Characterization of calcium signaling pathways in human preadipocytes

Intracellular free Ca²⁺ (Ca) is an important regulator of many cellular activities; however, Ca²⁺ signaling is not well studied in human preadipocytes. The purpose of the present study was to characterize Ca²⁺ signal pathways using a confocal scanning technique and RT‐PCR. It was found that spontaneous Ca oscillations were observed in 12.1% preadipocytes, and number of cells with Ca²⁺ oscillations was increased to 47.9% by 1% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry mainly via stored‐operated Ca²⁺ (SOC) entry. They were suppressed by the SOC entry channel blocker La³⁺, the phospholipase C (PLC) inhibitor U73122, the inositol trisphosphate receptor (IP3R) blocker 2‐amino‐ethoxydiphenyl borate, or the sarcoplasmic/endoplasmic reticulum Ca²⁺ pump (SERCA) inhibitors thapsigargin and cyclopiazonic acid, but not by ryanodine. The IP3R activator thimerosal increased Ca oscillations. In addition, the plasma membrane Ca²⁺ pump (PMCA) inhibitor carboxyeosin and Na⁺–Ca²⁺ exchanger (NCX) inhibitor Ni²⁺ both suppressed Ca²⁺ oscillations. RT‐PCR revealed that the mRNAs for IP3R1‐3, SERCA1,2, NCX3 and PMCA1,3,4, Ca_V1.2, and TRPC1,4,6, STIM1 and Orai1 (for SOC entry channels) were significant in human preadipocytes. The present study demonstrates that multiple Ca²⁺ signal pathways are present in human preadipocytes, and provides a basis for investigating how Ca²⁺ signals regulate biological and physiological activities of human preadipocytes. J. Cell. Physiol. 220: 765–770, 2009. © 2009 Wiley‐Liss, Inc.     

Functional ion channels and cell proliferation in 3T3-L1 preadipocytes

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation.     

Multi-state, 4-aminopyridine-sensitive ion channels in human spermatozoa

Although ion channels are known to be pivotal to sperm function, the technical difficulty of applying electrophysiological techniques to spermatozoa has prevented significant progress in this area. This is due to the cell size and angular shape in combination with their motility. Using a refined technique, specifically for patch clamping spermatozoa, we have made recordings from human cells. This technique permitted approaches which enable functional analysis of sperm ion channels, including acquisition of inside-out patches, generation of averaged currents, and observation of the effects of pharmacological manipulation during prolonged recordings. As well as a low conductance (7 pS) channel and a 25-pS channel, the most striking finding was the presence of very high conductance, 4-aminopyridine-sensitive multistate channels resembling the non-selective cation channel of sea urchin and mouse spermatozoa. Application of 2 mM 4-aminopyridine (a dose sufficient to cause channel blockade) caused an instant and dramatic transition of motility in the sperm population increasing hyperactivated motility by more than 10-fold as assessed by computer-assisted semen analysis. Combined application of patch clamp and pharmacological investigation of mature sperm cells and will permit rapid advances in our understanding the role of ion channels in sperm function.     

Resting and activation-dependent ion channels in human mast cells

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca(2+)-activated K(+) current exhibiting weak inward rectification, which polarized the cells to around -40 mV and a smaller outwardly rectifying Ca(2+)-independent Cl(-) conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca(2+)-activated K(+) currents and Ca(2+)-independent Cl(-) currents developing in approximately 50% of cells. In both cell types, the K(+) current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca(2+)-activated K(+) channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C(4) release, indicating that the Ca(2+)-activated K(+) current may enhance, but is not essential for, the release of these mediators.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes.

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

Influence of ion channels on the proliferation of human chondrocytes

The goal of the study was to examine connections between ion channel activity and the proliferation of human chondrocytes. Chondrocytes were isolated form human osteoarthritic knee joint cartilage. In this study the concentration-dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-aminopyridine (4-AP), 4',4' diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), verapamil (vp) and lidocaine (lido) on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry and the measurement of (3)H-thymidine incorporation as measure for the cell proliferation. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The maximal measurable effects of the membrane potential were listed with 0.25 mmol/l verapamil (-18%) and 0.1 mmol/l lidocaine (+20%). When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. After 12 days culture duration, lidocaine and 4-AP cause an increase of the DNA synthesis rate being a limited effect. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation. To gain knowledge of the regulation of chondrocyte proliferation via ion channel modulators could serve the research of new osteoarthritis treatment concepts.     

