Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.

L-type and T-type Ca2+ current in cultured ventricular guinea pig myocytes.

The aim of this investigation was to study L-type and T-type Ca(2+) current (I(CaL) and I(CaT)) in short-term cultured adult guinea pig ventricular myocytes. The isolated myocytes were suspended in serum-supplemented medium up to 5 days. Using whole-cell patch clamp techniques ICaL and ICaT were studied by applying voltage protocols from different holding potentials (-40 and -90 mV). After 5 days in culture the myocytes still showed their typical rod shaped morphology but a decline in cell membrane capacitance (26 %). The peak density of ICaT was reduced significantly between day 0 (-1.6+/-0.37 pA/pF, n=9) and day 5 (-0.4+/-0.13 pA/pF, n=11), whereas peak ICaL density revealed no significant differences during culturing. The I(CaT)/I(CaL) ratio dropped from 0.13 at day 0 to 0.05 at day 5. Compared with day 0 I(CaL) the steady state inactivation curve of day 1, day 3 and day 5 myocytes was slightly shifted to more negative potentials. Our data indicate that guinea pig ventricular L-type and T-type Ca(2+) channels are differently regulated in culture.

Effects of K+ channel openers on I K(ATP) of human atrial myocytes at physiological temperatures.

The aim of this study was to investigate the effects of the potassium channel openers (PCOs) cromakalim and pinacidil on the ATP-dependent potassium current I(K)(ATP) in human atrial myocytes. Cells were isolated from the right atrial appendage obtained during cardiac surgery. Membrane currents were studied with the patch-clamp technique in the whole-cell recording mode at 36 degrees -37 degrees C. Under physiological conditions (4.3 mmol/l ATP in the pipette solution, ATPi) I(K)(ATP) did not contribute to basal electrical activity. When ATPi was omitted from the pipette solution I(K)(ATP) activated with a time lag of 4.92+/-0.92 min (n=6) and was completely inhibited by glibenclamide. Using 4.3 mmol/l ATPi I(K)(ATP) at +30 mV was increased by 2.04+/-0.51, 7.24+/-1.65 and 13.22+/-3.71 pA/pF (n=7) with 10, 30 and 100 micromol/l cromakalim, respectively, and by 3.24+/-0.98 (n=6), 4.07+/-0.48 (n=10) and 3.46+/-1.23 pA/pF (n=6) with 10, 30 and 100 micromol/l pinacidil, respectively. Control current density was 5.39+/-0.47 pA/pF (n=39). Using 1 mmol/l ATPi I(K)(ATP) showed a more pronounced activation (4.81+/-3.28, n=6; 9.78+/-2.60, n=7; and 15.1+/-4.18 pA/pF, n=6; with 10, 30 and 100 micromol/l pinacidil, respectively). I(K)(ATP) activated by both compounds could be effectively inhibited by glibenclamide. Repetitive exposure to pinacidil (30 micromol/l at 4.3 mmol/l ATPi) caused a potentiation of I(K)(ATP). Current density at +30 mV was increased by 87% during the first and by 401% during the second pinacidil application (n=5). The data presented in this paper provide new information about electrophysiological characteristics of human atrial I(K)(ATP) and its modulation by the PCOs cromakalim and pinacidil and suggest species-dependent differences.

The sulphonylurea glibenclamide inhibits voltage dependent potassium currents in human atrial and ventricular myocytes.

1 It was the aim of our study to investigate the effects of the sulphonylurea glibenclamide on voltage dependent potassium currents in human atrial myocytes. 2 The drug blocked a fraction of the quasi steady state current (ramp response) which was activated positive to -20 mV, was sensitive to 4-aminopyridine (500 microM) and was different from the ATP dependent potassium current IK(ATP). 3 Glibenclamide dose dependently inhibited both, the peak as well as the late current elicited by step depolarization positive to -20 mV. The IC50 for reduction in charge area of total outward current was 76 microM. 4 The double-exponential inactivation time-course of the total outward current was accelerated in the presence of glibenclamide with a tau(fast) of 12.7+/-1.5 ms and a tau(slow) of 213+/-25 ms in control and 5.8+/-1.9 ms (P<0.001) and 101+/-20 ms (P<0.05) under glibenclamide (100 microM). 5 Our data suggest, that both repolarizing currents in human atrial myocytes, the transient outward current (Ito1) and the ultrarapid delayed rectifier current (IKur) were inhibited by glibenclamide. 6 In human ventricular myocytes glibenclamide inhibited Ito1 without affecting the late current. 7 Our data suggest that glibenclamide inhibits human voltage dependent cardiac potassium currents at concentrations above 10 microM.

Repolarizing currents in ventricular myocytes from young patients with tetralogy of Fallot.

OBJECTIVE: It was the aim of our study to describe repolarizing currents in ventricular myocytes isolated from children with tetralogy of Fallot. This is the first report on outward currents in ventricular myocytes from children. METHODS: Ventricular myocytes were isolated from tissue samples of the outflow tract of the right ventricle which were obtained during corrective surgery of tetralogy of Fallot. Action potentials and whole-cell currents were recorded with the patch clamp technique at a temperature of 36-37 degrees C. RESULTS: The mean resting potential was -71.7 +/- 1.92 mV, action potential amplitude was 110 +/- 2.96 mV and action potential duration at 90% repolarization was 794 +/- 99.5 ms (n = 12). In four out of 12 myocytes early afterdepolarizations (EADs) were observed. Upon hyperpolarization Ba(2+)-sensitive inward currents similar to the inward rectifier current (IKl) could be observed. The current density at -120 mV was -22.8 +/- 2.47 pA/pF (n = 14). A transient outward current (Itol) could be recorded in all myocytes studied, the current density varied from 0.3 to 8.6 pA/pF with a mean of 3.77 +/- 0.47 pA/pF at +40 mV (n = 38). Recovery of Itol from inactivation was fast (70% recovery within 100 ms), rate-dependent reduction amounted to 38.2% at 4 Hz. A delayed rectifier current was seen in only two out of 38 myocytes (rapid component IKr). CONCLUSIONS: The electrophysiological characteristics of right ventricular myocytes isolated from children with tetralogy of Fallot resemble in most cases subendocardial myocytes from adults. The most prominent difference is a fast recovery from inactivation as well as a small rate dependent reduction of Itol. The observed EADs may have clinical implications.

L-type calcium current in human ventricular myocytes at a physiological temperature from children with tetralogy of Fallot.

OBJECTIVE: The aim was to investigate the electrophysiological properties of the L-type calcium current (ICa,L) in ventricular myocytes at a physiological temperature (36-37 degrees C) isolated from children undergoing surgical repair of tetralogy of Fallot. METHODS: ICa,L was recorded with the patch-clamp technique in the single electrode whose-cell mode at a physiological calcium concentration (1.8 mmol/l) at 36-37 degrees C. RESULTS: Under these conditions, maximum current density averaged -5.80 +/- 0.45 pA/pF. ICa,L showed a bell-shaped current-voltage relationship: the current activated at -37.7 +/- 1.36 mV, peaked at +9.41 +/- 1.60 mV and reversed at +57.7 +/- 2.12 mV (n = 17). At +10 mV, time to peak of ICa,L was 5.23 +/- 0.46 ms. Membrane potentials for half-maximal steady-state activation and inactivation of ICa,L were -6.02 and -20.4 mV, respectively, the slope factors were 7.16 mV for steady-state activation and 6.49 mV for steady-state inactivation. ICa,L did not completely inactivate and showed a big window current between -45 and +40 mV. The inactivation of ICa,L showed a biexponential time course with a fast time constant ranging from 9.11 to 12.9 ms and a slow time constant ranging from 60.9 to 220 ms between -30 and +30 mV. Only the slow time constant showed a pronounced voltage dependency. The recovery from inactivation of ICa,L was biphasic with a fast time constant of 60.7 ms and a slow time constant of 619 ms. beta-Adrenergic stimulation with isoprenaline (1 mumol/l) increased the ICa,L density from -5.71 +/- 1.55 to -13.8 +/- 1.96 pA/pF (142%; P < 0.05) at +10 mV. CONCLUSIONS: The present study demonstrates that most of the electrophysiological properties of ICa,L in ventricular myocytes isolated from children with tetralogy of Fallot resemble those of adult ventricular cells. The existence of a big calcium window current could be involved in the occurrence of early afterdepolarizations which could lead to the high incidence of arrhythmias after surgical repair of tetralogy of Fallot.

Estimation of outward currents in isolated human atrial myocytes using inactivation time course analysis.

The aim was to investigate outward currents in single, isolated, human, atrial myocytes and to determine the relative contribution of individual current components to the total outward current. Currents were recorded using the whole-cell patch-clamp technique at 36-37 degreesC. Individual outward current components were estimated from recordings of total outward current using a mathematical procedure based on the inactivation time course of the respective currents. This method allows estimation of outward currents without the use of drugs or conditioning voltage-clamp protocols to suppress individual current components. A rapidly activating and partially inactivating total outward current was recorded when myocytes were voltage clamped at potentials positive to -20 mV (peak current density 24. 0+/-0.97 pA/pF at +40 mV; n=107 cells, 33 patients). This total outward current comprised three overlapping currents: a rapidly inactivating, transient, outward current (Ito1) a slowly and partially inactivating current (ultrarapid delayed rectifier, IKur) and a third current component which most probably reflects a non selective cation current (not characterized). The average current densities at +40 mV were 8.92+/-0.44 pA/pF for Ito1 and 15.1+/-0.72 pA/pF for IKur (n=107 cells). Recovery from inactivation was bi-exponential for both currents and was faster for Ito1. A slowly activating delayed rectifier current (IK) was not found. The current densities of peak Ito1 and IKur varied strongly between individual myocytes, even in those from the same patient. The ratio IKur/Ito1 was 0.5-6.9 with a mean of 1.98+/-0.11 (n=107 cells), suggesting that IKur is the main repolarizing current. The amplitudes of the total outward current, Ito1 and IKur, and the ratio of the latter two were independent of patient age (16-87 years).

