Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules.

Microtubule (MT) severing enzymes Katanin and Spastin cut the MT into smaller fragments and are being studied extensively usingin-vitroexperiments due to their crucial role in different cancers and neurodevelopmental disorders. It has been reported that the severing enzymes are either involved in increasing or decreasing the tubulin mass. Currently, there are a few analytical and computational models for MT amplification and severing. However, these models do not capture the action of MT severing explicitly, as these are based on partial differential equations in one dimension. On the other hand, a few discrete lattice-based models were used earlier to understand the activity of severing enzymes only on stabilized MTs. Hence, in this study, discrete lattice-based Monte Carlo models that included MT dynamics and severing enzyme activity have been developed to understand the effect of severing enzymes on tubulin mass, MT number, and MT length. It was found that the action of severing enzyme reduces average MT length while increasing their number; however, the total tubulin mass can decrease or increase depending on the concentration of GMPCPP (Guanylyl-(α,ß)-methylene-diphosphonate)-which is a slowly hydrolyzable analogue of GTP (Guanosine triphosphate). Further, relative tubulin mass also depends on the detachment ratio of GTP/GMPCPP and Guanosine diphosphate tubulin dimers and the binding energies of tubulin dimers covered by the severing enzyme.

The newborn Fmr1 knockout mouse: a novel model of excess ubiquinone and closed mitochondrial permeability transition pore in the developing heart.

BACKGROUND: Mitochondrial permeability transition pore (mPTP) closure triggers cardiomyocyte differentiation during development while pathological opening causes cell death during myocardial ischemia-reperfusion and heart failure. Ubiquinone modulates the mPTP; however, little is known about its mechanistic role in health and disease. We previously found excessive proton leak in newborn Fmr1 KO mouse forebrain caused by ubiquinone deficiency and increased open mPTP probability. Because of the physiological differences between the heart and brain during maturation, we hypothesized that developing Fmr1 KO cardiomyocyte mitochondria would demonstrate dissimilar features. METHODS: Newborn male Fmr1 KO mice and controls were assessed. Respiratory chain enzyme activity, ubiquinone content, proton leak, and oxygen consumption were measured in cardiomyocyte mitochondria. Cardiac function was evaluated via echocardiography. RESULTS: In contrast to controls, Fmr1 KO cardiomyocyte mitochondria demonstrated increased ubiquinone content and decreased proton leak. Leak was cyclosporine (CsA)-sensitive in controls and CsA-insensitive in Fmr1 KOs. There was no difference in absolute mitochondrial respiration or cardiac function between strains. CONCLUSION: These findings establish the newborn Fmr1 KO mouse as a novel model of excess ubiquinone and closed mPTP in the developing heart. Such a model may help provide insight into the biology of cardiac development and pathophysiology of neonatal heart failure. IMPACT: Ubiquinone is in excess and the mPTP is closed in the developing FXS heart. Strengthens evidence of open mPTP probability in the normally developing postnatal murine heart and provides new evidence for premature closure of the mPTP in Fmr1 mutants. Establishes a novel model of excess CoQ and a closed pore in the developing heart. Such a model will be a valuable tool used to better understand the role of ubiquinone and the mPTP in the neonatal heart in health and disease.

Novel Liposome Eencapsulated Guanosine Di Phosphate based Therapeutic Target against Anemia of Inflammation.

Hepcidin, master regulator of iron homeostasis, causes anemia under infectious and inflammatory conditions by reducing intestinal absorption of iron with decreased release of iron from macrophages and liver despite adequate iron stores leading to Anemia of Inflammation (AI). Many therapeutic trials have been carried out but none have been effective due to its adverse effects. In present study, we discover that Guanosine 5'-diphosphate (GDP) encapsulated in lipid vesicle (NH+) was found to inhibit NF-ҝB activation by limiting phosphorylation and degradation of IҝBα, thus, attenuating IL-6 secretion from macrophage cells. Moreover, the suppressed IL-6 levels down regulated JAK2/STAT3 pathway with decrease inflammation-mediated Hamp mRNA transcription (HepG2) and increase iron absorption (Caco2) in HepG2/Caco2 co-culture model. Analogous results were obtained in acute and chronic AI mice model thus, correcting haemoglobin level. These results proved NH + GDP as novel therapeutic agent to overcome limitations and suggests it as potential drug to ameliorate AI.

