Specific Ion Channel Blockers Research Articles

Stereoselective Interactions Between Local Anesthetics and Cardiac K+ Channels

Ionic channels are membrane proteins that can be blocked by many different types of drugs such as local anesthetics, antiarrhythmics, etc. Therefore, they are considered drug targets, whose topology, at the ion channel level, has been analyzed by studying the interactions of specific ion channel blockers and site-directed mutant ion channels. Stereoselective interactions are especially interesting because they can reveal three-dimensional relationships between drugs and channels with otherwise identical biophysical and physicochemical properties. Furthermore, stereoselectivity suggests direct and specific receptor-mediated action, and identification of such stereospecific interactions may have important clinical consequences. However, ≉25% of drugs used in clinical practice are racemic mixtures, the individual enantiomers of which frequently differ in both their pharmacodynamic and pharmacokinetic profile. Furthermore, these different effects induced by one of the enantiomers of a racemic drug may contribute to the undesired effects that can be similar or different to the pharmacological effect of the racemic drug. In other cases, the enantiomers on the molecular target are opposite. In this review, we focus on the stereoselective effects of bupivacaine on different Kv channels. Bupivacaine stereoselectively blocks Kv1.5 and Kv11.1 channels, whereas non-stereoselectively it blocks Kv2.1 and Kv4.3 channels.

Ion Channels in Cell Proliferation and Apoptotic Cell Death

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must--at some time point--increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl- and K+ channels. Besides regulating cytosolic Cl- activity, osmolyte flux and, thus, cell volume, most Cl- channels allow HCO3- exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K+ exit through K+ channels may decrease intracellular K+ concentration, which in turn favors apoptotic cell death. K+ channel activity further maintains the cell membrane potential, a critical determinant of Ca2+ entry through Ca2+ channels. Cytosolic Ca2+ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca2+ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.

Cyclic Mechanical Strain Regulates the PTHrP Expression in Cultured Chondrocytes via Activation of the Ca2+ Channel

The association between mechanical stimulation and chondrocyte homeostasis has been reported. However, the participation of PTHrP (parathyroid-hormone-related protein) in the mechano-regulation of chondrocyte metabolism remains unclear. We determined whether mechanical stimulation of chondrocytes induces the expression of PTHrP and, further, whether the mechano-modulation of PTHrP is dependent on the maturational status of chondrocytes. Cyclic mechanical strain was applied to rat growth plate chondrocytes at the proliferating, matrix-forming, and hypertrophic stages at 30 cycles/min. Cyclic mechanical strain significantly increased PTHrP mRNA levels in chondrocytes at the proliferating and matrix-forming stages only. The induction of PTHrP was dependent on loading magnitude at the proliferating stage. Using specific ion channel blockers, we determined that mechano-induction of PTHrP was inhibited by nifedipine, a Ca2+ channel blocker. These results suggest that mechanical induction of PTHrP possibly provides the environment for greater chondrocyte replication and matrix formation that would subsequently affect cartilage formation.

Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.

Human amylin (hAmylin), a 37-amino acid pancreatic peptide, and amyloid beta protein (A beta), a 39-43 amino acid peptide, abundantly deposited in the brains of Alzheimer's patients, induce neurotoxicity in hippocampal and cortical cultures. Although the mechanism of this neurotoxicity is unknown, both peptides are capable of modulating ion channel function that may result in a disruption of cellular homeostasis. In this study, we examined the effects of hAmylin on whole cell currents in chemically identified neurons from the rat basal forebrain and the interactions of hAmylin-induced responses with those of A beta. Whole cell patch-clamp recordings were performed on enzymatically dissociated neurons of the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Bath application of hAmylin (1 nM to 5 microM) resulted in a dose-dependent reduction in whole cell currents in a voltage range between -30 and +30 mV. Single-cell RT-PCR analysis reveal that all DBB neurons responding to hAmylin or A beta were cholinergic. Using specific ion channel blockers, we identified hAmylin and A beta effects on whole cell currents to be mediated, in part, by calcium-dependent conductances. Human amylin also depressed the transient outward (IA) and the delayed rectifier (IK) potassium currents. The hAmylin effects on whole cell currents could be occluded by A beta and vice versa. Human amylin and A beta responses could be blocked with AC187 (50 nM to 1 microM), a specific antagonist for the amylin receptor. The present study indicates that hAmylin, like A beta, is capable of modulating ion channel function in cholinergic basal forebrain neurons. Furthermore, the two peptides may share a common mechanism of action. The ability of an amylin antagonist to block the responses evoked by hAmylin and A beta may provide a novel therapeutic approach for Alzheimer's disease.

