细胞α1B-肾上腺素受体引起的Ca2+依赖性K+电流和Ca2+内流
作者：关永源 陶亮 贺华 韩启德 张幼怡 孙家钧
单位：关永源 陶亮 贺华 孙家钧 中山医科大学药理教研室； 广州，510089；韩启德 张幼怡 北京医科大学血管医学研究所； 北京，100083
关键词：钙通道；钾通道；受体；肾上腺素能α1；HEK-293细胞；电生理学
中山医科大学学报990402Study in the Ca2+-Dependent K+-Current and Ca2+Entry Inducedby Activation of α1B-Adrenoceptor Subtype in HEK-293 Cell
Guan Yongyuan1 Tao Liang1 He Hua1 Han Qide2 Zhang Youyi2 Sun Jiajun1
, http://www.100md.com
( 1 Department of Pharmacology, Sun Yat-sen University of Medical Sciences, Guangzhou, 510089
2 Institute of Vascular Medicine, Beijing Medical University, Beijing,100083)
Abstract Objective: To study the characterization of the outward K+-current and Ca2+ entry induced by activation of α1B-adrenoceptor subtype at the HEK-293 cells. Methods: The single K+-current was recorded from the cell attached configuration. The cytoplasmic Ca2+ was measured by fura-2 probe. Results: adrenaline evoked an outward K+-current with a conductance of 160 pS. The current was markedly inhibited by 50 μmol.L-1 chloroethylclonidine, 5 mmol.L-1 EGTA or 2 mmol.L-1 TEA. Nifedipine did not change this current and adrenaline-induced Ca2+ entry wihich was inhibited by 1 mmol.L-1 LaCl3. Conclusion: Activation of α1B-subtype receptor at HEK-293 cells evokes Ca2+ entry through the nifedipine resistant Ca2+ channel, followed by an outward K+-current.
, http://www.100md.com
Subject headings calcium channels; potassium channels; receptors, adrenergic, alpha-1; HEK-293 cell; electrophysiology
摘 要 目的：研究HEK-293细胞上α1B-肾上腺素受体亚型引起向外K+电流和Ca2+内流的特性。方法：细胞贴附式单通道记录K+通道和Fura-2荧光测定胞浆游离Ca2+浓度。结果：肾上腺素或苯肾上腺素可引发一电导为160 pS的外向K+电流。该电流可被50 μmol.L-1 氯乙醛可乐定(CEC)，5 mmol.L-1依他酸(EGTA)或2 mmol.L-1 四乙铵(TEA)抑制。硝苯吡啶(nifedipine)不改变该电流及α1B亚型引起的Ca2+内流；后者可被1 mmol.L-1 LaCl3抑制。结论：激活转染在HEK-293细胞上的α1B-受体亚型可引起通过硝苯吡啶不敏感Ca2+通道的Ca2+内流，并跟随产生-外向K+电流。
, http://www.100md.com
中图号 R 329.25
It has been suggested that α1-adrenoceptor (α1-AR)can be divided into α1A,α1B and α1D three subtypes. Because these α1-AR subtypes coexist in many tissues[1]，it is difficult to identify the functions of individual α1-AR subtype. Recently, a permanently transformed cell line derived from the human embryo renal cortical cells (HEK-293 cell line) has usually been used to express the cloned cDNA of various receptors[2～4]. HEK-293 cells were respectively transfected with α1A-,α1B- and α1D-AR subtypes. However, there is no report about the K+-currents induced by activation of α1B-AR in HEK-293 cells. In this study, we determined the Ca2+-dependent K+-currents and the change of intracellular Ca2+ concentration ([Ca2+]i),induced by activation of α1B-adrenoceptor subtype at the transfected HEK-293 cells.
, http://www.100md.com
1 Material and methods
1.1 Cell culture
Subclone of HEK-293 transfected with hamster α1B was kindly provided by Dr. Minneman. Subclones were maintained in DMEM containing 100 ml.L-1 calf serum, and propagated in the continued presence of selective antibiotic of 50 mg.L-1 hygromycin-pREP4.
