Water and ion channels : crucial in the initiation and progression of apoptosis in central nervous system?

Programmed cell death (PCD), is a highly regulated and sophisticated cellular mechanism that commits cell to isolated death fate. PCD has been implicated in the pathogenesis of numerous neurodegenerative disorders. Countless molecular events underlie this phenomenon, with each playing a crucial role in death commitment. A precedent event, apoptotic volume decrease (AVD), is ubiquitously observed in various forms of PCD induced by different cellular insults. Under physiological conditions, cells when subjected to osmotic fluctuations will undergo regulatory volume increase/decrease (RVI/RVD) to achieve homeostatic balance with neurons in the brain being additionally protected by the blood-brain-barrier. However, during AVD following apoptotic trigger, cell undergoes anistonic shrinkage that involves the loss of water and ions, particularly monovalent ions e.g. K+, Na+ and Cl-. It is worthwhile to concentrate on the molecular implications underlying the loss of these cellular components which posed to be significant and crucial in the successful propagation of the apoptotic signals. Microarray and real-time PCR analyses demonstrated several ion and water channel genes are regulated upon the onset of lactacystin (a proteosomal inhibitor)-mediated apoptosis. A time course study revealed that gene expressions of water and ion channels are being modulated just prior to apoptosis, some of which are aquaporin 4 and 9, potassium channels and chloride channels. In this review, we shall looked into the molecular protein machineries involved in the execution of AVD in the central nervous system (CNS), and focus on the significance of movements of each cellular component in affecting PCD commitment, thus provide some pharmacological advantages in the global apoptotic cell death.

a7-Nicotinic acetylcholine receptors mediate an A71-42-induced increase in the level of acetylcholinesterase in primary cortical neurones

The β-amyloid protein (Aβ) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Aβ have suggested that this increase may be due to a direct action of Aβ on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Aβ increases levels of AChE in primary cortical neurones. Aβ₁₋₄₂ was more potent than Aβ₁₋₄₀ in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Aβ peptides. The effect of Aβ₁₋₄₂ on AChE was blocked by inhibitors of α7 nicotinic acetylcholine receptors (α7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of α7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Aβ₁₋₄₂ on AChE is due to an agonist effect of Aβ₁₋₄₂ on the α7 nAChR.

Lamotrigine and the treatment of mania in bipolar disorder

Anticonvulsants, including valproate and carbamazepine, have established efficacy in the treatment of mania. The anticonvulsant, lamotrigine. has been reported to have antimanic and antidepressant efficacy, and mood-stabilising effects in case reports and preliminary open trials. The efficacy and tolerability of lamotrigine has been compared with olanzapine and lithium in a randomised, prospective, controlled fashion over a period of 4 weeks treatment in a total of 45 hospitalised patients with DSM-IV-defined mania. Significant improvements of a similar magnitude were observed for all treatment groups and lamotrigine was well tolerated. Mechanisms of action proposed to explain the antimanic activity of lamotrigine include inhibition of voltage-sensitive and use-dependent sodium channels, inhibition of glutamate release and calcium channel blockade. Platelet studies have indicated supersensitivity of glutamate receptors and increased intracellular calcium concentrations in patients with mania. Further clinical and mechanistic studies of lamotrigine use in mania are warranted.

Vasodilatory effects of homologous adrenomedullin 2 and adrenomedullin 5 on isolated blood vessels of two species of eel

In mammals, adrenomedullin (AM) is a potent vasodilator through signalling pathways that involve the endothelium. In teleost fishes, a family of five AMs are present (AM1/4, AM2/3 and AM5) with four homologous AMs (AM1, AM2/3 and AM5) recently cloned from the Japanese eel, Anguilla japonica. Both AM2 and AM5 have been shown to be strong in vivo vasodepressors in eel, but the mechanism of action of homologous AMs on isolated blood vessels has not been examined in teleost fish. In this study, both eel AM2 and AM5 caused a marked vasodilation of the dorsal aorta. However, only AM5 consistently dilated the small gonadal artery in contrast to AM2 that had no effect in most preparations. Neither AM2 nor AM5 had any effect when applied to the first afferent branchial artery; in contrast, eel ANP always caused a large vasodilation of the branchial artery. In the dorsal aorta, indomethacin significantly reduced the AM2 vasodilation, but had no effect on the AM5 vasodilation. In contrast, removal of the endothelium significantly enhanced the AM5 vasodilation only. In the gonadal artery, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) significantly reduced the AM5 vasodilation suggesting a role for soluble guanylyl cyclase in the dilation, but l-NNA and removal of the endothelium had no effect. The results of this study indicate that AM2 and AM5 have distinct vasodilatory effects that may be due to the peptides signalling via different receptors to regulate vascular tone in eel.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

ω-Conotoxin GVIA mimetics that bind and inhibit neuronal Cav2.2 ion channels

The neuronal voltage-gated N-type calcium channel (Ca_v2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a ¹²⁵I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca_v2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the ¹²⁵I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.

Potassium channel modulation by a toxin domain in matrix metalloprotease

Peptide toxins found in a wide array of venoms block K+ channels, causing profound physiological and pathological effects. Here we describe the first functional K+ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K+ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K+ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K+ channels by co-localizing with and trapping MMP23TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.