Pretreatment and analysis techniques development of TKIs in biological samples for pharmacokinetic studies and therapeutic drug monitoring.

Tyrosine kinase inhibitors (TKIs) have emerged as the first-line small molecule drugs in many cancer therapies, exerting their effects by impeding aberrant cell growth and proliferation through the modulation of tyrosine kinase-mediated signaling pathways. However, there exists a substantial inter-individual variability in the concentrations of certain TKIs and their metabolites, which may render patients with compromised immune function susceptible to diverse infections despite receiving theoretically efficacious anticancer treatments, alongside other potential side effects or adverse reactions. Therefore, an urgent need exists for an up-to-date review concerning the biological matrices relevant to bioanalysis and the sampling methods, clinical pharmacokinetics, and therapeutic drug monitoring of different TKIs. This paper provides a comprehensive overview of the advancements in pretreatment methods, such as protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE), micro-SPE (µ-SPE), magnetic SPE (MSPE), and vortex-assisted dispersive SPE (VA-DSPE) achieved since 2017. It also highlights the latest analysis techniques such as newly developed high performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) methods, capillary electrophoresis (CE), gas chromatography (GC), supercritical fluid chromatography (SFC) procedures, surface plasmon resonance (SPR) assays as well as novel nanoprobes-based biosensing techniques. In addition, a comparison is made between the advantages and disadvantages of different approaches while presenting critical challenges and prospects in pharmacokinetic studies and therapeutic drug monitoring.

The environmental sources of benzophenones: Distribution, pretreatment, analysis and removal techniques.

Benzophenones (BPs) have wide practical applications in real human life due to its presence in personal care products, UV-filters, drugs, food packaging bags, etc. It enters the wastewater by daily routine activities such as showering, impacting the whole aquatic system, then posing a threat to human health. Due to this fact, the monitoring and removal of BPs in the environment is quite important. In the past decade, various novel analytical and removal techniques have been developed for the determination of BPs in environmental samples including wastewater, municipal landfill leachate, sewage sludge, and aquatic plants. This review provides a critical summary and comparison of the available cutting-edge pretreatment, determination and removal techniques of BPs in environment. It also focuses on novel materials and techniques in keeping with the concept of "green chemistry", and describes on challenges associated with the analysis of BPs, removal technologies, suggesting future development strategies.

[Isolation, culture and validation of CD34+ vascular wall-resident stem cells from mice].

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34+ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34+ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34+ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca2+ release and extracellular Ca2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34+ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34+ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca2+ release from endoplasmic reticulum of CD34+ VW-SCs. Store-operated Ca2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca2+-free/Ca2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34+ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.

Assessment of mitochondrial dysfunction and implications in cardiovascular disorders.

Mitochondria play a pivotal role in cellular function, not only acting as the powerhouse of the cell, but also regulating ATP synthesis, reactive oxygen species (ROS) production, intracellular Ca2+ cycling, and apoptosis. During the past decade, extensive progress has been made in the technology to assess mitochondrial functions and accumulating evidences have shown that mitochondrial dysfunction is a key pathophysiological mechanism for many diseases including cardiovascular disorders, such as ischemic heart disease, cardiomyopathy, hypertension, atherosclerosis, and hemorrhagic shock. The advances in methodology have been accelerating our understanding of mitochondrial molecular structure and function, biogenesis and ROS and energy production, which facilitates new drug target identification and therapeutic strategy development for mitochondrial dysfunction-related disorders. This review will focus on the assessment of methodologies currently used for mitochondrial research and discuss their advantages, limitations and the implications of mitochondrial dysfunction in cardiovascular disorders.

Rapid UV-radiation synthesis of polyacrylate cryogel oil-sorbents with adaptable structure and performance.

Macro-porous poly(lauryl acrylate) cryogel sheets as oil-sorbents were prepared through UV-radiation cryo-polymerizations in 1, 4-dioxane at low temperatures (-5, -2 and 0 °C) within 30 min. The influences of total monomer concentration, crosslinking monomer amount and polymerization temperature on the formation of cryogels were studied. The chemical structure and porous morphology were characterized through the techniques of Fourier transform infrared spectroscopy, thermal gravimetric analysis, contact angle measurement and scanning electron microscopy, confirming the features of high hydrophobicity, macro-porosity and good thermal stability. As well, the comparison between conventional gels prepared at room temperature and cryogels at lower temperatures was made, showing the higher rate of cryo-polymerization than conventional polymerization under the same UV-radiation condition. The swelling investigation was carried out with several organic solvents and oils. Enhanced performance of oil absorption was observed for those cryogels considering the absorption capacity and absorption rate. Variation of initiator amount and acrylate monomers could also modulate the absorption capacity. Those cryogel oil-sorbents exhibited wide adaptability, good reusability and high-temperature tolerance. Thus, this rapid and low-cost fabrication opens out a novel pathway to prepare efficient oil-sorbents used in waste water treatment.

