Channel Protein Expression Research Articles

The clinnical significance of TRPM8 ion channels over-expressed in pancreatic cancer

Objective TRPM8 as a clinical biomarker and potential drug target may provide a gui-dance in accurately judging the malignant potential and prognosis of pancreatic cancer. Downregulating the expression of TRPM8 leads to inhibition of the proliferation of pancreatic cancer cells and this may provide a new target for personalized pancreatic cancer treatment. Methods The pancreatic cancer tissues of 77 patients and the normal pancreas tissues of 30 persons were collected and examined by immunohistochemistry. PCR, gene knockout, Western Blot, CCK8 and patch clamp experiments were performed on pancreatic cancer cell lines BxPC-3, PANC1 and normal pancreatic cell line HPCY-5. Results Immunohistochemistry and Western Blot experiments showed that the TRPM8 channel protein was highly expressed in cancer cells, and the high expression of channel proteins was related to the degree of tumor differentiation (P<0.05). The detection of the TRPM8 mRNA in pancreatic cancer cell lines BxPC-3 and PANC1 by QPCR showed absolutely high expressions relative to the normal cell line HPCY-5. The results of patch clamp and CCK8 showed that the membrane currents and proliferation of tumor cells were significantly decreased after TRPM8 gene knockout, and the difference is significant (P<0.05). Conclusions The TRPM8 ion channel was higher in pancreatic cancer cells than that in normal pancreatic cells. Overexpression of TRPM8 was closely related to tumor cell proliferation and invasion ability. Key words: Pancreatic cancer; TRPM8; Western Blot; Patch clamp

Angelica sinensis Polysaccharides Ameliorate Stress-Induced Premature Senescence of Hematopoietic Cell via Protecting Bone Marrow Stromal Cells from Oxidative Injuries Caused by 5-Fluorouracil.

Myelosuppression is the most common complication of chemotherapy. Decline of self-renewal capacity and stress-induced premature senescence (SIPS) of hematopoietic stem cells (HSCs) induced by chemotherapeutic agents may be the cause of long-term myelosuppression after chemotherapy. Whether the mechanism of SIPS of hematopoietic cells relates to chemotherapeutic injury occurred in hematopoietic microenvironment (HM) is still not well elucidated. This study explored the protective effect of Angelica sinensis polysaccharide (ASP), an acetone extract polysaccharide found as the major effective ingredients of a traditional Chinese medicinal herb named Chinese Angelica (Dong Quai), on oxidative damage of homo sapiens bone marrow/stroma cell line (HS-5) caused by 5-fluorouracil (5-FU), and the effect of ASP relieving oxidative stress in HM on SIPS of hematopoietic cells. Tumor-suppressive doses of 5-FU inhibited the growth of HS-5 in a dose-dependent and time-dependent manner. 5-FU induced HS-5 apoptosis and also accumulated cellular hallmarks of senescence including cell cycle arrest and typical senescence-associated β-galactosidase positive staining. The intracellular reactive oxygen species (ROS) was increased in 5-FU treated HS-5 cells and coinstantaneous with attenuated antioxidant capacity marked by superoxide dismutase and glutathione peroxidase. Oxidative stress initiated DNA damage indicated by increased γH2AX and 8-OHdG. Oxidative damage of HS-5 cells resulted in declined hematopoietic stimulating factors including stem cell factor (SCF), stromal cell-derived factor (SDF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), however, elevated inflammatory chemokines such as RANTES. In addition, gap junction channel protein expression and mediated intercellular communications were attenuated after 5-FU treatment. Significantly, co-culture on 5-FU treated HS-5 feeder layer resulted in less quantity of human umbilical cord blood-derived hematopoietic cells and CD34+ hematopoietic stem/progenitor cells (HSPCs), and SIPS of hematopoietic cells. However, it is noteworthy that ASP ameliorated SIPS of hematopoietic cells by the mechanism of protecting bone marrow stromal cells from chemotherapeutic injury via mitigating oxidative damage of stromal cells and improving their hematopoietic function. This study provides a new strategy to alleviate the complication of conventional cancer therapy using chemotherapeutic agents.

