Indirectly gated Cl(-)-dependent Cl(-) channels sense physiological changes of extracellular chloride in the leech

The maintenance of ion homeostasis requires adequate ion sensors. In leeches, 34 nephridial nerve cells (NNCs) monitor the Cl(-) concentration of the blood. After a blood meal, the Cl(-) concentration of leech blood triples and is gradually restored to its normal value within 48 h after feeding. As previously shown in voltage-clamp experiments, the Cl(-) sensitivity of the NNCs relies on a persistent depolarizing Cl(-) current that is turned off by an increase of the extracellular Cl(-) concentration. The activation of this Cl(-)-dependent Cl(-) current is independent of voltage and of extra- and intracellular Ca(2+). The transduction mechanism is now characterized on the single-channel level. The NNC's sensitivity to Cl(-) is mediated by a slowly gating Cl(-)-dependent Cl(-) channel with a mean conductance of 50 pS in the cell-attached configuration. Gating of the Cl(-) channel is independent of voltage, and channel activity is independent of extra- and intracellular Ca(2+). Channel activity and the macroscopic current are reversibly blocked by bumetanide. In outside-out patches, changes of the extracellular Cl(-) concentration do not affect channel activity, indicating that channel gating is not via direct interaction of extracellular Cl(-) with the channel. As shown by recordings in the cell-attached configuration, the activity of the channels under the patch is instead governed by the Cl(-) concentration sensed by the rest of the cell. We postulate a membrane-bound Cl(-)-sensing receptor, which-on the increase of the extracellular Cl(-) concentration-closes the Cl(-) channel via a yet unidentified signaling pathway.     

Persistent activation of GABA(A) receptor/Cl(-) channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons

Whole cell patch-clamp recordings using Cl(-)-filled pipettes revealed more negative levels of baseline current and associated current variance in embryonic rat hippocampal neurons co-cultured on a monolayer of astrocytes than those cultured on poly-D-lysine. These effects were mimicked by culturing neurons on poly-D-lysine in astrocyte-conditioned medium (ACM). The baseline current and variance decreased immediately in all cells after either local perfusion with saline or exposure to bicuculline, an antagonist of GABA at GABA(A) receptor/Cl(-) channels. Baseline current and variance in all cells reached a nadir at approximately 0 mV, the calculated equilibrium potential for Cl(-). Perfusion of ACM rapidly induced a sustained current in neurons, which also reversed polarity at approximately 0 mV. Bicuculline attenuated or eliminated the ACM-induced current at a concentration that completely blocked micromolar GABA-induced current. Quantitative analyses of spontaneously occurring fluctuations superimposed on the ACM-induced current revealed estimated unitary properties of the underlying channel activity similar to those calculated for GABA's activation of GABA(A) receptor/Cl(-) channels. Bicuculline-sensitive synaptic-like transients, which reversed at approximately 0 mV, were also detected in neurons cultured in ACM, and these were immediately eliminated along with the negative baseline current and superimposed current fluctuations by perfusion. Furthermore bicuculline-sensitive synaptic-like transients were rapidly and reversibly triggered when ACM was acutely applied. ACM induced an increase in cytoplasmic Ca(2+) in cultured embryonic hippocampal neurons that was completely blocked by bicuculline and strychnine. We conclude that astrocytes release diffusible substances, most likely GABA, that persistently activate GABA(A) receptor/Cl(-) channels in co-cultured neurons.     

H1-receptor dependent increase in platelet aggregation is mediated by intracellular calcium

The influence of histamine on human platelet function was studied by measuring the uptake of [³H]-histamine and by evaluating the effect of histamine on Ca²⁺ influx and on intracellular Ca²⁺ levels. The uptake of histamine by platelets is significantly reduced by mepyramine and unaffected by cimetidine. Histamine was found to increase both the [⁴⁵Ca]²⁺ uptake by resting and stimulated platelets, and the intracellular Ca²⁺ levels in Fura 2-AM loaded platelets. H₁-antihistaminics significantly reduced these processes, suggesting a role for histamine, which in platelets promotes aggregation by acting through H₁-receptors modulating intracellular Ca²⁺ levels.     