Azimilide and dofetilide produce similar electrophysiological and proarrhythmic effects in a canine model of Torsade de Pointes arrhythmias

Torsade de Pointes arrhythmias are a feared proarrhythmic effect of (antiarrhythmic) drugs. In dogs with chronic complete AV-block bradycardia-induced volume overload leads to electrical remodeling, which includes increased susceptibility to drug-induced Torsade de Pointes arrhythmias. The IKr channel blocker, dofetilide (Tikosyn™, 0.025 mg/kg/5 min), and the less specific ion channel blocker, azimilide (5 mg/kg/5 min), were compared in nine anesthetized dogs at 4 and 6 weeks of AV-block in a randomized cross-over design. Dosages were based on our own dose-dependence studies and on anti-arrhythmic dosages reported in the literature. Monophasic action potential catheters were placed endocardially in both the left and right ventricle to measure action potential duration, visualize early afterdepolarizations, and to assess interventricular dispersion of repolarization (i.e. left ventricular monophasic action potential duration (at 100%) minus right ventricular monophasic action potential duration (at 100%). Cycle length of idioventricular rhythm, QT-time and the occurrence of drug-induced Torsade de Pointes arrhythmias were determined using the surface electrocardiogram (ECG). Before drug administration, the electrophysiological parameters were identical at 4 and 6 weeks. Both azimilide and dofetilide increased monophasic action potential duration, cycle length of idioventricular rhythm, and QT-time. Dissimilar lengthening of left ventricular and right ventricular monophasic action potential duration increased the interventricular dispersion significantly from 55 to 110 ms for both drugs. All dogs had early afterdepolarizations, while, in the majority, ectopic ventricular beats developed (dofetilide 8/9 and azimilide 7/9). Torsade de Pointes arrhythmias incidence was comparable for dofetilide (6/9) and azimilide (5/9). In conclusion, azimilide and dofetilide show similar electrophysiological and proarrhythmic effects in our canine model with a high incidence of Torsade de Pointes arrhythmias.

Induction of potassium channels in mouse brain microglia: cells acquire responsiveness to pneumococcal cell wall components during late development

Lipopolysaccharides derived from cell walls of Gram-negative bacteria have proven a useful tool to simulate bacterial infection of the central nervous system. Rapid activation of microglia within the brain parenchyma as well as in vitro has thereby been shown to be an early event upon bacterial or lipopolysaccharide challenges. Less is known about microglial responses to a contact with Gram-positive bacteria, such as Streptococcus pneumoniae, a lethal pathogen causing meningitis with a 30% mortality rate. In the present study, we compared lipopolysaccharide-induced microglial activation in vitro with that induced by preparations of pneumococcal cell walls. As a readout of microglial activation, we studied by patch-clamp recording the expression of outward rectifying potassium currents ( I k + OR), which are known to be induced by lipopolysaccharide. We found that pneumococcal cell walls and lipopolysaccharide induced a similar type of I k + OR. Stimulation of I k + OR by pneumococcal cell walls and lipopolysaccharide involved protein synthesis since it was not induced in the presence of cycloheximide. Pharmacological characterization of the pneumococcal cell wall- and lipopolysaccharide-induced currents with specific ion channel blockers indicated for both cases expression of the charybdotoxin/margatoxin-sensitive Kv1.3 subtype of the Shaker family of voltage-dependent potassium channels. Activation of the outward currents by pneumococcal cell walls depended on the developmental stage: while lipopolysaccharide triggered I k + OR in both embryonal and postnatal microglial cells, pneumococcal cell walls had only a marginal effect on embryonal cells. This, however, does not imply that embryonic microglial cells are unresponsive to pneumococcal cell walls. In both embryonic and postnatal cells, (i) the amplitude of the constitutively expressed inward rectifying potassium current was significantly reduced, (ii) tumor necrosis factor- α was released and (iii) the cells changed their morphology, similarly as it was induced by lipopolysaccharide treatment. Thus, embryonic microglial cells are sensitive to pneumococcal cell wall challenges, but respond with a distinctly different pattern of physiological reactions. The expression of I k + OR could thus be a suitable tool to study signalling cascades selectively involved in the activation of microglia by Gram-negative and -positive cell wall components and to functionally distinguish between populations of microglial cells.