Smart MMP2-Responsive Nanoprobe for Activatable Fluorescence Imaging-Guided Local Triple-Combination Therapies with Single Light.

Elaborately designed stimuli-responsive smart systems simultaneously enabling activatable imaging and selective treatment are highly desirable for precise diagnosis and therapy of cancer. Herein, such a smart theranostic nanoprobe composed of hollow gold nanospheres (HAuNs), photosensitizer (PS), matrix metalloproteinase 2 (MMP2) substrate peptide, and model drug doxorubicin (DOX) was designed. In the design, HAuNs served as the acceptor of Förster resonance energy transfer (FRET), photothermal therapy (PTT) reagent, and nanocarrier. The fluorescence and 1O2 generation of PS were inhibited by HAuNs through FRET effect, avoiding phototoxicity to normal tissues during circulation. Meanwhile, owing to the MMP2-triggered peptide cleavage, the PS could be efficiently activated in a tumor for selective fluorescence imaging and photodynamic therapy (PDT). The recovered fluorescence could be applied for detecting MMP2, locating tumor in vivo, and further guiding the local triple-combination therapies including PDT, PTT, and chemotherapy. The synergistic treatments of activated PDT, PTT, and controlled DOX release were achieved with single light, which provided the best therapeutic effects with enhanced stability and remarkably reduced nonspecific toxicity of PS and anticancer drug. This study helps to design novel stimuli-responsive systems for precise molecular sensing and site-specific cancer treatment.

Gold Nanoparticles Grafted with PLL-b-PNIPAM: Interplay on Thermal/pH Dual-Response and Optical Properties.

Narrowly distributed poly(l-lysine-b-N-isopropylacrylamide) (PLL-b-PNIPAM) was prepared through ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N-carboxy-α-amino anhydride and atom transfer radical polymerization of NIPAM, followed with the removal of ε-benzyloxycarbonyl group. Then gold nanoparticles (AuNPs) grafted with PLL-b-PNIPAM (PNIPAM-PLL-AuNPs) were obtained by the reduction of chloroauric acid with sodium citrate in the presence of PLL-b-PNIPAM. PNIPAM-PLL-AuNPs and its precursors were thoroughly characterized by proton magnetic resonance spectroscope, Fourier transform infrared spectroscope, UV-vis spectroscope, transmission electron microscopy, dynamic light scattering, thermogravimetric analysis, and circular dichroism. The obtained PNIPAM-PLL-AuNPs exhibited high colloid stability even at strong alkaline (pH = 12) and acidic (pH = 2) conditions. The thermal and pH dual-responsive behaviors of the grafting PLL-b-PNIPAM chains was observed to be affected by AuNPs, while not for the secondary structure of PLL chains. Correspondingly, the surface plasmon resonance (SPR) of AuNPs was found to be sensitive to both pH value and temperature. A blue shift in the SPR happened both with increasing pH value and increasing temperature. The stimuli-response was reversible in heating-cooling cycles. The gold nanoparticles with both pH and temperature response may have potential applications in biomedical areas and biosensors.

Sodium Tanshinone II-A Sulfonate (DS-201) Induces Vasorelaxation of Rat Mesenteric Arteries via Inhibition of L-Type Ca2+ Channel.

