Characterization of potassium currents activated by purinergic receptors in dissociated mouse aorta myocytes

Le but de ce travail a été de caractériser les courants potassiques activés par la stimulation des récepteurs purinergiques chez des myocytes d'aorte thoracique de souris dispersés en aigu. Les travaux menés dans notre laboratoire ont mis en évidence 3 courants distincts induits par l'ATP sur ces myocytes: 1) un courant entrant P2X porté par du Na⁺ et du Ca²⁺, 2) des courants oscillants potassium et chlore qui dépendent du Ca²⁺ cytosolique, 3) un courant potassique sortant se développant très lentement. Ce travail de thèse a démontré que ce dernier courant n'était que partiellement inhibé par l'application simultanée de nombreux bloqueurs de canaux potassiques, indiquant qu'une partie de ce courant provient de canaux potassiques insensibles aux bloqueurs. Le fait que ce courant soit inhibé par une diminution de la valeur du pH extracellulaire suggère que le courant provient d'un sous-type de canaux potassiques nommés TASK

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.</p

Constitutive and evoked release of ATP in adult mouse olfactory epithelium

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin–luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration

Protein kinase A and C regulate leak potassium currents in freshly isolated vascular myocytes from the aorta.

We tested the hypothesis that protein kinase A (PKA) inhibits K2P currents activated by protein kinase C (PKC) in freshly isolated aortic myocytes. PDBu, the PKC agonist, applied extracellularly, increased the amplitude of the K2P currents in the presence of the "cocktail" of K(+) channel blockers. Gö 6976 significantly reduced the increase of the K2P currents by PDBu suggesting the involvement of either α or β isoenzymes of PKC. We found that forskolin, or membrane permeable cAMP, did not inhibit K2P currents activated by the PKC. However, when PKA agonists were added prior to PDBu, they produced a strong decrease in the K2P current amplitudes activated by PKC. Inhibition of PDBu-elicited K2P currents by cAMP agonists was not prevented by the treatment of vascular smooth muscle cells with PKA antagonists (H-89 and Rp-cAMPs). Zn(2+) and Hg(2+) inhibited K2P currents in one population of cells, produced biphasic responses in another population, and increased the amplitude of the PDBu-elicited K(+) currents in a third population of myocytes, suggesting expression of several K2P channel types. We found that cAMP agonists inhibited biphasic responses and increase of amplitude of the PDBu-elicited K2P currents produced by Zn(2+) and Hg(2). 6-Bnz-cAMp produced a significantly altered pH sensitivity of PDBu-elicited K2P-currents, suggesting the inhibition of alkaline-activated K2P-currents. These results indicate that 6-Bnz-cAMP and other cAMP analogs may inhibit K2P currents through a PKA-independent mechanism. cAMP analogs may interact with unidentified proteins involved in K2P channel regulation. This novel cellular mechanism could provide insights into the interplay between PKC and PKA pathways that regulate vascular tone

Effects of mercury and zinc on PDBU-elicited K⁺ currents.

<p>Outward K⁺ currents were elicited by linear voltage ramps varying from −100 to 100 mV. Data are displayed in concatenated pattern. K⁺ currents are shown in the presence of PDBu (1 µM) and the cocktail of K⁺ channel blockers. Application of mercury (10 µM) and zinc (50 µM) is shown by the horizontal line bar above each trace. Panel B and D: Currents recorded from five VSMC representing each type of response were analyzed. The area under curve (AUC) calculated from the K⁺ currents elicited by linear voltage ramps (examples shown in Panel A and C) were normalized to the membrane capacitance (pF) and plotted against time. To illustrate biphasic response VSMC with pronounced two phases were especially chosen.</p

Comparison of the current-voltage relationships obtained by subtraction of the control currents from PDBu-elicited K⁺ currents with the cocktail of K⁺-channel blockers for four experimental conditions (Panel A).