1.2 Single channel recording
, 百拇医药
Standard patch clamp techniques[5] were used for recording the membrane currents in the α1B transfected HEK-293 cells. Single K+-current was made from the cell attached configuration at 30 ℃. The pipette solution contained:KCl 139 mmol.L-1,MgCl2 0.5 mmol.L-1, CaCl2 0.1 mmol.L-1, HEPES 10, egtazic acid (EGTA) 0.09 mmol.L-1, glucose 10 mmol.L-1, pH 7.4 with KOH. The extracellular solution contained: NaCl 130 mmol.L-1, MgCl2 1.2 mmol.L-1, CaCl2 1.8 mmol.L-1, KCl 5.4 mmol.L-1, HEPES 10 mmol.L-1,glucose 5.2 mmol.L-1, pH 7.4 with NaOH. The pipettes had outside tip diameters between 1～2 μm and had resistance values in the range 4～8 mΩ. A patch clamp amplifier (Axopatch-1D; Axon Instruments, Inc.) was used to record currents with a lowpass filter set at 3 kHz.
, http://www.100md.com
Data were recorded from patches with apparent activity from only one channel as evident from single open levels over the 15 s recording periods. A measure of the channel open probability was found by summing the individual open times and dividing this value by the total recording time at a specified potential.
1.3 Intracellular Ca2+ concentration measuring
Cells (103 cells.L-1) were incubated with 1 μmol.L-1 fura-2/AM for 40 min at 37 ℃. The extracellular fura-2/AM was washed out with HEPES solution containing NaCL 110 mmol.L-1, KCl 5.4 mmol.L-1, MgCl2 1.0 mmol.L-1, CaCl2 1.0 mmol.L-1, HEPES 20 mmol.L-1, pH 7.4. The [Ca2+]i was monitored by a RF-5000 fluorescence spectrophotometer with dual excitation at 340 nm/380 nm and emission at 500 nm. [Ca2+]i was calculated from the formula as following:[Ca2+]i=Kd Sf380/b380 (R-Rmin)/(Rmax-R). Where, Kd is 225 nmol.L-1 in the cytoplasmic environment; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm; R is the fluorescence ratio (340 nm/380 nm) of the intracellular fura-2; Rmax and Rmin are the maximal and minimal fluorescence ratios which are obtained by additions of Triton X-100 (final concentration is 0.9 ml.L-1) and EGTA (final concentration is 3 mmol.L-1) respectively.
, http://www.100md.com
Nifedipine (Sigma) and fura-2/AM (Sigma) were dissolved in ethanol at 10 mmol.L-1 and in DMSO at 1 μmol.L-1 stock solutions respectively, and stored in dark. These stock solutions were freshly diluted to desired concentrations with demineralized water. Other reagents were obtained from Sigma Company.
2 Results
2.1 Signal channel recording
4.5 μmol.L-1 adrenaline or 20 μmol.L-1 phenylephrine evoked an outward K+ current which was significantly inhibited by 50 μmol.L-1 chloroethylclonidine (CEC), a selective α1B-adrenoceptor subtype blocker (fig.1).
, 百拇医药
Fig.1 The outward K+-current was recorded by cell attached configuration with a pipette potential of -70 mV
A. 4.5 μmol.L-1 Adrenaline (Adr) increased the channel open probability that was decreased by 50 μmol.L-1 chloroethylclonidine (CEC). B. The histogram of the channel open time distribution with time constant of 0.45 ms. C. The histogram of the channel close time distribution with the time constants of 0.96 and 7.98 ms
, 百拇医药
The opening time distribution of this K+ channel required an one component fit with time constant of 0.45 ms, and the channel close time distribution required a two components fit with time constants of 0.96 and 7.98 ms. This current was voltage-dependent and had a conductance of 160 pS(fig.2).