Background: We previously have proved that sodium tanshinone II-A sulfonate (DS-201), a derivative of traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), is an opener and vasodilator of BKCa channel in the vascular smooth muscle cells (VSMCs). Vascular tension is closely associated with Ca2+ dynamics and activation of BKCa channel may not be the sole mechanism for the relaxation of the vascular tension by DS-201. Therefore, we hypothesized that the vasorelaxing effect of DS-20 may be also related to Ca2+ channel and cytoplasmic Ca2+ level in the VSMCs. Methods: Arterial tension was measured by Danish Myo Technology (DMT) myograph system in the mesentery vessels of rats, intracellular Ca2+ level by fluorescence imaging system in the VSMCs of rats, and L-type Ca2+ current by patch clamp technique in Ca2+ channels transfected human embryonic kidney 293 (HEK-293) cells. Results: DS-201 relaxed the endothelium-denuded artery rings pre-constricted with PE or high K+ and the vasorelaxation was reversible. Blockade of K+ channel did not totally block the effect of DS-201 on vasorelaxation. DS-201 suppressed [Ca2+]i transient induced by high K+ in a concentration-dependent manner in the VSMCs, including the amplitude of Ca2+ transient, the time for Ca2+ transient reaching to the [Ca2+]i peak and the time to remove Ca2+ from the cytoplasm. DS-201 inhibited L-type Ca2+ channel with an EC50 of 59.5 µM and at about 40% efficacy of inhibition. However, DS-201did not significantly affect the kinetics of Ca2+ channel. The effect of DS-201 on L-type Ca2+ channel was rate-independent. Conclusion: The effect of DS-201 on vasorelaxation was not only via activating BKCa channel, but also blocking Ca2+ channel and inhibiting Ca2+ influx in the VSMCs of rats. The results favor the use of DS-201 and Danshen in the treatment of cardiovascular diseases clinically.

[Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers].

This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BKCa) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N2-weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BKCa channel into PLBMs, single channel characteristics of BKCa were studied by patch clamp method. The results showed that i) the single channel conductance of BKCa was (206.8 ± 16.9) pS; ii) the activities of BKCa channel were voltage dependent; iii) in the bath solution without Ca2+, there was almost no BKCa channel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKCa channels were activated in a Ca2+ concentration dependent manner; v) when [Ca2+] was increased from 1 µmol/L to 100 µmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BKCa was -30 mV when [K+] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BKCa into PLBMs showed similar electrophysiological characteristics to natural BKCa channels, so the PLBMs with incorporated BKCa can be used in the studies of pharmacology and dynamics of BKCa channel.

Different Effects of Hypertension and Age on the Function of Large Conductance Calcium- and Voltage-Activated Potassium Channels in Human Mesentery Artery Smooth Muscle Cells.

BACKGROUND: Large-conductance calcium- and voltage-activated potassium channels (BKC a channels) play important roles in the maintenance of vascular tone, and their dysregulation is associated with abnormal vascular relaxation and contraction. We tested the changes in BKC a channel properties in patients at different ages to assess the effects of hypertension and aging on the functional changes of BKC a channels. METHODS AND RESULTS: Patch clamp was performed to detect the activities of BKC a channels in freshly isolated human mesenteric artery smooth muscle cells from younger patients (aged ≤45 years) without hypertension, older patients (aged ≥65 years) without hypertension, and older patients with hypertension. The expression of mRNA and protein from BKC a channels was evaluated by reverse transcription polymerase chain reaction and Western blot analysis, respectively. Results showed that the whole-cell current density, spontaneous transient outward current, and Ca(2+) sensitivity of the artery smooth muscle cells were significantly decreased in the older patients with hypertension; the decreases were insignificant in the older patients without hypertension, although a clear tendency to have spontaneous transient outward current was detected in these patients. The expression of both mRNA and protein of BKC a subunits α and ß1 was significantly decreased in the older patients with hypertension but not in the older patients without hypertension compared with the younger patients without hypertension. CONCLUSIONS: Our findings demonstrate for the first time that hypertension is an important factor for the pathological alteration of the properties of BKC a channels in human mesenteric artery smooth muscle cells, and aging itself may also be a factor in these changes in the cells.

Comb-Type Grafted Hydrogels of PNIPAM and PDMAEMA with Reversed Network-Graft Architectures from Controlled Radical Polymerizations.

Dual thermo- and pH-responsive comb-type grafted hydrogels of poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) and poly(N-isopropylacrylamide) (PNIPAM) with reversed network-graft architectures were synthesized by the combination of atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization and click chemistry. Two kinds of macro-cross-linkers with two azido groups at one chain-end and different chain length [PNIPAM⁻(N3)2 and PDMAEMA⁻(N3)2] were prepared with N,N-di(ß-azidoethyl) 2-halocarboxylamide as the ATRP initiator. Through RAFT copolymerization of DMAEMA or NIPAM with propargyl acrylate (ProA) using dibenzyltrithiocarbonate as a chain transfer agent, two network precursors with different content of alkynyl side-groups [P(DMAEMA-co-ProA) and P(NIPAM-co-ProA)] were obtained. The subsequent azido-alkynyl click reaction of macro-cross-linkers and network precursors led to the formation of the network-graft hydrogels. These dual stimulus-sensitive hydrogels exhibited rapid response, high swelling ratio and reproducible swelling/de-swelling cycles under different temperatures and pH values. The influences of cross-linkage density and network-graft architecture on the properties of the hydrogels were investigated. The release of ceftriaxone sodium from these hydrogels showed both thermal- and pH-dependence, suggesting the feasibility of these hydrogels as thermo- and pH-dependent drug release devices.

