Effects of diadenosine polyphosphates,ATP and angiotensin II on membrane voltage and membrane conductances of rat mesangial cells

Diadenosine polyphosphates have been shown to influence renal perfusion pressure. As mesangial cells may contribute to these effects we investigated the effects of diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A) and diadenosine hexaphosphate (Ap₆A) on membrane voltage (V _m) and membrane conductance (g _m) in mesangial cells (MC) of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats in primary and long-term culture. We applied the patch-clamp technique in the fast-whole-cell configuration to measure V _m and g _m. To compare the effects of diadenosine polyphosphates with hitherto known agonists we also tested adenosine 5-triphosphate (ATP) and angiotensin II (Ang II). As there was no significant difference in the V _m values in MC of WKY (–42±1 mV, n=70) and SHR rats (–45±2 mV, n=99) as well as in the agonist-induced changes of V _m, all data were pooled. The V _m of all the cells was –44±1 mV (n=169) and g _m was 15.9±1.8 nS (n=141). Ion-exchange experiments showed the presence of a K⁺ and a non-selective cation conductance in resting MC whereas a Cl^– conductance or a Na⁺selective conductance could not be observed. Ap₃A, Ap₄A, Ap₅A, AP₆A and ATP each at a concentration of 5 mol/l, led to a significant depolarization of V _m by 5±2 mV (n=14), 7±1 mV (n=25), 3±1 mV (n=23), 2±1 mV (n=16), and 14±2 mV (n=23), respectively. For Ap₄A, the most potent diadenosine polyphosphate, we determined the half-maximally effective concentration (EC ₅₀) as 6 mol/l (n=5–25), for ATP as 2 mol/l (n=9–37), and for Ang II as 8 nmol/l (n=6–18). Ap₄A 100 mol/l increased g _m significantly by 55±20% (n=16), 100 mol/l ATP by 135±60% (n=18). The diadenosine polyphosphates examined were able to depolarize V _m (Ang II >ATP> Ap₄A>Ap₃A>Ap₅A>Ap₆A) by activation of a Cl^– conductance and a non-selective cation conductance, as do ATP or Ang II.     

Effect of vasoactive intestinal peptide,carbachol and other agonists on the membrane voltage of pancreatic duct cells

The regulation of pancreatic exocrine secretion involves hormonal, neural and neurohormonal components. Many agonists are known to be effective in pancreatic acinar cells, but less is known about the ducts. Therefore, we wanted to investigate the influence of various agonists on isolated perfused pancreatic ducts and, as a physiological response, we measured the basolateral membrane voltage of the duct cells (V _bl) with microelectrodes. Pancreatic ducts were dissected from pancreas of normal rats and bathed in a HCO₃ ^–-containing solution. Under control conditions, the average V _bl was between -50 and -70 mV. Vasoactive intestinal peptide (VIP) and carbachol (CCH) reversibly depolarized V _bl when applied to the bath. VIP (9×10^–9 mol/l) depolarized V _bl from -72±3 mV to -53±3 mV (n=20) and CCH (10^–5 mol/l) from -62±3 to -35±4mV (n=10). Furthermore, a decrease of the Cl^– concentration in the lumen led to an increase of VIP-induced depolarization of V _bl, suggesting that a luminal Cl^– conductance was increased. Cholecystokinin (CCK, 10^–10-10^–7 mol/l) and bombesin (10^–8, 10^–5 mol/l), which stimulate pancreatic exocrine secretion in acini or whole glands, showed no significant effect on V _bl of the duct cells tested in our preparation (n=7, 6). Neurotensin (10^–8 mol/l) had a marked depolarizing effect in two out of ten cases; V _bl depolarized from about -65 mV to-29 mV and the effect was reversible. Substance P (2×10^–7 mol/l), alone or in combination with secretin, had no effect on V _bl of the tested duct cells (n=11). We propose that the basolateral membrane of pancreatic duct cells possesses receptors for VIP, acetylcholine and neurotensin. CCK, bombesin and substance P had no detectable effects on V _bl of the duct cells tested, which could be due to the lack of corresponding receptors on these cells, or due to the absence of electrophysiologically detectable effects, in spite of receptor presence.Preliminary reports of the present study were presented at the 70th and 71st Meetings of the German Physiological Society, Germany, September 1991, March 1992     

