Characterization of lamotrigine inhibition of Na+ channels in rat hippocampal neurones

1. Lamotrigine (LTG), a new antiepileptic drug, requires long depolarizations to inhibit Na⁺ currents. This suggests either slow binding of LTG to the fast inactivated state or selective binding of LTG to the slow inactivated state of Na⁺ channels. To differentiate between these possibilities and to characterize further the action of LTG, we studied the affinity and kinetics of LTG binding to the Na⁺ channels in acutely dissociated hippocampal neurones of the rat.
2. LTG inhibited more Na⁺ currents at more depolarized holding potentials. The inhibitory effect at various holding potentials could be described by one-to-one binding curves, which yielded an apparent dissociation constant of ∼7 μM for LTG binding to the inactivated channels (K_I), and a dissociation constant more than 200 times larger for LTG binding to the resting channels. A similar value of K_I (∼9 μM) was also derived from the LTG concentration-dependent shift of the inactivation curve.
3. The recovery of LTG-bound inactivated Na⁺ channels was faster than the recovery of normal (drug-free) slow inactivated channels. Moreover, the binding kinetics of LTG onto the inactivated channels were faster than the development of the slow inactivated state, and were linearly correlated with LTG concentrations, with a binding rate constant of ∼10,000 M⁻¹ s⁻¹. These findings suggest that LTG chiefly binds to the fast inactivated state rather than the slow inactivated state.
4. We conclude that LTG, in therapeutic concentrations and at relatively depolarized membrane potentials, may potently inhibit Na⁺ currents by slow binding to the fast inactivated state of Na⁺ channels. Like phenytoin, the slow binding rates may explain why LTG effectively inhibits seizure discharges, yet spares most normal neuronal activities.

     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Isoform-specific regulation of the Na+-K+ pump by adenosine in guinea pig ventricular myocytes

Aim:

The present study investigated the effect of adenosine on Na⁺-K⁺ pumps in acutely isolated guinea pig (Cavia sp.) ventricular myocytes.

Methods:

The whole-cell, patch-clamp technique was used to record the Na⁺-K⁺ pump current (I_p) in acutely isolated guinea pig ventricular myocytes.

Results:

Adenosine inhibited the high DHO-affinity pump current (I_h) in a concentration-dependent manner, which was blocked by the selective adenosine A₁ receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists staurosporine, GF 109203X or the specific δ isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A₁ receptor agonist CCPA and a specific activator peptide of PKC-δ, PP114. In contrast, the selective A_2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and A₃ receptor agonist Cl-IB-MECA did not affect I_h. Application of the selective A_2A receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 and A₃ receptor antagonist MRS1191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced I_h inhibition.

Conclusion:

The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of I_h via adenosine A₁ receptors and the PKC-δ isoform.     

Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume,intracellular pH and migration of human polymorphonuclear leucocytes

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pH_i) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pH_i and migration of fMLP stimulated PMNs.
2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.
3. Inhibition of the Na⁺/H⁺ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling.
4. Step omission of extracellular Na⁺ caused intracellular acidification, which was accelerated by subsequent addition of gastric H⁺/K⁺ ATPase inhibitor SCH 28080, or by omission of extracellular K⁺ ions. In addition Na⁺ removal caused cell swelling, which was further enhanced by fMLP.
5. H⁺/K⁺ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV.
6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na⁺/H⁺ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.
7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.
8. Western analysis with a monoclonal anti gastric H⁺/K⁺ATPase β-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.
9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na⁺/H⁺ exchanger and a K⁺-dependent H⁺-extruding mechanism, presumably an H⁺/K⁺ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

     

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

1. Two types of Ca²⁺ channel α₁-subunits were co-expressed in Xenopus oocytes with the Ca²⁺ channel α₂- and β₁-subunits. The Ba²⁺ current through the α_1Cα₂β and the α_1Bα₂β channels had electrophysiological and pharmacological properties of L- and N-type Ca²⁺ channels, respectively.
2. Amlodipine had a strong blocking action on both the L-type and N-type Ca²⁺ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca²⁺ channel was comparable to that on the L-type Ca²⁺ channel. At −100 mV holding potential, the IC₅₀ values for amlodipine block on the L-type and N-type Ca²⁺ channel were 2.4 and 5.8 μM, respectively.
3. The blocking action of amlodipine on the N-type Ca²⁺ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca²⁺ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.
4. The time course of block development by amlodipine was similar for L-type and N-type Ca²⁺ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca²⁺ channel.