Regulation of Coronary Arterial Large Conductance Ca2+-Activated K+ Channel Protein Expression and Function by n-3 Polyunsaturated Fatty Acids in Diabetic Rats

Aim: The objective of this study was to examine the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on coronary arterial large conductance Ca²⁺-activated K⁺ (BK) channel function in coronary smooth muscle cells (SMCs) of streptozotocin-induced diabetic rats. Methods: The effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on coronary BK channel open probabilities were determined using the patch clamp technique. The mRNA and protein expressions of BK channel subunits were measured using qRT-PCR and Western blots. The coronary artery tension and coronary SMC Ca²⁺ concentrations were measured using a myograph system and fluorescence Ca²⁺ indicator. Results: Compared to nondiabetic control rats, the BK channel function was impaired with a reduced response to EPA and DHA in freshly isolated SMCs of diabetic rats. Oral administration of n-3 PUFAs had no effects on protein expressions of BK channel subunits in nondiabetic rats, but significantly enhanced those of BK-β₁ in diabetic rats without altering BK-α protein levels. Moreover, coronary ring tension induced by iberiotoxin (a specific BK channel blocker) was increased and cytosolic Ca²⁺ concentrations in coronary SMCs were decreased in diabetic rats, but no changes were found in nondiabetic rats. Conclusions: n-3 PUFAs protect the coronary BK channel function and coronary vasoreactivity in diabetic rats as a result of not only increasing BK-β₁ protein expressions, but also decreasing coronary artery tension and coronary smooth muscle cytosolic Ca²⁺ concentrations.

Wnt and inflammatory pathway mediated differential expression of water channel protein in glaucomatous trabecular meshwork

Wnt and inflammatory pathway mediated differential expression of water channel protein in glaucomatous trabecular meshwork

Role of adenosine triphosphate-sensitive potassium channel in hydrogen sulfide-induced inhibition of high glucoseinduced osteoblast damage

The aim of this study is to identify the role of adenosine triphosphate-sensitive potassium channel (KATP) in hydrogen sulfide (H₂S)-induced inhibition of high glucose (HG)-induced osteoblast damage. Osteoblasts from rat mandible were cultured and identified. The osteoblasts were then treated with HG, H₂S, KATP channel opener pinacidil (Pia), and KATP channel blocker glibenclamide (Gli). Western blot method was performed to detect the expression of KATP channel protein. CCK8, reverse transcriptase polymerase chain reaction (RT-PCR) , and image analysis were used to determine the effects of H₂S-KATP on the proliferation, differentiation, and mineralization of osteoblasts. The expression of KATP channel protein in osteoblasts was significantly decreased under the influence of HG. H₂S pretreatment significantly inhibited HG on KATP channel protein down-regulation. Moreover, H₂S pretreatment significantly inhibited the effect of HG on the proliferation of osteoblasts, thereby preventing HG-induced inhibition of osteoblasts differentiation and mineralization. Meanwhile, the KATP channel blocker effectively blocked the H₂S on osteoblasts and had a protective effect. Through the KATP channel, H₂S inhibited osteoblasts damage induced by HG.

Moderate exercise training does not prevent the reduction in myocardial L-type Ca2+ channels protein expression at obese rats.

