Citral inhibits the nociception in the rat formalin test: Effect of metformin and blockers of opioid receptor and the NO-cGMP-K+ channel pathway

The objective of the present study was to scrutinize the effect of nitric oxide (NO), cGMP, potassium channel blockers and metformin on the citral-produced peripheral antinociception. The rat paw 1% formalin test was used to assess nociception and antinociception. Rats were treated with local peripheral administration of citral (10-100 µg/paw). The antinociception of citral (100 µg/paw) was evaluated with and without the local pretreatment of naloxone, NG-L-nitro-arginine methyl ester (L-NAME, a NO synthesis inhibitor), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor), metformin, opioid receptors antagonists, and K+ channel blockers. Injection of citral in the rat paw significantly decreased the nociceptive effect of formalin administration during the two phases of the test. Local pretreatment of the paws with L-NAME and ODQ did not reduced the citral-induced antinociception. Glipizide or glibenclamide (Kir6.1-2; ATP-sensitive K+ channel blockers), tetraethylammonium or 4-aminopyridine (KV; voltage-gated K+ channel blockers) or charybdotoxin (KCa1.1; big conductance calcium-activated K+ channel blocker) or apamin (KCa2.1-3; small conductance Ca2+-activated K+ channel antagonist), or metformin, but not the opioid antagonists, reduced the antinociception of citral. Citral produced peripheral antinociception during both phases of the formalin test. These effects were due to the activation of K+ channels and a biguanide-dependent mechanism.

SK Channels Modulation Accelerates Equilibrium Recovery in Unilateral Vestibular Neurectomized Rats

We have previously reported in a feline model of acute peripheral vestibulopathy (APV) that the sudden, unilateral, and irreversible loss of vestibular inputs induces selective overexpression of small conductance calcium-activated potassium (SK) channels in the brain stem vestibular nuclei. Pharmacological blockade of these ion channels by the selective antagonist apamin significantly alleviated the evoked vestibular syndrome and accelerated vestibular compensation. In this follow-up study, we aimed at testing, using a behavioral approach, whether the antivertigo (AV) effect resulting from the antagonization of SK channels was species-dependent or whether it could be reproduced in a rodent APV model, whether other SK channel antagonists reproduced similar functional effects on the vestibular syndrome expression, and whether administration of SK agonist could also alter the vestibular syndrome. We also compared the AV effects of apamin and acetyl-DL-leucine, a reference AV compound used in human clinic. We demonstrate that the AV effect of apamin is also found in a rodent model of APV. Other SK antagonists also produce a trend of AV effect when administrated during the acute phase of the vertigo syndrome. Conversely, the vertigo syndrome is worsened upon administration of SK channel agonist. It is noteworthy that the AV effect of apamin is superior to that of acetyl-DL-leucine. Taken together, these data reinforce SK channels as a pharmacological target for modulating the manifestation of the vertigo syndrome during APV.

Single-Channel Permeability from Markovian Milestoning, Umbrella Sampling, and Voltage Simulations

Permeations of ions and small molecules through membrane channels have diverse functions within cells. Various all-atom molecular dynamics (MD) simulations methods have been developed for computing free energy and crossing rate of permeants. However, a systemic comparison across different methods is scarce. Here, using a carbon nanotube as a model of small conductance (~2 pS) ion channel, we systemically compared three classes of MD-based approaches for computing single-channel permeability for potassium ion: equilibrium free energy-based approach using umbrella sampling, rare-even sampling approach using Markovian milestoning, and steady-state approach using applied voltages. The consistent kinetic results from all three methods demonstrated the robustness of MD-based methods in computing ion channel permeation. Two solvent boundary conditions are tested for milestoning and yield consistent forward and backward mean first passage time (MFPT). The advantages and disadvantages of each method are discussed, with the focus on the future applications of milestoning in more complex systems.

Endothelium-Derived Hyperpolarizing Factor (EDHF) Mediates Acetylsalicylic Acid (Aspirin) Vasodilation of Pregnant Rat Mesenteric Arteries

Acetylsalicylic acid (aspirin) exhibits a broad range of activities, including analgesic, antipyretic, and antiplatelet properties. Recent clinical studies also recommend aspirin prophylaxis in women with a high risk of pre-eclampsia, a major complication of pregnancy characterized by hypertension. We investigated the effect of aspirin on mesenteric resistance arteries and found outdiscovered the molecular mechanism underlying this action. Aspirin (10−12–10−6 M) was tested on pregnant rat mesenteric resistance arteries by a pressurized arteriography. Aspirin was investigated in the presence of several inhibitors of: (a) nitric oxide synthase (L-NAME 2 × 10−4 M); (b) cyclooxygenase (Indomethacin, 10−5 M); (c) Ca2+-activated K+ channels (Kca): small conductance (SKca, Apamin, 10−7 M), intermediate conductance (IKca, TRAM34, 10−5 M), and big conductance (BKca, paxilline, 10−5 M); and (d) endothelial-derived hyperpolarizing factor (high KCl, 80 mM). Aspirin caused a concentration-dependent vasodilation. Aspirin-vasodilation was abolished by removal of endothelium or by high KCl. Furthermore, preincubation with either apamin plus TRAM-34 or paxillin significantly attenuated aspirin vasodilation (p < 0.05). For the first time, we showed that aspirin induced endothelium-dependent vasodilation in mesenteric resistance arteries through the endothelial-derived hyperpolarizing factor (EDHF) and calcium-activated potassium channels. By activating this molecular mechanism, aspirin may lower peripheral vascular resistance and be beneficial in pregnancies complicated by hypertension.

Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

Mechanosensitive channel gating by delipidation

The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-β-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.

Ginkgo biloba L. Prevents Hypobaric Hypoxia–Induced Spatial Memory Deficit Through Small Conductance Calcium-Activated Potassium Channel Inhibition: The Role of ERK/CaMKII/CREB Signaling

Hypobaric hypoxia (HH) is a stressful condition, which is more common at high altitudes and can impair cognitive functions. Ginkgo biloba L. leaf extract (GBE) is widely used as herbal medicine against different disorders. Its ability to improve cognitive functions, reduce oxidative stress, and promote cell survival makes it a putative therapeutic candidate against HH. The present study has been designed to explore the effect of GBE on HH-induced neurodegeneration and memory impairment as well as possible signaling mechanisms involved. 220–250 gm (approximately 6- to 8-week-old) Sprague Dawley rats were randomly divided into different groups. GBE was orally administered to respective groups at a dose of 100 mg/kg/day throughout the HH exposure, i.e., 14 days. Memory testing was performed followed by hippocampus isolation for further processing of different molecular and morphological parameters related to cognition. The results indicated that GBE ameliorates HH-induced memory impairment and oxidative damage and reduces apoptosis. Moreover, GBE modulates the activity of the small conductance calcium-activated potassium channels, which further reduces glutamate excitotoxicity and apoptosis. The exploration of the downstream signaling pathway demonstrated that GBE administration prevents HH-induced small conductance calcium-activated potassium channel activation, and that initiates pro-survival machinery by activating extracellular signal–regulated kinase (ERK)/calmodulin-dependent protein kinase II (CaMKII) and the cAMP response element–binding protein (CREB) signaling pathway. In summary, the current study demonstrates the beneficial effect of GBE on conditions like HH and provides various therapeutic targets involved in the mechanism of action of GBE-mediated neuroprotection.