Human BEC upregulate SUR1-TRPM4 channels that are involved in tPA-induced phasic secretion of MMP-9.

<p><b>A–D</b>: Macroscopic currents induced by recombinant tPA (rtPA) in activated but not in non-activated (CTR) human BEC; currents were blocked by glibenclamide (Glib) and 9-phenanthrol (9-PHE); n = 6–10 cells/condition; **, <i>P</i><0.01. <b>E</b>: Gelatin zymography showing that after activation, rtPA and PAR1-agonist TFLLR induce <i>phasic</i> secretion of MMP-9 in human BEC that is inhibited by glibenclamide; the bar graphs represent densitometric measurements of total MMP-9; the lanes and the bars in the graph are aligned for the different conditions: PAR1-agonist (TFLLR), rtPA and glibenclamide (Glib); n = 4; *, <i>P</i><0.05.</p

SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells

<div><p>Background</p><p>Hemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1—transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca²⁺-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process.</p><p>Methods</p><p>Cerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied <i>in vitro</i>, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways.</p><p>Results</p><p>Cerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused <i>de novo</i> expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca²⁺-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion.</p><p>Conclusions</p><p>tPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.</p></div

Direct versus indirect actions of ghrelin on hypothalamic NPY neurons

<div><p>Objectives</p><p>Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons.</p><p>Materials and methods</p><p>Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca²⁺ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus.</p><p>Results</p><p>Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca²⁺]_c, with an EC₅₀ ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP₃) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na⁺]_o suppressed activation, suggesting a role for Na⁺-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons <i>in situ</i>. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca²⁺]_c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca²⁺ channels, respectively, while Ni²⁺, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca²⁺ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca²⁺ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action <i>in vivo</i>.</p><p>Conclusions</p><p>NPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons <i>in situ</i>. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na⁺ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca²⁺ channels, which could contribute to NPY neuronal activity <i>in situ</i>.</p></div

Primer sequences for RT-PCR.

<p>Primer sequences for RT-PCR.</p

Additional file 1:Figure S1. of Heterogeneity of aquaporin-4 localization and expression after focal cerebral ischemia underlies differences in white versus grey matter swelling

Validation of Aqp4 antibody. (PDF 1092 kb

rtPA and PAR1-agonist cause secretion of MMP-9 from activated endothelial cells that is reduced by SUR1 inhibition.

<p><b>A</b>: RT-PCR showing that activation of murine BEC upregulates MMP-9 but not MMP-2 mRNA; n = 5. <b>B</b>: Gelatin zymography showing that non-activated murine BEC secrete minimal MMP-9, and that <i>tonic</i> secretion of MMP-9 and MMP-2, both pro and active forms, following overnight activation is not affected by glibenclamide; n = 5. <b>C</b>: Gelatin zymography showing that after activation, rtPA and PAR1-agonist TFLLR induce <i>phasic</i> secretion of MMP-9 that is inhibited by glibenclamide; n = 5; **, <i>P</i><0.01. <b>D</b> <i>left</i>: Immunoblot showing suppression of SUR1 after infection with shAbcc8 lentiviral vector; CTR, untreated cells; Scr, lentiviral vector with scrambled shRNA. <b>D</b> <i>right</i>: Gelatin zymography showing that after overnight activation, <i>phasic</i> secretion of MMP-9 induced by TFLLR is inhibited by pretreatment of the cells with shRNA against <i>Abcc8</i> (*); n = 3.</p

SUR1-TRPM4 channel upregulation <i>in vitro</i>.

<p><b>A</b>: Activity of the <i>Abcc8</i> promoter, the <i>Trpm4</i> promoter and a positive control (four consecutive NF-κB consensus sequences), under basal conditions and after stimulation by TNF (20 ng/mL); 3 replicates; *, <i>P</i><0.05; **, <i>P</i><0.01. <b>B</b>: Murine BEC were exposed to TNF (20 ng/mL) and lysate was analyzed by RT-PCR 16 hours later; transcripts for <i>Abcc8</i> and <i>Trpm4</i> were upregulated compared to control (CTR); the induction of <i>Abcc8</i> mRNA by TNF was reduced by the NF-κB inhibitor, PDTC (pyrrolidinedithiocarbamate); β-actin mRNA was used as a loading control; representative of 3 replicates. <b>C</b>: Macroscopic and single channel (inside-out patch) currents induced by ATP depletion in activated but not in non-activated (CTR) murine BEC; currents were blocked by glibenclamide (Glib) and 9-phenanthrol (9-PHE). <b>D</b>: Single channel current in an inside-out patch with 4 single channel levels during changes in bath solution from 145 mM Cs⁺/1 μM Ca²⁺, 145 mM Cs⁺/0 μM Ca²⁺,145 mM Cs⁺/1 μM Ca²⁺, 0 mM Cs⁺/75 mM Ca²⁺, 145 mM Cs⁺/1 μM Ca²⁺. <b>E</b>: Quantification of macroscopic currents at –50 mV induced by ATP depletion or diazoxide in activated or non-activated (CTR) murine BEC; currents were blocked by glibenclamide and by gene deletion of <i>Abcc8</i> (SUR1KO) and gene suppression of <i>Trmp4</i> (siRNA); cells/condition for each bar: 35, 12, 14, 19, 11, 11; 24, 19; 9, 13; **, <i>P</i><0.01; ***, <i>P</i><0.001. <b>F</b>: Immunoblots for pCaMKII and CaMKII, with densitometric quantification of pCaMKII in murine BEC without activation, under control conditions (CT) and after exposure to the Ca²⁺ ionophore (CI), A23187 (5 μM × 10 minutes), and in activated BEC exposed to TNF in the absence and presence of glibenclamide (Glib); both bands shown had molecular masses of 50 kD, corresponding to pCaMKIIα and CaMKIIα; pCaMKII normalized to levels induced by A23187; n = 5.</p

Plasmin opens the SUR1-TRPM4 channel.