Gi/o protein-coupled receptors in dopamine neurons inhibit the sodium leak channel NALCN.

Dopamine (D2) receptors provide autoinhibitory feedback onto dopamine neurons through well-known interactions with voltage-gated calcium channels and G protein-coupled inwardly-rectifying potassium (GIRK) channels. Here, we reveal a third major effector involved in D2R modulation of dopaminergic neurons - the sodium leak channel, NALCN. We found that activation of D2 receptors robustly inhibits isolated sodium leak currents in wild-type mice but not in NALCN conditional knockout mice. Intracellular GDP-ßS abolished the inhibition, indicating a G protein-dependent signaling mechanism. The application of dopamine reliably slowed pacemaking even when GIRK channels were pharmacologically blocked. Furthermore, while spontaneous activity was observed in nearly all dopaminergic neurons in wild-type mice, neurons from NALCN knockouts were mainly silent. Both observations demonstrate the critical importance of NALCN for pacemaking in dopaminergic neurons. Finally, we show that GABA-B receptor activation also produces inhibition of NALCN-mediated currents. Therefore, we identify NALCN as a core effector of inhibitory G protein-coupled receptors.

The Importance of Calcium Ions for Determining Mitochondrial Glycerol-3-Phosphate Dehydrogenase Activity When Measuring Uncoupling Protein 1 (UCP1) Function in Mitochondria Isolated from Brown Adipose Tissue.

Glycerol-3-phosphate is an excellent substrate for FAD-linked mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) in brown adipose tissue mitochondria and is regularly used as the primary substrate to measure oxygen consumption and reactive oxygen consumption by these mitochondria. mGPDH converts cytosolic glycerol-3-phosphate to dihydroxyacetone phosphate, feeding electrons directly from the cytosolic side of the mitochondrial inner membrane to the CoQ-pool within the inner membrane. mGPDH activity is allosterically activated by calcium, and when calcium chelators are present in the mitochondrial preparation medium and/or experimental incubation medium, calcium must be added to insure maximal mGPDH activity. It was demonstrated that in isolated brown adipose tissue mitochondria (1) mGPDH enzyme activity is maximal at free calcium ion concentrations in the 350 nM-1 µM range, (2) that ROS production also peaks in the 10-100 nM range in the presence of a UCP1 inhibitory ligand (GDP) but wanes with further increasing calcium concentration, and (3) that oxygen consumption rates peak in the 10-100 nM range with rates being maintained at higher calcium concentrations. This article provides easy-to-follow protocols to facilitate the measurement of mGPDH-dependent UCP1 activity in the presence of calcium for isolated brown adipose tissue mitochondria.

Lipopolysaccharide-induced inflammation in monocytes/macrophages is blocked by liposomal delivery of Gi-protein inhibitor.

BACKGROUND: Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. The involvement of Gi protein in mediating LPS effects has been demonstrated in murine macrophages and various cell types of human origin. PURPOSE: The aim of the present work was to evaluate the potential of a Gi-protein inhibitor encapsulated in liposomes in reducing the inflammatory effects induced by LPS in monocytes/macrophages. MATERIALS AND METHODS: Guanosine 5'-O-(2-thiodiphosphate) (GOT), a guanosine diphosphate analog that completely inhibits G-protein activation by guanosine triphosphate and its analogs, was encapsulated into liposomes and tested for anti-inflammatory effects in LPS-activated THP1 monocytes or THP1-derived macrophages. The viability of monocytes/macrophages after incubation with different concentrations of free GOT or liposome-encapsulated GOT was assessed by MTT assay. MAPK activation and production of IL1ß, TNFα, IL6, and MCP1 were assessed in LPS-activated monocytes/macrophages in the presence or absence of free or encapsulated GOT. In addition, the effect of free or liposome-encapsulated GOT on LPS-stimulated monocyte adhesion to activated endothelium and on monocyte chemotaxis was evaluated. RESULTS: We report here that GOT-loaded liposomes inhibited activation of MAPK and blocked the production of the cytokines IL1ß, TNFα, IL6, and MCP1 induced by LPS in monocytes and macrophages. Moreover, GOT encapsulated in liposomes reduced monocyte adhesion and chemotaxis. All demonstrated events were in contrast with free GOT, which showed reduced or no effect on monocyte/macrophage activation with LPS. CONCLUSION: This study demonstrates the potential of liposomal GOT in blocking LPS proinflammatory effects in monocytes/macrophages.