Thermosensitive TRP ion channels mediate cytosolic calcium response in human synoviocytes

The transient receptor potential (TRP) channels are important membrane sensors, responding to thermal, chemical, osmotic, or mechanical stimuli by activation of calcium and sodium fluxes. In this study, three distinct TRP channels were detected and their role established in mediating cytosolic free calcium concentration ([Ca²⁺]_cyt) response in tumor-derived SW982 synoviocytes and primary cultures of human synovial cells from patients with inflammatory arthropathies. As shown by fura-2 ratio measurements while cells were incubated in a temperature-regulated chamber, significant [Ca²⁺]_cyt elevation was elicited by rapid changes in bath temperature, application of TRPV1 receptor agonists capsaicin and resiniferatoxin, or a cold receptor stimulator, icilin. Temperature thresholds for calcium response were determined to be 12 ± 1°C for cold and 28 ± 2°C for heat activation. Temperature increases or decreases beyond these thresholds resulted in a significant rise in the magnitude of [Ca²⁺]_cyt spikes. Observed changes in [Ca²⁺]_cyt were completely abolished in calcium-free medium and thus resulted from direct calcium entry through TRP channels rather then by activation of voltage-dependent calcium channels. Two heat sensitive channels, TRPV1 and TRPV4, and a cold-sensitive channel, TRPA1, were detected by RT-PCR. Minimal mRNA for TRPV3 or TRPM8 was amplified. The RT-PCR results support the data obtained with the [Ca²⁺]_cyt measurements. We propose that the TRP channels are functionally expressed in human synoviocytes and may play a critical role in adaptive or pathological changes in articular surfaces during arthritic inflammation. transient receptor potential channels; vanilloid receptors; arthritis     

Divalent ion trapping inside potassium channels of human T lymphocytes

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation.     

Mitochondrial ion channels

Ion channels are proteins, which facilitate the ions flow throught biological membranes. In recent years the structure as well as the function of the plasma membrane ion channels have been well investigated. The knowledge of intracellular ion channels however is still poor. Up till now, the calcium channel described in endoplasmatic reticulum and mitochondrial porine are the examples of intracellular ion channels, which have been well characterized. The mitochondrial potassium channels: regulated by ATP (mitoK(ATP)) and of big conductance activated by Ca2+ (mitoBK(Ca)), which were described in inner mitochondrial membrane, play a key role in the protection of heart muscle against ischemia. In this review the last date concerning the mitochondrial ion channels as well as they function in cell metabolism have been presented.     

ATP敏感钾通道参与大鼠前脂肪细胞增殖和分化

为了探讨ATP敏感钾通道在前脂肪细胞增殖分化中作用,本实验用逆转录实时定量PCR方法检测大鼠前脂肪细胞和诱导5d获得的脂肪细胞中该通道磺脲类受体2（sulphonylurea receptor2,SUR2）mRNA表达,探讨该通道阻滞剂格列本脲和激动剂二氮嗪对前脂肪细胞中SUR2mRNA表达的影响;MTT检测前脂肪细胞增殖;流式细胞仪检测细胞周期;油红O染色法检测细胞内脂质含量;Image-Pro Plus5．0软件测量细胞直径;逆转录PCR检测过氧化物酶体增殖物激活受体-γ（peroxisome proliferator-activatedreceptor-γ PPAR-γ）mRNA表达。结果显示：前脂肪细胞及诱导5d获得的脂肪细胞均有SUR2mRNA表达,且后者明显高于前者;格列本脲抑制前脂肪细胞SUR2mRNA表达,剂量依赖性地促进前脂肪细胞增殖,增加G2／M＋S期细胞百分比,增加细胞脂质含量,使脂肪细胞直径增大,增加PPAR-γ mRNA的表达;二氮嗪在这些方面的作用与格列本脲相反。以上结果提示,ATP敏感钾通道在前脂肪细胞增殖和分化中可能起调节作用,PPAR-γ可能参与这些作用。