Effect Of Potassium Channel Blockade Research Articles

Electrophysiology and Live Fluorescence Imaging to Monitor the Effects of Potassium Channel Blockade on Lipopolysaccharide-Induced Immune Signaling

Studies have shown that ion-channel function in immune cells such as macrophages can influence pathogen-induced immune signaling. Thus, ion channels are viewed by some researchers as potential therapeutic targets for developing novel strategies for regulating immune response on demand when standard anti-pathogen therapies such as antibiotics and vaccinations fall short. However, the direct contribution of ion-channel function to the complex and interconnected signaling pathways in immune response has proved elusive, largely due to the difficulty in tracking multiple signaling nodes in these pathways in real-time. Toward this end, we tracked the real-time inflammatory response to E. coli derived lipopolysaccharide (LPS) in a mouse macrophage-like cell-line (RAW 264.7) with electrophysiology to measure potassium channel currents and live imaging with fluorescent fusion reporters of crucial events involved in immune signaling. We developed two reporter constructs: 1) GFP fused to the NFκB transcription factor subunit RelA (GFP-RelA) to track early ( 2 hours) cytokine induction. In RAW264.7 cells, a 100 nM LPS challenge produces two waves of GFP-RelA translocation from the cytoplasm to the nucleus while gradually increasing the expression of mCherry (TNFα promoter activity). Continuous exposure of LPS-challenged cells to the BK- and Kv-channel blocker tetraethylammonium modifies the translocation dynamics of GFP-RelA and the induction of the TNFα promoter in a dose-dependent manner. Thus we provide evidence in support of a BK- and/or Kv-channel contribution to both early and later LPS induced inflammatory signaling.

Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers

Cultured cerebellar granule neurons (CGN) are commonly used to assess neurotoxicity, but are routinely maintained in supraphysiological (25 mM) extracellular K + concentrations [K +] o. We investigated the effect of potassium channel blockade on survival of CGN derived from Swiss-Webster mice in supraphysiological (25 mM) and physiological (5.6 mM) [K +] o. CGN were cultured for 5 days in 25 mM K +, then in 5.6 mM K + or 25 mM K + (control). Viability, assayed 24 h later by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and by lactate dehydrogenase (LDH) release, was ∼50% in 5.6 mM K + versus 25 mM K + ( p < .001). Potassium channel blockers, 2 mM 4-aminopyridine (4-AP), 2 mM tetraethylammonium (TEA) or 1 mM Ba 2+, individually afforded limited protection in 5.6 mM K +. However, survival in 5.6 mM K + with a combination of 4-AP, TEA and Ba 2+ was similar to survival in 25 mM K + without blockers ( p < .001 versus 5.6 mM K + alone). CGN survival in 25 mM K + was attenuated 25% by 2 μM nifedipine ( p > .001), but nifedipine did not attenuate neuroprotection by K + channel blockers. Together, these results suggest that the survival of CGN depends on the K + permeability of the membrane rather than the activity of a particular type of K + channel, and that the mechanism of neuroprotection by K + channel blockers is different from that of elevated [K +] o.

Sodium reabsorption in thick ascending limb of Henle's loop: effect of potassium channel blockade in vivo.

1. Based on previous in vitro studies, inhibition of K(+) recycling in thick ascending limb (TAL) is expected to lower Na(+) reabsorption through (i) reducing the luminal availability of K(+) to reload the Na(+)-2Cl(-)-K(+) cotransporter and (ii) diminishing the lumen positive transepithelial potential difference which drives paracellular cation transport. 2. This issue was investigated in anaesthetized rats employing microperfusion of Henle's loop downstream from late proximal tubular site with K(+)-free artificial tubular fluid in nephrons with superficial glomeruli. 3. The unselective K(+) channel blocker Cs(+) (5 - 40 mM) dose-dependently increased early distal tubular delivery of fluid and Na(+) with a maximum increase of approximately 20 and 185%, respectively, indicating predominant effects on water-impermeable TAL. 4. The modest inhibition of Na(+) reabsorption in response to the 15 mM of Cs(+) but not the enhanced inhibition by 20 mM Cs(+) was prevented by luminal K(+) supplementation. Furthermore, pretreatment with 20 mM Cs(+) did not attenuate the inhibitory effect of furosemide (100 microM) on Na(+)-2Cl(-)-K(+) cotransport. 5. Neither inhibitors of large (charybdotoxin 1 microM) nor low (glibenclamide 250 microM; U37883A 100 microM) conductance K(+) channels altered loop of Henle fluid or Na(+) reabsorption. 6. The intermediate conductance K(+) channel blockers verapamil and quinine (100 microM) modestly increased early distal tubular Na(+) but not fluid delivery, indicating a role for this K(+) channel in Na(+) reabsorption in TAL. As observed for equieffective concentrations of Cs(+) (15 mM), Na(+) reabsorption was preserved by K(+) supplementation. 7. The results indicate that modest inhibition of K(+) channels lowers the luminal availability of K(+) and thus transcellular Na(+) reabsorption in TAL. More complete inhibition lowers paracellular Na(+) transport probably by reducing or even abolishing the lumen positive transepithelial potential difference. Under the latter conditions, transcellular Na(+) transport may be restored by paracellular K(+) backleak.