Increase in the intracellular free calcium concentration is not an obligatory early event in lipopeptide-induced B-cell activation

We recently showed that synthetic lipopeptides, analogues of the N-terminal region of bacterial lipoprotein, induce DNA synthesis in B lymphocytes in the absence of enhanced phosphatidylinositol 4,5-bisphosphate hydrolysis and protein kinase C translocation. Here we demonstrate that lipopeptides are capable of inducing enhanced expression of MHC class II molecules and early increases in the intracellular free calcium concentration ([Ca2+]i) in B cells. However, they do not effect T cells. The increase in [Ca2+]i seen in B cells is due primarily to Ca2+ release from intracellular stores. Since lipopeptides differ in their capability to induce early increases in [Ca2+]i and since the calcium response does not correlate with the ability of lipopeptides to induce proliferation and expression of MHC class II molecules, we suggest that this biochemical event may not be essential for lipopeptide-mediated B-cell activation.     

An increase in the cerebral infarction area during fatigue is mediated by il-6 through an induction of fibrinogen synthesis

OBJECTIVES:

Our study aimed to investigate the impact of fatigue on the severity of stroke and to explore the underlying mechanisms.

METHODS:

Fatigued male rats underwent middle cerebral artery occlusion and the infarcted brain area was determined. Then, coagulation parameters were assessed in the fatigued group and a control group. In addition, the level of fibrinogen was determined in rats deprived of sleep for various numbers of days. To study whether interleukin-6 was involved in fibrinogen synthesis during fatigue, we also measured levels of interleukin-6 in rats deprived of sleep for various numbers of days. Furthermore, brain injury by middle cerebral artery occlusion was measured in wild-type mice, interleukin-6^-/- mice and wild-type mice treated with bezafibrate.

RESULTS:

More severe cerebral infarction was observed in the fatigued rats, resulting in an infarct ratio of 23.4%. The infarct ratio was significantly increased in the fatigued rats compared with that in the control group (8%, p<0.05). The level of fibrinogen was increased significantly in the fatigued rats compared with that in the control group. In addition, a marked reduction in fibrinogen level was observed in the fatigued interleukin-6^-/- mice compared to their wild-type counterparts, whereas no difference was observed between fatigued wild-type mice and interleukin-6^-/- rats treated with recombinant human interleukin-6. The reduction in brain injury due to middle cerebral artery occlusion during fatigue was observed in interleukin-6^-/- mice and wild-type mice treated with bezafibrate.

CONCLUSION:

Fatigue could increase stroke severity and was associated with the interleukin-6-induced expression of fibrinogen.     

T-dependent B-cell activation is signalled by an early increase in potassium influx

When splenic lymphocytes from RNC nu/ + mice were enriched for T cells by lectin purification, mixed to constant cell density with splenocytes from syngeneic nude mice, and cultured with Con A, a proliferative response ensued which was greater than that expected from the T cells alone. This was shown both by incorporation of ³H-thymidine after 48 h of culture and by uptake of potassium (measured as ⁸⁶Rb) after 15 h. Analysis of metaphase chromosomes stained with Hoechst dye 33258 from co-cultured T-enriched and nude lymphocytes (from female and male donors, respectively) mixed in the proportion 1:4 showed that 40 % of the mitotic cells came from nude spleen. About half of the blast cells in such mixtures stained strongly with fluorescein-coupled goat anti-mouse immunoglobulin; T-cell blasts did not stain under these conditions. Treatment of the nu/nu cells with anti-Thy 1.2 and complement had no effect on their subsequent proliferation in coculture.B lymphocytes from nude mouse spleen were therefore activated to proliferate in this system. This B-cell activation can be detected by increased potassium uptake 15 h after the initiation of co-culture. Thus the increased monovalent cation flux (previously demonstrated when B and T lymphocytes were separately stimulated) also occurs when B cells are stimulated through cooperation with mitogen-activated T cells, and is also detectable early in culture. Tdependent activation of B cells is therefore detectable considerably earlier than by conventional assays (such as plaque formation).     

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Ca_v channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Ca_v channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca²⁺ through Ca_v channels contributes to the regulation of hLEC proliferation, identifying Ca_v channel blockers as potential therapeutic substances in ocular diseases.     

Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-

It has been suggested that the GABA(A) receptor-mediated depolarization in immature neurons depends on a high intracellular Cl(-) concentration maintained by Na-K-Cl cotransporter isoform1 (NKCC1). We previously found that activation of the GABA(A) receptor led to stimulation of NKCC1. This stimulation could be a result of GABA(A) receptor-mediated Cl(-) efflux. However, a loss of intracellular Cl(-) is associated with cell shrinkage, membrane depolarization, as well as a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). To determine which cellular mechanism is underlying NKCC1 stimulation, we investigated changes of intracellular Cl(-) content, [Ca(2+)](i), cell volume, and NKCC1 activity following GABA(A) receptor activation. The basal levels of intracellular (36)Cl were 0.70 +/- 0.04 micromol/mg protein. The intracellular (36)Cl content decreased to 0.53 +/- 0.03 micromol/mg protein in response to 30 microM muscimol (P < 0.05). The loss of intracellular (36)Cl was blocked by 10 microM bicuculline. Muscimol triggered a rise in [Ca(2+)](i), but did not cause cell shrinkage. In contrast, 10-50 mM [Cl(-)](o) or hypertonic HEPES-MEM resulted in reversible cell shrinkage (P < 0.05). Moreover, the GABA-mediated stimulation of NKCC1 activity was not abolished either by removal of extracellular Ca(2+) or BAPTA-AM. An increase in phosphorylation of NKCC1 was detected under both 10 mM [Cl(-)](o) and muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl(-), but not a subsequent rise in [Ca(2+)](i) or shrinkage, leads to stimulation of NKCC1. This stimulation may be an important positive feedback mechanism to maintain intracellular Cl(-) level and GABA function in immature neurons.     

Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na(+)-dependent Cl(-)-HCO3- exchange.

1. We have measured intracellular pH (pHi) in freshly isolated pyramidal neurones from the CA1 region of the rat hippocampus using the fluorescent indicator 2',7'-bis(carboxy-ethyl)-5-(and-6)-carboxyfluorescein (BCECF). 2. The neurones selected by our isolation procedure, when studied in the nominal absence of CO2-HCO3-, had a mean steady-state pHi of 6.81 +/- 0.02 (n = 163). The recovery of pHi from acid loads was very slow. The rate of recovery from acid loads was reduced by Na+ removal, but only very slightly inhibited by 1 mM amiloride. 3. The addition of 5% CO2-25 mM HCO3- caused steady-state pHi to increase from 6.74 +/- 0.05 to 7.03 +/- 0.03 (n = 28). In the presence of 5% CO2-25 mM HCO3-, the rate of pHi recovery from acid loads was much faster than in its absence. 4. The HCO(3-)-induced alkalinization was reversible, and did not occur in the absence of extracellular Na+ or in the presence of DIDS (4,4'-diisothiocyanatostilbene- 2,2'-disulphonic acid). 5. In the absence of external Cl-, successive exposures to CO2-HCO3- elicited alkalinizations that were progressively reduced in rate and amplitude. This effect, presumably due to gradual depletion of internal Cl-, was rapidly reversed by returning Cl- to the external medium. 6. We conclude that the major acid-extrusion mechanism in pyramidal CA1 neurones is the Na(+)-dependent Cl(-)-HCO3- exchanger. The Na(+)-dependent mechanism that operates in the nominal absence of HCO3- is far less active.     

AngII-induced glomerular mesangial cell proliferation inhibited by losartan via changes in intracellular calcium ion concentration

This study investigated the changes in intracellular [Ca²⁺]ⁱ (intracellular calcium ion concentration) and TRPC6 (transient receptor potential channel 6) expression during angiotensin II (AngII)-induced glomerular mesangial cell (GMC) proliferation, as well as the inhibitory effect of losartan. GMC cultures were split into four groups treated for 24 h: Group N (blank control group), Group A (10^?7 mol/L AngII), Group LT (10^?7 mol/L AngII and 10^?5 mol/L losartan), and Group Pred (10^?7 mol/L AngII and 10^?5 mol/L prednisone). GMCs proliferation was measured by the MTT and trypan blue assays. The distribution of TRPC6 was monitored by immunofluorescence, the expression of TRPC6 was detected by RT-PCR and Western blotting, and [Ca²⁺]ⁱ was measured by laser scanning confocal microscopy. The results showed that the maximal proliferation of GMCs was induced by treatment with 10^?7 mol/L AngII for 24 h. In Group A, the distribution of TRPC6 was not uniform in the cell membrane, there was increased accumulation of this protein within the cytoplasm, and the increased expression of TRPC6 and [Ca²⁺]ⁱ was consistent with the proliferation of cells. In Group LT, losartan inhibited the proliferation of GMCs significantly, the levels of TRPC6 and [Ca²⁺]ⁱ were diminished, and the distribution of TRPC6 was improved. Prednisone also significantly inhibited the proliferation of GMCs and had no effects on the expression of TRPC6 and [Ca²⁺]ⁱ in Group Pred. These findings suggested that AngII could enhance the expression of TRPC6, increase [Ca²⁺]^i, and demonstrate a time–dose–response relationship with the proliferation of GMCs, while losartan reversed the effect of AngII on GMC proliferation.     

