The endogenous production of hydrogen sulfide (H2S) and its physiological functions, including membrane hyperpolarization and smooth muscle cell relaxation, position this gas well in the family of gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). In this study, we demonstrate that H2S at physiologically relevant concentrations induced apoptosis of human aorta smooth muscle cells (HASMCs). Exposure of HASMCs to H2S did not induce necrosis as verified with Trypan blue exclusion and LDH release analysis. After inhibiting endogenous H2S production, exogenous H2S induced much more significant apoptosis, which was not altered by the presence of albumin or glutathione. H2S treatment increased the activities of ERK and p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase activity. Suppression of extracellular signal-regulated kinase (ERK) activity, but not of p38 activity, inhibited the H2S-induced apoptosis of HASMCs. The activation of ERK by H2S in HASMCs was accompanied by increased caspase-3 activity. Inhibition of caspase-3 by AC-DEVD-CHO attenuated the H2S-induced cell apoptosis. Inhibition of ERK by U0126 decreased caspase-3 activity, whereas AC-DEVD-CHO did not alter ERK activity. In conclusion, exogenous H2S induces apoptosis of HASMCs, which is significantly affected by the endogenous H2S level. Of the three investigated MAPKs, only ERK played an active role in mediating H2S-induced apoptosis of HASMCs by activating caspase-3. These findings may help reveal novel mechanisms for many diseases linked to H2S-related abnormal cellular proliferation and apoptosis.
.Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the "nonexcitable" glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ϳ70, 10, and Ͻ5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEAsensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel ␣-subunits with accessory, possibly Ca 2ϩ -sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.
The human embryonic kidney cells (HEK-293) have been widely used as one mammalian expression system in the study of voltage-gated K+ (Kv) channels. Understanding the endogenous Kv channels in these cells is the prerequisite for the characterization of the heterogeneously expressed Kv channels in these cells. In the present study we screened the transcriptional expression of different Kv genes in HEK-293 cells using reverse transcribed DNApolymerase chain reaction (RT-PCR) method. Among 16 Kv genes examined in native HEK-293 cells 10 Kv genes were reproducibly amplified, including those Kv a subunits encoding for the delayed rectifier (IK) [Kv1.1, Kv1.2, Kv1.3, Kv1.6, and Kv3.1], and for the transient outward Kv channels (IA) [Kv1.4, Kv3.3, Kv3.4, and Kv4.1] as well as a Kvbeta2 subunit. The whole-cell outward rectifier IK currents in the native HEK-293 cells were recorded (203 +/- 13 pA at +30 mV, n = 82) with the patch-clamp technique. In about 42% of the examined cells, IA coexisted with IK currents. IK currents were inhibited by tetraethylammonium chloride (TEA) at 1 and 10 mM by 39.5 and 48.4%, respectively. A 39.6% inhibition of IK currents was also observed in the presence of4-aminopyridine (4-AP, 5 mM). Interestingly, both TEAand 4-AP also inhibited IA currents. 4-acelamido-4'-isothiocyanalostilbene-2, 2'-disulfonic acid (1 mM), a Cl- channel blocker, had no effect on the endogenous outward currents. We concluded that multiple endogenous Kv genes were expressed in native HEK-293 cells, which possessed significant endogenous IK and IA currents with unique pharmacological properties.
ABSTRACT. Postmenopausal osteoporosis, a common type of osteoporosis in women, has become a serious public health issue. Astragalus polysaccharides (APS), possessing various pharmacological activities, are the active ingredients of Radix Astragali. It can be advantageous in the treatment of postmenopausal osteoporosis. In the present study, we evaluated the potential therapeutic effects of APS on postmenopausal osteoporosis by using a mice model induced by ovariectomy (OVX). Forty-eight female 6-week-old outbred ICR mice were randomly divided into six groups (N = 8): Sham group, OVX group, 17 b-estradiol (E2, 0.1 mg/kg)-treated OVX group, and APS (at three doses: 100, 200, and 400 mg/kg)-treated OVX groups. The effect of APS on the bone mineral density (BMD) was determined using dualenergy X-ray absorptiometry. The serum levels of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), osteocalcin, and tumor necrosis factor (TNF)-a were measured using ELISA kits. The results revealed that APS exerted significant antiosteoporotic activity by increasing the BMD considerably in a dosedependent manner. APS treatment reduced the serum RANKL levels considerably and increased the serum OPG levels, thereby lowering the ratio of RANKL/OPG. Furthermore, APS also markedly reduced osteocalcin and TNF-a concentration in OVX-induced postmenopausal osteoporosis mice model. These results showed that APS exerts a protective effect on bone loss in OVX mice. The molecular mechanism underlying this effect be the reduction of bone resorption and inhibition of osteoclastogenesis. Our findings suggest that APS may be a potential strategy for the prevention and treatment of postmenopausal osteoporosis.
KATP channels are a complex of regulatory sulfonylurea receptor subunits and the pore-forming inward rectifiers such as Kir6.1. Using the whole-cell patch-clamp technique, we investigated the interaction of nicotine with the Kir6.1 subunit as well as the underlying mechanism. Stable expression of Kir6.1 in HEK-293 cells yielded a detectable inward rectifier KATP current. This inward current was significantly inhibited by PNU-37883A and by a specific anti-Kir6.1 antibody. Nicotine at 30 and 100 microM increased Kir6.1 currents by 42 +/- 11.8% and 26.2 +/- 14.6%, respectively (n = 4-6, P < 0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (P < 0.05). Nicotine at 100 microM increased the production of superoxide anion (O2) by 20.3 +/- 5.7%, whereas at 1 mM it significantly decreased the production of O2 by 37.7 +/- 4.3%. Coapplication of hypoxanthine (HX) and xanthine oxidase (XO) to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents (P < 0.05). The stimulatory effect of HX/XO on Kir6.1 current was abolished by tempol, a scavenger of O2. Tempol also abolished the stimulatory effect of 30 muM nicotine on Kir6.1 currents. In conclusion, nicotine stimulates Kir6.1 channel at least in part through the production of O2.
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