     

Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity

Background and purpose:

Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na⁺ channel. However, long-term effects of bepridil on the Na⁺ channel remain unclear. We explored the long-term effect of bepridil on the Na⁺ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na_v1.5 channel.

Experimental approach:

Na⁺ currents were recorded by whole-cell voltage-clamp technique. Na⁺ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis.

Key results:

Treatment of cardiomyocytes with 10 µmol·L⁻¹ bepridil for 24 h augmented Na⁺ channel current (I_Na) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca²⁺/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I_Na increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na_v1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na_v1.5 were not modified. Bepridil and W-7 did not increase I_Na any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner.

Conclusions and implications:

We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na_v1.5 α-subunit, which in turn increased Na⁺ current.     

Blockade of N-type Ca2+ current by cilnidipine (FRC-8653) in acutely dissociated rat sympathetic neurones

1. The inhibitory effects of cilnidipine (FRC-8653) and various organic Ca²⁺ channel blockers on high voltage-activated Ba²⁺ currents (HVA I_Ba) in rat sympathetic neurones were examined by means of the conventional whole-cell patch-clamp recording mode under voltage-clamped conditions.
2. HVA I_Ba was classified into three different current components with subtype selective peptide Ca²⁺ channel blockers. No ω-Agatoxin IVA-sensitive (P-type) or ω-conotoxin MVIIC-sensitive (Q-type) current components were observed. Most (>85%) I_Ba was found to consist of ω-conotoxin GVIA-sensitive N-type components.
3. The application of cilnidipine inhibited HVA I_Ba in a concentration-dependent manner. The K_d value for cilnidipine was 0.8 μM. Cilnidipine did not shift the current-voltage (I-V) relationship for HVA I_Ba, as regards the threshold potential and peak potential where the amplitude reached a maximum.
4. High concentrations of three hypotensive Ca²⁺ channel blockers, nifedipine, diltiazem and verapamil, all inhibited HVA I_Ba in a concentration-dependent manner. The K_d values for nifedipine, diltiazem and verapamil were 131, 151 and 47 μM, respectively. A piperazine-type Ca²⁺ channel blocker, flunarizine, showed a relatively potent blocking action on I_Ba. The K_d value was about 3 μM.
5. These results thus show that cilnidipine potently inhibits the sympathetic Ca²⁺ channels which predominantly consist of an ω-Cg-GVIA-sensitive component. This blockade of the N-type Ca²⁺ channel, as well as the L-type Ca²⁺ channel by cilnidipine suggests that it could be used therapeutically for treatment of hypersensitive sympathetic disorders associated with hypertension.

     

Regulation of apical and basolateral K+ conductances in the rat colon

1. Apical administration of an ionophore, nystatin, and basolateral depolarization by K⁺ were used to investigate the regulation of apical and basolateral electrogenic transport pathways for K⁺ in the rat proximal and distal colon.
2. Administration of nystatin (100 μg ml⁻¹ at the mucosal side), in the presence of Na⁺ and in the presence of a serosally directed K⁺ gradient, stimulate a large increase in short-circuit current (I_SC) and tissue conductance in both colonic segments. This response was composed of a pump current generated by the Na⁺-K⁺-ATPase and of a current across a quinine-sensitive basolateral K⁺ conductance.
3. The pump current, measured as Na⁺-dependent or scilliroside-sensitive current in the absence of a K⁺ gradient, was significantly greater in the distal than in the proximal colon. The pump current was unaltered by pretreatment of the tissue with forskolin (5×10⁻⁶ mol l⁻¹).
4. The current across the basolateral K⁺ conductance, measured as current in the presence of a serosally directed K⁺ gradient either in the absence of Na⁺ or in the presence of scilliroside, was increased by the cholinoreceptor agonist, carbachol (5×10⁻⁵ mol l⁻¹), but inhibited by forskolin (5×10⁻⁶ mol l⁻¹).
5. Basolateral K⁺ depolarization induced a negative I_SC in both colonic segments, which was inhibited by the K⁺ channel blocker quinine (10⁻³ mol l⁻¹ at the mucosal side), but was resistant to tetraethylammonium (5×10⁻³ mol l⁻¹ at the mucosal side). This K⁺ current across an apical K⁺ conductance was stimulated in both colonic segments by carbachol, whereas forskolin had no effect, although control experiments revealed that forskolin was still able to open an apical Cl⁻ conductance under these conditions.
6. These results demonstrate that an increase in intracellular Ca²⁺ concentration induced by carbachol causes an increase in the basolateral and the apical K⁺ conductance, thereby inducing K⁺ secretion in parallel with an indirect support for Cl⁻ secretion due to the hyperpolarization of the cell membrane. In contrast, the dominating effect of an increase in the intracellular cyclic AMP concentration is inhibition of a basolateral K⁺ conductance; a mechanism which might contribute to the inhibition of K⁺ absorption.

     

Inhibition of delayed rectifier K+ channels by phenytoin in rat neuroblastoma cells

1. The action of the anticonvulsant drug phenytoin on K⁺ currents was investigated in neuroblastoma cells by whole-cell voltage-clamp recording.
2. Neuroblastoma cells expressed an outward K⁺ current with a voltage- and time-dependence which resembled the delayed-rectifier K⁺ current found in other cells. When added to the standard external solution at concentrations ranging between 1 and 200 μM, phenytoin reduced the current (n=65). Inhibition was concentration-dependent with a half-maximal inhibitory concentration of 30.9±0.8 μM.
3. The K⁺ current inhibition by phenytoin was voltage-dependent with block by phenytoin being relieved by depolarization.
4. The times taken to reach steady-state inhibition and complete recovery from inhibition were about 20 s. Neither the activation and inactivation rates of the K⁺ current nor the K⁺ channel availability were significantly altered by the blocking drug. A use-dependent block was observed at phenytoin concentrations of 10, 25 and 50 μM.
5. These results suggest that phenytoin affects K⁺ currents and that this effect might lead to a reduction in neuronal excitability.

     

Activation by high potassium of a novel voltage-operated Ca2+ channel in rat spleen

1. High potassium produced a concentration-dependent contraction in rat isolated spleen.
2. The high potassium-induced contraction of rat spleen was abolished in Ca²⁺-free Krebs solution containing 1 mM EGTA, and the subsequent addition of 3 mM Ca²⁺ restored the high potassium-induced contraction to the control level.
3. Nifedipine, verapamil, diltiazem, Cd²⁺, Ni²⁺, Co²⁺, R-(+)-Bay K 8644 and pimozide inhibited and relaxed high potassium-induced contraction of rat spleen with IC₅₀ and EC₅₀ values much higher than those values in rat aorta.
4. In addition, high potassium-stimulated contraction of rat spleen was insensitive to ω-conotoxin GVIA, ω-conotoxin MVIIC and ω-agatoxin IVA.
5. The high potassium-induced contraction of rat spleen was also unaffected by tetrodotoxin (TTX), prazosin, chloroethylclonidine (CEC), yohimbine, propranolol, atropine, diphenhydramine, cimetidine, ketanserin, 3-tropanyl-indole-3-carboxylate, saralasin, indomethacin, nordihydroguaiaretic acid, GR32191B, domperidone, naloxone, chlorpromazine, suramin, (±)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX), L-659,877, L-703,606, lorglumide, PD 135,158 N-methyl-D-glucamine, benextramine, amiloride, dantrolene, TMB-8, econazole, staurosporine and neomycin.
6. Forskolin and sodium nitroprusside relaxed high potassium-induced contraction of rat spleen with EC₅₀ values of 0.55±0.04 and 20.0±2.7 μM, respectively.
7. It is concluded that high potassium may activate a novel, pharmacologically uncharacterized voltage-operated Ca²⁺ channel in rat spleen.

     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

Neuroregulation of mucus secretion by opioid receptors and KATP and BKCa channels in ferret trachea in vitro