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity.

Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRasG12C using GDP equipped with an electrophilic group at the ß-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such ß-modified derivatives for Ras of at least 104 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.

Characterization of serotonin-induced inhibition of excitatory synaptic transmission in the anterior cingulate cortex.

Excitatory synaptic transmission in central synapses is modulated by serotonin (5-HT). The anterior cingulate cortex (ACC) is an important cortical region for pain perception and emotion. ACC neurons receive innervation of projecting serotonergic nerve terminals from raphe nuclei, but the possible effect of 5-HT on excitatory transmission in the ACC has not been investigated. In the present study, we investigated the role of 5-HT on glutamate neurotransmission in the ACC slices of adult mice. Bath application of 5-HT produced dose-dependent inhibition of evoked excitatory postsynaptic currents (eEPSCs). Paired pulse ratio (PPR) was significantly increased, indicating possible presynaptic effects of 5-HT. Consistently, bath application of 5-HT significantly decreased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. After postsynaptic application of G protein inhibitor GDP-ß-S, 5-HT produced inhibition of eEPSCs was significantly reduced. Finally, NAN-190, an antagonist of 5-HT1A receptor, significantly reduced postsynaptic inhibition of 5-HT and abolished presynaptic inhibition. Our results strongly suggest that presynaptic as well as postsynaptic 5-HT receptor including 5-HT1A subtype receptor may contribute to inhibitory modulation of glutamate release as well as postsynaptic responses in the ACC.

Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

Ethanol Disinhibits Dorsolateral Striatal Medium Spiny Neurons Through Activation of A Presynaptic Delta Opioid Receptor.

The dorsolateral striatum mediates habit formation, which is expedited by exposure to alcohol. Across species, alcohol exposure disinhibits the DLS by dampening GABAergic transmission onto this structure's principal medium spiny projection neurons (MSNs), providing a potential mechanistic basis for habitual alcohol drinking. However, the molecular and circuit components underlying this disinhibition remain unknown. To examine this, we used a combination of whole-cell patch-clamp recordings and optogenetics to demonstrate that ethanol potently depresses both MSN- and fast-spiking interneuron (FSI)-MSN GABAergic synaptic transmission in the DLS. Concentrating on the powerfully inhibitory FSI-MSN synapse, we further show that acute exposure of ethanol (50 mM) to striatal slices activates delta opioid receptors that reside on FSI axon terminals and negatively couple to adenylyl cyclase to induce a long-term depression of GABA release onto both direct and indirect pathway MSNs. These findings elucidate a mechanism through which ethanol may globally disinhibit the DLS.

[THE PARTICIPATION OF THE ELEMENTS OF CYTOSKELETON IN THE MOBILIZATION OF Ca2+ FROM INTRACELLULAR STORES OF INTACT AND DEVITRIFIED PORCINE OOCYTES].

