Interleukin-10 release from astrocytes suppresses neuronal apoptosis via the TLR2/NFκB pathway in a neonatal rat model of hypoxic-ischemic brain damage.

The biological function of interleukin-10 (IL-10) and the relationship between IL-10 secretion and the Toll-like receptor 2 (TLR2) expression levels in the central nervous system following hypoxic-ischemic brain damage (HIBD) are poorly understood. Here, we intend to elucidate the biological function and mechanism of IL-10 secretion following HIBD. In this study, we used a neonatal rat model of HIBD and found that rats injected with adeno-associated virus-IL-10-shRNA (short hairpin RNA) exhibited partially impaired learning and memory function compared to rats administered adeno-associated virus-control-shRNA. In vitro oxygen-glucose deprivation (OGD) induced IL-10 release from astrocytes but not from neurons. Pretreatment with exogenous recombinant IL-10 alleviated OGD-mediated apoptosis of neurons but not astrocytes. In addition, we also observed that hypoxic injury induced a marked increase in IL-10 expression in astrocytes as a result of activation of the TLR2/phosphorylated nuclear factor kappa B (p-NFκB) p65 signaling cascade; furthermore, this effect disappeared upon small interfering RNA targeting rat TLR2 gene (siTLR2) treatment. Pyrrolidinedithiocarbamate, an inhibitor of NFκB activation, reduced the IL-10 expression levels in both OGD-injured astrocytes in vitro and the hippocampi of HIBD rats in vivo but did not significantly affect TLR2 expression. Furthermore, a luciferase assay revealed that p-NFκB p65 could bind the -1700/-1000 bp proximal region of the IL-10 gene promoter to regulate IL-10 secretion from astrocytes and that this interaction could be controlled by OGD treatment. These data suggest that HIBD induces IL-10 secretion from astrocytes to exert a paracrine-induced anti-apoptotic effect on injured neurons via the TLR2/NFκB signaling pathway, which may improve learning and memory dysfunction after ischemic injury.

Effects of ion channels on proliferation in cultured human cardiac fibroblasts.

Our previous study demonstrated that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts, including a large-conductance Ca(2+)-activated K(+) current (BKCa), a volume-sensitive chloride current (I(Cl.vol)), and voltage-gated sodium currents (I(Na)). The present study was designed to examine the possible involvement of these ion channels in proliferation of cultured human cardiac fibroblasts using approaches of cell proliferation assay, whole-cell patch voltage-clamp, siRNA and Western blot analysis. It was found that the blockade of BKCa with paxilline (1-3µM) or I(Cl.vol) with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium (DIDS, 100-200µM), but not I(Na) with tetrodotoxin (0.1-10µM), remarkably suppressed proliferation in human cardiac fibroblasts. Knockdown of KCa1.1 or Clcn3 with specific siRNAs significantly reduced BKCa or I(Cl.vol) current, mRNA and channel protein levels, and inhibited growth of human cardiac fibroblasts. Flow cytometry analysis showed accumulation of cardiac fibroblasts at G0/G1 phase and reduced cell number in S phase after inhibition of BKCa or I(Cl.vol) with channel blockers or knock down of the corresponding channels with specific siRNAs; these effects were accompanied by a decreased expression of cyclin D1 and cyclin E. The present results demonstrate the novel information that BKCa and I(Cl.vol) channels, but not I(Na) channels, are involved in the regulation of proliferation in cultured human cardiac fibroblasts by promoting cell cycle progression via modulating cyclin D1 and cyclin E expression.

Characterization of ion channels in human preadipocytes.

Ion channels participate in regulation of cell proliferation. However, though preadipocyte (the progenitor of fat cell) is a type of highly proliferating cells, ion channel expression and their role in proliferation is not understood in human preadipocytes. The present study was designed to characterize ion channels using whole-cell patch clamp technique, RT-PCR, and Western blotting. It was found that a 4-aminopyridine- (4-AP) sensitive transient outward K(+) current (I(to)) was present in a small population of (32.0%) cells, and an outward "noisy" big conductance Ca(2+)-activated K(+) current (I(KCa)) was present in most (92.7%) preadipocytes. The noisy current was inhibited by the big conductance I(KCa) channel blocker paxilline (1 microM), and enhanced by the Ca(2+) ionophore A23187 (5 microM) and the big conductance I(KCa) channel activator NS1619 (10 microM). RT-PCR and Western blot revealed the molecular identities (i.e., KCa1.1 and Kv4.2) of the functional ionic currents I(KCa) and I(to). Blockade of I(KCa) or I(to) with paxilline or 4-AP reduced preadipocyte proliferation, and similar results were obtained with specific siRNAs targeting to KCa1.1 and Kv4.2. Flow cytometric analysis showed ion channel blockade or knockdown of KCa1.1 or Kv4.2 with specific siRNA increased the cell number of G0/G1 phase. The present study demonstrates for the first time that two types of functional ion channel currents, I(to) and big conductance I(KCa), are present in human preadipocytes and that these two types of ion channels participate in regulating proliferation of human preadipocytes.