1. Opioid agonists inhibit neurogenic mucus secretion in the airways. The mechanism of the inhibition is unknown but may be via opening of potassium (K⁺) channels. We studied the effect on neurogenic secretion in ferret trachea in vitro of the OP₁ receptor (formerly known as δ opioid receptor) agonist [D-Pen^2,5]enkephalin (DPDPE), the OP₂ receptor (formely κ) agonist U-50,488H, the OP₃ receptor (formerly μ) agonist [D-Ala², N-Me-Phe, Gly-ol⁵]enkephalin (DAMGO), the ATP-sensitive K⁺ (K_ATP) channel inhibitor glibenclamide, the large conductance calcium activated K⁺ (BK_Ca) channel blocker iberiotoxin, the small conductance K_Ca (SK_Ca) channel blocker apamin, the K_ATP channel opener levcromakalim, a putative K_ATP channel opener RS 91309, and the BK_Ca channel opener NS 1619. Secretion was quantified by use of ³⁵SO₄ as a mucus marker.
2. Electrical stimulation increased tracheal secretion by up to 40 fold above sham-stimulated levels. DAMGO or DPDPE (10 μM each) significantly inhibited neurogenic secretion by 85% and 77%, respectively, effects which were reversed by naloxone. U-50,488H had no significant inhibitory effect on neurogenic secretion, and none of the opioids had any effect on ACh-induced or [Sar⁹]substance P-induced secretion.
3. Inhibition of neurogenic secretion by DAMGO or DPDPE was reversed by iberiotoxin (3 μM) but not by either glibenclamide or apamin (0.1 μM each). Iberiotoxin alone did not affect the neurogenic secretory response.
4. Levcromakalim, RS 91309 or NS 1619 (3 nM–3 μM) inhibited neurogenic secretion with maximal inhibitions at 3 μM of 68%, 72% and 96%, respectively. Neither levcromakalim nor RS 91309 at any concentration tested significantly inhibited acetylcholine (ACh)-induced secretion, whereas inhibition (60%) was achieved at the highest concentration of NS 1619, a response which was blocked by iberiotoxin.
5. Inhibition of neurogenic secretion by levcromakalim (3 μM) or RS 91309 (30 nM) was inhibited by glibenclamide but not by iberiotoxin. In contrast, inhibition by NS 1619 (30 nM and 3 μM) was blocked by iberiotoxin but not by glibenclamide.
6. We conclude that, in ferret trachea in vitro, OP₁ or OP₃ opioid receptors inhibit neurogenic mucus secretion at a prejunctional site and that the mechanism of the inhibition is via opening of BK_Ca channels. Direct opening of BK_Ca channels or K_ATP channels also inhibits neurogenic mucus secretion. In addition, opening of BK_Ca channels inhibits ACh-evoked secretion of mucus. Drugs which open BK_Ca channels may have therapeutic anti-secretory activity in bronchial diseases in which neurogenic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

     

SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil

1. The classical ATP sensitive K⁺ (K_ATP) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K⁺ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K⁺ channel openers, such as pinacidil and nicorandil.
2. In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the K_ATP channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method.
3. In the cell-attached recordings (145 mM K⁺ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K⁺ channel in both the transfected cells.
4. In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR_2B/Kir6.2 and SUR_2A/Kir6.2 channels (EC₅₀ of ∼2 and ∼10 μM, respectively). On the other hand, nicorandil activated the SUR_2B/Kir6.2 channel >100 times more potently than the SUR_2A/Kir6.2 (EC₅₀ of ∼10 μM and >500 μM, respectively).
5. Thus, nicorandil, but not pinacidil, preferentially activates the K_ATP channels containing SUR_2B. Because SUR_2A and SUR_2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR_2B may play a critical role in the action of nicorandil on the vascular type classical K_ATP channel.

     

Effects of the 5-lipoxygenase activating protein inhibitor MK886 on voltage-gated and Ca2+-activated K+ currents in rat arterial myocytes

1. The effects on the voltage-gated (I_K) and Ca²⁺ activated (I_K,Ca) K⁺ currents in rat arterial myocytes of the 5-lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated.
2. In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration-dependent reduction of the I_K, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886.
3. MK886 application led to a concentration-dependent increase in the amplitude of the TEA-sensitive I_K,Ca current and single channel activity in RPAMs in whole cell and inside-out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4–5 fold increase was observed at 10 μM MK886. MK886 also increased I_K,Ca in rat mesenteric arterial myocytes (RMAMs).
4. L538,916, an analogue of MK886 which does not block FLAP, had no effect on either I_K or I_K,Ca at a concentration of 10 μM.
5. Leukotriene C₄ (100 nM) had no effect on either I_K or I_K,Ca in RPAMs. MK886 produced its usual increase in I_K,Ca and also blocked I_K, in the presence of leukotriene C₄. Similarly, leukotriene E₄ (100 nM) did not alter the amplitude of I_K. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 μM) did not affect I_K in RPAMs, and did not affect the response to MK886.
6. Arachidonic acid (10 μM) enhanced I_K,Ca in both RPAMs and RMAMs.
7. The results show that MK886 markedly affects both I_K and I_K,Ca in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K⁺ channels, and mimic its effects. Alternatively, MK886 might act via non-selective effects on other arachidonic acid metabolites which could modify K⁺ channel function.

     

Blowing off acid: a new tool to study Na+/HCO3- co-transport

Investigation of the physiological functions and possible pathological roles of Na(+)/HCO(3)(-) co-transport in the heart has been hampered by uncertainty over the molecular identity of cardiac Na(+)/HCO(3)(-) co-transporter(s) and the absence of selective pharmacological inhibitors. In their paper published in this issue, Ch'en and colleagues describe the extensive characterization of S0859 as a high-affinity inhibitor of Na(+)/HCO(3)(-) co-transport in cardiac myocytes (Ch'en et al., 2008). The availability of S0859 provides a powerful new tool to investigate the (patho)physiological significance of Na(+)/HCO(3)(-) co-transport in the heart and other tissues.     

Acetylcholine modulation of high-voltage-activated calcium channels in the neurones acutely dissociated from rat paratracheal ganglia

1. The modulation of high-voltage-activated (HVA) Ca²⁺ channels by acetylcholine (ACh) was studied in the paratracheal ganglion cells acutely dissociated from 2-week-old Wistar rats by use of the nystatin perforated patch recording configuration under voltage-clamp conditions.
2. ACh inhibited the HVA Ca²⁺ currents in a concentration- and voltage-dependent manner.
3. The inhibition was mimicked by a muscarinic agonist, oxotremorine. Pirenzepine and methoctramine produced parallel shifts to the right in the ACh concentration-response curves. Schild analysis of the ACh concentration-ratios yield pA₂ values for pirenzepine and methoctramine of 6.85 and 8.57, respectively, suggesting the involvement of an M₂ receptor.
4. Nifedipine, ω-conotoxin-GVIA and ω-conotoxin-MVIIC reduced the HVA I_Ca by 16.8, 59.2 and 6.3%, respectively. A current insensitive to all of these Ca²⁺ antagonists, namely `R-type'', was also observed. The results indicated the existence of L-, N-, P/Q-, and R-type Ca²⁺ channels.
5. The ACh-sensitive current component was markedly reduced in the presence of ω-conotoxin-GVIA, but not with both nifedipine and ω-conotoxin-MVIIC. ACh also inhibited the R-type HVA I_Ca remaining in saturating concentrations of nifedipine, ω-conotoxin-GVIA and ω-conotoxin-MVIIC.
6. The inhibitory effect of ACh was prevented by pretreatment with pertussis toxin.
7. It was concluded that ACh selectively reduces both the N- and R-type Ca²⁺ channels, by activating pertussis toxin sensitive G-protein through the M₂ muscarinic receptor in paratracheal ganglion cells.

     

Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurones

1. The effect of adenosine 5′-triphosphate (ATP) on glutamatergic synaptic transmission in hippocampus was examined by an indicator of intracellular Ca²⁺ oscillations. These oscillations were postsynaptic responses by glutamate released from presynaptic sites. ATP completely inhibited the oscillations in a concentration-dependent manner.
2. The ATP-induced inhibition was mediated via P2-purinoceptors since ATP exhibited the inhibitory action even in the presence of P1-purinoceptor antagonists. Also non-hydrolysable ATP analogues and uridine 5′-triphosphate (UTP) inhibited the oscillation.
3. The rank order of agonist potency of ATP analogues for inhibition of the Ca²⁺ oscillation was as follows: 2-methyl-thio-adenosine 5′-triphosphate⩾ATP>adenosine 5′-O-(3-thiotriphosphate)>UTP>α,β-methylene-adenosine 5′-triphosphate. These inhibitory effects were insensitive to suramin. Judging from this rank order of potency, the inhibitory P2-purinoceptor could be assigned to a subclass of GTP-binding protein coupled-type receptors.
4. The site of action of ATP was thought to be presynaptic since ATP did not affect the postsynaptic Ca²⁺ responses by glutamate. These results suggest the existence of a presynaptic inhibitory P2-receptor that inhibits glutamate release in the hippocampus.

