1.PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine.
The Korean Journal of Physiology and Pharmacology 2008;12(5):259-265
[Ca2+]i transients by reverse mode of cardiac Na+/Ca2+ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca2+ transients ('rundown'). Rundown of NCX1 was independent of membrane PIP2 depletion. Although the activation of protein kinase C (PKC) was observed during the Ca2+ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.
Forskolin
;
Fura-2
;
Membranes
;
Naphthalenes
;
Protein Kinase C
;
Staurosporine
2.Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium.
Seong Ho OK ; Seong Chun KWON ; Sebin KANG ; Mun Jeoung CHOI ; Ju Tae SOHN
Korean Journal of Anesthesiology 2014;67(6):404-411
BACKGROUND: Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta. METHODS: Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca2+]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.
Animals
;
Aorta*
;
Calcium*
;
Endothelium*
;
Fura-2
;
Gadolinium
;
Mepivacaine
;
Nifedipine
;
Rats*
;
Sarcoplasmic Reticulum
;
Vasoconstriction
;
Verapamil
3.Effects of glucosamine on Ca2+ signaling and K+ channel currents in T lymphocytes.
Eun Young SUH ; Bo PANG ; Sung Joon KIM ; Woo Kyung KIM
Korean Journal of Medicine 2010;79(5):536-542
BACKGROUND/AIMS: Glucosamine is widely taken as a functional food, and some studies reported its anti-inflammatory effects. K+ channels and intracellular signal play important roles in the activation of immune cells such as T lymphocytes. Therefore we aimed to examine the effects of glucosamine on the cell physiological parameters. METHODS: In Jurkat-T lymphocytes, intracellular [Ca2+] ([Ca2+]i) was measured using fura-2 fluorimetry, and voltage-gated K+ current (I(Kv)) was measured using whole-cell clamp technique. Ca2+-activated K+ current (I(Kca)) was measured in HEK293 cells over expressing SK4 using inside-out patch clamp technique. RESULTS: An acute application of glucosamine (0.5 mM) affected neither the increase in [Ca2+]i induced by CD3 stimulation (anti-CD3 Ab, 5 microgram/mL) nor the I(Kv) in Jurkat-T cells. A chronic stimulation of with anti-CD3 Ab (5 microgram/mL, 24~36 hr) largely increased the amplitude of IKv. However, the combined treatment with glucosamine (0.1 mM) did not block the increase of I(Kv). The I(KCa) in SK4-overexpressing cells was slightly decreased by glucosamine (0.5 mM). CONCLUSIONS: While glucosamine had a minor inhibitory effect on SK4 K+ channels, the anti-inflammatory effects of glucosamine could not be explained by the effects on the Ca2+ signaling in T lymphocytes.
Calcium
;
Fluorometry
;
Functional Food
;
Fura-2
;
Glucosamine
;
HEK293 Cells
;
Ion Channels
;
Lymphocytes
;
T-Lymphocytes
4.Effects of Noradrenaline on the Membrane Potential of Prostatic Neuroendocrine Cells of Rat.
Jun Hee KIM ; Sun Young SHIN ; Dae Yong UHM ; Sung Joon KIM
The Korean Journal of Physiology and Pharmacology 2003;7(1):47-52
The prostate gland contains numerous neuroendocrine cells that are believed to influence the function of the prostate gland. Our recent study demonstrated the expression of both alpha 1- and alpha 2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively, in rat prostate neuroendocrine cells (RPNECs). In this study, the effects of NA on the resting membrane potential (RMP) of RPNECs were investigated using a whole-cell patch clamp method. Fresh RPNECs were dissociated from the ventral lobe of rat prostate and identified from its characteristic shape; round or oval shape with dark cytoplasm. Under zero-current clamp conditions with KCl pipette solution, the resting membrane potential (RMP) of RPNECs was between 35 mV and 85 mV. In those RPNECs with relatively hyperpolarized RMP (< 60 mV), the application of noradrenaline (NA, 1 micro M) depolarized the membrane to around 40 mV. In contrast, the RPNECs with relatively depolarized RMP (> 45 mV) showed a transient hyperpolarization and subsequent fluctuation at around 40 mV on application of NA. Under voltage clamp conditions (holding voltage, 40 mV) with CsCl pipette solution, NA evoked a slight inward current (< 20 pA). NA induced a sharp increase of cytosolic Ca2+ concentration ([Ca2+]c), measured by the fura-2 fluorescence, and the voltage clamp study showed the presence of charybdotoxin-sensitive Ca2+ -activated K+ currents. In summary, adrenergic stimulation induced either depolarization or hyperpolarization of RPNECs, depending on the initial level of RMP. The inward current evoked by NA and the Ca2+ -activated K+ current might partly explain the depolarization and hyperpolarization, respectively.