Joint action of theophylline and guanosine diphosphate leads to the additional release of Ca2+ from intracellular stores of oocytes with intact microfilaments and microtubules, additional release of Ca2+ was not observed in oocytes after the joint action of prolactin and guanosine triphosphate. In the presence of an inhibitor of polymerization microfilaments cytochalasin D additional release of Ca2+ not detected in oocytes treated by theophylline and guanosine diphosphate, but treatment of oocyte by prolactin with guanosine triphosphate leads to the additional release of Ca2+ from intracellular stores. Nocodazole had no effect on the release of Ca2+ from intracellular stores of oocytes treated by theophylline with guanosine diphosphate or prolactin with guanosine triphosphate. The joint action of prolactin and guanosine triphosphate on devitrified oocytes with destroyed microfilaments (as a result of defrosting) leads to additional Ca2+ exit from the intracellular stores. The obtained data expand ideas concerning features of regulation of intracellular processes in porcine oocytes.

Activation of 5-HT2A/C receptor reduces glycine receptor-mediated currents in cultured auditory cortical neurons.

Glycine receptors (GlyRs) permeable to chloride only mediate tonic inhibition in the cerebral cortex where glycinergic projection is completely absent. The functional modulation of GlyRs was largely studied in subcortical brain regions with glycinergic transmissions, but the function of cortical GlyRs was rarely addressed. Serotonin could broadly modulate many ion channels through activating 5-HT2 receptor, but whether cortical GlyRs are subjected to serotonergic modulation remains unexplored. The present study adopted patch clamp recordings to examine functional regulation of strychnine-sensitive GlyRs currents in cultured cortical neurons by DOI (2,5-Dimethoxy-4-iodoamphetamine), a 5-HT2A/C receptor agonist. DOI caused a concentration-dependent reduction of GlyR currents with unchanged reversal potential. This reduction was blocked by the selective receptor antagonists (ritanserin and risperidone) and G protein inhibitor (GDP-ß-s) demonstrated that the reducing effect of DOI on GlyR current required the activation of 5-HT2A/C receptors. Strychnine-sensitive tonic currents revealed the inhibitory tone mediated by nonsynaptic GlyRs, and DOI similarly reduced the tonic inhibition. The impaired microtube-dependent trafficking or clustering of GlyRs was thought to be involved in that nocodazole as a microtube depolymerizing drug largely blocked the inhibition mediated by 5-HT2A/C receptors. Our results suggested that activation of 5-HT2A/C receptors might suppress cortical tonic inhibition mediated by GlyRs, and the findings would provide important insight into serotonergic modulation of tonic inhibition mediated by GlyRs, and possibly facilitate to develop the therapeutic treatment of neurological diseases such as tinnitus through regulating cortical GlyRs.

Postsynaptic Depolarization Enhances GABA Drive to Dorsomedial Hypothalamic Neurons through Somatodendritic Cholecystokinin Release.

Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca(2+) channels in sensory neurons via distinct mechanisms.

Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the µ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-ß-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.

H2O2 release from the very long chain acyl-CoA dehydrogenase.

Enhanced mitochondrial generation of oxidants, including hydrogen peroxide (H2O2), is related to a large number of pathological conditions, including diet-induced obesity and steatohepatosis. Indeed, we have previously shown that high fat diets increase the generation of H2O2 in liver mitochondria energized by activated fatty acids. Here, we further study fatty-acid induced H2O2 release in liver mitochondria, and determine the characteristics that regulate it. We find that this production of H2O2 is independent of mitochondrial inner membrane integrity and insensitive to purine nucleotides. On the other hand, palmitate-induced H2O2 production is strongly enhanced by high fat diets and is pH-sensitive, with a peak at a matrix pH of ~8.5. Using recombinantly expressed human very long chain acyl-CoA dehydrogenase, we are able to demonstrate that palmitate-induced H2O2 release may be ascribed to the activity of this enzyme alone, acting as an oxidase. Our results add to a number of findings indicating that sources outside of the electron transport chain can generate significant, physiopathologically relevant, amounts of oxidants in mitochondria.

Orexin-A differentially modulates AMPA-preferring responses of ganglion cells and amacrine cells in rat retina.

