Computational Modeling to Explain Why 5,5-Diarylpentadienamides are TRPV1 Antagonists.

Several years ago, the crystallographic structures of the transient receptor potential vanilloid 1 (TRPV1) in the presence of agonists and antagonists were reported, providing structural information about its chemical activation and inactivation. TRPV1's activation increases the transport of calcium and sodium ions, leading to the excitation of sensory neurons and the perception of pain. On the other hand, its antagonistic inactivation has been explored to design analgesic drugs. The interactions between the antagonists 5,5-diarylpentadienamides (DPDAs) and TRPV1 were studied here to explain why they inactivate TRPV1. The present work identified the structural features of TRPV1-DPDA complexes, starting with a consideration of the orientations of the ligands inside the TRPV1 binding site by using molecular docking. After this, a chemometrics analysis was performed (i) to compare the orientations of the antagonists (by using LigRMSD), (ii) to describe the recurrent interactions between the protein residues and ligand groups in the complexes (by using interaction fingerprints), and (iii) to describe the relationship between topological features of the ligands and their differential antagonistic activities (by using a quantitative structure-activity relationship (QSAR) with 2D autocorrelation descriptors). The interactions between the DPDA groups and the residues Y511, S512, T550, R557, and E570 (with a recognized role in the binding of classic ligands), and the occupancy of isoquinoline or 3-hydroxy-3,4-dihydroquinolin-2(1H)-one groups of the DPDAs in the vanilloid pocket of TRPV1 were clearly described. Based on the results, the structural features that explain why DPDAs inactivate TRPV1 were clearly exposed. These features can be considered for the design of novel TRPV1 antagonists.

Scorpion Toxins and Ion Channels: Potential Applications in Cancer Therapy.

Apoptosis, a genetically directed process of cell death, has been studied for many years, and the biochemical mechanisms that surround it are well known and described. There are at least three pathways by which apoptosis occurs, and each pathway depends on extra or intracellular processes for activation. Apoptosis is a vital process, but disturbances in proliferation and cell death rates can lead to the development of diseases like cancer. Several compounds, isolated from scorpion venoms, exhibit inhibitory effects on different cancer cells. Indeed, some of these compounds can differentiate between healthy and cancer cells within the same tissue. During the carcinogenic process, morphological, biochemical, and biological changes occur that enable these compounds to modulate cancer but not healthy cells. This review highlights cancer cell features that enable modulation by scorpion neurotoxins. The properties of the isolated scorpion neurotoxins in cancer cells and the potential uses of these compounds as alternative treatments for cancer are discussed.

NaStEP, an essential protein for self-incompatibility in Nicotiana, performs a dual activity as a proteinase inhibitor and as a voltage-dependent channel blocker.

The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.

Antimicrobial Peptides From Lycosidae (Sundevall, 1833) Spiders.

Antimicrobial peptides (AMPs) have been found in all organism taxa and may play an essential role as a host defense system. AMPs are organized in various conformations, such as linear peptides, disulfide bond-linked peptides, backbone-linked peptides and circular peptides. AMPs apparently act primarily on the plasma membrane, although an increasing number of works have shown that they may also target various intracellular sites. Spider venoms are rich sources of biomolecules that show several activities, including modulation or blockage of ion channels, anti-insect, anti-cancer, antihypertensive and antimicrobial activities, among others. In spider venoms from the Lycosidae family there are many linear AMPs with a wide range of activities against several microorganisms. Due to these singular activities, some Lycosidae AMPs have been modified to improve or decrease desirable or undesirable effects, respectively. Such modifications, especially with the aim of increasing their antibiotic activity, have led to the filing of many patent applications. This review explores the abundance of Lycosidae venom AMPs and some of their derivatives, and their use as new drug models.

Scorpion venomics: a 2019 overview.

Introduction: A few scorpions are dangerous to humans. Their medical relevance was the initial driving force for venom research. By classical biochemistry and molecular cloning, several venom peptides and their coding transcripts were characterized, mainly those related to toxins. The discovery of other components with novel activities and potential applications has revitalized the interest in the field in the last decade and a half. Nontoxic scorpion species have also attracted major interest.Areas covered: Advances in the identification of scorpion venom components via high-throughput venomics (genomics, transcriptomics and proteomics) up to 2019 are summarized. A classification system for venom-related transcripts and proteins, together with an intuitive systematic nomenclature for RNAseq-generated transcripts are proposed. Venom components classified as Na+, K+, Ca2+, Cl- and TRP channel toxins, enzymes, protease inhibitors, host defense peptides and other peptidic molecules are briefly reviewed, giving a comprehensive picture of the venom.Expert opinion: Modern high-throughput technologies applied to scorpion venom studies have resulted in a dramatic increase in both, the number and diversity of available sequences, leading to a deeper understanding of the composition of scorpion venoms. Still, many newly-discovered venom constituents remain to be characterized, to complete the puzzle of scorpion venoms.

