BackgroundATP-sensitive potassium (KATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons.ResultsCell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i.This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit.ConclusionNO activates KATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate KATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of KATP current even after decrease of this current by painful-like nerve injury.

PEE could prevent the development of neuroinflammation and related POCD in aged rats by reversion of a proinflammatory phenotype of hippocampal microglia.

Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca 2ϩ accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca 2ϩ channel inhibition and K ϩ channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K ϩ current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-␣, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K ϩ channel activation and protection against ischemia-reperfusion injury.

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BackgroundATP-sensitive potassium (KATP) channels in neurons mediate neuroprotection, they regulate membrane excitability, and they control neurotransmitter release. Because loss of DRG neuronal KATP currents is involved in the pathophysiology of pain after peripheral nerve injury, we characterized the distribution of the KATP channel subunits in rat DRG, and determined their alterations by painful axotomy using RT-PCR, immunohistochemistry and electron microscopy.ResultsPCR demonstrated Kir6.1, Kir6.2, SUR1 and SUR2 transcripts in control DRG neurons. Protein expression for all but Kir6.1 was confirmed by Western blots and immunohistochemistry. Immunostaining of these subunits was identified by fluorescent and confocal microscopy in plasmalemmal and nuclear membranes, in the cytosol, along the peripheral fibers, and in satellite glial cells. Kir6.2 co-localized with SUR1 subunits. Kir6.2, SUR1, and SUR2 subunits were identified in neuronal subpopulations, categorized by positive or negative NF200 or CGRP staining. KATP current recorded in excised patches was blocked by glybenclamide, but preincubation with antibody against SUR1 abolished this blocking effect of glybenclamide, confirming that the antibody targets the SUR1 protein in the neuronal plasmalemmal membrane.In the myelinated nerve fibers we observed anti-SUR1 immunostaining in regularly spaced funneled-shaped structures. These structures were identified by electron microscopy as Schmidt-Lanterman incisures (SLI) formed by the Schwann cells. Immunostaining against SUR1 and Kir6.2 colocalized with anti-Caspr at paranodal sites.DRG excised from rats made hyperalgesic by spinal nerve ligation exhibited similar staining against Kir6.2, SUR1 or SUR2 as DRG from controls, but showed decreased prevalence of SUR1 immunofluorescent NF200 positive neurons. In DRG and dorsal roots proximal to axotomy SLI were smaller and showed decreased SUR1 immunofluorescence.ConclusionsWe identified Kir6.2/SUR1 and Kir6.2/SUR2 KATP channels in rat DRG neuronal somata, peripheral nerve fibers, and glial satellite and Schwann cells, in both normal state and after painful nerve injury. This is the first report of KATP channels in paranodal sites adjacent to nodes of Ranvier and in the SLI of the Schwann cells. After painful axotomy KATP channels are downregulated in large, myelinated somata and also in SLI, which are also of smaller size compared to controls.Because KATP channels may have diverse functional roles in neurons and glia, further studies are needed to explore the potential of KATP channels as targets of therapies against neuropathic pain and neurodegeneration.

These results show that isoflurane impairs insulin secretion and glucose utilization. The mechanism of action responsible for these effects may involve a decrease in glucose-induced inhibition of adenosine triphosphate-sensitive potassium channel activity in pancreatic beta cells.

Background The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca2+ signaling in primary sensory neurons after injury. We therefore examined the effect of injury on intracellular Ca2+ stores of the endoplasmic reticulum, which critically regulate the Ca2+ signal and neuronal function. Methods Intracellular Ca2+ levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats following L5 spinal nerve ligation, compared to neurons from control animals. Results Endoplasmic reticulum Ca2+ stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca2+-free bath solution that removes the contribution of store-operated membrane Ca2+ channels, and after blockade of both the mitochondrial, sarco-endoplasmic Ca2+-ATPase, and the plasma membrane Ca2+ ATPase pathways. Ca2+ released by the sarco-endoplasmic Ca2+-ATPase blocker thapsigargin and by the Ca2+-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca2+ stores in axotomized neurons were not expanded by neuronal activation by K+ depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca2+-ATPase was normal. Luminal Ca2+ concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation. Conclusions Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca2+ stores and a reduced luminal Ca2+ concentration. Depletion of Ca2+ stores may contribute to the pathogenesis of neuropathic pain.

Painful axotomy decreases KATP channel current (IKATP) in primary afferent neurons. Because cytosolic Ca 2؉ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca 2؉ -calmodulin (CaM)-Ca 2؉ /CaM-dependent kinase II (CaMKII) regulates IK ATP in large DRG neurons. Immunohistochemistry identified the presence of K ATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200 -positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK ATP by ionomycin, a Ca 2؉ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K ATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca 2؉ -evoked K ATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK ATP by glybenclamide, a selective KATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K ATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK ATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via K ATP channel inhibition. Unitary current conductance and sensitivity of K ATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.calcium ͉ calmodulin ͉ potassium channels ͉ dorsal root ganglia ͉ neuropathic pain A fter peripheral nerve injury, phenotypic changes in axons and the corresponding somata in the dorsal root ganglia (DRG) lead to membrane hyperexcitability, which results in neuropathic pain (1, 2). These alterations include decreased Ca 2ϩ influx via voltage-gated calcium channels (VGCC), reduced [Ca 2ϩ ] i , and diminished Ca 2ϩ -induced Ca 2ϩ release (3, 4). Additionally, ATPsensitive potassium (K ATP ) channels in large axotomized DRG neurons from rats with hyperalgesia exhibit decreased opening (5, 6). K ATP channels in cardiac myocytes and pancreatic ␤-cells are modulated by cytosolic Ca 2ϩ (7,8), but it remains unknown whether Ca 2ϩ affects neuronal K ATP channels.Cytosolic Ca 2ϩ regulates neuronal channels and other targets via multiple mechanisms (9), including the Ca 2ϩ -calmodulin (CaM)-Ca 2ϩ /CaM-dependent kinase II (CaMKII) pathway (10). CaMKII, which is abundant in neurons (11), assembles into a dodecameric holoenzymatic structure (12, 13). Ca 2ϩ /CaM activates CaMKII subunits, triggering autophosphorylation at T286 residues (14) that increase the affinit...