Young Min Bae

Contribution of Ca2+‐activated K+ channels and non‐selective cation channels to membrane potential of pulmonary arterial smooth muscle cells of the rabbit

1 Using the perforated patch‐clamp or whole‐cell clamp technique, we investigated the contribution of Ca2+‐activated K+ current (IK(Ca)) and non‐selective cation currents (INSC) to the membrane potential in small pulmonary arterial smooth muscle cells of the rabbit. 2 The resting membrane potential (Vm) was ‐39·2 ± 0·9 mV (n= 72). It did not stay at a constant level, but hyperpolarized irregularly, showing spontaneous transient hyperpolarizations (STHPs). The mean frequency and amplitude of the STHPs was 5·6 ± 1·1 Hz and ‐7·7 ± 0·7 mV (n= 12), respectively. In the voltage‐clamp mode, spontaneous transient outward currents (STOCs) were recorded with similar frequency and irregularity. 3 Intracellular application of BAPTA or extracellular application of TEA or charybdotoxin suppressed both the STHPs and STOCs. The depletion of intracellular Ca2+ stores by caffeine or ryanodine, and the removal of extracellular Ca2+ also abolished STHPs and STOCs. 4 Replacement of extracellular Na+ with NMDG+ caused hyperpolarization Vm of without affecting STHPs. Removal of extracellular Ca2+ induced a marked depolarization of Vm along with the disappearance of STHPs. 5 The ionic nature of the background inward current was identified. The permeability ratio of K+ : Cs+ : Na+ : Li+ was 1·7 : 1·3 : 1 : 0·9, indicating that it is a non‐selective cation current (INSC). The reversal potential of this current in control conditions was calculated to be ‐13·9 mV. The current was blocked by millimolar concentrations of extracellular Ca2+ and Mg2+. 6 From these results, it was concluded that (i) hyperpolarizing currents are mainly contributed by Ca2+‐activated K+ (KCa) channels, and thus STOCs result in transient membrane hyperpolarization, and (ii) depolarizing currents are carried through NSC channels.

p38 Mitogen-Activated Protein Kinase Contributes to the Diminished Aortic Contraction by Endothelin-1 in DOCA-Salt Hypertensive Rats

Abstract—We investigated whether the diminished contractile responsiveness to endothelin-1 (ET-1) is associated with the altered activation of mitogen-activated protein kinase (MAPK) in aortic smooth muscles from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. ET-1 dose-dependently increased contractions in aortic smooth muscle strips, and the contractions were significantly attenuated in tissues from DOCA-salt hypertensive rats compared with those from sham-operated rats. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was elevated by ET-1, with the magnitude and time-course being similar between strips. Although ET-1 also increased the phosphorylation of p38 MAPK in both strips, the increment was markedly lower in the strips from DOCA-salt hypertensive rats compared with sham-operated controls. 5-Hydroxytryptamine (5-HT) increased vascular contraction and phosphorylation of both MAPK isoforms; these were greater in DOCA-salt hypertensive rats than in sham-operated rats. ET-1 also increased the phosphorylation of caldesmon, an actin-binding protein, in sham-operated and DOCA-salt hypertensive rats. However, the increment was markedly lower in the strips from DOCA-salt hypertensive rats compared with sham-operated controls. The phosphorylation of MAPK isoforms and caldesmon elevated by ET-1 was inhibited by PD098059, an inhibitor of ERK1/2 kinase, and SB203580, an inhibitor of p38 MAPK, respectively. These results suggest that ET-1 and 5-HT induce contraction by activating the MAPK pathway in rat aortic smooth muscle and that the diminished responsiveness to ET-1 in the DOCA-salt hypertensive rat may be, in part, mediated by the decrease of caldesmon phosphorylation after the decreased activation of p38 MAPK.