The pacemaker current I(f) in single human atrial myocytes and the effect of beta-adrenoceptor and A1-adenosine receptor stimulation.

1. We used single human atrial myocytes to study I(f) occurrence, properties and pharmacological modulation. Cells were obtained by chunk enzymatic digestion from samples of right atrial appendages of patients undergoing corrective cardiac surgery. 2. Patch-clamped cells in the whole-cell configuration were superfused with a modified Tyrode solution to reduce contamination by interfering currents and to amplify I(f). The average cell membrane capacitance was 85.06+/-2.41 pF (n=531). Data were consistent with the geometrical dimensions of the cells (length 94.2+/-1.89 microm, width 17.9+/-0.42 microm, n=126). 3. When hyperpolarizing to -120 mV from a holding potential of -40 mV, 252 of 306 tested cells (82%) expressed a hyperpolarization-activated inward current (I(f) density=3.77+/-0.25 pA pF(-1)); the current was considered to be present in a given cell if its density at -120 mV was larger than 0.5 pA pF(-1). 4. Current activation was sigmoidal and fitted a Boltzmann model; the average activation curve (n=25) showed a maximum current amplitude of 205.97+/-19.94 pA, corresponding to 3.87+/-0.63 pA pF(-1), voltage of half-maximal activation (V(1/2)) at -86.68+/-2.19 mV and a slope of -11.39+/-0.69 mV. The reversal potential of I(f) measured by tail-current analysis was -13.07+/-1.92 mV (n=6). The addition of CsCl (5 mM) fully and reversibly blocked the current. 5. In the presence of the beta-adrenoceptor agonist isoprenaline (Iso, 1 microM), V(1/2) was significantly shifted toward less negative potentials by 6.06+/-1.96 mV (n=16, P=0.0039). The selective A1-adenosine receptor agonist cyclopentyladenosine (CPA, 1 microM) caused a statistically significant shift of V(1/2) toward more negative potentials with respect to the control curve, both in the absence (-7.37+/-1.83 mV, P=0.0005, n=11) and in the presence of 1 microM Iso (-4.97+/-1.78, P=0.031, n=6). 6. These results demonstrate that a current with the properties of I(f) described in cardiac primary and secondary pacemakers occurs in the majority of human atrial cells. While the pathophysiological relevance of I(f) in human atrial tissue remains to be defined, our data clearly show that it is modulated through stimulation of beta-adrenoceptors and A1-adenosine receptors.

Effects of the class III antiarrhythmic drug ambasilide on outward currents in human atrial myocytes.

We have studied the inhibitory influence of the class III antiarrhythmic drug ambasilide (LU 47110) on the transient outward current Ito1 and the sustained current Iso following inactivation of Ito1, in human atrial myocytes. The two currents are separated by a mathematical procedure based on the amplitudes and time constants of the biexponential inactivation of the total outward current. The frequency dependence, the recovery from inactivation and the kinetics of activation and inactivation are described. Ambasilide reversibly and concentration dependently inhibited Ito1, Iso and the sodium current INa. Concentration required for half maximal inhibition (IC50) for the effects on Ito1 and Iso were 23.3 mumol/l and 45.7 mumol/l respectively, concentrations shown by others to be effective in terminating and preventing fibrillation in a dog atrial arrhythmia model. Ambasilide not only reduced the amplitude of Ito1 and Iso but also accelerated the time course of inactivation from 14.22 to 6.69 ms and from 202.3 to 87.9 ms respectively. The amplitude of Ito1 showed only a small dependence on stimulation frequency characteristic for human atrial myocytes, whereas Iso was reduced significantly at higher stimulation frequencies. Ambasilide did not change these relationships (0.1-4 Hz) and therefore did not show the reverse use-dependence known from other class III antiarrhythmic agents and which is an important property for a prospective antiarrhythmic drug. The lack of an effect of ambasilide on both steady-state activation and inactivation of Ito1, and the time constant of recovery from inactivation, suggests that ambasilide acts by changing conductance rather than by influencing the gating mechanism. The described characteristics make ambasilide an interesting substance in the group of class III antiarrhythmic drugs.