Animals
;
Cytoplasm
;
Cytosol
;
Fluorescence
;
Fura-2
;
Membrane Potentials*
;
Membranes*
;
Neuroendocrine Cells*
;
Norepinephrine*
;
Prostate
;
Rats*
5.Buffering Contribution of Mitochondria to the Ca2+i Increase by Ca2+ Influx through Background Nonselective Cation Channels in Rabbit Aortic Endothelial Cells.
Young Chul KIM ; Sang Jin LEE ; Ki Whan KIM
The Korean Journal of Physiology and Pharmacology 2005;9(1):29-35
To prove the buffering contribution of mitochondria to the increase of intracellular Ca2+ level ([Ca2+]i) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial Ca2+ entry and Ca2+ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM (R340/380) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh (10microM) increased R340/380 by 1.1+/-0.15 (mean+/-S.E., n=6). When the external Na+ was totally replaced by NMDG+, R340/380 was increased by 1.19+/-0.17 in a reversible manner (n=27). NMDG+-induced [Ca2+]i increase was followed by oscillatory decay after [Ca2+]i reached the peak level. The increase of [Ca2+]i by NMDG+ was completely suppressed by replacement with Cs+. When 1microM CCCP was applied to bath solution, the ratio of [Ca2+]i was increased by 0.4+/-0.06 (n=31). When 1microM CCCP was used for pretreatment before application of NMDG+, oscillatory decay of [Ca2+]i by NMDG+ was significantly inhibited compared to the control (p < 0.05). In addition, NMDG+-induced increase of [Ca2+]i was highly enhanced by pretreatment with 2microM CCCP by 320+/-93.7%, compared to the control (mean+/-S.E., n=12). From these results, it is concluded that mitochondria might have buffering contribution to the [Ca2+]i increase through regulation of the background NSCC in RAECs.
Acetylcholine
;
Baths
;
Carbonyl Cyanide m-Chlorophenyl Hydrazone
;
Endothelial Cells*
;
Fluorescence
;
Fura-2
;
Mitochondria*
6.Effect of Parathyroid Hormone on the intracellular pH Regulation of Osteoblast-like Cells
Chong Il YOO ; Jung Yoon LEE ; Hae Rhan BAE ; Kyung Taek KIM ; Byeong Hwan KIM
The Journal of the Korean Orthopaedic Association 1995;30(2):203-215
Intracellular pH regulation of osteoblasts is of a great importance in the process of bone formation and resorption, and has been suggested to be mediated via intracellular Ca2+ and cAMP messenger systems. To elucidate the mechanism of modulation of intracellular pH by parathyroid hormone and PMA(Phorbo1-12-myristate-13-acetate), effects of these agonists on the individual transporter system, Na+-H+ antiporter and Cl−-HCO3-(−OH−) exchanger, were investigated. Intracellular pH and Ca2+ were measured by using the fluorescent dye BCECF and fura-2, respectively, in UMR-106 cell monolayer grown on glass coverslip. Addition of tumor promotor, PMA(luM) caused 0.14 unit pH rise of resting intracellular pH(pHi) and 38% increase of the initial rate of pHi recovery after cytosolic acid load. Perfusion of Cl−-free solution resulted in rapid cytosolic alkalinization of which the rate was increased 26% by preincubation of PMA. Ca2+ ionophore, ionomycin (1uM) decreased resting pHi by 0.17 unit, but had no effect on the initial rate of pHi recovery after cytosolic acid load. However, the addition of ionomycin augmented the initial rate of pHi increase after Cl−-depletion outside the cells by 34% over the control. Stimulation of cells with parathyroid hormone(10-8M) caused an initial acidification (0.27 unit) followed by cytosolic alkalinization, with inhibiting effect on the initial rate of pHi recovery after acid load (42%). But parathyroid hormone did not have any significant effect on the rate of pHi increase after Cl−-depletion. PMA caused a sustained increase of intracellular Ca2+, of which the peak depended on the concentration of Ca2+ in extracellular medium. Ionomycin caused a transient increase of Ca2+ but PTH had no significant increase of intracellular Ca2+ in the concentration range of 10-6M to 10-12M tested. 