Effects of cell differentiation on ion conductances and membrane voltage in LLC-PK1 cells

LLC-PK1 cells serve as a widely used model for the renal proximal tubule. Until now, little has been found out about their membrane voltage (V _m) and ionic conductances (g). Several studies have shown changes in cell properties during differentiation and ageing. The aim of this study was to examine the relationship between V _m or g and the age of these cells. Therefore, we investigated single cells, subconfluent and confluent monolayers of LLC-PK1 cells aged 1–8 days with the slow-whole-cell patch-clamp technique. The V _m of all cells was-34±2 mV (n=75) and the membrane conductance (g _m) was 2.3±0.3 nS (n=30). V _m in cells aged up to 2 days was-24±3 mV (n=22) whereas V _m in cells aged 5–8 days was -50±3 mV (n=15). An increase of extracellular K⁺ from 3.6 to 18.6 mmol/l led to a depolarization in all cells of 4±1 mV (n=31) and an increase of g _m by 17±13% (n=15). Complete replacement of extracellular Na⁺ by N-methyl-D-glucamine (NMDG) led to a hyperpolarization of 19±2 mV (n=38) and g_m was lowered by 27±14% (n=17). A reduction in extracellular Cl^– from 147 to 32 mmol/l showed no significant effect on V _m (n=16) or g _m (n=11). Amiloride (10 mol/l) had no significant effect on V _m (n=13) or g _m (n=7). The reduction of the extracellular osmolarity from 290 to 160 mosmol/l led to a hyperpolarization of 11±1 mV (n=18) and an increase in g _m by 326±117% (n=12). There was no significant correlation between g _m and cell age. LLC-PK1 cells used in this study have a K⁺ conductance and a non-selective cation conductance in parallel. With increasing age, LLC-PK1 cells became more and more conductive for K⁺ and lost their nonselective cation conductance. There is no evidence for a significant amiloride-sensitive Na⁺ or Cl^– conductance in these cells. The K⁺ conductance could be activated by osmotically induced cell swelling.     

Effect of various routine cytopreparatory techniques on normal urothelial cells and their nuclei.

Nuclear and cytoplasmic sizes of cells in permanent, stained smear preparations differ from those in unfixed unstained cells. In air-dried MGG-stained smears the area of the nucleus is 50% larger and that of the cytoplasm 30% larger. In wet-fixed Papanicolaou-stained smears the nucleus is 10--30% and the cytoplasm is 15--55% smaller. The shrinkage in the wet fixation method is dependent on the concentration of the ethyl alcohol applied. The staining method has relatively little influence on nuclear and cytoplasmic size. The three-dimensional appearance of the smeared, stained cells is also dependent on the cytopreparatory technique applied:in the methods with air-drying the nuclei and cells are flat and in the wet fixation method more spherical. In the methods with air-drying the nuclear:cytoplasmic ratio is larger than that seen with the wet fixation methods.     