Dynamic responses and vibration control of the transmission tower-line system: a state-of-the-art review.

This paper presented an overview on the dynamic analysis and control of the transmission tower-line system in the past forty years. The challenges and future developing trends in the dynamic analysis and mitigation of the transmission tower-line system under dynamic excitations are also put forward. It also reviews the analytical models and approaches of the transmission tower, transmission lines, and transmission tower-line systems, respectively, which contain the theoretical model, finite element (FE) model and the equivalent model; shows the advances in wind responses of the transmission tower-line system, which contains the dynamic effects under common wind loading, tornado, downburst, and typhoon; and discusses the dynamic responses under earthquake and ice loads, respectively. The vibration control of the transmission tower-line system is also reviewed, which includes the magnetorheological dampers, friction dampers, tuned mass dampers, and pounding tuned mass dampers.

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs.

Large conductance Ca(2+)-activated K(+) channel (BKCa) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BKCa channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K(+) currents (STOC, a component pattern of BKCa currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BKCa currents) via an IP3 receptor- and sarcoplasmic Ca(2+) mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BKCa channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BKCa channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BKCa channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca(2+) sensitivity of the channels.

Rhynchophylline-induced vasodilation in human mesenteric artery is mainly due to blockage of L-type calcium channels in vascular smooth muscle cells.

Rhynchophylline (Rhy) is a pharmacologically active substance isolated from Uncaria rhynchophylla which has been used to treat cardiovascular diseases and has drawn considerable attention in recent years for its antihypertensive activities. We investigated the actions of Rhy on endothelium-denuded human mesenteric artery by tension measurement and its actions on high conductance Ca(2+)-activated K(+) channels (BKCa) currents and calcium currents (ICa) in freshly isolated smooth muscle cells using perforated patch clamp technique. Intracellular Ca(2+) level was measured in Fura-2-loaded cells. Rhy inhibited both the KCl and BayK-evoked mesenteric artery constrictions in a dose-dependent manner. K(+) channel blockers (TEA, glibenclamide, IbTX, and 4-AP) did not affect the vasorelaxing effect of Rhy. Rhy inhibited L-type voltage-gated Ca(2+) current (ICa,L) but had no significant effect on macroscopic BKCa current. Rhy preincubation markedly reduced the elevation of [Ca(2+)]i level induced by KCl depolarization. Caffeine-stimulated [Ca(2+)]i elevation was also decreased to some extent by pretreatment with Rhy for 20 min. Our results show that Rhy relaxes smooth muscles of human mesenteric resistance arteries, mainly due to inhibition of Ca(2+) influx by blockage of L-type Ca(2+) channels and thereby the decrease in [Ca(2+)]i.

[Angiotensin II activates large-conductance Ca(2+)-activated potassium channels in human mesenteric artery smooth muscle cells].