Enhancement of receptor-operated cation current and TRPC6 expression in arterial smooth muscle cells of deoxycorticosterone acetate-salt hypertensive rats

Objectives In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, altered reactivity of blood vessels to vasoactive agonists is frequently associated with an elevation in blood pressure. Canonical transient receptor potential (TRPC) channels are believed to encode receptor-operated cation channels (ROC), the activation of which is involved in smooth muscle depolarization and vasoconstriction. The aims of the present study were to investigate whether the ROC current is increased in DOCA-hypertensive rats and determine whether aldosterone directly enhances the expression of TRPC. Methods The nystatin-perforated patch-clamp technique was used for the recording of receptor-stimulated ion currents in mesenteric arterial smooth muscle cells, which were enzymatically dispersed from sham-operated and DOCA-salt hypertensive rats. Expressions of TRPCs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blot analysis. Results Receptor-stimulated currents activated by 5-hydroxytryptamine (serotonin) and norepinephrine were increased significantly in the mesenteric arterial smooth muscle cells of DOCA-salt hypertensive rats compared to sham-operated rats. Ion-substitution experiments revealed that the enhanced currents were cation currents (ROC currents). Enhanced expression of TRPC6 in mesenteric arteries from DOCA-salt hypertensive rats was demonstrated by real-time RT-PCR. Up-regulation of TRPC6 by aldosterone treatment in vitro was also observed in A7r5 cells by RT-PCR and in western blots. Conclusion These results suggest that aldosterone enhances TRPC6 expression and ROC currents in vascular smooth muscle cells, and that this may in turn contribute to altered vascular reactivity and to hypertension.

Gene Transfer of Redox Factor-1 Inhibits Neointimal Formation: Involvement of Platelet-Derived Growth Factor-β Receptor Signaling via the Inhibition of the Reactive Oxygen Species–Mediated Syk Pathway

The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (Ref-1) in vascular smooth muscle cells has yet to be clearly elucidated. Therefore, we attempted to determine the roles of Ref-1 in the migration induced by platelet-derived growth factor (PDGF)-BB and in its signaling in rat aortic smooth muscle cells (RASMCs). Cellular migration, superoxide (O2−·) production, Rac-1 activity, and neointima formation were determined in cells transfected with adenoviruses encoding for Ref-1 (AdRef-1) and small interference RNA of Ref-1. Overexpression of Ref-1 induced by treatment with RASMCs coupled with AdRef-1 inhibited the migration induced by PDGF-BB. PDGF-BB also increased the phosphorylation of the PDGF&bgr; receptor, spleen tyrosine kinase (Syk), mitogen-activated protein kinase, and heat shock protein 27, but these increases were significantly inhibited by AdRef-1 treatment. PDGF-BB increased O2−· production and Rac-1 activity, and these were diminished in cells transfected with AdRef-1. In contrast, RASMC migration, phosphorylation of Syk and O2−· production in response to PDGF-BB were increased by the knock down of Ref-1 with small interference RNA. The phosphorylation of PDGF&bgr; receptor in response to PDGF-BB was inhibited completely by the Syk inhibitor and was partly attenuated by a NADPH oxidase inhibitor. PDGF-BB increased the sprout outgrowth of the aortic ring ex vivo, which was inhibited in the AdRef-1–infected RASMCs as compared with the controls. Balloon injury–induced neointimal formation was significantly attenuated by the gene transfer of AdRef-1. These results indicate that Ref-1 inhibits the PDGF-mediated migration signal via the inhibition of reactive oxygen species–mediated Syk activity in RASMCs.

Modulation of voltage-dependent K+ channel by redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit

Abstract It has been suggested that hypoxic pulmonary vasoconstriction (HPV) results from the depolarization that is induced by the suppression of K+ current in pulmonary arterial smooth muscle cells (PASMC). We tested the hypothesis that the effect of the cellular redox potential on voltage-sensitive K+ (Kv) current is involved in HPV as a primary sensing mechanism. Kv current in PASMC and ear arterial smooth muscle cells (EASMC) of the rabbit was recorded using the whole-cell patch-clamp technique, and the effect of redox agents [dithiothreitol, DTT and 2,2’-dithio-bis(5-nitropyridine), DTBNP] was tested. Kv current was decreased by DTT, but increased by DTBNP. DTT accelerated the inactivation kinetics, but did not affect steady-state activation and inactivation, whereas DTBNP accelerated activation kinetics. Kv current has a non-inactivating window in the range of from –40 mV to +10 mV. The resting membrane potential measured using the nystatin-perforated-patch method, however, lay between –50 mV and –30 mV and was not depolarized by 5 mM 4-aminopyridine. The membrane-impermeable oxidizing agent DTNB has no effect on Kv current, suggesting that redox modulation sites are intracellular sulphydryl groups. In EASMC, Kv current was decreased by DTT, but increased by DTBNP, indicating that the redox-potential-induced modulation of Kv current in EASMC and in PASMC is the same. It is therefore concluded that Kv current is modulated by the cellular redox potential, but that this modulation is not involved in HPV as a primary sensing mechanism.

Staurosporine inhibits voltage-dependent K+ current through a PKC-independent mechanism in isolated coronary arterial smooth muscle cells.

We examined the effects of the protein kinase C (PKC) inhibitor staurosporine (ST) on voltage-dependent K+ (KV) channels in rabbit coronary arterial smooth muscle cells. ST inhibited the KV current in a dose-dependent manner with a Kd value of 1.3 μM. The inhibition of the KV current by ST was voltage-dependent between −30 and +10 mV. The additive inhibition of the KV current by ST was voltage-dependent throughout the activation voltage range. The rate constants of association and dissociation of ST were 0.63 μM−1 s−1 and 0.92 s−1, respectively. ST produced use-dependent inhibition of the KV current. ST shifted the activation curve to more positive potentials but did not have any significant effect on the voltage dependence of the inactivation curve. ST did not have any significant effects on other types of K+ channel. Another PKC inhibitor, chelerythrine, and PKA inhibitor peptide (PKA-IP) had little effect on the KV current. These results suggest that ST interacts with KV channels that are in the closed state and that ST inhibits KV channels in the open state in a manner that is phosphorylation-independent and voltage-, time-, and use-dependent.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca2+-activated K+ channels and inward rectifier K+ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.

Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase.

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.

Laser-induced thermoelastic effects can evoke tactile sensations

Humans process a plethora of sensory information that is provided by various entities in the surrounding environment. Among the five major senses, technology for touch, haptics, is relatively young and has relatively limited applications largely due to its need for physical contact. In this article, we suggest a new way for non-contact haptic stimulation that uses laser, which has potential advantages such as mid-air stimulation, high spatial precision, and long working distance. We demonstrate such tactile stimulation can be enabled by laser-induced thermoelastic effects by means of physical and perceptual studies, as well as simulations. In the physical study, the mechanical effect of laser on a human skin sample is detected using low-power radiation in accordance with safety guidelines. Limited increases (< ~2.5 °C) in temperature at the surface of the skin, examined by both thermal camera and the Monte Carlo simulation, indicate that laser does not evoke heat-induced nociceptive sensation. In the human EEG study, brain responses to both mechanical and laser stimulation are consistent, along with subjective reports of the non-nociceptive sensation of laser stimuli.

Generation of functional cardiomyocytes from mouse induced pluripotent stem cells

BACKGROUND Induced pluripotent stem (iPS) cells allow derivation of autologous differentiated cells for cell therapy. The purpose of this study was to compare the cardiac differentiation potential of mouse iPS cells with embryonic stem (ES) cells and demonstrate that they could produce functional cardiomyocytes. METHODS iPS cells were prepared from mouse embryonic fibroblasts by lentiviral mediated expression of four transcription factors (Oct4/Sox2/Klf4/C-myc). To induce cardiac cell differentiation, iPS-S-6 or D3-ES cells were induced to form embryoid bodies (EBs) using a two-medium culture protocol, then plated onto gelatin-coated plates and maintained in DMEM. RESULTS Following classification of the generation periods of contracting EBs into early (d8-d11), middle (d12-d15) and late (d16-20), iPS cells in the early period exhibited characteristics similar to ES cells. In iPS cells from the middle period group, the ratio of contracting EBs was significantly increased compared to ES cells, and the difference persisted in cells from the late period group (p<0.05). The percentage of contracting EBs formed from iPS and ES cells were 44.8% and 33.3%, respectively. In addition, iPS cell-derived cardiomyocytes exhibited mRNA expression of cardiac mesoderm markers such as GATA4 and NKX2.5, and cardiomyocyte markers such as α1s, α1c, α-MHC, β-MHC, Cx40, TnI, TnT, ANF and Hey2. Single cardiomyocytes exhibited typical cross-striated myofibrillar organization, and electrophysiological studies revealed functional cardiac-specific voltage-gated Na(+), Ca(2+) and K(+) channels. CONCLUSIONS These results demonstrate that functional cardiomyocytes can be generated from iPS cells, and suggest that these cells may be useful for the treatment of cardiovascular disease.