Preeclamptic serum and soluble fms-like tyrosine kinase-1 suppress endothelial inward rectifier potassium currents.

INTRODUCTION: Inward rectifier K+ (Kir) channel, a major factor determining endothelial membrane potential, regulates Ca2+ influx and vasodilator release, which is impaired in preeclamptic blood vessels. Previously, human umbilical vein endothelial cell (HUVEC) Kir currents were shown to decrease after incubating in preeclamptic plasma. We aimed to demonstrate whether sFlt-1, which is high in preeclamptic blood, could inhibit Kir channel function and expression. METHODS: HUVECs were cultured in regular medium, regular medium with added sFlt-1, or serum from preeclampsia patients or normal pregnant women (Control, sFlt-1, PE, or NP, respectively). Using whole-cell patch clamp technique, we identified Kir currents with the Kir blocker 2 mM BaCl2 and compared the currents among groups. The expression of Kir 2.1 and 2.2 channels were determined using immunofluorescent staining. RESULTS: sFlt-1 and PE groups exhibited similar Kir currents, while NP group possessed significantly larger currents, similar to Control group currents. Moreover, sFlt-1 and sFlt-1/PlGF ratio showed strong negative correlation with Kir currents (r = -0.71 and -0.70, respectively; P < 0.05). There were no significant differences in mean fluorescence intensity representing Kir 2.1 and 2.2 channels expression in all four groups. DISCUSSION: This is the first report to demonstrate sFlt-1 inhibition against Kir currents, which could lead to maternal endothelial dysfunction and hypertension seen in preeclampsia. However, channel expression was unaffected by sFlt-1 incubation, suggesting dysfunctions of channel or other processes (e.g., membrane translocation). The present data could pave the way for novel therapies targeting sFlt-1 or Kir to alleviate hypertension in preeclampsia.

Hesperetin Relaxes Depolarizing Contraction in Human Umbilical Vein by Inhibiting L-Type Ca2+ Channel.

OBJECTIVE: To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca2+ channel (LTCC) and related signaling pathway. METHODS: Isometric tension recording was performed in HUV rings pre-contracted with K+. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping in A7r5 cells, a rat vascular smooth muscle cell line, was performed to study the effect of hesperetin on LTCC current. RESULTS: After depolarizing precontraction, hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7% ± 4.3% compared to matching vehicle, osmolality, and time controls (P<0.0001). Importantly, hesperetin competitively inhibited BayK8644-induced contraction, shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49-2.40] in vehicle control to 11.30 nmol/L (95% CI 5.45-23.41) in hesperetin (P=0.0001). Moreover, hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase, adenylyl cyclase, PDE3, PDE4, and PDE5 (P<0.01), while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (P>0.05). However, simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin's effect (P>0.05). In whole-cell patch-clamping, hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7% ± 5.8% (P=0.0104). CONCLUSIONS: Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC, and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.

Ferulic acid enhances insulin secretion by potentiating L-type Ca2+ channel activation.

OBJECTIVE: To investigate the effect of ferulic acid, a natural compound, on pancreatic beta cell viability, Ca2+ channels, and insulin secretion. METHODS: We studied the effects of ferulic acid on rat insulinoma cell line viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. The whole-cell patch-clamp technique and enzyme-linked immunosorbent assay were also used to examine the action of ferulic acid on Ca2+ channels and insulin secretion, respectively. RESULTS: Ferulic acid did not affect cell viability during exposures up to 72 h. The electrophysiological study demonstrated that ferulic acid rapidly and concentration-dependently increased L-type Ca2+ channel current, shifting its activation curve in the hyperpolarizing direction with a decreased slope factor, while the voltage dependence of inactivation was not affected. On the other hand, ferulic acid have no effect on T-type Ca2+ channels. Furthermore, ferulic acid significantly increased insulin secretion, an effect inhibited by nifedipine and Ca2+-free extracellular fluid, confirming that ferulic acid-induced insulin secretion in these cells was mediated by augmenting Ca2+ influx through L-type Ca2+ channel. Our data also suggest that this may be a direct, nongenomic action. CONCLUSION: This is the first electrophysiological demonstration that acute ferulic acid treatment could increase L-type Ca2+ channel current in pancreatic ß cells by enhancing its voltage dependence of activation, leading to insulin secretion.

Ethanol enhances endothelial ionic currents and nitric oxide release via intermediate-conductance calcium-activated potassium channel.

AIMS: Ethanol is known to induce NO release and coronary vasorelaxation. Evidence suggests that K+ channels, especially a Ca2+-activated K+ channel (KCa), may regulate endothelial NO production. We aimed to investigate the ethanol effect on K+ currents in human coronary artery endothelial cells (HCAECs), identify the K+ channel type/subtype and signaling pathway involved, and demonstrate the relevance to ethanol-induced NO release. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. NO production were measured using the fluorescent probe, 2,3-diaminonaphthalene. KEY FINDINGS: We found that ethanol significantly potentiated HCAEC current (maximal increase to 155.68 ±â€¯18.93%, 20 mM ethanol, +80 mV; mean ±â€¯SEM, n = 9). Ethanol-induced current was significantly inhibited by blockers of IKCa or SKCa (intermediate- or small-conductance KCa), but not by blocking other K+ channels. When other known HCAEC channels were inhibited except IKCa, 20 mM ethanol significantly increased IKCa current to 198 ±â€¯25.11% (n = 6), but it could not enhance SKCa current that was similarly isolated. Moreover, ethanol-induced NO release was prevented by blocking IKCa channel, adenosine A2A receptor (A2AR), Gs protein, or protein kinase A (PKA). SIGNIFICANCE: This study was the first to demonstrate that acute ethanol exposure could activate endothelial IKCa channel, via A2AR-Gs-PKA signaling, leading to increased whole-cell current and NO release, which could be an important mechanism underlying ethanol-induced NO release and vasodilation.

Loss-of-function mutations of SCN10A encoding NaV1.8 α subunit of voltage-gated sodium channel in patients with human kidney stone disease.

Human kidney stone disease (KSD) causes significant morbidity and public health burden worldwide. The etiology of KSD is heterogeneous, ranging from monogenic defects to complex interaction between genetic and environmental factors. However, the genetic defects causing KSD in the majority of affected families are still unknown. Here, we report the discovery of mutations of SCN10A, encoding NaV1.8 α subunit of voltage-gated sodium channel, in families with KSD. The region on chromosome 3 where SCN10A locates was initially identified in a large family with KSD by genome-wide linkage analysis and exome sequencing. Two mutations (p.N909K and p.K1809R) in the same allele of SCN10A co-segregated with KSD in the affected family. Additional mutation (p.V1149M) of SCN10A was identified in another affected family, strongly supporting the causal role of SCN10A for KSD. The amino acids at these three positions, N909, K1809, and V1149, are highly conserved in vertebrate evolution, indicating their structural and functional significances. NaV1.8 α subunit mRNA and protein were found to express in human kidney tissues. The mutant proteins expressed in cultured cells were unstable and causing reduced current density as analyzed by whole-cell patch-clamp technique. Thus, loss-of-function mutations of SCN10A were associated with KSD in the families studied.

Testosterone rapidly increases Ca2+-activated K+ currents causing hyperpolarization in human coronary artery endothelial cells.

Testosterone has endothelium-dependent vasodilatory effects on the coronary artery, with some reports suggesting endothelial ion channel involvement. This study employed the whole-cell patch clamp technique to investigate the effect of testosterone on ion channels in human coronary artery endothelial cells (HCAECs) and the mechanisms involved. We found that 0.03-3µM testosterone significantly induced a rapid, concentration-dependent increase in total HCAEC current (EC50, 71.96±1.66nM; maximum increase, 59.13±8.37%; mean±SEM). The testosterone-enhanced currents consisted of small- and large-conductance Ca2+-activated K+ currents (SKCa and BKCa currents), but not Cl- and nonselective cation currents. Either a non-permeant testosterone conjugate or the non-aromatizable androgen dihydrotestosterone (DHT) could increase HCAEC currents as well. The androgen receptor antagonist flutamide prevented this testosterone, testosterone conjugate, and DHT effect, while the estrogen receptor antagonist fulvestrant did not. Incubating HCAECs with pertussis toxin or protein kinase A inhibitor H-89 largely inhibited the testosterone effect, while pre-incubation with phospholipase C inhibitor U-73122, prostacyclin inhibitor indomethacin, nitric oxide synthase inhibitor L-NAME or cytochrome P450 inhibitor MS-PPOH, did not. Finally, testosterone application induced HCAEC hyperpolarization within minutes; this effect was prevented by SKCa and BKCa current inhibitors apamin and iberiotoxin. This is the first electrophysiological demonstration of androgen-induced KCa current increase, leading to hyperpolarization, in any endothelial cell, and the first report of SKCa as a testosterone target. Our data show that testosterone rapidly increased whole-cell HCAEC SKCa and BKCa currents via a surface androgen receptor, Gi/o protein, and protein kinase A. This mechanism may explain rapid testosterone-induced coronary vasodilation seen in vivo.

Ginsenoside Re enhances small-conductance Ca(2+)-activated K(+) current in human coronary artery endothelial cells.