Characterization of calcium signaling pathways in human preadipocytes.

Intracellular free Ca2+ (Ca(i)2+) 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(i)2+ 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(i)2+ 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(i)2+ 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(V)1.2, 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.

Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors.

The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X2aR, P2X2bR, and P2X3R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences of P2X2aR and P2X2bR. Chimeric P2X2a/V60-F301X3R and P2X2b/V60-F301X3R inherited the P2X3R ligand-selective profile, whereas the potency of agonists for P2X2a/V60-R180X3R was in between those observed at parental receptors. Furthermore, P2X2a/V60-F301X3R and P2X2a/V60-R180X3R desensitized in a P2X2aR-specific manner, and P2X2b/V60-F301X3R desensitized with rates comparable with those of P2X2bR. In striking contrast to parental receptors, the rates of decay in P2X2a/V60-F301X3R and P2X2b/V60-F301X3R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X2a/V60-R180X3R-expressing cells. However, the recovery from desensitization of P2X2a/V60-F301X3R and P2X2a/V60-R180X3R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.

Molecular dissection of purinergic P2X receptor channels.

The P2X receptors (P2XRs) are a family of ATP-gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. P2XRs are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X(1)-P2X(7). All subunits possess intracellular N- and C-termini, two transmembrane domains, and a relatively large extracellular ligand-binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of extracellular ATP leads to deactivation and resensitization of receptors. Activated P2XRs generate inward currents caused by Na(+) and Ca(2+) influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage-gated calcium entry in excitable cells. No crystal structures are available for P2XRs and these receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of P2XRs, including heteromerization, chimerization, mutagenesis, and biochemical studies.

Signaling by purinergic receptors and channels in the pituitary gland.

Adenosine 5'-triphosphate is frequently released by cells and acts as an agonist for G protein-coupled P2Y receptors and ligand-gated P2X cationic channels in numerous tissues. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors. In the pituitary gland, adenosine 5'-triphosphate is released from the endings of magnocellular hypothalamic neurons and by anterior pituitary cells through pathway(s) that are still not well characterized. This gland also expresses several members of each family of purinergic receptors. P2X and adenosine receptors are co-expressed in the somata and nerve terminals of vasopressin-releasing neurons as well as in some secretory pituitary cells. P2X receptors stimulate electrical activity and modulate InsP(3)-dependent calcium release from intracellular stores, whereas adenosine receptors terminate electrical activity. Calcium-mobilizing P2Y receptors are expressed in pituicytes, folliculo-stellate cells and some secretory cells of the anterior pituitary.

Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors.

Purinergic P2X receptors are ion-conducting channels composed of three subunits, each having two transmembrane domains and intracellular amino (N) and carboxyl (C) termini. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post-transcriptional modifications on receptor architecture and function remains poorly understood. In this study, we focused on mouse pituitary P2X2 receptors. In this tissue, progressive splicing of the P2X2a C terminus generated two functional subunit variants, P2X2b and P2X2e, which exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the intersubunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C termini of P2X2a receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C termini and between C- and N termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X2b or P2X2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X2b receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in the cytoplasmic intersubunit interactions and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.

Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-3 in hypothalamic and pituitary cells.

Hypothalamic and pituitary cells express G protein-coupled adenosine and P2Y receptors and cation-conducting P2X receptor-channels, suggesting that extracellular ATP and other nucleotides may function as autocrine and/or paracrine signaling factors in these cells. Consistent with this hypothesis, we show that cultured normal and immortalized pituitary and hypothalamic cells release ATP under resting conditions. RT-PCR analysis also revealed the presence of transcripts for ecto-nucleotidase eNTPDase 1-3 in these cells. These enzymes were functional as documented by degradation of endogenously released and exogenously added ATP. Blocking the activity of eNTPDases by ARL67156 led to an increase in ATP release in perifused pituitary cells and inhibition of degradation of extracellularly added ATP. Furthermore, the addition of apyrase, a soluble ecto-nucleotidase, and the expression of recombinant mouse eNTPDase-2, enhanced degradation of both endogenously released and exogenously added ATP. The released ATP by resting hypothalamic cells was sufficient to activate and desensitize high-affinity recombinant P2X receptors, whereas facilitation of ATP metabolism by the addition of apyrase protected their desensitization. These results indicate that colocalization of ATP release sites and ecto-nucleotidase activity at the plasma membrane of hypothalamic and pituitary cells provides an effective mechanism for the operation of nucleotides as extracellular signaling molecules.

Dependence of purinergic P2X receptor activity on ectodomain structure.

Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X(4)R > P2X(2a)R = P2X(2b)R P2X(7)R. Chimeras having the ectodomain Ile(66)-Tyr(310) sequence of P2X(2)R and Val(61)-Phe(313) sequence of P2X(7)R in the backbone of P2X(4)R were expressed but were non-functioning channels. P2X(2a) + X(4)R and P2X(2b) + X(4)R chimeras having the Val(66)-Tyr(315) ectodomain sequence of P2X(4)R in the backbones of P2X(2a)R and P2X(2b)R were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X(2b)R and P2X(2a)R, chimeric P2X(2b) + X(4)R desensitized more rapidly than P2X(2a) + X(4)R, and the rate of desensitization of P2X(2a)+X(4)R increased by substituting its Arg(371)-Pro(376) intracellular C-terminal sequence with the Glu(376)-Gly(381) sequence of P2X(4)R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.

Ca(2+)-mobilizing endothelin-A receptors inhibit voltage-gated Ca(2+) influx through G(i/o) signaling pathway in pituitary lactotrophs.

In excitable cells, receptor-induced Ca(2+) release from intracellular stores is usually accompanied by sustained depolarization of cells and facilitated voltage-gated Ca(2+) influx (VGCI). In quiescent pituitary lactotrophs, however, endothelin-1 (ET-1) induced rapid Ca(2+) release without triggering Ca(2+) influx. Furthermore, in spontaneously firing and depolarized lactotrophs, the Ca(2+)-mobilizing action of ET-1 was followed by inhibition of spontaneous VGCI caused by prolonged cell hyperpolarization and abolition of action potential-driven Ca(2+) influx. Agonist-induced depolarization of cells and enhancement of VGCI upon Ca(2+) mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin (PTX). Activation of adenylyl cyclase by forskolin and addition of cell-permeable 8-bromo-cAMP did not affect ET-1-induced sustained inhibition of VGCI, suggesting that the cAMP-protein kinase A signaling pathway does not mediate the inhibitory action of ET-1 on VGCI. Consistent with the role of PTX-sensitive K(+) channels in ET-1-induced hyperpolarization of control cells, but not PTX-treated cells, ET-1 decreased the cell input resistance and activated a 5 mM Cs(+)-sensitive K(+) current. In the presence of Cs(+), ET-1 stimulated VGCI in a manner comparable with that observed in PTX-treated cells, whereas E-4031, a specific blocker of ether-a-go-go-related gene-like K(+) channels, was ineffective. Similar effects of PTX and Cs(+) were also observed in GH(3) immortalized cells transiently expressing ET(A) receptors. These results indicate that signaling of ET(A) receptors through the G(i/o) pathway in lactotrophs and the subsequent activation of inward rectifier K(+) channels provide an effective and adenylyl cyclase-independent mechanism for a prolonged uncoupling of Ca(2+) mobilization and influx pathways.

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels.

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

Role of nucleotide P2 receptors in calcium signaling and prolactin release in pituitary lactotrophs.

Anterior pituitary cells express nucleotide-gated G protein-coupled P2 receptors (P2YRs) and cation-conducting channels (P2XRs). However, the identification of P2 receptors subtypes and their native ligands, and the distribution and function of these receptors within the secretory and non-secretory pituitary cells has been incompletely characterized. The focus in this study was on lactotroph subpopulation of cells. ATP and ADP, but not UTP and UDP, triggered calcium signaling in a majority (85%) of lactotrophs and prolactin release in mixed pituitary cells. Consistent with the role of P2 receptors in signaling and secretion, the actions of ATP and ADP were abolished in the presence of apyrase, an ectonucleotidase. Transcripts for Gq-coupled calcium-mobilizing P2Y1R, P2Y2R, P2Y4R, and P2Y6R, as well as Gi-coupled P2Y12R, were identified in mixed anterior pituitary cells. The ligand-selectivity profile of calcium mobilization-dependent signaling and prolactin secretion and the blockade of these responses by pyridoxal 5-phosphate 6-azophenyl-2',4'-disulphonic acid indicated that P2Y1R mediates the stimulatory action of ATP and ADP. Within the channels expressed in anterior pituitary (P2X2R, P2X3R, P2X4R, and P2X7R), the P2X4R subtype provides a major pathway for calcium influx-dependent signaling and prolactin secretion. This conclusion was based on comparison of native to recombinant channels with respect to their ligand preference, sensitivity to pyridoxal 5-phosphate 6-azophenyl-2',4'-disulphonic acid, and the rates of calcium signal desensitization.

Purinergic P2X(2) receptor desensitization depends on coupling between ectodomain and C-terminal domain.

The wild-type P2X(2) purinergic receptor (P2X(2a)R) and its splice form lacking the intracellular Val(370)-Gln(438) C-terminal sequence (P2X(2b)R) respond to ATP stimulation with comparable EC(50) values and peak current/calcium responses but desensitize in a receptor-specific manner. P2X(2a)R desensitizes slowly and P2X(2b)R desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X(2a)R and P2X(2b)R. Both receptors showed similar EC(50) values (estimated from the peak calcium response) and IC(50) values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP