Decrease In Calcium Current Research Articles

NOD-1 Stimulation Induces Cardiac Dysfunction and Calcium Handling Impairment in a Mice Model

The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, e.g. pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory diseases. Recent studies have established a relationship between the innate immune response and some cardiovascular diseases. In this regard, nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. NLRs activation promotes a cascade of molecular signaling events that ultimately lead to inflammation and cell death. NOD1 is a NLR-subfamily that upon activation results in transcription of inflammatory genes. NLR dysfunction has been described in a variety of maladies, including chronic inflammation, autoimmunity and cancer predisposition; however less information is available related to the contribution of these mediators to the cardiac disease progress. Our results showed that mice cardiac tissues express NOD1 and the activation of this NLR with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction. At the cellular level, NOD1 stimulation induces a decrease in calcium current (ICaL) and [Ca2+]I transients. In conclusion our results demonstrates that NLR-NOD1 specific activation can induce several pathological processes involved in some cardiac diseases.

The Two Intracellular Ca2+Release Channels, Ryanodine Receptor and Inositol 1,4,5-Trisphosphate Receptor, Play Different Roles during Fertilization in Ascidians

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs ofPhallusia mammillatausing whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3(10 μMperfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15–20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes.

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.

Modulation of ionic currents by dopamine in an interneurone of the respiratory central pattern generator of Lymnaea stagnalis.

Dopamine elicits alternating bursts of activity in the respiratory interneurones of the snail Lymnaea stagnalis. One of the neurones (VD4) was isolated in culture, and the effects of dopamine on both membrane voltage and current were studied utilising the whole-cell tight-seal recording technique. Dopamine had little effect on resting potentials near -60 mV, nor did it affect spike threshold or input resistance measured near -60 mV. However, it did alter the excitability of the cell, changing the response to current injection from one of repetitive spiking to one of rapid accommodation. Under voltage-clamp, VD4 responded to dopamine (EC50 = 92 nmol l-1) with increased net outward current at all potentials more positive than -60 mV. This was due primarily to an increase in voltage-gated potassium current and a decrease in calcium current. A reduction of Cd(2+)-sensitive outward current, possibly calcium-gated potassium current, was also evident at potentials more positive than +60 mV. The physiological actions of dopamine on these cells in vivo are consistent with the inhibitory mechanisms presented in this study.

Acetylcholine reduces the slow calcium current in embryonic skeletal muscle cells in culture.

Xenopus skeletal muscle cells when grown in culture develop a slow inward calcium current that is sensitive to dihydropyridines. Acetylcholine (ACh, 10 microM) applied through a puffer pipette caused a large inward current in these cells (at the holding potential) through the nicotinic receptor channels and reduced the inward calcium current (during a step depolarization to 0 mV). After the ACh application was discontinued the holding current rapidly returned to pre-ACh levels (20 s) whereas the calcium current showed a slow, partial recovery to pre-ACh levels. Outward potassium current was also reduced during the application of ACh but recovered completely after ACh was discontinued. The effect of ACh on the calcium current was not mimicked by muscarine (100 microM) and was absent when 10 micrograms/ml alpha-bungarotoxin was added to the bath suggesting that the decrease in calcium current was mediated by current through the nicotinic receptor.

Action potential conduction between guinea pig ventricular cells can be modulated by calcium current.

We used cell pairs electrically coupled with relatively high intercellular resistance to investigate the involvement of calcium current in the origin of the source current during the conduction process of the action potential (AP). Three interventions were used to reduce the calcium current: a specific calcium channel blocker [nifedipine (NIF)], premature stimulation, and increments in the frequency of stimulation of the cell. The ionic membrane current (Iion) after the peak of the AP of the stimulated cell was positive and small when the cell was uncoupled. However, when the stimulated cell was coupled to a cell model or to another cell, Iion during this period became negative and large to supply the coupling current. A rapid early repolarization of the AP occurred in the stimulated cell because of the removal of charge from the stimulated cell. NIF decreased the magnitude of the net negative Iion during this period and caused a more rapid early repolarization in the stimulated cell. NIF increased the delay between the activations of two coupled cells at a given coupling resistance (Rc) but decreased the longest delay that could be produced without conduction failure for a given cell pair. The highest Rc below which conduction of AP occurred was also decreased by NIF. Premature stimulation and an increase of the stimulation frequency also caused an increase in the extent of the early repolarization and increased the delay between two cell activations at a given Rc. Conduction block occurred with sufficient prematurity or at a sufficiently high frequency of stimulation even though activation of the stimulated cell occurred for each stimulus. The Iion that flows during the early plateau phase of the AP in the stimulated cell became negative and significantly large by coupling two cardiac cells together. This current flow is a major component needed to supply the coupling current through the intercellular resistance. The decrease of calcium current caused a decrease in the magnitude of this net inward ionic current, resulting in an increase of the rate of early repolarization and an increase in the conduction delay between two cells at a given Rc. These results suggest the involvement of calcium current in the conduction process when cells are coupled at relatively high Rc.