Effect of potassium channel blockade and alpha 2-adrenoceptor activation on the release of nitric oxide from non-adrenergic non-cholinergic nerves.

1. Using a superfusion bioassay cascade, we studied the effect of K+ channel blockers and alpha 2-adrenoceptor agents on the release of a transferable factor, previously characterized as nitric oxide (NO) or a nitric oxide-related substance (NO-R), in response to non-adrenergic non-cholinergic (NANC) nerve stimulation in the canine ileocolonic junction (ICJ). 2. The non-selective K+ channel blockers, 4-aminopyridine (4-AP, 50 microM) and tetraethylammonium (TEA, 1 mM) and the more selective blocker of Ca(2+)-activated K+ channels, charybdotoxin (Leiurus quinquestriatus venom (LQV), 0.4 microgram ml-1), significantly enhanced the release of NO-R induced by low frequency stimulation (2-4 Hz). In the presence of 4-AP and TEA, the release of NO-R was nearly abolished by tetrodotoxin (2 microM), and by L-NG-nitroarginine (L-NOARG, 0.1 mM). Relaxations induced by direct injection of exogenous NO (5-50 pmol) or nitroglycerin (GTN, 10-30 pmol) onto the rabbit aortic detector ring were not affected. 3. The alpha 2-adrenoceptor agonist, UK-14,304 (0.3 microM) inhibited the release of NO-R induced by low (2-4 Hz), but not that induced by high (16 Hz), frequency stimulation. This inhibitory effect was completely reversed by the alpha 2-adrenoceptor antagonist, yohimbine (0.3 microM). Neither UK-14,304 nor yohimbine affected the relaxations induced by exogenous NO (5 pmol) or GTN (10 pmol) on the aortic detector ring.3+

Potential beneficial effects of potassium channel blockade by tedisamil in isolated perfused working rat heart with coronary artery ligation

Tedisamil, a potassium channel blocker, is known to produce bradycardia. The hypothesis tested was that tedisamil-induced bradycardia should improve the mechanical and metabolic properties of the ischemic myocardium. Using isolated perfused rat heart with coronary-artery ligation, tedisamil was compared with alinidine, verapamil, and propranolol. The end points were myocardial mechanical function and oxygen uptake. In coronary-ligated hearts, tedisamil (1.83×10-6 M) decreased heart rate, increased left ventricular dP/dtmax, and increased pressure power production. Efficiency of work rose with tedisamil. Alinidine (7×10-6 M) also decreased heart rate without altering left ventricular dP/dtmax, power production, or myocardial efficiency. Verapamil (1.5×10-7 M) did not alter heart rate but decreased left ventricular dP/dtmax, while power production and myocardial efficiency also fell. Propranolol (1×10-5 M) caused changes similar to verapamil, except that the heart rate also fell. Dobutamine (1.18×10-7 M) increased cardiac output and oxygen uptake without altering mechanical efficiency. Of the drugs tested and in the concentrations used in the coronary ligated rat heart, it was only tedisamil that had the capacity to cause less decrease in mechanical work and in myocardial efficiency than in controls.

Effect of potassium-channel blockade on refractoriness and ventricular vulnerability during acute ischemia

Effect of potassium-channel blockade on refractoriness and ventricular vulnerability during acute ischemia

Effects of potassium channel blockade on endogenous glutamate release from cerebellar slices

The effects of potassium channel blockade on the spontaneous release of endogenous glutamate from rat cerebellar slices was studied. Tetrapentylammonium (TPeA), 4-aminopyridine and quinine all increased the spontaneous release of glutamate. The effect of TPeA and 4-AP was potentiated in the absence of Ca 2+ from the perfusing fluid. In normal artificial cerebrospinal fluid (ACSF) the Ca 2+ channel antagonist, verapamil, mimicked the effects of TPeA seen in Ca 2+-free ACSF. The increased release of glutamate produced by TPeA under Ca 2+-free conditions was inhibited by the sodium channel blocker, tetrodotoxin, and by Ruthenium red, which inhibits mobilizftion of mitochondrialn Ca 2+. The results suggest that external Ca 2+ is not required in the TPeA-induced release of glutamate. It is proposed that the prolongation of depolarization seen with potassium channel blocking drugs enables sufficient sodium to enter the neurone and release Ca 2+ from intraneuronal stores in order to facilitate transmitter release.