Fig.2 The current was voltage-dependent
On the left:the current was recorded in the different pipette potentials. On the right:l-V relationship curve
, 百拇医药
The current was inhibited by 2 mmol.L-1 TEA(a Ca2+-dependent K+ channel blocker) and completely blocked by 5 mmol.L-1 EGTA. However, 0.2 μmol.L-1 nifedipine by which Ca2+ entry induced by depolarization was blocked[6] did not significantly inhibit this current (fig.3 and tab.1). Before and after addition of nifedipine, the channel opening probabilities were 0.057±0.002 and 0.058±0.002 respectively (n=4; P>0.05).
, http://www.100md.com
Fig.3 The current was recorded with a pipette potential of -60 mV
On the left, A, B, and C express the current recorded pretreatment, 1 μmol.L-1 nifedipine and 2 mmol.L-1 TEA respectively. Nifedipine did not significantly change the current which was markedly inhibited by TEA. On the right, the current was recorded before (A) and after (B) addition of 5 mmol.L-1 EGTA
, http://www.100md.com
2.2 [Ca2+]i measuring
In the HEK-293 cells, the resting [Ca2+]i was (23.9±1.3) nmol.L-1. 50 mmol.L-1 KCl did not increase [Ca2+]i. In Ca2+-free medium, 1 μmol.L-1 adrenaline evoked a transient increase in [Ca2+]i, which was due to the intracellular calcium release, from (23.9±1.3) nmol.L-1 to (293.3±85.4) nmol.L-1 (n=4,P<0.01). Subsequent addition of CaCl2 (the final concentration was 1.5 mmol.L-1 in the medium) further induced an increase in [Ca2+]i (due to the extracellular Ca2+ entry) to (456.6±52.6) nmol.L-1, then gradually decreased to a sustained level of (156.8±29.7) nmol.L-1 (n=4). 1 mmol.L-1 nifedipine did not reduce the [Ca2+]i induced by addition of CaCl2, whereas 1 mmol.L-1 LaCl3 significantly decreased [Ca2+]i from (156.8±29.7) nmol.L-1 to (84.9±4.6) nmol.L-1 (n=1,P<0.01; Fig.4).
, 百拇医药
Table 1 The effects of nifedipine, CEC, EGTA and Mn2+on the adrenalin-induced open probability of K+ channel Drugs
n
Pretreatment
Treatment
Nifedipine
4
0.057±0.002
0.058±0.001
CEC
, 百拇医药
3
0.061±0.010
0.009±0.001
TEA
4
0.051±0.012
0.010±0.005
EGTA
4
0.049±0.005
0
Mn2+
, http://www.100md.com
3
0.065±0.008
0
Fig.4 Adrenaline (Adr) induced a transient increase in [Ca2+]i under condition of Ca2+-free medium
Subsequent addition of CaCl2 further increased the [Ca2+]i; 1 μmol.L-1 nifedpinde (Nif) did not reduced [Ca2+]i whereas LaCl3 significantly decreased it
, 百拇医药
Addition of EGTA (the concentration in medium was 3 mmol.L-1) completely blocked the Ca2+ entry and made the [Ca2+]i turn to resting level.
3 Discussion
Recently, the HEK-293 cell line was used to express cDNA of ATP-sensitive potassium channels for studying the characterization of these channels[7,8]. In this study, although we did not transfect any cDNA of potassium channels in the α1B-HEK-293 cells, activation of α1B-adrenoceptor in HEK-293 cells evoked an outward K+-current with a conductance of 160 pS. It indicates that HEK-293 cells can express some kind of K+ channel. Because the EGTA blocked the current, this K+-current was thought to be dependent on the extracellular Ca2+ entry. Nifedipine, a L-subtype voltage-dependent Ca2+ channel (VDC) blocker, did not change this current. In the experiments of fura-2 probe, it was shown that nifedipie did not decrease the elevation of [Ca2+]i induced by activation of α1B-subtype, whereas, LaCl3 significantly inhibited the increase in [Ca2+]i induced by Ca2+ entry. This data is consistent with the results from single channel recording experiments, and suggests that the Ca2+ entry induced by α1B-adrenoceptor subtype in HEK-293 cell be not mediated by L-subtype of VDC.