10-8M PTH increased cAMP levels by about 10-fold and 10-10M PTH did by 1.6-fold. PMA, which increased cytosolic Ca2+ concentration, also had an stimulatory effect on cAMP production in the concentration range of 10-5M to 10-6M by 2-fold. These findings suggest that in UMR-106 cells Ca2+ and cAMP can influence pHi by altering the activity of pHi regulatory transporter system, and parathyroid hormones modulate pHi by inhibiting Na+-H+ antiporter via intracellular increase of cAMP, which is probably accounts for the inhibitory effect of parathyroid hormone on the proliferation of osteoblasts.
Cytosol
;
Fura-2
;
Glass
;
Hydrogen-Ion Concentration
;
Ion Transport
;
Ionomycin
;
Osteoblasts
;
Osteogenesis
;
Parathyroid Hormone
;
Perfusion
7.A study of the Ca2+ and the apoptosis of the KB cell lines after 10 Gy irradiation.
Je Woon MOON ; Sam Sun LEE ; Min Suk HEO ; Tae Won PARK ; Dong Soo YOU
Journal of Korean Academy of Oral and Maxillofacial Radiology 1999;29(1):105-117
PURPOSE: Ionizing radiations have been reported as an apoptosis initiating stimulus in various cells and it has established that sustained elevations in [Ca2+] can lead to DNA fragmentation by Ca2+-dependent endonucleases, ultimately resulting in apoptotic cell death. The previous experiments have been reported by using primarily thymocytes and lymphocytes and the change of [Ca2+] was measured only by minutes or hours respectively. We need to evaluate [Ca2+] in both several minutes and hours after irradiation of radiation of radiation therapy and verify the apoptotic cells. MATERIALS AND METHODS: We have measured [Ca2+] in human gingival epitheloid cancer cell with 10 Gy irradiation, at minutely intervals and hourly intervals using digitized video-intensified fluorescence microscopy and the fluorescent Ca2+ indicator dye, fura-2. In order to find out that the transient rise in [Ca2+] could induced apoptosis, cells were incubated for 1 hour at 37 degrees C with TdT enzyme, rinsed and resuspended containing fluorescence and observed under a confocal fluorescence microscope. MTT assay was done to determine cell activity and LDH assay was done to determine the amount of necrotic cells. RESULTS: After irradiation, the transient and temporal increasing of [Ca2+] in the KB cells was founded. Though, there was no change in the intracellular [Ca2+] at 30 minutes and 2 hours after irradiation. We could detect of DNA fragmented cells at 4 hours after 10 Gy irradiated cells. There were no significant differences between 4 hour, 1 day, 3 day cells. There were no significant differences in MTT and LDH assay between the irradiated group and the control group after 4 hours and 1 day. Though after 3 days there were differences in MTT and LDH assay between the irradiated group was significantly decreased than the control group, in LDH assay the number of necrotic cell death of the irradiated was higher than the control group. CONCLUSION: In KB cells there were incipient and temporal increasing of the [Ca2+] with 10 Gy irradiation and the apoptosis was founded from 4 hours later which was earlier than seeing of the change of the amount of the cellular ability and necrosis.
Apoptosis*
;
Cell Death
;
DNA
;
DNA Fragmentation
;
Endonucleases
;
Fluorescence
;
Fura-2
;
Humans
;
KB Cells*
;
Lymphocytes
;
Microscopy, Fluorescence
;
Necrosis
;
Radiation, Ionizing
;
Thymocytes
8.Effects of Estrogen on Intracellular Calcium-Related T-Lymphocyte Function.