     

Preservation of mitochondrial function may contribute to cardioprotective effects of Na+/Ca2+ exchanger inhibitors in ischaemic/reperfused rat hearts

BACKGROUND AND PURPOSE: Na+/Ca2+ exchanger (NCX) inhibitors are known to attenuate myocardial reperfusion injury. However, the exact mechanisms for the cardioprotection remain unclear. The present study was undertaken to examine the mechanism underlying the cardioprotection by NCX inhibitors against ischaemia/reperfusion injury. EXPERIMENTAL APPROACH: Isolated rat hearts were subjected to 35-min ischaemia/60-min reperfusion or 20-min ischaemia/60-min reperfusion. NCX inhibitors (3-30 microM KB-R7943 (KBR) or 0.3-1 microM SEA0400 (SEA)) were given for 5 min prior to ischaemia (pre-ischaemic treatment) or for 10 min after the onset of reperfusion (post-ischaemic treatment). KEY RESULTS: With 35-min ischaemia/60-min reperfusion, pre- or post-ischaemic treatment with KBR or SEA neither enhanced post-ischaemic contractile recovery nor attenuated ischaemia- or reperfusion-induced Na+ accumulation and damage to mitochondrial respiratory function. With the milder model (20-min ischaemia/reperfusion), pre- or post-ischaemic treatment with 10 microM KBR or 1 microM SEA significantly enhanced the post-ischaemic contractile recovery, associated with reductions in reperfusion-induced Ca2+ accumulation, damage to mitochondrial function, and decrease in myocardial high-energy phosphates. Furthermore, Na+ influx to mitochondria in vitro was enhanced by increased concentrations of NaCl. KBR (10 microM) and 1 microM SEA partially decreased the Na+ influx. CONCLUSIONS AND IMPLICATIONS: The NCX inhibitors exerted cardioprotective effects during relatively mild ischaemia. The mechanism may be attributable to prevention of mitochondrial damage, possibly mediated by attenuation of Na+ overload in cardiac mitochondria during ischaemia and/or Ca2+ overload via the reverse mode of NCX during reperfusion.     

Tandospirone-induced K+ current in acutely dissociated rat dorsal raphe neurones

1. The effects of tandospirone (TDS) on dissociated rat dorsal raphe neurones were investigated using the patch-clamp method.
2. Under current-clamp conditions, TDS hyperpolarized the cell membrane, resulting in the reduction of firing rates.
3. Under voltage-clamp conditions, TDS induced an inward rectifying K⁺ current in a concentration-dependent manner.
4. The TDS-induced K⁺ currents (I_TDS) were mimicked by 8-OH-DPAT, a 5-HT_1A agonist. The I_TDS was blocked by spiperone, a 5-HT_1A receptor antagonist, in a concentration-dependent manner.
5. N-Ethylmaleimide, an agent which uncouples between the receptor and the G-protein, irreversibly blocked the I_TDS.
6. In neurones perfused intracellularly with a pipette-solution containing GTP using the conventional whole-cell patch recording, the I_TDS showed a gradual rundown. When the neurones were perfused with GTPγS, TDS activated the inwardly rectifying K⁺ current in an irreversible manner.
7. In the inside-out patch recording mode, TDS-activated single K⁺ channel currents (i_TDS) which also showed an inward rectification. When the GDP in cytosolic side was completely replaced with GTP, the open probability of i_TDS significantly increased.
8. These results indicate that the activation of 5-HT_1A receptors by TDS directly opens the inward rectifying K⁺ channels via a G-protein mediated process.

     

Negative inotropic effects of endothelin-1 in mouse cardiomyocytes: evidence of a role for Na+-Ca2+ exchange

Endothelin-1 (ET-1) is a peptide hormone produced within the myocardium which may modulate myocardial contractility in a paracrine-autocrine fashion. In the majority of species, ET-1 has a direct positive inotropic effect on the myocardium that involves both increased myofilament Ca(2+) sensitivity and increased Ca(2+) transients. Ca(2+) entry through reverse-mode Na(+)-Ca(2+) exchange, involving both indirect effects via elevation of intracellular [Na(+)] and direct activation of the Na(+)-Ca(2+) exchanger, have been suggested to contribute to the increase in Ca(2+) transients. Conversely, mouse cardiomyocytes show an exclusively negative inotropic response to ET-1. Here, Nishimaru and colleagues present novel evidence that the negative inotropic effect of ET-1 in mouse cardiomyocytes involves both a reduction in myofilament Ca(2+) sensitivity and increased Ca(2+) extrusion, via Na(+)-Ca(2+) exchange. Data obtained using the selective Na(+)-Ca(2+) exchange blocker, SEA0400, suggest that a re-assessment of the role of the exchanger in Ca(2+)-handling by mouse cardiomyocytes may be necessary.