Effect of bradykinin and histamine on the membrane voltage,ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (V _m), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37°C. The mean value of V _m was –41±0.5 mV (n=189). BK (10^–6 mol/l, n=29) and Hist (10^–5 mol/l, n= 55) induced a rapid transient hyperpolarization by 15±1 mV and 18±1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6±1 mV (BK 10^–6 mol/l) and 7±1 mV (Hist 10^–5 mol/l). The ED₅₀ was about 5×10^–8 mol/l for BK and 5×10^–7 mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K⁺ concentration from 3.6 to 30 mmol/l depolarized V _m by 8±1 mV and completely inhibited the hyperpolarizing effect of both agents (n=11). Reduction of extracellular Cl^– concentration from 145 to 30 mmol/l led to a depolarization by 2 ±1 mV (n=25). In 30 mmol/l Cl^– the depolarizations induced by BK (10^–7 mol/l) and Hist (10^–6 mol/l) were augmented to 9±2 mV (n=14) and to 10±2 mV (n=11), respectively. Ba²⁺ (5 mmol/l) depolarized V _m by 19±5 mV (n=6) and completely inhibited the hyperpolarization induced by BK (10^–6 mol/l, n=3) and reduced that of Hist (10^–5 mol/l) markedly (n=3). Preincubation with the K⁺ channel blocker charybdotoxin (1–10 nmol/l) for 3 min had no significant effect on V _m, but reduced markedly the BK(10^–6 mol/l, n=11) and Hist-(10^–5 mol/l, n=6) induced hyperpolarizations. In 10 out of 31 experiments in the cell attached nystatin patch configuration big K⁺ channels with a conductance of 247±17 pS were found. The open probability of these K⁺ channels was increased 3- to 5-fold during the hyperpolarization induced by BK (10^–7 mol/l) or Hist (10^–5 mol/l, both n= 4). In excised inside/out patches this K⁺ channel had a mean conductance of 136±8.5 pS (n=10, clamp voltage 0 mV). The channel was outwardly rectifying and its open probability was increased when Ca²⁺ on the cytosolic side was greater than 0.1 mol/l. The data indicate that BK and Hist activate a and a in hGEC. The hyperpolarization is induced by the activation of a Ca²⁺-dependent maxi K⁺ channel.     

Effect of ATP,carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts

The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca²⁺ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca²⁺]_i, in perfused rat pancreatic ducts using the Ca²⁺-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, V _bl, of individual cells. The resting basal [Ca²⁺]_i was relatively high, corresponding to 263±28 nmol/l, and it decreased rapidly to 106±28 nmol/l after removal of Ca²⁺ from the bathing medium (n=31). Carbachol increased [Ca²⁺]_i in a concentration-dependent manner. At 10 mol/l the fura-2 fluorescence ratio increased by 0.49±0.06 (n=24), corresponding to an increase in [Ca²⁺]_i by 111±15 nmol/l (n=17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67±0.06 and 1.01±14 (n=46; 12), corresponding to a [Ca²⁺]_i increase of 136±22 nmol/l and 294±73 nmol/l respectively (n= 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized V _bl from –62±3 mV to-70±3 mV, an effect which was in some cases only transient (n=7). This effect of ATP was different from that of carbachol, which depolarized V_bl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of V _bl (n=4). Several other putative agonists of pancreatic HCO ₃ ^– secretion were also tested for their effects on [Ca²⁺]_i. Bombesin (10 nmol/l) increased the fura-2 fluorescence ratio by 0.24±0.04 (n=8), neurotensin (10 nmol/l) by 0.25±0.04 (n=6), substance P (0.1 mol/l) by 0.22±0.06 (n=6), and cholecystokinin (10 nmol/l) by 0.14±0.03 (n=7). Taken together, our studies show that Ca²⁺ homeostasis plays a role in pancreatic ducts. The most important finding is that carbachol and ATP markedly increase [Ca²⁺]_i, but their different electrophysiological responses indicate that intracellular signalling pathways may differ.Preliminary reports of the present study have been presented at the 72nd Meeting of the German Physiological Society, March 1993     

Cell growth on immobilized cell-growth factor. II. Adhesion and growth of fibroblast cells on poly(methyl methacrylate) membrane immobilized with proteins of various kinds.

The surface of poly(methyl methacrylate) membrane was partially hydrolysed and the carboxyl groups produced were coupled with various protein molecules with water-soluble carbodiimide. The immobilized proteins were a cell-growth factor insulin, cell adhesion factors fibrinogen and fibronectin, and serum proteins albumin and gamma-globulin. The insulin-immobilized poly(methyl methacrylate) membrane strongly accelerated the growth and slightly accelerated the adhesion of fibroblast cells. The immobilized fibronectin and fibrinogen enhanced the cell adhesion, and the former also accelerated the cell growth. The immobilized albumin and gamma-globulin influenced the adhesion and growth of cells very little. It was found that various proteins specifically influence the adhesion and growth of cells in an immobilized state.     

Effect of various substances on colchicine uptake by cells sensitive and resistant to it

The effect of various substances on the uptake of [³H]colchicine by L and L-53 cells, resistant to colcemid and colchicine was investigated. Vinblastin, to which L-53 cells possess cross resistance, increases accumulation of labeled colchicine in L cells threefold, and in L-53 cells fivefold. Substances lowering the ATP level in the cells (oligomycin, etc.) increase the uptake of colchicine by L and L-53 cells by 2–4 times. Colchicine uptake by resistant cells is increased in the presence of these substances more than in sensitive L cells. Lumicolchicine, a structural analog of colchicine which does not bind with tubulin, the protein of the microtubules, stimulates colchicine uptake approximately equally by L and L-53 cells.A. N. Belozerskii Interfaculty Research Laboratory of Molecular Biology and Bioorganic Chemistry; Department of Molecular Biology, Faculty of Biology Moscow University. Laboratory of Cytogenetics, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 343–345, September, 1979.     

Inhibition of lymphoid cell DNA synthesis by metal allergens at various concentrations. Effect on short-time cultured nonadherent cells compared to nonseparated cells

Metal allergen concentrations inhibiting lymphoid cell DNA synthesis were tested on short-time cultured nonadherent and nonseparated thymic and splenic cells. With all tested metal allergens, the DNA-synthesis-inhibiting effect was more marked for nonadherent compared to nonseparated splenic cells. In thymocytes, a stronger inhibition of nonadherent cells was seen after addition of mercuric chloride. A protective or modifying role of adherent cells towards inhibiting or toxic effects of metal allergens in vitro is postulated.     

Effect of cholinergic agonists on resting membrane potential of earthworm body wall muscle cells

Carbachol in a concentration of 5×10^-8 mol/liter does not hyperpolarize, and in a concentration of 5×10^-6 mol/liter depolarizes the membrane of somatic muscle cell in earthworm. d-Tubocurarine, -bungarotoxin, atropine, and hexamethonium added to the incubation medium did not abolish the carbachol-induced decrease in resting membrane potential. Each of these drugs alone had no effect on resting membrane potential in muscle cells. Presumably, the acetylcholine-sensitive receptor-channel membrane complex in earthworm muscle cell differs from acetylcholine receptor in skeletal muscle fibers and peripheral neurons of vertebrates.     

Effect of ionic medium on carbacholine-induced membrane depolarization in lumbricus terrestris somatic muscle cells

Carbacholine depolarizes Lumbricus terrestris myocyte membrane. Addition of verapamil, tetrodotoxin, removal of K⁺, Cl^-, and Ca²⁺, and replacement of Ca²⁺ with Mn²⁺ in the bathing solution did not prevent the carbacholine-induced decrease in resting potential, but in a sodium-free medium carbacholine was ineffective. It was hypothesized that depolarization of the sarcolemma resulting from activation of cholinomimetic-sensitive channel-receptor complex of the Lumbricus terrestris somatic muscle cells is primarily determined by sodium permeability of the membrane.     

PSMA对LNCaP细胞生长、迁移及ERK蛋白活性的影响

目的: 利用我们已经成功沉默前列腺特异性膜抗原(PSMA)的LNCaP前列腺癌细胞株,探讨PSMA对LNCaP细胞磷酸化胞外信号调节激酶(ERK)及细胞生长、迁移的影响,为进一步研究PSMA在前列腺癌发展中的作用提供理论基础。方法: 实验对象包括携带可稳定抑制PSMA表达siRNA慢病毒的LNCaP细胞组(实验组),携带对任何基因无干扰作用siRNA慢病毒的LNCaP细胞组(空转组),同时建立未进行处理的普通LNCaP细胞组(对照组),分别对在一般培养基及添加ERK蛋白上游抑制剂的3组细胞,使用Western blotting和细胞免疫化学的方法检测MAPK/ERK蛋白活性,并用MTT描绘细胞生长曲线、Transwell观察细胞迁移情况。结果: 在一般培养基的3组细胞中,Western blotting提示实验组磷酸化ERK蛋白表达明显低于对照组和空转组;免疫细胞化学结果显示实验组染色明显比对照组和空转组弱,阳性细胞数较少;MTT绘制生长曲线,得到实验组细胞的增殖生长能力较对照组、空转组降低;Transwell结果提示实验组较对照组和空转组细胞的增殖迁移能力降低。在ERK磷酸化被抑制的情况下,3组细胞磷酸化ERK蛋白均低表达,MTT及Transwell检测显示其生长迁移能力都处于低水平,且与在一般培养基中实验组的效应类似。结论: 初步发现PSMA可能通过上调前列腺癌LNCaP细胞ERK蛋白的活性,从而在其生长、迁移中起正向调节作用。     

Effect of membrane potential on the passive transport of TI+ in human red blood cells

It was earlier found that TI⁺ can easily penetrate the red cell membrane. The main finding of this work is that TI⁺ can be used for studying alterations in the membrane potential of human red blood cells exposed to various experimental conditions. It was shown that after inhibiting active transport by ouabain, both the rate of transmembrane movement and the cell/medium distribution of TI⁺ were in a good agreement with the expected changes in membrane potential. Alterations in membrane potential were induced by modifying the cation permeability of the red cell membrane and by varying the cation concentration gradient across the membrane, which were achieved: 1) by incubation in an electrolyte-free sucrose solution, 2) by addition of valinomycin or 3) by addition of propranolol. Changes in cation permeability were followed by means of ⁸⁶Rb tracer. Hyperpolarization of the red cell membrane led to accelerated influx and retarded efflux of TI⁺. The opposite effect was obtained by depolarization. Quantification of the results was made using the Nernst equation and the cell/medium concentration ratio of TI⁺ at equilibrium. The calculations show that the membrane potential of the propranolol-treated cells increased to about -20 mV, negative inside. The mechanism of the propranolol effect is briefly discussed.     

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection. I. Immunoglobulin expression on human lymphoblastoid cells established from various lymphoid cells

Forty-seven lymphoblastoid cell lines were established from human fetal lymphoid tissues, cord blood lymphocytes (CBL) and adult peripheral blood lymphocytes (PBL) by Epstein-Barr virus (EBV) infection. Their surface immunoglobulin (sIg), intracytoplasmic immunoglobulin (cIg) expression, and immunoglobulin (Ig) content in the culture supernatant were tested. Expression of sIgM, sIgG and sIgA were predominant on fetus-derived cell lines, while sIgD was the most prominent on CBL-derived cells. Though cIg expression did not vary between cell lines of different origin, Ig content in the culture supernatant differed greatly. Fetus- and CBL-derived cells secreted IgM exclusively, but PBL-derived cells secreted not only IgM, but also IgG and IgA abundantly. These results indicate that the lymphoblastoid cells established by EBV infection reflect the Ig phenotype of the cell from which they originated.     

