Antinociceptive Effects of Aza-Bicyclic Isoxazoline-Acylhydrazone Derivatives in Different Models of Nociception in Mice

Background: In a study recently published by our research group, the compounds isoxazoline-acylhydrazone derivatives R-99 and R-123 presented promising antinociceptive activity. However, the mechanism of action of this compound is still unknown. Objective: This study aimed to assess the mechanisms involved in the antinociceptive activity of these compounds in chemical models of pain. Methods: Animals were orally pretreated and evaluated in the acetic acid-, formalin-, capsaicin-, carrageenan- and Complete Freund's Adjuvant (CFA)-induced pain models in mice. The effects of the compounds after pretreatment with naloxone, prazosin, yohimbine, atropine, L-arginine, or glibenclamide were studied, using the acetic acid-induced writhing test to verify the possible involvement of opioid, α1-adrenergic, α2-adrenergic or cholinergic receptors, and nitric oxide or potassium channels pathways, respectively. Results: R-99 and R-123 compounds showed significant antinociceptive activity on pain models induced by acetic acid, formalin, and capsaicin. Both compounds decreased the mechanical hyperalgesia induced by carrageenan or CFA in mice. The antinociceptive effects of R-99 and R-123 on the acetic acid-induced writhing test were significantly attenuated by pretreatment with naloxone, yohimbine or atropine. R-99 also showed an attenuated response after pretreatment with atropine and glibenclamide. However, on the pretreatment with prazosin, there was no change in the animals' response to both compounds. Conclusion: R-99 and R-123 showed antinociceptive effects related to mechanisms that involve, at least in part, interaction with the opioid and adrenergic systems and TRPV1 pathways. The compound R-99 also interacts with the cholinergic pathways and potassium channels.

Alternative Targets for Modulators of Mitochondrial Potassium Channels

Mitochondrial potassium channels control potassium influx into the mitochondrial matrix and thus regulate mitochondrial membrane potential, volume, respiration, and synthesis of reactive oxygen species (ROS). It has been found that pharmacological activation of mitochondrial potassium channels during ischemia/reperfusion (I/R) injury activates cytoprotective mechanisms resulting in increased cell survival. In cancer cells, the inhibition of these channels leads to increased cell death. Therefore, mitochondrial potassium channels are intriguing targets for the development of new pharmacological strategies. In most cases, however, the substances that modulate the mitochondrial potassium channels have a few alternative targets in the cell. This may result in unexpected or unwanted effects induced by these compounds. In our review, we briefly present the various classes of mitochondrial potassium (mitoK) channels and describe the chemical compounds that modulate their activity. We also describe examples of the multidirectional activity of the activators and inhibitors of mitochondrial potassium channels.

Mitochondrial potassium channels: A novel calcitriol target

Background Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane. Methods The effects of calcitriol on the potassium ion current were measured using the patch-clamp method modified for the inner mitochondrial membrane. Molecular docking experiments were conducted in the Autodock4 program. Additionally, changes in gene expression were investigated by qPCR, and transcription factor binding sites were analyzed in the CiiiDER program. Results For the first time, our results indicate that calcitriol directly affects the activity of the mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa) from the human astrocytoma (U-87 MG) cell line but not the mitochondrial calcium-independent two-pore domain potassium channel (mitoTASK-3) from human keratinocytes (HaCaT). The open probability of the mitoBKCa channel in high calcium conditions decreased after calcitriol treatment and the opposite effect was observed in low calcium conditions. Moreover, using the AutoDock4 program we predicted the binding poses of calcitriol to the calcium-bound BKCa channel and identified amino acids interacting with the calcitriol molecule. Additionally, we found that calcitriol influences the expression of genes encoding potassium channels. Such a dual, genomic and non-genomic action explains the pleiotropic activity of calcitriol. Conclusions Calcitriol can regulate the mitochondrial large-conductance calcium-regulated potassium channel. Our data open a new chapter in the study of non-genomic responses to vitamin D with potential implications for mitochondrial bioenergetics and cytoprotective mechanisms.