By activating their receptors (OX1R and OX2R) orexin-A/B regulate wake/sleeping states, feeding behaviors, but the function of these peptides in the retina remains unknown. Using patch-clamp recordings and calcium imaging in rat isolated retinal cells, we demonstrated that orexin-A suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-preferring receptor-mediated currents (AMPA-preferring currents) in ganglion cells (GCs) through OX1R, but potentiated those in amacrine cells (ACs) through OX2R. Consistently, in rat retinal slices orexin-A suppressed light-evoked AMPA-preferring receptor-mediated excitatory postsynaptic currents in GCs, but potentiated those in ACs. Intracellular dialysis of GDP-ß-S or preincubation with the Gi/o inhibitor pertussis toxin (PTX) abolished both the effects. Either cAMP/the protein kinase A (PKA) inhibitor Rp-cAMP or cGMP/the PKG blocker KT5823 failed to alter the orexin-A effects. Whilst both of them involved activation of protein kinase C (PKC), the effects on GCs and ACs were respectively eliminated by the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor and phosphatidylcholine (PC)-PLC inhibitor. Moreover, in GCs orexin-A increased [Ca(2+)]i and the orexin-A effect was blocked by intracellular Ca(2+)-free solution and by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. In contrast, orexin-A did not change [Ca(2+)]i in ACs and the orexin-A effect remained in intracellular or extracellular Ca(2+)-free solution. We conclude that a distinct Gi/o/PI-PLC/IP3/Ca(2+)-dependent PKC signaling pathway, following the activation of OX1R, is likely responsible for the orexin-A effect on GCs, whereas a Gi/o/PC-PLC/Ca(2+)-independent PKC signaling pathway, following the activation of OX2R, mediates the orexin-A effect on ACs. These two actions of orexin-A, while working in concert, provide a characteristic way for modulating information processing in the inner retina.

Phenylephrine enhances glutamate release in the medial prefrontal cortex through interaction with N-type Ca2+ channels and release machinery.

α1 -adrenoceptors (α1 -ARs) stimulation has been found to enhance excitatory processes in many brain regions. A recent study in our laboratory showed that α1 -ARs stimulation enhances glutamatergic transmission via both pre- and post-synaptic mechanisms in layer V/VI pyramidal cells of the rat medial prefrontal cortex (mPFC). However, a number of pre-synaptic mechanisms may contribute to α1 -ARs-induced enhancement of glutamate release. In this study, we blocked the possible post-synaptic action mediated by α1 -ARs to investigate how α1 -ARs activation regulates pre-synaptic glutamate release in layer V/VI pyramidal neurons of mPFC. We found that the α1 -ARs agonist phenylephrine (Phe) induced a significant enhancement of glutamatergic transmission. The Phe-induced potentiation was mediated by enhancing pre-synaptic glutamate release probability and increasing the number of release vesicles via a protein kinase C-dependent pathway. The mechanisms of Phe-induced potentiation included interaction with both glutamate release machinery and N-type Ca(2+) channels, probably via a pre-synaptic Gq /phospholipase C/protein kinase C pathway. Our results may provide a cellular and molecular mechanism that helps explain α1 -ARs-mediated influence on PFC cognitive functions. Alpha1 -adrenoceptor (α1 -ARs) stimulation has been reported to enhance glutamatergic transmission in layer V/VI pyramidal neurons of the rat medial prefrontal cortex (mPFC). We found that α1 -ARs agonist phenylephrine (Phe) increases pre-synaptic glutamate release probability and the number of released vesicles via interaction with both glutamate release machinery and N-type Ca(2+) channels. Our results may provide a cellular and molecular mechanism that helps explain α1 -ARs-mediated influence on PFC cognitive functions. Gq, Gq protein; PLC, phospholipase C; PKC, protein kinase C; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-d-aspartate; Glu, glutamate; Phe, phenylephrine.

Detection of UCP1 protein and measurements of dependent GDP-sensitive proton leak in non-phosphorylating thymus mitochondria.

Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.

Enhancement of GABA-activated currents by arginine vasopressin in rat dorsal root ganglion neurons.

A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹°-1 × 10⁻5 mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻6 mol/L). Also it could be removed by intracellular dialysis of either GDP-ß-S (5 × 10⁻4mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻6 mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.

Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.