<p><b>A</b>: Control experiment showing requirement for proteolytic activity; change in intracellular Ca²⁺ concentration (ΔF/F₀) induced by recombinant tPA (rtPA) in activated murine BEC, in the absence and presence of the non-specific serine threonine protease inhibitors (PI’s), aprotinin and leupeptin; representative of 5–7 cells per condition. <b>B</b>: Control experiment showing non-involvement of NMDA (N-methyl-D-aspartate) receptor; change in intracellular Ca²⁺ induced by glutamate plus glycine (Glu+Gly) in non-activated (CTR) and in activated BEC; representative of 7 cells per condition. <b>C</b>: In activated murine BEC, rtPA fails to induce SUR1-TRPM4 current in the presence of tranexamic acid (TXA), although Ca²⁺ influx via A23187 activates the channel; exogenous plasmin induces SUR1-TRPM4 current blocked by glibenclamide (Glib); bar graph showing SUR1-TRPM4 currents under the conditions indicated; illustrative currents during ramp pulses are also shown; cells/condition for each bar: 25, 11, 8, 5, 5. <b>D</b>: In activated murine BEC, rtPA fails to induce SUR1-TRPM4 current in the presence of PAR1-antagonist RWJ56110, although Ca²⁺ influx via A23187 activates the channel; PAR1-agonist SFLLRN induces SUR1-TRPM4 current that is blocked by glibenclamide; bar graph showing SUR1-TRPM4 currents under the conditions indicated; illustrative currents during ramp pulses are also shown; cells/condition for each bar: 25, 12, 11, 5, 5. <b>E</b>: Plasmin induces Ca²⁺ influx (ΔF/F₀) in activated but not non-activated (CTR) murine BEC; 5–8 cells per condition. <b>F</b>: rtPA induces phosphorylation of ERK1/2 (p42/44 MAPK); bar graph showing >20% increase in p-ERK42 and p-ERK44 due to rtPA; n = 3; **, <i>P</i><0.01.</p

SUR1-TRPM4 channel opening by tPA requires Ca²⁺ influx via TRPC3.

<p><b>A</b>: Macroscopic currents during 200-ms step pulses (#1,3,5) and ramp pulses (–100 to +100 mV; 4 mV/msec) (#2,4), induced by recombinant tPA (rtPA) in activated murine BEC, recorded initially using extracellular solution with 1.8 mM Ca²⁺, and after switching to extracellular solution containing 0 mM Ca²⁺; <i>right</i>: illustrative currents during ramp pulses after addition of rtPA, before and after switch to 0 Ca²⁺; the difference current is also shown (thick line). <b>B</b>: rtPA-induced current in activated BEC was not blocked by ruthenium red (RR), but was blocked by Gd⁺³ and Pyr3; illustrative currents during ramp pulses after addition of rtPA, before and after Gd⁺³ or Pyr3 are also shown; <i>bar graph</i>: rtPA-induced currents in activated BEC in the presence of 1.8 mM Ca²⁺, 0 mM Ca²⁺; 1.8 mM Ca²⁺ plus RR or SKF-96365 or Pyr3 or Gd³⁺; cells/condition for each bar: 25, 7, 5, 7, 5, 7. <b>C</b>: Change in intracellular Ca²⁺ concentration (ΔF/F₀) induced by rtPA in activated but not in non-activated (Ctr) BEC; the rtPA-induced increase in Ca²⁺ was blocked by pretreatment with SKF-96365 but not RR; <i>bar graph</i>: mean change at 10–12 minutes in intracellular Ca²⁺ concentration induced by rtPA in non-activated and activated BEC, in the presence of RR; SKF-96365; Gd³⁺; cells/condition for each bar: 10, 8, 10, 10, 10; ***, <i>P</i><0.001.</p

K_ATP channel modulators have SUR1 dependent effects on isolated NPY-GFP neurons.

<p>(A) Diazoxide (200 μM) did not affect ghrelin action. (B) When applied alone, diazoxide (200 μM) mimics the effect of ghrelin, while glibenclamide (1μM) reduced the stimulatory effect of ghrelin. (C-D) Neither diazoxide (200 μM) nor glibenclamide (1μM) affected ghrelin action in SUR1^-/- mice lacking SUR1. Ghrelin was applied at 200 pM as shown (red bars). (E) Comparison of the percentages of all ghrelin-activated cells in preparations of neurons isolated from SUR1^-/- versus NPY-GFP mice shows the mean values are not significantly different, p > 0.7, using an unpaired, two-tailed t-test. About two-thirds of the activated neurons in NPY-GFP mouse preparations were GFP-positive. Values are the means ± S.D. from four NPY-GFP and six SUR1^-/- preparations totaling more than 1000 cells.</p