fMLP-dependent activation of Akt and ERK1/2 through ROS/Rho A pathways is mediated through restricted activation of the FPRL1 (FPR2) receptor

Objective and design

The objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte.

Materials or subjects

Freshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP.

Treatment

Time-course and dose–response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases.

Methods

Freshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media.

Results

fMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H₂O₂ release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H₂O₂-induced activation of Akt and RhoA-GTPase.

Conclusion

Collectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.

     

GTP and GDP activation of K+ channels that can be inhibited by ATP

K⁺ channels that can be inhibited by intracellular ATP have been found in many different cell types. In the insulin-secreting pancreatic islet cells these channels are of crucial importance for stimulus-secretion coupling as glucose stimulation closes the ATP-sensitive channels which leads to depolarization and firing of Ca²⁺ action potentials. We now demonstrate that nucleotides other than ATP also influence the gating of these K⁺ channels. In contrast to the action of ATP, GTP (10 M – 1 mM) and GDP (100 M to 1 mM) evoke dose-dependent channel activation and this effect is immediately reversible. Phosphorylation is not directly involved as non-hydrolysable GTP-and GDP-analogues also evoke channel opening. ATP reversibly inhibits opening of the GTP- or GDP- activated K⁺ channels.     

Murine coronavirus-induced oligodendrocyte apoptosis is mediated through the activation of the Fas signaling pathway

We previously showed that infection of rat oligodendrocytes by ultraviolet light-inactivated mouse hepatitis virus (MHV) resulted in apoptosis, suggesting that the apoptosis is triggered during cell entry. To further characterize the earliest apoptotic signaling events, here we treated cells with an antibody specific to the MHV receptor prior to and during virus infection or with an antibody specific to MHV spike protein following virus binding. Both treatments blocked virus infection and apoptosis, indicating that virus-receptor binding is necessary but not sufficient for the apoptosis induction. Furthermore, virus infection significantly increased the formation of the "death-receptor complexes" consisting of Fas, Fas-associated death domain and procaspase-8, but did not induce the complexes involving the tumor necrosis factor receptor and its associated death domain, demonstrating the specific activation of the Fas signaling pathway. Moreover, virus infection did not alter the abundance of the individual proteins of the complexes, suggesting that the activation of the Fas signaling pathway was at the post-translational level. Treatment with a Fas/Fc chimera, which blocks Fas-Fas ligand-mediated apoptosis, inhibited the formation of the complexes and blocked the activation of caspase-8 and apoptosis in MHV-infected cells. It also inhibited the release of cytochrome c from mitochondria and the activation of caspase-9. These results demonstrate that oligodendrocyte apoptosis is triggered by MHV infection during cell entry through the activation of the Fas signaling pathway.     

An antibody to BP 180 kDa antigen is able to induce an increase of intracellular Ca2+ concentration in DJM-1 (human squamous cell carcinoma) cells

To elucidate the induction of transmembrane signal transduction of keratinocytes by bullous pemphigoid (BP) antibodies, we assessed the intracellular Ca2+ concentration ([Ca2+]i) in DJM-1 cells (a squamous cell carcinoma cell line). The time course of fluo-3-fluorescence intensity was investigated, and transient increase of fluorescence intensity in DJM-1 cells was observed when the DJM-1 cells were incubated with rabbit IgG anti-human BP 180kDa antigen (BP 180). No increase of fluorescence intensity was observed when the cells were incubated with rabbit IgG anti-human BP 230 kDa antigen (BP 230). This transient increase of fluorescence intensity was inhibited by U-73122, a specific inhibitor of phospholipase C. These results indicate the possibility that an antibody to BP 180 induces transmembrane signal transduction in keratinocytes.     