Authors have showed that obesity implicates cardiac dysfunction associated with myocardial L‐type calcium channels (LTCCs) activity impairments, as well as moderate exercise training (MET) seems to be an important therapeutic tool. We tested the hypothesis that MET promotes improvements on LTCCS activity and protein expression at obesity induced by unsaturated high‐fat diets, which could represent a protective effects against development of cardiovascular damage. Male Wistar rats were randomized in control (C, n = 40), which received a standard diet and obese (Ob; n = 40), which received high‐fat diet. After 20 weeks, the animals were assigned at four groups: control (C; n = 12); control submitted to exercise training (ET; n = 14); obese (Ob; n = 10); and obese submitted to exercise training (ObET; n = 11). ET (5 days/week during 12 weeks) began in the 21th week and consisted of treadmill running that was progressively increased to reach 60 min. Final body weight (FBW), body fat (BF), adiposity index (AI), comorbidities, and hormones were evaluated. Cardiac remodeling was assessed by morphological and isolated papillary muscles function. LTCCs activity was determined using specific blocker, while protein expression of LTCCs was evaluated by Western blot. Unsaturated high‐fat diet promoted obesity during all experimental protocol. MET controlled obesity process by decreasing of FBW, BF, and AI. Obesity implicated to LTCCs protein expression reduction and MET was not effective to prevent this condition. ET was efficient to promote several improvements to body composition and metabolic parameters; however, it was not able to prevent or reverse the downregulation of LTCCs protein expression at obese rats.

Effects of calcium channel on ovarian cancer cells.

The aim of the present study was to examine the effects of calcium channel protein on ovarian cancer cells. The expression of calcium channel protein in normal ovarian cells and ovarian cancer cells was detected by fluorescence quantitative PCR. Subsequently, the ovarian cancer cells were added to calcium channel protein activator media at various concentrations of 0, 1, 4, 8, 12, 16 and 20 mmol/l. The concentration of calcium ion in different samples was produced, and using an MTT assay, ovarian cancer cell activity in various samples was detected. Finally, a flow cytometer was used to explore the apoptosis rate. It was found that there was a significant difference between the expression of calcium channel protein in normal ovarian tissue and ovarian cancer cells (P<0.05), as well as a significant difference of calcium concentration among various samples (P<0.05). When the concentrations of calcium channel activator were 1, 4, 8, 12, 16 and 20 mmol/l, the values of the ovarian cancer cell inhibition rates were 4.6, 21.3, 48.3, 67.9, 52.8 and 31.8%, respectively. It showed that the calcium channel activator inhibited the proliferation of ovarian cancer cells to a certain extent, in a dose-dependent manner, especially when the concentration was at 12 mmol/l at which the intracellular calcium concentration was similar to that in normal ovarian cells. In conclusion, calcium ions play an important role in promoting cell proliferation of ovarian cancer cells, and they were involved in apoptosis of ovarian cancer cells to some extent, which regulates apoptosis by controlling the content of intracellular calcium.

Expression and Localization of Equine Tissue-Specific Divalent Ion-Transporting Channel Proteins

Divalent ions such as calcium, iron, and copper have roles in physiological functions such as maintaining ionic homeostasis. Ion channels, present in the cell membrane, control the flow of ions across the cell membrane. Hyperkalemic periodic paralysis, iron-deficiency anemia, and horse colic are related to ion channel malfunctions. Equine ion channels have not been studied as much as those in other species. In the present study, we focused on expression and localization of calcium, iron, and copper ion channels in equine duodenum, heart, kidney, liver, lung, ovary, uterus, and testis tissues. Gene and protein expressions were quantified by using real-time polymerase chain reaction and western blotting, respectively. Localization of each channel was identified via immunohistochemical analysis. Gene (mRNA) and protein levels of Nckx3 and Trpv2 were high in uterus tissue. Ferroportin (Ireg1) mRNA levels varied, but its protein level was notably high in liver. Hephaestin (Heph) mRNA level was high in duodenum, whereas its protein level was high in liver. Nckx3 was highly expressed in ovary, uterus, and testis. Trpv2 was expressed in duodenum, liver, lung, and kidney. Ferroportin was expressed in duodenum, liver, ovary, and uterus. Hephaestin was expressed in duodenum, ovary, and testis. Copper transporter 1 (Ctr1) was expressed in all tissues, whereas Atp7a was expressed in duodenum, kidney, and testis. Each protein was expressed in their respective structures, including gland, epithelium, and myocyte. Taken together, the regulation of each ion channel is important to secretion, absorption, and contraction in the various organ. The results of this study could form a basis for elucidating Nckx3-, Trpv2-, Ireg1-, Heph-, Ctr1-, and Atp7a-related diseases in horse.

