• Journal of Chest Surgery
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• v.22 no.4
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• pp.548-561
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• 1989

In single atrial cells isolated from the rabbit the properties of inward current of Na-Ca exchange were investigated using the whole cell voltage clamp technique. The current was recorded during repolarization following brief 2 ms depolarizing pulse to +40 mV from a holding potential of * 70 mV. Followings are the results obtained: 1. When stimulated every 30 seconds, the inward currents were activated and reached peak values 6-12 ms after the beginning of depolarizing pulse. The mean current amplitude was 342 pA/cell. 2. The current decayed spontaneously from the peak activation and the time course of the relaxation showed two different phases fast and slow phase. The time constants were 10-18 ms and 60-140 ms, respectively. 3. The recovery of inward current was tested by paired pulse of various intervals. The peak current recovered exponentially with time constant of 140 ms and 1 p M isoprenaline accelerated the recovery process. 4. Relaxation time course was also affected by pulse interval and time constant of the fast phase was reduced almost linearly according to the decrease of pulse interval between 30 sec and 1 sec. 5. The peak activation was increased in magnitude by long prepulse stimulation, 5 p M Bay K, 1 p M isoprenaline or internal and external application of c-AMP. 6. The relaxation time constant of the fast phase was prolonged by 5 p M Bay K or c-AMP, and shortened by isoprenaline. However the time course of the slow relaxation phase was not so much changed. From the above results, it could be concluded that increase of the calcium current by Bay K or c-AMP results in the potentiation and prolongation of intracellular calcium transient, and the facilitation of Ca uptake by SR might be a mechanism of shortening the time constant of current relaxation by short interval stimulation or isoprenaline.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.1
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• pp.65-71
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• 2013

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body $Mg^{2+}$ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-${\kappa}B$ ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of $Mg^{2+}$. Knock-down of TRPM7 by siTRPM7 reduced intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increases by 0 mM $[Mg^{2+}]_e$ in HEK293 cells and inhibited the generation of RANKL-induced $Ca^{2+}$ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced $[Ca^{2+}]_i$ oscillations that triggers the late stages of osteoclastogenesis.

• The Korean Journal of Physiology
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• v.24 no.2
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• pp.377-388
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• 1990

Ca movements during the late plateau phase in rabbit atrium implicate Na-Ca exchange. In single atrial cells isolated from the rabbit the properties of the inward current of Na-Ca exchange were investigated using the whole cell voltage clamp technique. The inward currents were recorded during repolarization following brief 2 ms depolarizing pulse to +40 mV from a holding potential of -70 mV. Followings are the results obtained: 1) When stimulated every 30 sec, the inward currents were activated and reached peak values $6{\sim}12\;ms$ after the beginning of depolarizing pulse. The mean current amplitude was 342 pA/cell. 2) The current decayed spontaneously from the peak activation and the timecourse of the relaxation showed two different phases: fast and slow phase. 3) The recovery of the inward current was tested by paired pulse of various interval. The peak current recovered exponentialy with a time course similar to that of Ca current recovery. 4) Relaxation timecourse was also affected by pulse interval and time constant was reduced almost linearly according to the decrease of pulse interval between 30 sec and 1 sec. 5) The peak inward current was increased by long prepulse stimulation, Bay K, isoprenaline or c-AMP. 6) The relaxation time constant of the inward current was prolonged by Bay K or c-AMP, and shortened by isoprenaline. From the above results, it could be concluded that increase of the calcium current potentiates and prolongs intracellular calcium transients, while shortening of the timecourse by isoprenaline or short interval stimulations might be due to the facilitation of Ca uptake by SR.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.134-139
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• 2021

Under physiological conditions, calcium (Ca²⁺) regulates essential functions of polarized secretory cells by the stimulation of specific Ca²⁺ signaling mechanisms, such as increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the store-operated Ca²⁺ entry (SOCE) and the receptor-operated Ca²⁺ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP₃) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca²⁺ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca²⁺ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2^-/-) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2^-/- mice. Furthermore, the response of Ca²⁺ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2^-/- mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca²⁺]_i via SOCE and ROCE in mouse pancreatic acinar cells.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.44-51
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• 2012