The aim of present study was to explore the vasodilatation mechanism of angiotensin II (AngII) at the molecular level by investigating the effect of AngII on large-conductance Ca²âº-activated potassium channels (BK(Ca)) in human mesenteric artery smooth muscle cells. The effect of AngII on BK(Ca) was observed by using patch clamp single channel recording technique and amphotericin-perforated whole-cell recording technique. AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R) mRNA expression in human mesenteric artery was detected by RT-PCR. In cell-attached patch (Vm = +40 mV), AngII (100 nmol/L) had no significant effect on BK(Ca). After pretreatment with Valsartan (a specific inhibitor of AT1R, 10 µmol/L), 25, 100 and 250 nmol/L AngII stimulated BK(Ca) activity significantly in a dose response manner. After pretreatment of Valsartan, AngII (100 nmol/L) enhanced BK(Ca) open probability (NP(O)) from 0.010 ± 0.003 to 0.039 ± 0.015, decreased the mean close time (T(C)) of BK(Ca) markedly from (2 729.5 ± 808.6) ms to (487.7 ± 182.5) ms (n = 11, P < 0.05) , but AngII had no significant influences on the amplitude (Amp) and the mean open time (T(O)) of BK(Ca). Further PD123,319 (a specific inhibitor of AT2R) treatment prevented the stimulatory effect of AngII: PD123,319 decreased the NP(O) of BK(Ca) from 0.016 ± 0.003 to 0.004 ± 0.001 (n = 5, P < 0.05), but had no significant influences on Amp, T(O) and T(C) of BK(Ca). In addition, after pretreatment with Valsartan and PD123,319, AngII (100 nmol/L) had no significant effect on BK(Ca). In the amphotericin-perforated whole-cell patch-clamp configuration, after pretreatment with Valsartan, the current density of BK(Ca) at the voltage of -60 - +30 mV had no significant changes before and after adding 100 nmol/L AngII, but the current density of BK(Ca) at the voltage of +40 mV, +50 mV and +60 mV increased significantly after adding 100 nmol/L AngII, from (9.03 ± 2.23) pA/pF, (12.88 ± 2.55) pA/pF and (17.26 ± 2.84) pA/pF to (12.47 ± 2.22) pA/pF, (18.71 ± 2.51) pA/pF and (27.21 ± 3.12) pA/pF (n = 6, P < 0.05), respectively. Using RT-PCR, the AT1R mRNA and AT2R mRNA from isolated human mesenteric artery were detected. So we can draw a conclusion, AngII can stimulate BK(Ca) activity in human mesenteric artery smooth muscle cells after pretreatment with Valsartan, which is possibly mediated by AT2R.

Function of BKCa channels is reduced in human vascular smooth muscle cells from Han Chinese patients with hypertension.

Chronic hypertension is associated with an impaired vascular relaxation caused by an increased vascular tone; however, the underlying mechanisms are not fully understood in human patients. The present study was to investigate whether large-conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels are involved in dysfunctional relaxation of artery in Han Chinese patients with hypertension using the perforated patch clamp, inside-out single-channel, and macromembrane patch recording techniques to determine whole-cell current, spontaneous transient outward current, open probability, and Ca(2+) sensitivity and the reverse transcription polymerase chain reaction and Western blot analysis to examine the gene and protein expression of α-subunit (KCa1.1) and ß1-subunit (KCNMB1) of BK(Ca) channels in isolated human vascular smooth muscle cells and mesenteric arteries from normotensive and hypertensive patients. It was found that whole-cell current density, spontaneous transient outward current, and Ca(2+) sensitivity, but not single-channel open probability and slope conductance, were significantly decreased in vascular smooth muscle cells from patients with hypertension. Interestingly, mRNA and protein levels of KCNMB1, but not KCa1.1, were reduced in the arterial tissue from patients with hypertension. These results demonstrate for the first time that whole-cell current, spontaneous transient outward current, and Ca(2+) sensitivity of BK(Ca) channels are reduced in human vascular smooth muscle cells, which resulted from downregulation of ß1-subunit of the channel. This may account, at least in part, for the dysfunction of artery relaxation in Han Chinese patients with primary hypertension.

[ß-estradiol activates BK(Ca) in mesenteric artery smooth muscle cells of post-menopause women].

The aim of the present study was to study the effect of ß-estradiol (ß-E(2)) on the large-conductance Ca(2+)-activated potassium (BK(Ca)) channel in mesenteric artery smooth muscle cells (SMCs). The mesenteric arteries were obtained from post-menopause female patients with abdominal surgery, and the SMCs were isolated from the arteries using an enzymatic disassociation. According to the sources, the SMCs were divided into non-hypertension (NH) and essential hypertension (EH) groups. Single channel patch clamp technique was used to investigate the effect of ß-E(2) and ICI 182780 (a specific blocker of estrogen receptor) on BK(Ca) in the SMCs. The results showed the opening of BK(Ca) in the SMCs was voltage and calcium dependent, and could be blocked by IbTX. ß-E(2) (100 µmol/L) significantly increased open probability (Po) of BK(Ca) in both NH and EH groups. After ß-E(2) treatment, NH group showed higher Po of BK(Ca) compared with EH group. ICI 182780 could inhibit the activating effect of ß-E(2) on BK(Ca) in no matter NH or EH groups. These results suggest ß-E(2) activates BK(Ca) in mesenteric artery SMCs from post-menopause women via estrogen receptor, but hypertension may decline the activating effect of ß-E(2) on BK(Ca).