Genomic upregulation of cardiac Cav1.2\u03b1 and NCX1 by estrogen in women

BackgroundWomen have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts.MethodsPostmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs.ResultsIn women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1–2 days) increased ICa,L (31%, P < 0.05) and INCX (7.5-fold, − 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter ICa,L and INCX.ConclusionsThe results show that estrogen upregulates cardiac ICa,L and INCX in women through genomic mechanisms that account for sex differences in Ca2+ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.

The expression of water channel proteins during human salivary gland development: a topographic study of aquaporins 1, 3 and 5

Some members of aquaporin family (AQP) plays crucial functions in salivary synthesis and secretion. These proteins expression has already been reported during salivary gland formation, however no previous studies in human developing glands have been performed. We evaluated AQP1, 3 and 5 expression through the stages of human salivary gland morphogenesis and discuss the possible role of AQP for glandular maturation. Human salivary glands derived from foetuses aged between 14 and 25 weeks were submitted to immunohistochemistry. At the bud stage, membrane expression of AQP1, 3 and 5 were observed within the epithelial bud cells presenting a similar apicolateral pattern, also found at the pseudoglandular stage, present within the terminal portions of future acini, while AQP5 was also particularly strong at the apical membrane of pre-acinar and pre-ductal cells. AQP5 was co-localised with Cytokeratin 7. Similar AQP1, 3 and 5 expression were observed at the following canalicular stage, where distinct and strongly luminal and acinar AQP5 expression is present. During the final terminal bud stage, AQP1 was only identified in serous acini, myoepithelial and endothelial cells, while differentiated mucous acinar cells and ducts were negative. AQP3 was detected at apicolateral membranes of both mucous and serous acini. AQP5 also showed a diffuse expression in mucous and serous acini, in addition to strong apical membrane expression within lumen of intercalated ductal cells. This topographic analysis of AQP1, 3 and 5 revealed differences in the expression pattern throughout salivary gland developmental stages, suggesting different roles for each protein in human glandular maturation.

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

BackgroundThe inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations.MethodsMolecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively.ResultsPA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM).Conclusions1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Oxidative stress promotes myocardial fibrosis by upregulating KCa3.1 channel expression in AGT-REN double transgenic hypertensive mice.

The intermediate-conductance Ca2+-activated K+ (KCa3.1) channels play a pivotal role in the cardiac fibroblast proliferation and inflammatory reaction during the progression of myocardial fibrosis. However, the relationship between KCa3.1 expression and oxidative stress, the important factor of promoting fibrosis, has not been clearly established. This study was designed to investigate whether the role of oxidative stress in promoting myocardial fibrosis is related to KCa3.1 channel by using biochemical approaches. It was found that mean blood pressure, plasma Ang II level, and myocardium malondialdehyde (MDA) content of angiotensinogen-renin (AGT-REN) double transgenic hypertension (dTH) mice were higher than those in wild-type (WT) mice of the same age (4, 8 and 12months) and were significantly increased with age. However, plasma Ang (1-7) level and myocardium superoxide dismutase (SOD) activity showed a downward trend and were lower than those of the same-aged WT mice (4, 8 and 12months). In addition, protein expression of myocardium KCa3.1 channel in 4-, 8-, and 12-month-old dTH mice were significantly higher than that of the same-aged WT mice and gradually increased with age. TRAM-34, a blocker of KCa3.1 channel, and losartan mitigated the myocardial structural and functional damage by inhibiting collagen deposition and decreasing the expression of β-MHC. After intervention of ROS scavenger N-acetyl cysteine (NAC) and NADPH inhibitor apocynin (Apo) in 6-month-old dTH mice for 4weeks, myocardial oxidative stress level was reduced and KCa3.1 channel protein expression was decreased. Meanwhile, Apo inhibited the myocardium p-ERK1/2/T-ERK protein expression in dTH mice, and after blockage of ERK1/2 pathway with PD98059, the KCa3.1 protein expression was reduced. These results demonstrate for the first time that KCa3.1 channel is likely to be a critical target on the oxidative stress for its promoting role in myocardial fibrosis, and the ERK1/2 pathway may be involved in the regulation of oxidative stress to KCa3.1.

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR 2.1 backward trafficking.

Drug‐induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK 1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late‐endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK 1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK‐KWGF cells, both drugs dose‐ and time‐dependently increased KIR2.1 expression (2.0 ± 0.2‐fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3‐fold with dronedarone: 5 μM, 24 hrs) and late‐endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co‐expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS‐7, END‐2, MES‐1 and EPI‐7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR 2.1 upon 24‐hrs treatment, whereas dronedarone tended to increase IKIR 2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK 1 by inhibiting KIR2.1 degradation.

Effects of HCN2 in the development of peripheral neuropathic pain in rats

To explore the effects of hyperpolarization-activated cyclic nucleotide-gated channels 2(HCN2) in the formation of peripheral neuropathic pain in rats. Twenty-four healthy adult rats were divided into two groups randomly(n=12):the sham group rats were only isolated the left L4, L5 spinal nerve, the spinal nerve ligation(SNL) group was separated the spinal nerve and performed the corresponding ligation. The behavioral experiments were tested 7 days after operation; The model rats were randomly divided into 3 groups(n=6):① negative group(Saline), intra-plantar injection of saline in left hindpaws; ② positive group(gabapentin, GBPT), intraperitoneal injection of gabapentin; ③ experimental group(ZD7288), intra-plantar injection of ZD7288 in left hindpaws. The behavioral experiments were tested before injection and 1 h, 4 h, 24 h and 48 h after injection; Obtaining the dorsal root ganglion(DRG) of the control group (before operation), sham group and the SNL group(n=6), using qPCR and Western blot to analyze the mRNA and protein of HCN2 in rats' DRG. The rat model of neuropathic pain was successfully established. Compared with saline group, GBPT group and ZD7288 group could significantly reduce the symptoms of neuropathic pain in rats after injection 1 h (P<0.01), and there was no difference between GBPT group and ZD7288 group. Compared with control group and sham group, the expression of HCN2 mRNA in SNL group's DRG was significantly increased (P<0.01), and the expression of HCN2 channel protein was also increased significantly (P<0.05). HCN2 is involved in the development of peripheral neuropathic pain and is likely to be a potential new target for the treatment of neuropathic pain.

Chronic Femoral Artery Ligation Exaggerates the Exercise Pressor Reflex but Not Selective Activation of the Muscle Mechanoreflex in Decerebrate Rats

Mechanical and metabolic stimuli arising within contracting skeletal muscles reflexly increase sympathetic nervous system activity, blood pressure, and heart rate. This reflex, termed the exercise pressor reflex, is exaggerated in a rat model of limb ischemia in which a femoral artery is chronically ligated. We recently found that the mechanically‐sensitive component of the exercise pressor reflex contributes to its exaggeration in rats with a ligated femoral artery despite no difference in mechano‐gated channel protein expression in lumbar dorsal root ganglia (DRG) tissue. This suggests that metabolite‐induced sensitization of mechanoreceptors underlies the exaggerated mechanically‐sensitive component of the exercise pressor reflex in ligated rats. This prompted us to seek the physiological evidence in support of the anatomical evidence that ligation does not increase mechano‐gated channel expression in DRG tissue.ObjectiveWe used a within‐rat comparison experimental design to test the hypothesis that the increase in blood pressure, heart rate, and renal sympathetic nerve activity (RSNA) during selective activation of the muscle mechanoreflex (a purely mechanical stimulus) would not be different when evoked from a freely perfused hindlimb compared a ligated hindlimb.MethodsFifteen male Sprague‐Dawley rats had their left femoral artery ligated 72 hours before the experiment. In decerebrate, unanesthetized rats the muscle mechanoreflex was selectively activated by passively stretching the Achilles tendon for 30 s. In five of those fifteen rats, the exercise pressor reflex was evoked by electrically‐stimulating the sciatic nerves for 30 s which produced static hindlimb muscle contractions.ResultsIn 10 rats there were no differences between the peak pressor (freely perfused: 13±2, ligated: 14±3 mmHg, p=0.801), cardioaccelerator (freely perfused: 12±2, ligated: 11±2 bpm, p=0.672), or RSNA (freely perfused: 40±7, ligated: 46±6%, p=0.377) responses evoked by tendon stretch of the freely perfused and ligated hindlimbs. In five additional rats, the exercise pressor reflex was greater when evoked from the contracting ligated hindlimb (28±3 mmHg) compared to the contracting freely perfused hindlimb (21±3 mmHg, p=0.014). In those same five rats, however, in which femoral artery ligation exaggerated the exercise pressor reflex there was no difference in the peak pressor response to tendon stretch between hindlimbs (freely perfused: 16±2, ligated: 15±3 mmHg, p=0.581).ConclusionWe found that the reflex pressor, cardioaccelerator, and RSNA responses to tendon stretch (a purely mechanical stimulus) were not exaggerated by femoral artery ligation, even in rats in which the exercise pressor reflex was confirmed to be exaggerated by ligation. These findings indicate that the ligation‐induced exaggeration of the mechanically‐sensitive component of the exercise pressor reflex reported previously must be due to a metabolite‐induced sensitization of mechano‐gated receptors.Support or Funding InformationDepartmental funds

Pharmacological Modulation of Kir3.1 and Kir3.4 Cell Surface Expression

BackgroundSmall molecules have been shown to pharmacologically modulate the cell surface expression of ion channel proteins. We thus investigated the effects of the antimalarial aminoquinolines Chloroquine and Primaquine on the cell surface density of Kir3.1/Kir3.4 proteins which mediate the acetylcholine activated inward rectifier potassium current (IKACh) in the heart. We compared the effects of Chloroquine and Primaquine to those of known lysosomal inhibitors‐ ammonium chloride (NH4Cl) and Leupeptin‐ on the surface density of the IKACh proteins. We hypothesized that long term Chloroquine treatment increases the surface expression of Kir3.1/Kir3.4 independently of the lysosomal pathway.MethodsExperiments were performed in human embryonic kidney (HEK293) cells stably transfected with Kir3.1 and Kir3.4. ATTO‐488 conjugated TertiapinQ (a potent blocker of IKACh) was used to label the IKACh proteins in live cells, and flow cytometry was used to quantify their expression on the cell surface after 24 hours treatment with 1, 10 and 50 μM Chloroquine, 10 μM Primaquine, 10 mM NH4Cl, and 300 μM Leupeptin. Endosomal/lysosomal pH was also measured in flow cytometry, using the pH‐sensitive pHrodo Green Dextran 10,000 MW dye conjugate. Western blotting was done after overnight treatment with Chloroquine and Leupeptin to determine the total levels of Kir3.1 and Kir3.4, and of MAP LC3α/β, a protein known to undergo lysosomal degradation. Results are reported as averages ± SD.ResultsIn flow cytometry experiments, Chloroquine at 1 μM, 10 μM and 50 μM showed a dose dependent increase in Kir3.1/Kir3.4 surface expression (5.6 % ± 6 vs. control, n.s.; 31.7% ± 18, p&lt;0.01 vs. control, and 54.4% ± 18, p&lt;0.01 vs. control). Moreover, treatment with 10 μM Primaquine, a compound structurally similar to Chloroquine, did not show a significant increase compared to vehicle treatment. The lysosomal inhibitor Leupeptin (300 μM) did not significantly increase cell surface IKACh channels expression compared to control. On the other hand, disruption of lysosomal pH with 10 mM NH4Cl significantly increased cell surface expression of Kir3.1/Kir3.4, but the increase was less pronounced compared to that of Chloroquine (24% ± 18 vs 32.7% ± 11.7, p&lt;0.05). We subsequently examined the influence of Chloroquine, Primaquine, NH4Cl and Leupeptin on the endosomal/lysosomal pH. Endosomal/lysosomal pH was significantly increased (indicated by a decrease in pH‐sensitive pHrodo Green Dextran fluorescence) by NH4Cl and Primaquine versus control (1003 A.U. ± 133 and 1063 A.U. ± 115, vs 1368 A.U. ± 165, p&lt;0.05) but not by Chloroquine and Leupeptin (1520 A.U. ± 199 and 1424 A.U. ± 151, vs 1368 A.U. ± 165, n.s.). Finally, in western blots, Chloroquine and Leupeptin did not significantly affect Kir3.1 and Kir3.4 total protein levels, however, Chloroquine significantly increased MAP LC3α/β levels.ConclusionLong term Chloroquine treatment promotes cell surface expression of Kir3.1 and Kir3.4. Accumulation of the IKACh mediating proteins at the cell surface appears to be independent of the possible effects of Chloroquine on lysosomal function.Support or Funding InformationFunded in part by NIH grant R01HL129136 to SFN

Cyclophosphamide-induced HCN1 channel upregulation in interstitial Cajal-like cells leads to bladder hyperactivity in mice

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are confirmed to be expressed in bladder interstitial Cajal-like cells (ICC-LCs), but little is known about their possible role in cystitis-associated bladder dysfunction. The present study aimed to determine the functional role of HCN channels in regulating bladder function under inflammatory conditions. Sixty female wild-type C57BL/6J mice and sixty female HCN1-knockout mice were randomly assigned to experimental and control groups, respectively. Cyclophosphamide (CYP)-induced cystitis models were successfully established in these mice. CYP treatment significantly enhanced HCN channel protein expression and Ih density and significantly altered bladder HCN1 channel regulatory proteins. Carbachol (CCH) and forskolin (FSK) exerted significant effects on bladder ICC-LC [Ca2+]i in CYP-treated wild-type (WT) mice, and HCN1 channel ablation significantly decreased the effects of CCH and FSK on bladder ICC-LC [Ca2+]i in both naive and CYP-treated mice. CYP treatment significantly potentiated the spontaneous contractions and CCH (0.001–10 μM)-induced phasic contractions of detrusor strips, and HCN1 channel deletion significantly abated such effects. Finally, we demonstrated that the development of CYP-induced bladder overactivity was reversed in HCN1−/− mice. Taken together, our results suggest that CYP-induced enhancements of HCN1 channel expression and function in bladder ICC-LCs are essential for cystitis-associated bladder hyperactivity development, indicating that the HCN1 channel may be a novel therapeutic target for managing bladder hyperactivity.

Effect of Qibai Pingfei capsule medicated serum on protein expressions of KATP channel in pulmonary arterial smooth muscle cells via nitric oxide

To investigate the ATP-sensitive potassium channel (KATP channel) protein expressions during different periods under hypoxia condition and explore the effect of Qibai Pingfei capsule medicated serum (hereinafter referred to as QBPF) on the correlation between the protein expressions of KATP channel and nitric oxide in rat pulmonary arterial smooth muscle cells(PASMCs). Qibai Pingfei capsules were given to SD rats via continuous gavage for 10 days to obtain QBPF. Primary rats PASMCs were cultured by the direct adherent culture method. Western blot was applied to detect the protein expression levels of KATP channel (Kir6.1 and SUR2B) in PASMCs. Then the noncompetitive inhibitor of NO synthase--Nω-nitro-L-arginine methyl ester(L-NAME) and KATP channel inhibitor--glyburide(GLYB) were applied respectively to evaluate the effect of QBPF on the protein expressions of KATP channel. The protein expressions of Kir6.1 and SUR2B were increased after 6-hour hypoxia treament, peaked at the 24-hour hypoxia treament, and decreased in both 48-hour and 72-hour hypoxia groups. Especially, QBPF could further up-regulate the Kir6.1 and SUR2B protein expressions under 24-hour hypoxia condition; however, such up-regulation effect could be blocked by KATP channel inhibitor GLYB and NO specific inhibitor L-NAME, indicating that QBPF played the role of opening KATP channel. The regulatory mechanism was probably associated with up-regulating KATP channel protein expression via NO relative pathway, involving pulmonary vasodilation, and thus relieving the occurence and development of COPD.

Selective Loss of Smaller Spines in Schizophrenia.

Decreased density of dendritic spines in adult schizophrenia subjects has been hypothesized to result from increased pruning of excess synapses in adolescence. In vivo imaging studies have confirmed that synaptic pruning is largely driven by the loss of large or mature synapses. Thus, increased pruning throughout adolescence would likely result in a deficit of large spines in adulthood. Here, the authors examined the density and volume of dendritic spines in deep layer 3 of the auditory cortex of 20 schizophrenia and 20 matched comparison subjects as well as aberrant voltage-gated calcium channel subunit protein expression linked to spine loss. Primary auditory cortex deep layer 3 spine density and volume was assessed in 20 pairs of schizophrenia and matched comparison subjects in an initial and replication cohort (12 and eight pairs) by immunohistochemistry-confocal microscopy. Targeted mass spectrometry was used to quantify postsynaptic density and voltage-gated calcium channel protein expression. The effect of increased voltage-gated calcium channel subunit protein expression on spine density and volume was assessed in primary rat neuronal culture. Only the smallest spines are lost in deep layer 3 of the primary auditory cortex in subjects with schizophrenia, while larger spines are retained. Levels of the tryptic peptide ALFDFLK, found in the schizophrenia risk gene CACNB4, are inversely correlated with the density of smaller, but not larger, spines in schizophrenia subjects. Consistent with this observation, CACNB4 overexpression resulted in a lower density of smaller spines in primary neuronal cultures. These findings require a rethinking of the overpruning hypothesis, demonstrate a link between small spine loss and a schizophrenia risk gene, and should spur more in-depth investigations of the mechanisms that govern new or small spine generation and stabilization under normal conditions as well as how this process is impaired in schizophrenia.

Beneficial Effect of Renal Denervation on Ventricular Premature Complex Induced Cardiomyopathy.

BackgroundFrequent ventricular premature complexes (VPCs) can lead to the development of dilated cardiomyopathy and sudden cardiac death. Renal artery sympathetic denervation (RDN) may protect the heart from remodeling. This study aimed to investigate the effect of frequent VPCs on structural and electrical properties and whether RDN can protect the heart from remodeling.Methods and ResultsEighteen rabbits were randomized to control (n=6), VPC (n=6), and VPC‐RDN (n=6) groups. Surgical and chemical RDNs were approached through bilateral retroperitoneal flank incisions in the VPC‐RDN group. Pacemakers were implanted to the left ventricular apex to produce 50% VPC burden for 5 weeks in the VPC and VPC‐RDN groups. In addition, ventricular myocardium was harvested for western blot and trichrome stain. Echocardiographic results showed left ventricular enlargement after 5‐week pacing in the VPC group, but not in the VPC‐RDN group, when compared to baseline. In biventricles, ion channel protein expressions of Nav1.5, Cav1.2, Kir2.1, and SERCA2 were similar among 3 groups. However, the degree of biventricular fibrosis was extensive in the VPC group, compared to the control and VPC‐RDN groups. Importantly, ventricular fibrillation inducibility was higher in the VPC group (41%) when comparing to the control (13%; P<0.05) and VPC‐RDN groups (13%; P<0.05), respectively.ConclusionsFrequent VPCs are associated with the development of cardiac structural remodeling and high ventricular fibrillation inducibility. RDN prevents cardiac remodeling and the occurrence of ventricular arrhythmia through antifibrosis.