Calcium ions play an important role in the establishment and maintenance of pregnancy, but molecular and cellular regulatory mechanisms of calcium ion action in the uterine endometrium are not fully understood in pigs. Previously, we have shown that calcium regulatory molecules, transient receptor potential vanilloid type 5 (TRPV6) and calbindin-D9k (S100G), are expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner, and that estrogen of conceptus origin increases endometrial TRPV6 expression. However, regulation of S100G expression in the uterine endometrium and conceptus expression of S100G has been not determined during early pregnancy. Thus, we investigated regulation of S100G expression by estrogen and interleukin-$1{\beta}$ (IL1B) in the uterine endometrium and conceptus expression of S100G during early pregnancy in pigs. We obtained uterine endometrial tissues from day (D) 12 of the estrous cycle and treated with combinations of steroid hormones, estradiol-$17{\beta}$ ($E_2$) and progesterone ($P_4$), and increasing doses of IL1B. Real-time RT-PCR analysis showed that $E_2$ and IL1B increased S100G mRNA levels in the uterine endometrium, and conceptuses expressed S100G mRNA during early pregnancy, as determined by RT-PCR analysis. To determine if endometrial expression of S100G mRNA during the implantation period was affected by the somatic cell nuclear transfer (SCNT) procedure, we compared S100G mRNA levels in the uterine endometrium from gilts with SCNT-derived conceptuses with those from gilts with conceptuses derived from natural mating on D12 of pregnancy. Real-time RT-PCR analysis showed that levels of S100G mRNA in the uterine endometrium from gilts carrying SCNT-derived conceptuses was significantly lower than those from gilts carrying conceptuses derived from natural mating. These results showed that S100G expression in the uterine endometrium was regulated by estrogen and IL1B of conceptus origin, and affected by the SCNT procedure during early pregnancy. These suggest that conceptus signals regulate S100G, an intracellular calcium transport protein, for the establishment of pregnancy in pigs.

• The Korean Journal of Physiology
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• v.22 no.1
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• pp.13-29
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• 1988

In order to elucidate the regulatory mechanism of intracellular calcium ion concentrations, contractions or contractures induced by $Na^{+}-removal$, calcium-application or ouabain-treatment as an index of $Na^+/Ca^{2+}$ exchange activity were studied in atrial muscle or vascular smooth muscle (aorta and renal artery) of the rabbit. The magnitude of low sodium contractures in atrial trabeculae increased with sigmoid shape when external sodium concentrations were reduced to sodium-free condition, whereas that of calcium contracture intensified in a parabolic pattern when external calcium concentrations were elevated to 8 mM. $Na^{+}-removal$ contractures were induced in a duration-dependent manner to $K^{+}-free$ exposure and same findings were observed with ouabain treatment. $Na^{+}-free$ contractures were not affected by verapamil treatment, but stimulated by $100{\mu}M\;Mn^{2+}$ and inhibited by high concentrations of $Mn^{2+}\;(2{\sim}8mM)$ in a dose-dependent manner. Ryanodine which is known to suppress the release of calcium from internal store abolished spontaneous twitch contractions induced by $K^{+}-free$ solution, but had no effect on the development $Na^{+}-free$ contractures. Na-free contractures were not always induced in vascular smooth muscle preparations. Contractures by $O\;mM\;Na^+$ were usually seen in aorta, but not often in renal artery.$50\;mM\;K^+$, noradrenaline (NA) and angiotensin II (AII) always evoked very large contraction in all preparations of vascular smooth muscle. Contractures developed by $O\;mM\;Na^+$ were not sensitive to verapamil treatment as in atrial trabeculae, but were abolished by $100{\mu}M\;Mn^{2+}$. In contrast to $Na^{+}-free$ contractures, $Mn^{2+}(100{\mu}M)$ had no effect on the contractures induced by NA or 50 mM$K^+$. Caffeine in the concentration of 10 mM evoked transient contracture in the distal renal artery. The rate of spontaneous relaxation in caffeine contracture was dependent upon the concentrations of external sodium, and had double component of relaxation when the rate of relaxation was plotted in the semilogarithmic scale of relative tension versus time. Especially late components of relaxation had more direct relation to $Na^+$ concentrations. It could be concluded that $Na^+/Ca^{2+}$ exchange mechanism in the heart has a large capacity, inhibited by $Mn^{2+}$ but not by verapamil and ryanodine, while $Na^+/Ca^{2+}$ exchange system in vascular smooth muscle has a very low capacity especially in small artery, inhibited by low concentration of $Mn^{2+}\;(100{\mu}M)$ but not affected by verapamil and ryanodine.

