransmitter release and stimulate ryanodine-sensitive Ca(2+) channels. However, the effect of delta-HCH on neuronal activity remains unclear, although it may enhance the gamma-aminobutyric acid-induced current. Its effects on ionic currents were investigated in rat pituitary GH(3) cells and human neuroblastoma IMR-32 cells. In GH(3) cells, delta-HCH increased the amplitude of Ca(2+)-activated K(+) current (I(K(Ca))). delta-HCH (100 microM) slightly inhibited the amplitude of voltage-dependent K(+) current. delta-HCH (30 microM) suppressed voltage-dependent L-type Ca(2+) current (I(Ca, L)), whereas gamma-HCH (30 microM) had no effect on I(Ca, L). In the inside-out configuration, delta-HCH applied intracellularly did not change the single channel conductance of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels; however, it did increase the channel activity. The delta-HCH-mediated increase in the channel activity is mainly mediated by its increase in the number of long-lived openings. delta-HCH reversibly increased the activity of BK(Ca) channels in a concentration-dependent manner with an EC(50) value of 20 microM. delta-HCH also caused a left shift in the midpoint for the voltage-dependent opening. In contrast, gamma-HCH (30 microM) suppressed the activity of BK(Ca) channels. Under the current-clamp mode, delta-HCH (30 microM) reduced the firing rate of spontaneous action potentials; however, gamma-HCH (30 microM) increased it. In neuroblastoma IMR-32 cells, delta-HCH also increased the amplitude of I(K(Ca)) and stimulated the activity of intermediate-conductance K(Ca) channels. This study provides evidence that delta-HCH is an opener of K(Ca) channels. The effects of delta-HCH on these channels may partially, if not entirely, be responsible for the underlying cellular mechanisms by which delta-HCH affects neuronal or neuroendocrine function.

Action Potentials↗

PubMed2000

Regulation of Ca(2+)-activated K+ currents by ciglitazone in rat pituitary GH3 cells.

BACKGROUND: Ciglitazone, an antidiabetic agent of the thiazolidinedione family, is known to be an activator of the peroxisome-proliferator activator receptor (PPAR)-gamma. The underlying mechanism of ciglitazone actions on ionic currents in neuroendocrine cells remains unclear. METHODS: The effects of ciglitazone on ionic currents were investigated in rat pituitary GH3 cells using the whole-cell and inside-out configurations of the patch-clamp technique. RESULTS: In GH3 cells, ciglitazone at 3-300 mumol/L caused a reversible increase in the amplitude of the Ca(2+)-activated K+ current (IK(Ca)) with a half-maximal concentration of 16 mumol/L. Under the inside-out patch recording mode, ciglitazone applied intracellularly increased the activity of the large-conductance Ca(2+)-activated K+ (BKCa) channels, but did not affect their single-channel conductance. However, troglitazone (30 mumol/L) caused a reduction in the channel activity. The ciglitazone-induced change in the kinetic behavior of BKCa channels is due to an increase in mean open time and a decrease in mean closed time, whereas the troglitazone-induced decrease in the channel activity is related to a decrease in mean open time and an increase in mean closed time. Ciglitazone caused a left shift in the midpoint for voltage-dependent opening. The ciglitazone-stimulated activity of BKCa channels is independent of internal Ca2+. Under the current clamp mode, ciglitazone (30 mumol/L) hyperpolarized the membrane potential. CONCLUSIONS: This study shows that in addition to its activation of PPAR-gamma, ciglitazone can stimulate the activity of BKCa channels expressed in GH3 cells. These effects may affect membrane potentials and contribute to the ciglitazone-induced change in the functional activity of neurons or neuroendocrine cells.

Adenosine Triphosphate↗

PubMed1999

A further investigation of ATP-induced calcium mobilization in MDCK cells.

We have previously reported that La3+ inhibited the ATP-induced rise in intracellular Ca2+ levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Here we further investigated the ATP-induced Ca2+ signal. ATP caused a rise in [Ca2+]i dose-dependently between 1 microM-1 mM. The rises induced by 10 microM-1 mM ATP were inhibited by Ca2+ removal. The pleateau phase of the ATP response was primarily maintained by Ca2+ influx because it was reduced or eliminated by Ca2+ removal. ATP failed to elevate [Ca2+]i after the endoplasmic reticulum Ca2+ store had been depleted by 2,5-di-tert-butylhydroquinone or cyclopiazonic acid, suggesting that the ATP-induced Ca2+ influx was capacitative Ca2+ entry. Capacitative Ca2+ entry was directly measured by addition of 5 mM CaCl2 to cells pretreated with ATP (0.1 mM) in Ca(2+)-free medium. This capacitative Ca2+ entry was inhibited by econazole (25 microM) or SKF96365 (50 microM). The ATP response was significantly enhanced by extracellular alkalization to pH 8 or pretreatment with gly-phe-beta-naphthylamide. Pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP) or extracellular Na+ removal had no enhancement, implicating that efflux via plasmalemmal Ca2+ pumps (but not Na+/Ca2+ exchange) and buffering by lysosomes (but not mitochondria) might be involved in the decay of the ATP response.

Adenosine Triphosphate↗