Expression of Kv4.2 and Kv4.3 potassium channels in human umbilical veins from normal, diabetic and hypertensive pregnancies

Objective: A substantial line of evidence indicates that Kv4.2 and Kv4.3 channels are the major components of rapid transient-outward potassium currents (A-type currents). It is speculated that those currents may be involved in the maintenance of the membrane potential, as well as in the regulation of propagation and frequency of action potentials. However, very little is known about the presence and function of A-type currents in human vascular smooth muscles such as human umbilical vein (HUV). Having in mind its crucial role in the proper fetal oxygenation the aim of the study was to determine whether Kv4.2 and Kv4.3 potassium channels are present in HUV smooth muscle and to investigate potential alterations of their expression during maternal pathological conditions - gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH). Materials and methods: Healthy, diabetic and hypertensive pregnancies were subjects of this investigation. Each group was consisted of 6 HUV samples obtained from 6 normal pregnancies, 6 pregnancies with GDM, and 6 pregnancies with PIH. After pharmacology analysis, immunohistochemistry and Western blot were performed. Results: Immunohistochemistry revealed similar expression pattern of both, Kv4.2 and Kv4.3 subunits, in HUV smooth muscle in all groups of patients. Results obtained by Western blot were in agreement with immunohistochemical staining. The expression of Kv4.2 and Kv4.3 subunits was not significantly different between the groups. Conclusion: Collectively, this is the first study that demonstrated presence of Kv4.2 and Kv4.3 potassium channels in the HUV smooth muscle and their preservation during the course of GDM and PIH. These channels are most likely major components of rapid A-type currents that may be relevant for maternal-fetus blood flow and hence fetal development. Also, they may represent sensors for detecting hemodynamic and/or metabolic changes in the local environment.

Activation of TRESK background potassium channels by cloxyquin exerts protective effects against excitotoxic-induced brain injury and neuroinflammation in neonatal rats

The excitotoxicity is a common pathological mechanism of perinatal brain injuries (PBI), however neuroinflammation resulting in PBI is both a cause and a consequence of excitotoxicity. TRESK background potassium channels are an important regulator of neuronal excitability. We therefore investigated effects of activation of TRESK channels by selective activator cloxyquin on excitotoxic-induced brain injury and neuroinflammation involving brain mast cells and inflammatory cytokines in neonatal rats. An excitotoxic model mimicking human perinatal brain lesions was established via intracerebral injection of the glutamatergic agonist ibotenate to into newborn rats. P5 rat pups were intraperitoneally pretreated with vehicle, three different doses of cloxyquin (0.2, 1 and 5 mg/kg), or NMDA receptor antagonist MK-801 (positive control) 30 minutes prior to intracerebral injection of 10 µg ibotenate. Rat pups were sacrificed one or five days after the injury. Coronal brain sections were stained with cresyl-violet for histopathological examinations, and with toluidine-blue for brain mast cells assessments. Concentrations of activin A, IL-1β, IL-6 and IL-10 in brain homogenates were measured using ELISA. Cloxyquin dose-dependently ameliorated ibotenate-induced impairments in the cortical and white matter, and suppressed ibotenate-induced activation and number of brain mast cells. Moreover, cloxyquin dose-dependently reduced concentrations of activin A, IL-1β and IL-6 in the brain tissue induced by ibotenate while it elevated IL-10 level. Our findings reveal for the first time that cloxyquin, a selective activator of TRESK channels, dose-dependently exerted protective effects against excitotoxic-induced neonatal brain injury and neuroinflammation. TRESK channels may be a promising new target for the treatment of PBIs.

The Molecular Basis for the Calcium-Dependent Slow Afterhyperpolarization in CA1 Hippocampal Pyramidal Neurons

Neuronal signal transmission depends on the frequency, pattern, and timing of spike output, each of which are shaped by spike afterhyperpolarizations (AHPs). There are classically three post-spike AHPs of increasing duration categorized as fast, medium and slow AHPs that hyperpolarize a cell over a range of 10 ms to 30 s. Intensive early work on CA1 hippocampal pyramidal cells revealed that all three AHPs incorporate activation of calcium-gated potassium channels. The ionic basis for a fAHP was rapidly attributed to the actions of big conductance (BK) and the mAHP to small conductance (SK) or Kv7 potassium channels. In stark contrast, the ionic basis for a prominent slow AHP of up to 30 s duration remained an enigma for over 30 years. Recent advances in pharmacological, molecular, and imaging tools have uncovered the expression of a calcium-gated intermediate conductance potassium channel (IK, KCa3.1) in central neurons that proves to contribute to the slow AHP in CA1 hippocampal pyramidal cells. Together the data show that the sAHP arises in part from a core tripartite complex between Cav1.3 (L-type) calcium channels, ryanodine receptors, and IK channels at endoplasmic reticulum-plasma membrane junctions. Work on the sAHP in CA1 pyramidal neurons has again quickened pace, with identified contributions by both IK channels and the Na-K pump providing answers to several mysteries in the pharmacological properties of the sAHP.