• The Korean Journal of Physiology
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• v.28 no.2
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• pp.169-179
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• 1994

The effect of extracellular and intracellular pH on vascular tone and $^{45}Ca$ uptake were investigated in aortic strips and dispersed single aortic smooth muscle cells of spontaneously hypertensive rats (SHR) and aged-matched Wistar-Kyoto rats (WKY). The contraction produced by a change of extracellular pH (pHo) in the range of $6.5{\sim}8.3$ was estimated by comparison with the level of vascular tone at pH 7.4. Contraction was induced below pHo 6.5 in WKY, pHo 7.1 in SHR, and over pHo 8.0 on both strains. The amplitude of contraction induced by high pHo (over pHo 7.7) was similar in SHR and WKY, but that induced by low pHo (below pHo 7.1) in SHR was greater than that in WKY. Either high pHo- or low pHo-induced contractions in WKY and SHR were not induced in the Ca-free Tyrode's solution and were induced by the addition of Ca. $^{45}Ca$ uptake increased progressively as pHo was increased from 6.8 to 8.1 in the single aortic smooth muscle cells of WKY and SHR. $NH_4Cl$ induced a gradually developing contraction in a dose-dependent manner $(5\;mM{\sim}30\;mM)$ and the removal of $NH_4Cl$ induced transient contraction was followed by profound relaxation in the aortic rings of both strains. The contractions induced by $NH_4Cl$ or by the removal of $NH_4Cl$ in SHR were significantly greater than that in WKY. These contractions were not induced in Ca-free Tyrode's solution. $^{45}Ca$ uptake was increased by $NH_4Cl$ (20 mM) and was not changed by the removal of $NH_4Cl$ (20 mM) in the aortic strips of WKY and SHR. As a summary of above results, the vascular tone of SHR was more sensitive to the change pHi and pHo than that of WKY. The contractions induced by change of extracellular or intracellular pH depended on extracellular Ca in the aorta of SHR nnd WKY. However, the Ca uptake was in accord with the changes of contraction but increase in contraction by low pH was not accompanied by an increase in Ca uptake in both strains.

• Journal of Chest Surgery
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• v.32 no.7
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• pp.603-612
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• 1999