<p>1) 6-Bnz-cAMP (300 µM) was added to the bath solution after the PKC agonist PDBu (squares). 2) Cells were pretreated with 6-Bnz-cAMP (300 µM) for for 15–20 minutes before application of PDBu (diamonds). The two other current voltage relationships show PDBu-elicited K⁺ currents in the presence of cocktail of K⁺ channel blockers recorded from cells pretreated with PKA antagonist H-89 (1 µM). 3) Forskolin (1 µM) was applied to the bath solution before PDBu (circles). 4) Cells were dialyzed with 6-Bnz-cAMP (30 µM) and Rp-cAMPS (300 µM). 6-Bnz-cAMP (300 µM) was also added to the bath solution before application of PKC agonist PDBu (down-triangles). cAMP agonists inhibited significantly PDBu-elicited K⁺ current (P<0.01, by t-test). Panel B. Effects of extracellular pH on the PDBu-elicited K⁺ currents recorded in freshly isolated myocytes from the mouse aorta. Currents were recorded at different pH values and were normalized to membrane capacitance and to the maximum at pH 9. Response curves for control cells (squares) and for cells pretreated with 6-Bn-cAMP (circles). PDBu-elicited K⁺ currents with changes in extracellular pH are shown. 6-Bnz-cAMP significantly shifted the response curve to the low pH values (p<0.01 t-test). Panel C shows representative PDBu-elicited K⁺ currents, as well as the effect of pH = 6 (upper trace) and the effect of pH = 9 lower trace. Currents were elicited by step pulses to 0 mV and to 50 mV from holding potential of −60 mV.</p

Dose response inhibition of PDBu-elicited currents produced by zinc and mercury recorded in the presence of the “cocktail” of K⁺-channel blockers.

<p>K⁺ currents were elicited by linear voltage ramps varying from −100 to 100 mV. The area under curve (AUC) was calculated and normalized to membrane capacitance (pF), following which the data sets were normalized to the maximum value. Panel A: dose response curve of the inhibition of PDBu-elicited K⁺ current by mercury was fitted with logistic function. PDBu-elicited K⁺ currents were recorded in control cell (circles) and in cells pretreated with 6-Bnz-cAMp (300 µM) (squares). EC50 calculated for control cells was 7.6±1.2 µM and the slope was 2.3±0.3. EC50 calculated for cells pretreated with 6-Bnz-cAMP was 6.9±1.5 µM and the slope was 1.1±0.2. Panel B: dose response curve of the inhibition of PDBu-elicited K⁺ current by zinc was fitted with logistic function. PDBu-elicited K⁺ currents were recorded in control cell (circles) and in cells pretreated with 6-Bnz-cAMp (300 µM) (squares). EC50 calculated for control cells was 7.4±0.2 µM and the slope was 2.2±0.1. EC50 calculated for cells pretreated with 6-Bnz-cAMP was 7.9±1.4 µM and the slope was 1.2±0.2. Panels C and D: examples of PDBu-elicited K⁺ currents with the cocktail of K⁺ channel blockers in the bath solution. K⁺ currents were elicited by linear voltage ramps varying from −100 to 100 mV. Data are displayed in a concatenated pattern. The application of mercury (Hg) and zinc (Zn) is shown by the horizontal line bars above the panels. 6-Bnz-cAMP did not significantly alter EC50 of the Zn²⁺ and Hg²⁺ inhibited currents (P>0.05, by t-test).</p

Action of cAMP agonists on PDBu-elicited K⁺ currents recorded in freshly isolated myocytes from the mouse aorta.

<p>Panel A: Current-voltage relationship recorded in control (up triangles); after application of forskolin (1 µM) (filled squares); and after application of the “cocktail” of K⁺ channel blockers (circles). Forskolin significantly increased the amplitude of the K⁺ currents (*P<0.05, **P<0.01, ***P<0.001 by two-way ANOVA). K⁺ channel blockers significantly inhibited the forskolin-elicited K⁺ current (P<0.001, by two-way ANOVA). Panel B: Current voltage relations recorded in the control (squares); after application of the membrane permeable cAMP analog 6-Bnz-cAMP (300 µM) (circles); after application of the PKC agonist PDBu (up triangles); and after application of the “cocktail” of K⁺ channel blockers (down-triangles). The cocktail of K⁺ channel blockers significantly inhibited the 6-Bnz-cAMP-elicited K⁺ current (*P<0.01, **P<0.001, by two-way ANOVA). Panel C: example of the superimposed families of the currents used to build up the current-voltage relationships. Currents were elicited by voltage steps from −100 mV to 100 mV in increments of 10 mV from holding potential of −60 mV. K⁺ currents were recorded in the control (Contr.); after application of the membrane permeable cAMP analog 6-Bnz-cAMP (cAMP); after application of the PKC agonist PDBu (PDBu); and after application of the cocktail of K⁺ channel blockers (K+-Block.).</p