, 百拇医药
The present results indicate that activation of α1B-adrenoceptor in HEK-293 cells produces a Ca2+ entry through nifedipine-resistant Ca2+ channel, elevates [Ca2+]i, and then ,evokes an outward K+-current.
We have noted the data from other laboratories are distinct from ours. It was reported that the Ca2+ entry induced by α1B-adrenoceptor activation in rat medullary thyroid carcinoma 6-23 cells[9],Cos-1 cells[10] and rat irideal blood vessels[11] was voltage dependent. Nifedipine markedly inhibited this Ca2+ entry.
, 百拇医药
All results submit a proof for the fact that the mechanisms of Ca2+ movement induced by α1B-adrenoceptor are different from cell line to cell line. It appears that these differences are dependent on the nature of cells.
References
1 Ruffulo R R Jr. α-adrenoceptors. Pharmac Ther, 1994,61(1-2):1
2 Wang Z, Arden J, Sadee W. Basal phosphorylation of mu-opioid receptor is agonist modulated and Ca2+-depdendent. FEBS Lett, 1996,387(1):53
, http://www.100md.com
3 Tia S, Wang J F, Kotchabhakdi N, et al. Developmental changes of inhibitory synaptic currents in cerebellar granule neurons:role of GABAA receptor alpha 6 subunit. J Neurosci, 1996,16(11):3630
4 Moore R H, Sadovikoff N, Hoffenberg S, et al. Ligand-stimulated α2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes. J Cell Sci, 1995,108(Pt 9):2983
5 Hamill O P, Marty A, Neher E, et al. Improved patch-clamp techniques for highresolution current recording from cells and cell-free membrane patches. Pflügers Arch, 1981,391(2):85
, http://www.100md.com
6 Guan Y Y, Kwan C Y, Daneil E E. Evidence against the role of α1 adrenoceptor reserve in buffering the inhibitory effect on nifedipine on the contractility of canine vascular smooth muscle. Can J Physiol Pharmacol, 1990,68(10):1346
7 Sakura H, Ammala C, Smith P A, et al. Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic cells, brain, heart and skeletal muslce. FEBS Lett, 1995,377(3):338
, 百拇医药
8 Inagaki N, Tsura Y, Namba N, et al. Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle and heart. J Bio Chem, 1995,270(11):5691
9 Esbenshade T A, Theroux T L, Minneman K P. Increased voltage-dependent calcium influx produced by α1B-adrenergic receptor activation in rat medullary thyroid carcinoma 6-23 cells. Mol Pharmacol 1994,45(4):591
, 百拇医药
10 Perez D M, DeYoung M B, Graham R M. Coupling of expressed α1B and α1D adrenergic receptor to multiple signaling pathways is both G protein and (cell) type specific. Mol Pharmacol, 1993,44(4):784
11 Gould D J, Hill C E, α1B-receptors and intracellular Ca2+ mediate sympathetic nerve induced constriction of rat irideal blood vessels. J Auton Nerv Syst, 1994,50(2):139
(1999 - 06 - 24收稿 1999 - 09 - 29修回), 百拇医药
单位：关永源 陶亮 贺华 孙家钧 中山医科大学药理教研室； 广州，510089；韩启德 张幼怡 北京医科大学血管医学研究所； 北京，100083
关键词：钙通道；钾通道；受体；肾上腺素能α1；HEK-293细胞；电生理学
中山医科大学学报990402Study in the Ca2+-Dependent K+-Current and Ca2+Entry Inducedby Activation of α1B-Adrenoceptor Subtype in HEK-293 Cell
Guan Yongyuan1 Tao Liang1 He Hua1 Han Qide2 Zhang Youyi2 Sun Jiajun1
, http://www.100md.com
( 1 Department of Pharmacology, Sun Yat-sen University of Medical Sciences, Guangzhou, 510089
2 Institute of Vascular Medicine, Beijing Medical University, Beijing,100083)
Abstract Objective: To study the characterization of the outward K+-current and Ca2+ entry induced by activation of α1B-adrenoceptor subtype at the HEK-293 cells. Methods: The single K+-current was recorded from the cell attached configuration. The cytoplasmic Ca2+ was measured by fura-2 probe. Results: adrenaline evoked an outward K+-current with a conductance of 160 pS. The current was markedly inhibited by 50 μmol.L-1 chloroethylclonidine, 5 mmol.L-1 EGTA or 2 mmol.L-1 TEA. Nifedipine did not change this current and adrenaline-induced Ca2+ entry wihich was inhibited by 1 mmol.L-1 LaCl3. Conclusion: Activation of α1B-subtype receptor at HEK-293 cells evokes Ca2+ entry through the nifedipine resistant Ca2+ channel, followed by an outward K+-current.