Background : It has been documented that brief repetitive periods of ischemia and reperfusion (ischemic preconditioning, IP) enhances the recovery of post-ischemic contractile function and reduces infarct size after a longer period of ischemia. Many mechanisms have been proposed to explain this process. Recent studies have suggested that transient increase in the intracellular calcium may have triggered the activation of protein kinase C(PKC); however, there are still many controversies. Accordingly, the author performed the present study to test the hypothesis that preconditioning with high concentration of calcium before sustained subsequent ischemia(calcium preconditioning) mimics IP by PKC activation. Material and Method : The isolated hearts from the New Zealand White rabbits(1.5∼2.0 kg body weight) Method: The isolated hearts from the New Zealand White rabbits(1.5∼2.0 kg body weight) were perfused with Tyrode solution by Langendorff technique. After stabilization of baseline hemodynamics, the hearts were subjected to 45-minute global ischemia followed by a 120-minute reperfusion with IP(IP group, n=13) or without IP(ischemic control, n=10). IP was induced by single episode of 5-minute global ischemia and 10-minute reperfusion. In the Ca2+ preconditioned group, perfusate containing 10(n=10) or 20 mM(n=11) CaCl2 was perfused for 10 minutes after 5-minute ischemia followed by a 45-minute global ischemia and a 120-minute reperfusion. Baseline PKC was measured after 50-minute perfusion without any treatment(n=5). Left ventricular function including developed pressure(LVDP), dP/dt, heart rate, left ventricular end-diastolic pressure(LVEDP) and coronary flow(CF) was measured. Myo car ial cytosolic and membrane PKC activities were measured by 32P-${\gamma}$-ATP incorporation into PKC-specific pepetide. The infarct size was determined using the TTC (tetrazolium salt) staining and planimetry. Data were analyzed using one-way analysis of variance(ANOVA) variance(ANOVA) and Tukey's post-hoc test. Result: IP increased the functional recovery including LVDP, dP/dt and CF(p<0.05) and lowered the ascending range of LVEDP(p<0.05); it also reduced the infarct size from 38% to 20%(p<0.05). In both of the Ca2+ preconditioned group, functional recovery was not significantly different in comparison with the ischemic control, however, the infarct size was reduced to 19∼23%(p<0.05). In comparison with the baseline(7.31 0.31 nmol/g tissue), the activities of the cytosolic PKC tended to decrease in both the IP and Ca2+ preconditioned groups, particularly in the 10 mM Ca2+ preconditioned group(4.19 0.39 nmol/g tissue, p<0.01); the activity of membrane PKC was significantly increased in both IP and 10 mM Ca2+ preconditioned group (p<0.05; 1.84 0.21, 4.00 0.14, and 4.02 0.70 nmol/g tissue in the baseline, IP, and 10 mM Ca2+ preconditioned group, respectively). However, the activity of both PKC fractions were not significantly different between the baseline and the ischemic control. Conclusion: These results indicate that in isolated Langendorff-perfused rabbit heart model, calcium preconditioning with high concentration of calcium does not improve post-ischemic functional recovery. However, it does have an effect of limiting(reducing) the infart size by ischemic preconditioning, and this cardioprotective effect, at least in part, may have resulted from the activation of PKC by calcium which acts as a messenger(or trigger) to activate membrane PKC.

• The Korean Journal of Pharmacology
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• v.26 no.2
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• pp.127-133
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• 1990

Dibutyryl-cyclic AMP (db-cAMP) and forskolin were used to investigate vasodilating mechanism of cAMP in rabbit aorta. Db-cAMP and forskolin inhibited the development of contractile tension induced by norepinephrine (NE) concentration-dependently. However, high $K{^+}-induced$ contractile tension was inhibited less effectively by db-cAMP and forskolin. Db-cAMP and forskolin inhibited $^{45}Ca^{2+}$ uptake increased by NE. Forskolin seemed to inhibit $^{45}Ca^{2+}$ uptake increased by high $K{^+}$, but this inhibition was not significant statistically. Db-cAMP inhibited $Ca^{2+}-transient$ contraction by NE in $Ca^{2+}-free$ solution. In conclusion, it seems that cAMP blocks $Ca^{2+}$ influx through receptor operated $Ca^{2+}$ channels (ROCs), but that the effect of cAMP on $Ca^{2+}$ influx through voltage gated $Ca^{2+}$ channels (VGCs) is not clear in this experiment. Furthermore, cAMP is likely to inhibit calcium release from the intracellular stores.

• Journal of Ginseng Research
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• v.35 no.4
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• pp.471-478
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• 2011

Ginseng, the root of Panax ginseng, is one of the oldest herbal medicines. It has a variety of physiological and pharmacological effects. Recently, we isolated a subset of glycolipoproteins that we designated gintonin, and demonstrated that it induced transient change in intracellular calcium concentration $([Ca^{2+}]_i)$ in cells via G-protein-coupled receptor signaling pathway(s). The previous method for gintonin isolation included multiple steps using methanol, butanol, and other organic solvents. In the present study, we developed a much simple method for the preparation of gintonin from ginseng root using 80% ethanol extraction. The extracted fraction was designated edible gintonin. This method produced a high yield of gintonin (0.20%). The chemical characteristics of gintonin such as molecular weight and the composition of the extract product were almost identical as the gintonin prepared using the previous extraction regimen involving various organic solvents. We also examined the physiological effects of edible gintonin on endogenous $Ca^{2+}$-activated $Cl^-$ channel activity of Xenopus oocytes. The 50% effective dose was $1.03{\pm}0.3\;{\mu}g$/mL. Finally, since gintonin preparation through ethanol extraction is easily reproducible, gintonin could be commercially applied for ginseng-derived functional health food and/or drug following the confirmations of in vitro and in vivo physiological and pharmacological effects of gintonin.