Yoon Young KIM ; Hoon KIM ; Seung Yup KU ; Chang Suk SUH ; Seok Hyun KIM ; Young Min CHOI ; Sung Joon KIM
Tissue Engineering and Regenerative Medicine 2016;13(3):270-273
Regulation of immune cell function is an important in the field of hormone-related tissue engineering and regenerative medicine. In this sense, hormonal regulation of immune cell function is a critical issue to be solved. It has been known that ovarian sex hormone play an important roles in immune function, however, little has been known whether estrogen affects T-lymphocyte function. Human Jurkat T cells were treated with estradiol (E₂) at concentrations of 0, 10, 100, 1000 ng/mL, and calcium response was evaluated. Intracellular calcium concentrations after Fura-2 acetoxymethyl ester treatment show an increasing trend at higher E₂ concentrations although these alterations did not reach a statistical significance. The expression of calcium channel-related gene CACNA1C did not show any significant changes according to the concentration of E₂. Taken together, estrogen has an implication as a possible hormonal regulator of intracellular calcium release in human Jurkat T cells via non-genomic pathway. Further studies are necessary to investigate the combined effects of sex hormones and cytokines in both T- and B-lymphocytes.
B-Lymphocytes
;
Calcium
;
Calcium Channels
;
Cytokines
;
Estradiol
;
Estrogens*
;
Fura-2
;
Gonadal Steroid Hormones
;
Humans
;
Regenerative Medicine
;
T-Lymphocytes*
;
Tissue Engineering
9.A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.
Jeong Hoon LEE ; Jeong Mi HA ; Chae Hun LEEM
The Korean Journal of Physiology and Pharmacology 2015;19(4):373-382
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Psi(m) or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 microM. This 1 microM cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to ~30 microM in the presence of 1 microM cytosolic Ca2+. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.
Animals
;
Calcium
;
Coloring Agents
;
Cytosol
;
Fura-2
;
Hydrogen-Ion Concentration
;
Membrane Potential, Mitochondrial
;
Membrane Potentials
;
Mitochondria
;
Muscle Cells*
;
NAD*
;
Rats*
10.Mechanism of Acetylcholine-induced Endothelium-dependent Relaxation in the Rabbit Carotid Artery by M3-receptor Activation.
Yong Jin SONG ; Seong Chun KWON
The Korean Journal of Physiology and Pharmacology 2004;8(6):313-317
The present study were designed to characterize the action mechanisms of acetylcholine (ACh) -induced endothelium-dependent relaxation in arteries precontracted with high K (70 mM). For this, we simultaneously measured both muscle tension and cytosolic free Ca2 concentration ([Ca2 ]i), using fura-2, in endothelium-intact, rabbit carotid arterial strips. In the artery with endothelium, high K increased both [Ca2 ]i and muscle tension whereas ACh (10microM) significantly relaxed the muscle and increased [Ca2 ]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased [Ca2 ]i without relaxing the muscle. In the artery without endothelium, high K increased both [Ca2 ]i and muscle tension although ACh was ineffective. 4-DAMP (10 nM) or atropine (0.1microM) abolished ACh-induced increase in [Ca2 ]i and relaxation. The increase of [Ca2 ]i and vasorelaxation by ACh was siginificantly reduced by either 3microM gadolinium, 10microM lanthanum, or by 10microM SKF 96365. These results suggest that in rabbit carotid artery, ACh-evoked relaxation of 70 mM K -induced contractions appears to be mediated by the release of NO. ACh-evoked vasorelaxation is mediated via the M3 subtype, and activation of the M3 subtype is suggested to stimulate nonselective cation channels, leading to increase of [Ca2 ]i in endothelial cells.
Acetylcholine
;
Arteries
;
Atropine
;
Carotid Arteries*
;
Cytosol
;
Endothelial Cells
;
Endothelium
;
Fura-2
;
Gadolinium
;
Lanthanum
;
Muscle Tonus
;
Nitric Oxide
;
Nitroarginine
;
Relaxation*
;
Vasodilation