The technique of simultaneous recording calcium transients and spontaneous transient outward currents in arterial smooth muscle cells.

Laser scanning confocal microscopy (LSCM) and whole-cell perforated patch-clamp techniques were combined to study simultaneously the changes of intracellular signal molecules and membrane currents. Intracellular calcium transients and spontaneous transient outward currents (STOCs) were recorded simultaneously in freshly isolated mouse cerebral artery smooth muscle cells. The cells loaded with fluo-4/AM were scanned with the confocal line-scan mode. Triggering voltage pulses derived from an EPC-10 patch clamp amplifier triggered the confocal line scan. The results showed that STOCs and intracellular calcium transients could be simultaneously recorded in the same cell. This technique will be useful in studies of diseases caused by impairments of intracellular Ca(2+) signaling and related ionic channel activities, or vice versa.

Mechanism of intracellular signal transduction during injury of renal tubular cells induced by postasphyxial serum in neonates with asphyxia.

BACKGROUND: Renal injury is a severe and extremely common complication that occurs early in neonates with asphyxia. Reperfusion injury has been suggested as the cause of kidney damage during resuscitation of neonatal asphyxia. Previous studies have demonstrated that postasphyxial serum from neonates with asphyxia may result in apoptosis of renal tubular cells. However, the mechanisms that mediate renal tubular cell apoptosis induced by postasphyxial serum remain poorly understood. OBJECTIVES: In this report we investigate the intracellular signal transduction mechanisms that operate during injury of renal tubular cells induced by postasphyxial serum in neonates. METHODS: Cultured human renal proximal tubular cells HK-2 cell were exposed to 10% fetal calf serum (normal control), 20% postasphyxial serum or 20% postasphyxial serum with pyrrolidine dithiocarbamate (PDTC). The expression of both BAD and BAX in the cytoplasm was detected by immunohistochemistry. The mitochondria membrane potential (Deltapsim) was examined by confocal microscopy, and the release of the apoptogenic mitochondrial proteins cytochrome C and AIF was assessed by Western blot analysis. RESULTS: Loss of mitochondria membrane potential was detected in HK-2 cells treated with 20% postasphyxial serum as compared to cells in normal serum or PTDC-pretreated cells in 20% postasphyxial serum. A significant increase of Bad and Bax protein expression was also detected, along with the release of cytochrome C and AIF from mitochondria to cytosol in the postasphyxial serum treated cells, but not in the normal or PTDC-pretreated control cells. CONCLUSIONS: Our findings suggest that postasphyxial serum may induce renal tubular cell apoptosis through the mitochondrial pathway, and its intracellular signal transduction mechanism includes the activation of nuclear factor-kappaB.

Activation of high conductance Ca(2+)-activated K(+) channels by sodium tanshinoneII-A sulfonate (DS-201) in porcine coronary artery smooth muscle cells.

High conductance Ca(2+) activated K(+) channels (BK(Ca)) in vascular smooth muscles play important roles in controlling the vascular tone by determining the level of membrane potential and Ca(2+) influx through voltage gated Ca(2+) channels. Agents that alter the activity of Ca(2+) channels or BK(Ca) thus affect the vascular tone in both physiological and pathological conditions. Danshen, the dried root of Salvia miltiorrhiza, is a commonly used traditional Chinese medicine and is widely used as an effective remedy for cardiovascular and cerebral vascular diseases partly by its vasodilatation. Sodium tanshinoneII-A sulfonate (DS-201) is a water-soluble derivative of Tanshinone IIA, the main active component of Danshen. The purpose of this study was to explore possible mechanisms of vasodilative effects of DS-201 using porcine coronary artery smooth muscle. DS-201 induced relaxation of the coronary smooth muscle which had been contracted with 30 mM KCl, and the relaxation was inhibited by 100 nM iberiotoxin (IbTX), a specific BK(Ca) channel blocker. Using perforated whole-cell recordings and single channel recordings, effects of DS-201 on BK(Ca) were examined. The results showed that DS-201 activated BK(Ca). Extracellular application of DS-201 at 40, 80 microM under the whole-cell configuration induced increases of the BK(Ca) macroscopic currents by 43.6%, 42.1% respectively, and the spontaneous transient outward K(+) currents (STOCs) by 48.7%, 47.4% respectively. In inside-out patches, bath application of 20-150 muM of DS-201 activated BK(Ca) by 5.4-173.2 fold. These results indicate that the vasodilatation by DS-201 is related to activation of BK(Ca).