, http://www.100md.com
Subject headings calcium channels; potassium channels; receptors, adrenergic, alpha-1; HEK-293 cell; electrophysiology
摘 要 目的：研究HEK-293细胞上α1B-肾上腺素受体亚型引起向外K+电流和Ca2+内流的特性。方法：细胞贴附式单通道记录K+通道和Fura-2荧光测定胞浆游离Ca2+浓度。结果：肾上腺素或苯肾上腺素可引发一电导为160 pS的外向K+电流。该电流可被50 μmol.L-1 氯乙醛可乐定(CEC)，5 mmol.L-1依他酸(EGTA)或2 mmol.L-1 四乙铵(TEA)抑制。硝苯吡啶(nifedipine)不改变该电流及α1B亚型引起的Ca2+内流；后者可被1 mmol.L-1 LaCl3抑制。结论：激活转染在HEK-293细胞上的α1B-受体亚型可引起通过硝苯吡啶不敏感Ca2+通道的Ca2+内流，并跟随产生-外向K+电流。
, http://www.100md.com
中图号 R 329.25
It has been suggested that α1-adrenoceptor (α1-AR)can be divided into α1A,α1B and α1D three subtypes. Because these α1-AR subtypes coexist in many tissues[1]，it is difficult to identify the functions of individual α1-AR subtype. Recently, a permanently transformed cell line derived from the human embryo renal cortical cells (HEK-293 cell line) has usually been used to express the cloned cDNA of various receptors[2～4]. HEK-293 cells were respectively transfected with α1A-,α1B- and α1D-AR subtypes. However, there is no report about the K+-currents induced by activation of α1B-AR in HEK-293 cells. In this study, we determined the Ca2+-dependent K+-currents and the change of intracellular Ca2+ concentration ([Ca2+]i),induced by activation of α1B-adrenoceptor subtype at the transfected HEK-293 cells.
, http://www.100md.com
1 Material and methods
1.1 Cell culture
Subclone of HEK-293 transfected with hamster α1B was kindly provided by Dr. Minneman. Subclones were maintained in DMEM containing 100 ml.L-1 calf serum, and propagated in the continued presence of selective antibiotic of 50 mg.L-1 hygromycin-pREP4.
1.2 Single channel recording
, 百拇医药
Standard patch clamp techniques[5] were used for recording the membrane currents in the α1B transfected HEK-293 cells. Single K+-current was made from the cell attached configuration at 30 ℃. The pipette solution contained:KCl 139 mmol.L-1,MgCl2 0.5 mmol.L-1, CaCl2 0.1 mmol.L-1, HEPES 10, egtazic acid (EGTA) 0.09 mmol.L-1, glucose 10 mmol.L-1, pH 7.4 with KOH. The extracellular solution contained: NaCl 130 mmol.L-1, MgCl2 1.2 mmol.L-1, CaCl2 1.8 mmol.L-1, KCl 5.4 mmol.L-1, HEPES 10 mmol.L-1,glucose 5.2 mmol.L-1, pH 7.4 with NaOH. The pipettes had outside tip diameters between 1～2 μm and had resistance values in the range 4～8 mΩ. A patch clamp amplifier (Axopatch-1D; Axon Instruments, Inc.) was used to record currents with a lowpass filter set at 3 kHz.
, http://www.100md.com
Data were recorded from patches with apparent activity from only one channel as evident from single open levels over the 15 s recording periods. A measure of the channel open probability was found by summing the individual open times and dividing this value by the total recording time at a specified potential.
1.3 Intracellular Ca2+ concentration measuring
Cells (103 cells.L-1) were incubated with 1 μmol.L-1 fura-2/AM for 40 min at 37 ℃. The extracellular fura-2/AM was washed out with HEPES solution containing NaCL 110 mmol.L-1, KCl 5.4 mmol.L-1, MgCl2 1.0 mmol.L-1, CaCl2 1.0 mmol.L-1, HEPES 20 mmol.L-1, pH 7.4. The [Ca2+]i was monitored by a RF-5000 fluorescence spectrophotometer with dual excitation at 340 nm/380 nm and emission at 500 nm. [Ca2+]i was calculated from the formula as following:[Ca2+]i=Kd Sf380/b380 (R-Rmin)/(Rmax-R). Where, Kd is 225 nmol.L-1 in the cytoplasmic environment; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm; R is the fluorescence ratio (340 nm/380 nm) of the intracellular fura-2; Rmax and Rmin are the maximal and minimal fluorescence ratios which are obtained by additions of Triton X-100 (final concentration is 0.9 ml.L-1) and EGTA (final concentration is 3 mmol.L-1) respectively.
, http://www.100md.com
Nifedipine (Sigma) and fura-2/AM (Sigma) were dissolved in ethanol at 10 mmol.L-1 and in DMSO at 1 μmol.L-1 stock solutions respectively, and stored in dark. These stock solutions were freshly diluted to desired concentrations with demineralized water. Other reagents were obtained from Sigma Company.
2 Results
2.1 Signal channel recording
4.5 μmol.L-1 adrenaline or 20 μmol.L-1 phenylephrine evoked an outward K+ current which was significantly inhibited by 50 μmol.L-1 chloroethylclonidine (CEC), a selective α1B-adrenoceptor subtype blocker (fig.1).
, 百拇医药
Fig.1 The outward K+-current was recorded by cell attached configuration with a pipette potential of -70 mV
A. 4.5 μmol.L-1 Adrenaline (Adr) increased the channel open probability that was decreased by 50 μmol.L-1 chloroethylclonidine (CEC). B. The histogram of the channel open time distribution with time constant of 0.45 ms. C. The histogram of the channel close time distribution with the time constants of 0.96 and 7.98 ms
, 百拇医药
The opening time distribution of this K+ channel required an one component fit with time constant of 0.45 ms, and the channel close time distribution required a two components fit with time constants of 0.96 and 7.98 ms. This current was voltage-dependent and had a conductance of 160 pS(fig.2).
Fig.2 The current was voltage-dependent
On the left:the current was recorded in the different pipette potentials. On the right:l-V relationship curve
, 百拇医药
The current was inhibited by 2 mmol.L-1 TEA(a Ca2+-dependent K+ channel blocker) and completely blocked by 5 mmol.L-1 EGTA. However, 0.2 μmol.L-1 nifedipine by which Ca2+ entry induced by depolarization was blocked[6] did not significantly inhibit this current (fig.3 and tab.1). Before and after addition of nifedipine, the channel opening probabilities were 0.057±0.002 and 0.058±0.002 respectively (n=4; P>0.05).
, http://www.100md.com
Fig.3 The current was recorded with a pipette potential of -60 mV
On the left, A, B, and C express the current recorded pretreatment, 1 μmol.L-1 nifedipine and 2 mmol.L-1 TEA respectively. Nifedipine did not significantly change the current which was markedly inhibited by TEA. On the right, the current was recorded before (A) and after (B) addition of 5 mmol.L-1 EGTA
, http://www.100md.com
2.2 [Ca2+]i measuring
In the HEK-293 cells, the resting [Ca2+]i was (23.9±1.3) nmol.L-1. 50 mmol.L-1 KCl did not increase [Ca2+]i. In Ca2+-free medium, 1 μmol.L-1 adrenaline evoked a transient increase in [Ca2+]i, which was due to the intracellular calcium release, from (23.9±1.3) nmol.L-1 to (293.3±85.4) nmol.L-1 (n=4,P<0.01). Subsequent addition of CaCl2 (the final concentration was 1.5 mmol.L-1 in the medium) further induced an increase in [Ca2+]i (due to the extracellular Ca2+ entry) to (456.6±52.6) nmol.L-1, then gradually decreased to a sustained level of (156.8±29.7) nmol.L-1 (n=4). 1 mmol.L-1 nifedipine did not reduce the [Ca2+]i induced by addition of CaCl2, whereas 1 mmol.L-1 LaCl3 significantly decreased [Ca2+]i from (156.8±29.7) nmol.L-1 to (84.9±4.6) nmol.L-1 (n=1,P<0.01; Fig.4).
, 百拇医药
Table 1 The effects of nifedipine, CEC, EGTA and Mn2+on the adrenalin-induced open probability of K+ channel Drugs
n
Pretreatment
Treatment
Nifedipine
4
0.057±0.002
0.058±0.001
CEC
, 百拇医药
3
0.061±0.010
0.009±0.001
TEA
4
0.051±0.012
0.010±0.005
EGTA
4
0.049±0.005
0
Mn2+
, http://www.100md.com
3
0.065±0.008
0
Fig.4 Adrenaline (Adr) induced a transient increase in [Ca2+]i under condition of Ca2+-free medium
Subsequent addition of CaCl2 further increased the [Ca2+]i; 1 μmol.L-1 nifedpinde (Nif) did not reduced [Ca2+]i whereas LaCl3 significantly decreased it
, 百拇医药
Addition of EGTA (the concentration in medium was 3 mmol.L-1) completely blocked the Ca2+ entry and made the [Ca2+]i turn to resting level.
3 Discussion
Recently, the HEK-293 cell line was used to express cDNA of ATP-sensitive potassium channels for studying the characterization of these channels[7,8]. In this study, although we did not transfect any cDNA of potassium channels in the α1B-HEK-293 cells, activation of α1B-adrenoceptor in HEK-293 cells evoked an outward K+-current with a conductance of 160 pS. It indicates that HEK-293 cells can express some kind of K+ channel. Because the EGTA blocked the current, this K+-current was thought to be dependent on the extracellular Ca2+ entry. Nifedipine, a L-subtype voltage-dependent Ca2+ channel (VDC) blocker, did not change this current. In the experiments of fura-2 probe, it was shown that nifedipie did not decrease the elevation of [Ca2+]i induced by activation of α1B-subtype, whereas, LaCl3 significantly inhibited the increase in [Ca2+]i induced by Ca2+ entry. This data is consistent with the results from single channel recording experiments, and suggests that the Ca2+ entry induced by α1B-adrenoceptor subtype in HEK-293 cell be not mediated by L-subtype of VDC.
, 百拇医药
The present results indicate that activation of α1B-adrenoceptor in HEK-293 cells produces a Ca2+ entry through nifedipine-resistant Ca2+ channel, elevates [Ca2+]i, and then ,evokes an outward K+-current.
We have noted the data from other laboratories are distinct from ours. It was reported that the Ca2+ entry induced by α1B-adrenoceptor activation in rat medullary thyroid carcinoma 6-23 cells[9],Cos-1 cells[10] and rat irideal blood vessels[11] was voltage dependent. Nifedipine markedly inhibited this Ca2+ entry.
, 百拇医药
All results submit a proof for the fact that the mechanisms of Ca2+ movement induced by α1B-adrenoceptor are different from cell line to cell line. It appears that these differences are dependent on the nature of cells.
References
1 Ruffulo R R Jr. α-adrenoceptors. Pharmac Ther, 1994,61(1-2):1
2 Wang Z, Arden J, Sadee W. Basal phosphorylation of mu-opioid receptor is agonist modulated and Ca2+-depdendent. FEBS Lett, 1996,387(1):53
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