AIMS: Ginsenosides, active components in ginseng, have been shown to increase nitric oxide (NO) production in aortic endothelial cells. This effect was reversed by tetraethylammonium (TEA) inhibition of endothelial Ca(2+)-activated K(+) (KCa) channels. The objectives of this study, therefore, were to test 1) whether vasorelaxing ginsenoside Re could affect KCa current, an important regulator of NO production, in human coronary artery endothelial cells (HCAECs); and 2) whether small-conductance KCa (SKCa) channel was the channel subtype involved. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. KEY FINDINGS: Ginsenoside Re dose-dependently increased endothelial outward currents, with an EC50 of 408.90±1.59nM, and a maximum increase of 36.20±5.62% (mean±SEM; p<0.05). Apamin, an SKCa channel inhibitor, could block this effect, while La(3+), a nonselective cation channel (NSC) blocker, could not. When NSC channel, inward-rectifier K(+) channel, intermediate-, and large-conductance KCa channels were simultaneously blocked, ginsenoside Re could still increase outward currents significantly (35.49±4.22%; p<0.05); this effect was again abolished by apamin. Repeating the experiments when Cl(-) channel was additionally blocked gave similar results. Finally, we demonstrated that ginsenoside Re could hyperpolarize HCAECs; this effect was reversed by apamin. These data clearly indicate that ginsenoside Re increased HCAEC outward current via SKCa channel activation, and NSC channel was not involved. SIGNIFICANCE: This is the first report to demonstrate that ginsenoside Re could increase SKCa channel activity in HCAECs. This can be a mechanism mediating ginseng's beneficial actions on coronary vessels.

Role of meditation in reducing sympathetic hyperactivity and improving quality of life in lupus nephritis patients with chronic kidney disease.

BACKGROUND: Lupus nephritis is an important leading cause of chronic kidney disease (CKD) among the young population in Thailand. Systemic lupus erythematosus (SLE) is often characterized by the presence of sympathetic hyperactivity, which results in a perishing outcome. Some physiological studies reveal that meditation may reduce this autonomic dysfunction. The authors hypothesized that meditation could be beneficial in alleviating the sympathetic hyperactivity and improving quality of life in lupus nephritis patients with CKD. MATERIAL AND METHOD: The authors performed a prospective pilot study, which enrolled lupus nephritis patients and categorized enrollees into meditation group and control group. Method of meditation was instructed by an expert in Buddhist studies for a duration of 60 minutes every month. Participants in the intervention group were advised to meditate every day for 24 weeks. To evaluate change in sympathetic activity, normetanephrine level was measured at beginning and the end of study and compared between both groups. Quality of life was determined by SF-36. Heart rate variability was also assessed in meditation group. RESULTS: Thirty eligible patients were recruited into the study. Fifteen patients were stratified in the meditation group and 15 patients in the control group. After meditation for 6 months, serum normetanephrine level decreased, but without statistical significance (0.105 vs. 0.059, p = 0.28). The reduction in normetanephrine level was also observed in the control group (p = 0.11). In the aspect of quality of life, scores of physical and mental components improved significantly. In meditation group, physical component score increased from 21.4 (5.0-50.2) to 62.2 (51.8-88.4) points (p < 0.01) and mental score increased from 16.9 (4.4-46.0) to 72.4 (45.1-81.6) points (p < 0.01). Quality of life score in the meditation group significantly increased more than in control group (p < 0.01). The parameter of heart rate variability in time and frequency domain also improved in the meditation group. CONCLUSION: In lupus nephritis patients with CKD, meditation shows a trend of benefits in reducing sympathetic overactivity and improving quality of life. Our results support the important role of meditation as a valuable adjunctive treatment of lupus nephritis with CKD.

Effects of preeclamptic plasma on potassium currents of human umbilical vein endothelial cells.

Endothelial cell (EC) dysfunction in preeclampsia (PE) may be mediated by humoral factors secreted by placenta, thereby affecting the EC vasoactive compound production. Possible targets of these factors include potassium channels, which are important in EC membrane potential control, calcium influx, and vasoactive compound release. Alterations in potassium channel function may thus contribute to the pathogenesis of PE. The present study compared the effects of 10% plasma from PE, normal pregnant (NP), or nonpregnant women (NS) on potassium currents of human umbilical vein ECs (HUVECs), using whole-cell patch clamp technique, with HUVECs in conventional culture medium (10% fetal bovine serum) as controls. Cells of all groups were similar in morphology and whole-cell capacitance. The fraction of cells with inward rectifier potassium channel (IRK) current in PE plasma (41.2%) was significantly lower than those in NP and NS plasmas (76.9% and 59.1%, respectively), although the IRK current density was similar among groups. The outward current components included the calcium-sensitive potassium channels (K(Ca)) and were partially blocked by 100 nmol/L apamin and 200 nmol/L iberiotoxin. The fraction with outward current in PE plasma (100%) was significantly higher than those in NP and NS plasmas (76.9% and 81.8%). The findings indicate inhibition of IRK expression by PE plasma in HUVEC culture, while K(Ca) expression may be facilitated probably as a compensatory response to diminished IRK. These data suggest that potassium channels may be a target of the pathogenic factor/factors in the plasma of patients with PE and may play roles in the pathogenesis of this condition.