Prostaglandin E(2)-activated housekeeping Cl(-) channels in the basolateral membrane of rat gastric parietal cells.

Cl(-) channels in the basolateral membrane of parietal cells within isolated rat gastric glands were studied by the whole-cell patch-clamp technique. The membrane potential (E(m)) of non-stimulated parietal cells changed as a function of the basolateral extracellular Cl(-) concentration (46 mV per decade), but E(m) did not change significantly as a function of the K(+) concentration. The extracellular addition of prostaglandin E(2) (PGE(2); 10 microM) increased the whole-cell Cl(-) current. A bifunctional prostaglandin EP3 agonist/EP1 antagonist, 5(Z)-7-[(1S, 2S,3S,5R)-3-(trans-beta-styren)sulfonamido-6,6-dimethylbi cyclo-(3.1. 1)hept-2-yl]-5-heptenoic acid (ONO-NT-012; 10 microM), also increased the Cl(-) current. Basal Cl(-) currents and the PGE(2)- and ONO-NT-012-increased Cl(-) currents were voltage-independent and inhibited by a Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), at 500 microM. The single Cl(-) channel conductance was estimated to be 0.29 picosiemens (pS) by variance noise analysis. Both PGE(2) and ONO-NT-012 increased intracellular free Ca(2+) concentration in the fura-2-loaded parietal cell transiently. The present study has shown that housekeeping sub-pS Cl(-) channels are present in the basolateral membrane of rat parietal cell, and that the channels are regulated positively by PGE(2) via the EP3 receptor.     

Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels

Genetic ablation of the fibroblast growth factor (Fgf) 14 gene in mice or a missense mutation in Fgf14 in humans causes ataxia and cognitive deficits. These phenotypes suggest that the neuronally expressed Fgf14 gene is essential for regulating normal neuronal activity. Here, we demonstrate that FGF14 interacts directly with multiple voltage-gated Na⁺ (Nav) channel α subunits heterologously expressed in non-neuronal cells or natively expressed in a murine neuroblastoma cell line. Functional studies reveal that these interactions result in the potent inhibition of Nav channel currents ( I _Na) and in changes in the voltage dependence of channel activation and inactivation. Deletion of the unique amino terminus of the splice variant of Fgf14 , Fgf14-1b , or expression of the splice variant Fgf14-1a modifies the modulatory effects on I _Na, suggesting an important role for the amino terminus domain of FGF14 in the regulation of Na_v channels. To investigate the function of FGF14 in neurones, we directly expressed Fgf14 in freshly isolated primary rat hippocampal neurones. In these cells, the addition of FGF14-1a–GFP or FGF14-1b–GFP increased I _Na density and shifted the voltage dependence of channel activation and inactivation. In fully differentiated neurones, FGF14-1a–GFP or FGF14-1b–GFP preferentially colocalized with endogenous Nav channels at the axonal initial segment, a critical region for action potential generation. Together, these findings implicate FGF14 as a unique modulator of Nav channel activity in the CNS and provide a possible mechanism to explain the neurological phenotypes observed in mice and humans with mutations in Fgf14 .     

Inhibition of T-cell activation by syndecan-4 is mediated by CD148 through protein tyrosine phosphatase activity

Most coinhibitory receptors regulate T-cell responses through an ITIM that recruits protein tyrosine phosphatases (PTPs) to mediate inhibitory function. Because syndecan-4 (SD-4), the coinhibitor for DC-associated heparan sulfate proteoglycan integrin ligand (DC-HIL), lacks such an ITIM, we posited that SD-4 links with a PTP in an ITIM-independent manner. We show that SD-4 associates constitutively with the intracellular protein syntenin but not with the receptor-like PTP CD148 on human CD4(+) T cells. Binding to DC-HIL allowed SD-4 to assemble with CD148 through the help of syntenin as a bridge, and this process upregulated the PTP activity of CD148, which is required for SD-4 to mediate DC-HIL's inhibitory function. Using a mouse model, we found SD-4 to be located away from the immunological synapse formed between T cells and APCs during activation of T cells. These findings indicate that SD-4 is unique among known T-cell coinhibitors, in employing CD148 to inhibit T-cell activation at a site distal from the synapse.