Effect of spatial arrangement of the basement membrane on cultured pleomorphic adenoma cells. Study by immunocytochemistry and electron and confocal microscopy

In a cell line from human pleomorphic adenoma (AP2 cells) we studied the response of these cells to basement membrane proteins. The culture was characterized as myoepithelial-like by transmission electron microscopy and immunocytochemistry. AP2 cells were grown in contact with a reconstituted basement membrane (Matrigel). Cells grown on Matrigel showed conspicuous phenotypic alterations, depending on how the substrate was applied. Cells grown on the top of Matrigel developed a dendritic phenotype, exhibiting thin, long and intercommunicating cytoplasmic extensions resembling normal myoepithelial cells. Cells grown inside Matrigel formed multi-layered clusters. Light, confocal and transmission electron microscopy showed that these clusters were formed by double-layered epithelioid cells delimiting luminal spaces. The cells facing the lumen were cuboidal, showing microvilli at the apical plasmalemmal and junctional complexes. The spatial arrangement of basement membrane is a key modulator of morphogenetic changes and cytodifferentiation of tumour myoepithelial cell lineage in culture.     

Effect of various E. coli LPS chemotypes on apoptosis and activation of human neutrophils

We studied the effects of various Escherichia coli LPS chemotypes on the production of reactive oxygen species and regulation of apoptosis in human neutrophils. A correlation was found between the increase in chemiluminescence (Re-LPS<Ra-LPS<S-LPS) and lengthening of the polysaccharide chain in endotoxins. Shortening of the polysaccharide chain was associated with more pronounced inhibition of neutrophil apoptosis under the influence of endotoxins, which correlated with the increase in hydrophobicity of these chemotypes. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 136–138, August, 2006     

米醇溶蛋白及壳聚糖共混合膜对骨髓间充质干细胞成骨分化的影响

BACKGROUND: Some scholars have prepared zein/chitosan composite membrane based on blending methods, and preliminary evaluation of its physical and chemical properties shows that chitosan partly improves the mechanical properties and hydrophilic properties of zein. Therefore, zein/chitosan composite membrane presumably has good cytocompatibility, which is beneficial to osteogenic differentiation of bone marrow mesenchymal stem cells.     

cGMP对慢性低氧大鼠肺动脉平滑肌细胞膜电压门控钾通道的作用

目的:探讨cGMP对慢性低氧大鼠肺动脉平滑肌细胞(PASMC)膜电压门控钾通道(Kv通道)的作用, 为进一步阐明慢性低氧性肺动脉高压的发病机理提供理论依据。方法: Wistar大鼠, 随机分为对照组和慢性低氧组, 低氧组大鼠每天低氧(氧浓度10%±1%)8 h, 连续4周。单个大鼠PASMC的获得采用急性酶分离法(胶原酶Ⅰ型和木瓜蛋白酶)。采用全细胞膜片钳技术测定两组PASMC的静息膜电位(Em)和电压门控钾通道的钾离子电流(I_KV), 观察并比较cGMP (1 mmol/L) 以及cGMP和蛋白激酶G(PKG)抑制剂H-8 (1 mmol/L) 应用后两组PASMC I_KV的不同变化。结果:慢性低氧大鼠PASMC的静息膜电位和电压门控钾通道电流明显低于正常对照组。cGMP可抑制正常和慢性低氧大鼠PASMC +50 mV刺激时的峰值I_KV[正常组从(118.0±5.0)pA/pF下降到(89.9±16.5) pA/pF, n=6, P<0.05;慢性低氧组则从(81.0±5.0) pA/pF 下降到(56.8±9.1) pA/pF, n=6, P<0.05], 该抑制作用可被PKG的抑制剂H-8阻断[正常组(119.2±10.3) pA/pF vs (117.8±9.1)　pA/pF, n=6, P>0.05;慢性低氧组(96.8±6.2) pA/pF vs (98.0±2.2)　pA/pF, n=6, P>0.05]。结论:慢性低氧抑制肺动脉平滑肌细胞的电压门控钾通道。cGMP可能通过磷酸化作用而抑制正常和慢性低氧肺动脉平滑肌细胞的电压门控钾通道电流。