25-Hydroxycholesterol as a Signaling Molecule of the Nervous System.

Cholesterol is an essential component of plasma membrane and precursor of biological active compounds, including hydroxycholesterols (HCs). HCs regulate cellular homeostasis of cholesterol; they can pass across the membrane and vascular barriers and act distantly as para- and endocrine agents. A small amount of 25-hydroxycholesterol (25-HC) is produced in the endoplasmic reticulum of most cells, where it serves as a potent regulator of the synthesis, intracellular transport, and storage of cholesterol. Production of 25-HC is strongly increased in the macrophages, dendrite cells, and microglia at the inflammatory response. The synthesis of 25-HC can be also upregulated in some neurological disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, spastic paraplegia type 5, and X-linked adrenoleukodystrophy. However, it is unclear whether 25-HC aggravates these pathologies or has the protective properties. The molecular targets for 25-HC are transcriptional factors (LX receptors, SREBP2, ROR), G protein-coupled receptor (GPR183), ion channels (NMDA receptors, SLO1), adhesive molecules (α5ß1 and ανß3 integrins), and oxysterol-binding proteins. The diversity of 25-HC-binding proteins points to the ability of HC to affect many physiological and pathological processes. In this review, we focused on the regulation of 25-HC production and its universal role in the control of cellular cholesterol homeostasis, as well as the effects of 25-HC as a signaling molecule mediating the influence of inflammation on the processes in the neuromuscular system and brain. Based on the evidence collected, it can be suggested that 25-HC prevents accumulation of cellular cholesterol and serves as a potent modulator of neuroinflammation, synaptic transmission, and myelinization. An increased production of 25-HC in response to a various type of damage can have a protective role and reduce neuronal loss. At the same time, an excess of 25-HC may exert the neurotoxic effects.

Membrane cholesterol oxidation downregulates atrial ß-adrenergic responses in ROS-dependent manner.

Oxidation of membrane cholesterol is a hallmark of many pathological conditions, including cardiovascular diseases. Cholesterol could be oxidized in a result of free radical and enzymatic reactions. Here, we studied the effect of cholesterol oxidation by cholesterol oxidase (ChO) on responses to ß-AR stimulation in isolated mouse atria. Acute exposure to ChO led to partial cholesterol oxidation without a significant change in atrial membrane cholesterol content. Pretreatment with ChO itself did not affect contractions and Ca2+ transient amplitude. However, cholesterol oxidation markedly suppressed ß-AR-mediated increase in contractility and Ca2+ transient as well as NO levels. At the same time, ChO markedly facilitated ß-AR-induced reactive oxygen species (ROS) production. Antioxidant and protein kinase C inhibitor prevented the depressant action of ChO on ISO-dependent contractility, Ca2+ transient and NO production. Similar effects had a selective ß2-AR antagonist, which also suppressed the increase in ROS levels after ChO pretreatment. These results suggest that membrane cholesterol oxidation enhances ß2-AR-dependent elevation of ROS production, leading to suppression of ß-AR-mediated increase in contractility, Ca2+ transient and NO synthesis in mice atria. The oxidative cholesterol modification could contribute to disturbance in ß-AR signaling in pathological conditions.

Brain cholesterol metabolite 24-hydroxycholesterol modulates inotropic responses to ß-adrenoceptor stimulation: The role of NO and phosphodiesterase.

AIMS: 24-Hydroxycholesterol (24HC) is the main brain cholesterol metabolite, which level in the circulation is significantly changed under physiological and pathological conditions. Here, we have studied the effect of 24HC on the inotropic responses to ß-adrenoceptor (AR) stimulation. MAIN METHODS: Electrical stimulation-evoked contractions were recorded in isolated atria from mice. Fluorescent dyes, Fluo-4 and DAF-FM, were used for estimation of Ca2+ transient and NO production, respectively. KEY FINDINGS: We revealed that 24HC in the submicromolar range attenuated ß-AR-induced positive inotropy in isolated atria. This was accompanied by a decrease in Ca2+ transient and unchanged nitric oxide (NO) production. However, ß1-AR-induced positive inotropy and enhancement of Ca2+ transient were increased by 24HC due to suppression of NO production. Only ß2-AR-dependent inotropy and enhancement of Ca2+ transient were decreased by 24HC in a NO-independent manner. Inhibition of phosphodiesterase (PDE) suppressed effect of 24HC on ß2-AR-dependent contractility as well as on non-subtype specific ß-AR activation. Moreover, 24HC counteracted positive inopropic action of PDE inhibitors, IBMX and rolipam. Thus, 24HC modulates the effects of ß1- and ß2-AR stimulation via different mechanisms linked with change in activity of NO synthase or PDE, respectively. Under conditions of non-selective activation of ß-ARs, the depressant effect of 24HC related with ß2-AR-dependent signaling dominates. SIGNIFICANCE: We suggest that 24HC could serve as a modulator of atrial ß-AR signaling, contributing to regulation of contractility.

ß2-adrenoceptor agonist-evoked reactive oxygen species generation in mouse atria: implication in delayed inotropic effect.

Fenoterol, a ß2-adrenoceptor agonist, has anti-apoptotic action in cardiomyocytes and induces a specific pattern of downstream signaling. We have previously reported that exposure to fenoterol (5 µM) results in a delayed positive inotropic effect which is related to changes in both Ca2+ transient and NO. Here, the changes in reactive oxygen species (ROS) production in response to the fenoterol administration and the involvement of ROS in effect of this agonist on contractility were investigated in mouse isolated atria. Stimulation of ß2-adrenoceptor increases a level of extracellular ROS, while intracellular ROS level rises only after removal of fenoterol from the bath. NADPH-oxidase inhibitor (apocynin) prevents the increase in ROS production and the Nox2 isoform is immunofluorescently colocalized with ß2-adrenoceptor at the atrial myocytes. Treatments with antioxidants (N-acetyl-L-cysteine, NADPH inhibitors, exogenous catalases) significantly inhibit the fenoterol induced increase in the contraction amplitude, probably by attenuating Ca2+ transient and up-regulating NO production. ROS generated in a ß2-adrenoceptor-dependent manner can potentiate the activity of some Ca2+ channels. Indeed, inhibition of ryanodine receptors, TRPV-or L-type Ca2+- channels shows a similar efficacy in reduction of positive inotropic effect of both fenoterol and H2O2. In addition, detection of mitochondrial ROS indicates that fenoterol triggers a slow increase in ROS which is prevented by rotenone, but rotenone has no impact on the inotropic effect of fenoterol. We suggest that stimulation of ß2-adrenoceptor with fenoterol causes the activation of NADPH-oxidase and after the agonist removal extracellularly generated ROS penetrates into the cell, increasing the atrial contractions probably via Ca2+ channels.