Effect of specific ion channel blockers on cultured schwann cell proliferation

Mitogenesis in a variety of tissues is known to be inhibited by K+ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Using radiolabeled thymidine as a proliferation index we have examined what role, if any, specific K+ channels have in cultured Schwann cells that have been induced to proliferate by pre-exposure to mitogens. TEA and 4-AP are "broad-spectrum" in that they block a variety of different types of K+ channel. In contrast, we found that alpha-dendrotoxin (alpha-DTX), a specific blocker of the type 1 fast delayed rectifier current (the largest component of Schwann cell K+ current) does not affect proliferation, suggesting that type 1 current may not be involved in mitogenesis. This suggestion is supported by our finding that the values of the KD for the mitogenic effect (722 nM, 4-AP; 13 mM, TEA) are much larger than the corresponding electrophysiological values for type 1 channels (0.1 mM, 4-AP; 0.2 mM, TEA). Charybdotoxin (200 nM) and iberiotoxin (100 nM), inhibitors of Ca2+-activated K+ channels, cesium (5 mM), an inhibitor of inward rectifier channels, and furosemide (100 pM), which blocks Na+/K+/Cl- cotransport, all had no effect on proliferation. Interestingly, 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS), which blocks voltage-gated Cl- channels, reduced proliferation. In summary, broad-spectrum K+ channel blockers inhibit Schwann cell proliferation, but inhibitors specific for type 1, Ca2+-activated, and inward rectifier K+ channels do not. Whether the inhibition is mediated by type 2 K- channels, by an as yet unidentified Schwann cell K+ channel, or by another mechanism remains unclear.

Effect of specific ion channel blockers on cultured schwann cell proliferation

Effect of specific ion channel blockers on cultured schwann cell proliferation

A specific blocker reveals the presence and function of the hyperpolarization-activated cation current I h in peripheral mammalian nerve fibres

Electrotonic responses recorded extra- or intracellularly from peripheral nerve preparations show a “sag” to hyperpolarizing current pulses. [10, 11, 20]The biophysical nature of this “inward rectification” is still under discussion since the phenomenon has not been noted at voltage-clamped single nerve fibres, and since Cs +, which reduces inward rectification, is not a specific ion channel blocker. In this study, we found that low micromolar concentrations of ZD 7288, a specific blocker of the hyperpolarization-activated cationic current (I h) in the soma of central mammalian neurones, [8]result in a complete block of inward rectification in the electrotonic responses of isolated rat spinal dorsal roots. In addition, ZD 7288 enhanced the activity-dependent slowing of conduction seen in compound C fibre action potentials of isolated rat vagus nerves and augmented the post-tetanic hyperpolarization following trains of action potentials in unmyelinated and myelinated axons. The data suggest that ZD 7288 is a potent blocker and a useful research tool for the study of hyperpolarization-activated inward rectification (I h) of peripheral nerve preparations.

Segmental Difference of Water and Electrolyte Transport in Rat Colon in vivo

The segmental difference of water and electrolyte transport in the rat colon was studied in vivo. The proximal and distal colon segments were perfused separately but simultaneously at a constant rate with physiological solution, and net movements of water, sodium and chloride were determined. The effects of osmolality and sodium concentration of perfusate were assessed. The effect of a sodium channel blocker on the net transport of water and electrolytes was also studied in each colon segment. The net absorption of water, sodium and chloride correlated with the sodium concentration and osmolality of the perfusion solution in both colon segments and were dominant in the distal colon segment in each condition, compared with that in the proximal colon segment. The concentrations of three electrolytes in the collected fluid were almost the same as those of the perfusion solutions in both segments and these results indicated that water was transported isotonically through the colon lumen. Benzamil, a specific sodium ion channel blocker, inhibited net water and sodium absorption by 58.8% and 63.1% in the proximal colon segment and by 52.0% and 43.6% in the distal colon segment, respectively. These results suggest the existence of an electrogenic sodium transport mechanism and a paracellular pathway in normal (i.e., not treated with corticosteroids or sodium-depleted food) rats which has not been detected in in vitro studies with both apical and basolateral membrane vesicles.

Photoaffinity labeling of acetylcholine receptor in millisecond time scale

Photoaffinity labeling of acetylcholine receptors can be performed with a time resolution allowing to discriminate reaction sites within the receptor protein in its different functional states. This is achieved by a combination of a stopped-flow apparatus with a high energy pulse laser. The photoaffinity label used is the lipophilic cation [ 3H]TPMP + which has been shown to be a non-competitive antagonist and a specific ion channel blocker. AChR in its resting (channel closed) and active (channel open) state incorporates the label mainly into the α-polypeptide chain of the receptor. Only several hundred milliseconds after mixing AChR with agonist labeling of δ-chains becomes significant.