Effects of isoflurane on K+ and Ca2+ conductance in isolated smooth muscle cells of canine cerebral arteries.

Although isoflurane is a known cerebral vasodilator, the mechanism of isoflurane-induced vasodilation is not clear. The purpose of this study was to investigate the effects of 2.6% isoflurane (1.2 mM) on macroscopic calcium and potassium channel currents in voltage-clamped canine middle cerebral artery cells. Cells were dialyzed with K(+)-glutamate solution and superfused with Tyrode's solution for measurement of potassium current (n = 20). Stepwise depolarization from a holding potential of -60 mV to beyond -30 mV elicited an outward, slowly inactivating potassium current that was reduced 50% +/- 2% and 81% +/- 3% (mean +/- SEM) in the presence of 1 mM 4-aminopyridine and 30 mM tetraethylammonium, respectively. Calcium ionophore (A23187, 10 microM) increased the potassium current by 76% +/- 3%, suggesting calcium dependency. Isoflurane reduced the amplitude of the potassium current by 35% +/- 4%. Calcium current was measured in cells dialyzed with solution containing 130 mM Cs(+)-glutamate and superfused with solution containing 10 mM BaCl2 and 135 mM tetraethylammonium to pharmacologically isolate the calcium current (n = 13). Under these conditions, progressive depolarizing steps from -60 mV elicited an inward current that was maximally activated at +20 mV and essentially eliminated by 1 microM nifedipine. This current, resembling a long-lasting (L-type) Ca2+ channel current, was reduced 40% +/- 4% by isoflurane. The results of this study suggest that isoflurane acts directly at the vascular muscle membrane to suppress transmembrane calcium and potassium currents. The decrease in calcium current would cause vasodilation; however, the concomitant decrease in potassium current may partially antagonize the depressant effect of isoflurane mediated through calcium current reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine-induced changes in atrial action potential: contribution of Ca and K currents

In this study, we examined the relative contribution of the increase in acetylcholine-regulated potassium current (IK ACh) and decrease in calcium current (ICa) to the adenosine (Ado)-induced shortening of action potential duration (APD). In isolated guinea pig atrial myocytes, membrane potentials and currents were measured by the whole cell patch-clamp technique. ICa and IK ACh were individualized by blocking the K currents with Cs+ and ICa with Cd2+. The effects of Ado on membrane potential and currents were concentration dependent. Ado (10 microM) shortened APD at 0 mV and at 90% of repolarization (APD0,90) to 7 +/- 1 and 26 +/- 6 ms from control values of 23 +/- 3 and 89 +/- 6 ms, respectively. Concomitant with the changes in APD, Ado decreased ICa from -9.2 +/- 1.3 to -6.8 +/- 10 microA/microF (26% decrease) but increased IK ACh from +3.5 +/- 0.5 to +7.8 +/- 0.8 microA/microF (123% increase). When rundown of ICa was taken into account, the maximum decrease in ICa caused by Ado was 12%. The effect of Ado on ICa and IK ACh was not altered by treatment of the cells with either Cs+ or Cd2+. The shortening of ADP0,90 strongly correlated with the increase in IK ACh but minimally with the decrease in ICa. A 22% reduction in ICa caused by lowering extracellular Ca2+ concentration ([Ca2+]o) from 3.6 to 1.8 mM was associated with an 11 and 14% shortening of APD0 and APD90, respectively. In the same myocytes an 18% decrease in ICa by 10 microM Ado reduced APD0 and APD90 by 58 and 61%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of intracellular cyclic AMP injection on calcium current in identifiedHelix pomatia neurons

The effect of intracellular injection of cyclic AMP (cAMP) and extracellular application of theophylline on the inward calcium current was investigated in neurons RPa3 and LPa3 ofHelix pomatia. Iontophoretic injection of cyclic AMP (current 10–35 nA, duration about 1 min) led to a decrease in amplitude of the calcium current to a new stationary level, which depended on the injection current. After the end of injection the calcium current was restored to its initial level. Current-voltage characteristic curves of the calcium current were not shifted along the voltage axis by cAMP injection, indicating that the reduction in this current was connected with a change in maximal calcium conductance. An increase in the frequency of depolarizing shifts from 0.1 to 0.5 Hz caused a decrease in the calcium current but did not affect the time course of the decrease in calcium current in response to injection of cAMP or the time course of its recovery after the end of injection. Theophylline an inhibitor of cyclic nucleotide phosphodiesterase, in a concentration of 1 mM in the external solution, lowered the amplitude of the calcium current by 50–75% of its initial value. In 40% of neurons, abolition of the action of theophylline by rinsing was incomplete, but in the rest the effect of theophylline was irreversible. It is postulated on the basis of the results that cytoplasmic compounds take part in regulation of the calcium current of molluscan neurons. The possible physiological role of this process is discussed.