Mechanosensitive ion channel inhibitors promote the stiffening of the plasma membrane of mouse sensory neurons.

The mechanosensitivity of cells depends on the lipid-protein interactions of the plasma membrane. Affectations in the lipid region of the plasma membrane affect the transduction of mechanical forces, and any molecule that modifies the biophysical integrity of the lipid bilayer can alter the mechanical activity of the proteins inside the membrane. To understand whether inhibitors of mechanically activated ion channels affect the mechanical properties of the plasma membrane, we evaluated the rigidity of the membrane of sensory neurons of the DRG of mice using a variant of the scanning ion conductance microscopy method, which allows us to calculate the Young's modulus of individual cells before and after the perfusion of different doses of Gd3+, ruthenium red and GsMTx-4. Our results suggest that these molecules compromise the membrane by increasing the Young's modulus value, which indicates that the membrane becomes more rigid; these compounds act through different mechanisms and by a non-specific manner, each one shows a certain preference for specific cell subpopulations, depending on their cell size and their reactivity to isolectin B4. Our results support the idea that the biophysical properties that result from the interactions that arise in the membranes are part of the mechanotransduction process.

Inhibition of striatal cholinergic interneuron activity by the Kv7 opener retigabine and the nonsteroidal anti-inflammatory drug diclofenac.

Striatal cholinergic interneurons provide modulation to striatal circuits involved in voluntary motor control and goal-directed behaviors through their autonomous tonic discharge and their firing "pause" responses to novel and rewarding environmental events. Striatal cholinergic interneuron hyperactivity was linked to the motor deficits associated with Parkinson's disease and the adverse effects of chronic antiparkinsonian therapy like l-DOPA-induced dyskinesia. Here we addressed whether Kv7 channels, which provide negative feedback to excitation in other neuron types, are involved in the control of striatal cholinergic interneuron tonic activity and response to excitatory inputs. We found that autonomous firing of striatal cholinergic interneurons is not regulated by Kv7 channels. In contrast, Kv7 channels limit the summation of excitatory postsynaptic potentials in cholinergic interneurons through a postsynaptic mechanism. Striatal cholinergic interneurons have a high reserve of Kv7 channels, as their opening using pharmacological tools completely silenced the tonic firing and markedly reduced their intrinsic excitability. A strong inhibition of striatal cholinergic interneurons was also observed in response to the anti-inflammatory drugs diclofenac and meclofenamic acid, however, this effect was independent of Kv7 channels. These data bring attention to new potential molecular targets and pharmacological tools to control striatal cholinergic interneuron activity in pathological conditions where they are believed to be hyperactive, including Parkinson's disease.

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

Effect of fipronil on energy metabolism in the perfused rat liver.

Fipronil is an insecticide used to control pests in animals and plants that can causes hepatotoxicity in animals and humans, and it is hepatically metabolized to fipronil sulfone by cytochrome P-450. The present study aimed to characterize the effects of fipronil (10-50µM) on energy metabolism in isolated perfused rat livers. In fed animals, there was increased glucose and lactate release from glycogen catabolism, indicating the stimulation of glycogenolysis and glycolysis. In the livers of fasted animals, fipronil inhibited glucose and urea production from exogenous l-alanine, whereas ammonia and lactate production were increased. In addition, fipronil at 50µM concentration inhibited the oxygen uptake and increased the cytosolic NADH/NAD⁺ ratio under glycolytic conditions. The metabolic alterations were found both in livers from normal or proadifen-pretreated rats revealing that fipronil and its reactive metabolites contributed for the observed activity. The effects on oxygen uptake indicated that the possible mechanism of toxicity of fipronil involves impairment on mitochondrial respiratory activity, and therefore, interference with energy metabolism. The inhibitory effects on oxygen uptake observed at the highest concentration of 50µM was abolished by pretreatment of the rats with proadifen indicating that the metabolites of fipronil, including fipronil sulfone, acted predominantly as inhibitors of respiratory chain. The hepatoxicity of both the parent compound and its reactive metabolites was corroborated by the increase in the activity of lactate dehydrogenase in the effluent perfusate in livers from normal or proadifen-pretreated rats.

Cardioprotective effect of hyperthyroidism on the stunned rat heart during ischaemia-reperfusion: energetics and role of mitochondria.

NEW FINDINGS: What is the central question of this study? Hyperthyroidism is a cardiac risk factor, but thyroid therapy is used on myocardial stunning. What is the consequence of hyperthyroidism for mitochondrial metabolism and Ca(2+) handling of the postischaemic stunned heart? What is the main finding and its importance? Hyperthyroidism reduced stunning and improved muscle economy of the postischaemic rat heart. The activities of the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channel in hyperthyroid rat hearts were different from those in the euthyroid rat hearts. These findings contribute to the understanding of mitochondrial bioenergetics in pathology and support thyroid therapy in the stunning induced by ischaemia. Transient ischaemia and hyperthyroidism are cardiovascular risk factors. Nevertheless, 3,5,3'-triiodothyronine/thyroxine therapy has been used to revert myocardial stunning. We studied the influence of hyperthyroidism on the role played by mitochondria in myocardial stunning consequent to ischaemia-reperfusion. Rats were injected s.c. daily with 20 µg kg(-1) triiodothyronine for 15 days (HpT group). Isolated ventricles from either HpT or euthyroid (EuT) rats were perfused in a calorimeter, and left intraventricular pressure (in millimetres of mercury) and heat release (Ht; in milliwatts per gram) were measured. Stunning was evoked by 20 min of no-flow ischaemia and 45 min reperfusion. The HpT hearts developed higher postischaemic contractile recovery (PICR) and improved total muscle economy (P/Ht) with lower diastolic contracture (ΔLVEDP) than EuT hearts. Release of Ca(2+) from the sarcoplasmic reticulum during reperfusion with 10 mm caffeine in low-[Na(+) ] Krebs solution evoked a higher contracture in EuT than in HpT hearts. Blockade of the mitochondrial sodium-calcium exchanger with clonazepam increased ΔLVEDP and reduced P/Ht and PICR in HpT but not in EuT hearts. The clonazepam-induced dysfunction in HpT hearts was reduced by ciclosporin, suggesting a dependance on activation of the mitochondrial permeability transition pore. Blockade of the mitochondrial Ca(2+) uniporter with Ru360 reduced P/Ht and PICR to ∼10% in both HpT and EuT hearts. Blockade of mitochondrial K(+) channels with 5-hydroxydecanoate increased LVEDP and reduced PICR and P/Ht in HpT hearts, while it only increased LVEDP in EuT hearts. The results suggest that hyperthyroidism prevents the stunning with high dependence on the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channels. Both HpT and EuT hearts showed a similar and critical role of the uniporter. The HpT hearts have a slow sarcoplasmic reticulum Ca(2+) loss and low mitochondrial Ca(2+) uptake.

Antiepileptic drugs: Energy-consuming processes governing drug disposition.

Diffusion is not the main process by which drugs are disposed throughout the body. Translational movements of solutes given by different energy-consuming mechanisms are required in order to dispose them efficiently. Membrane transportation and cardiac output distribution are two effective processes to move the molecules among different body sites. Gastrointestinal-blood cycling constitutes a supplementary way to regulate the distribution of molecules between the non-hepatic organs and the liver. Any change in the relative supply of drug molecules among eliminating organs could modify their clearance from the body. Either the nonlinear phenytoin (PHT) pharmacokinetic response or the influence that carbamazepine (CBZ) exerts on PHT exposure could be explained throughout their efflux transporter inducer abilities. Cardiac output distribution difference between the individuals might also explain the dual CBZ-over-PHT interaction response. Finally, valproic acid (VPA) pharmacokinetics can be understood by adding to these mechanisms of transportation its ability to cross the mitochondrial membrane of the hepatocyte.

Pharmacological study of the one spike spherical neuron phenotype in Gymnotus omarorum.

The intrinsic properties of spherical neurons play a fundamental role in the sensory processing of self-generated signals along a fast electrosensory pathway in electric fish. Previous results indicate that the spherical neuron's intrinsic properties depend mainly on the presence of two resonant currents that tend to clamp the voltage near the resting potential. Here we show that these are: a low-threshold potassium current blocked by 4-aminopyridine and a mixed cationic current blocked by cesium chloride. We also show that the low-threshold potassium current also causes the long refractory period, explaining the necessary properties that implement the dynamic filtering of the self-generated signals previously described. Comparative data from other fish and from the auditory system indicate that other single spiking onset neurons might differ in the channel repertoire observed in the spherical neurons of Gymnotus omarorum.

Phα1ß toxin prevents capsaicin-induced nociceptive behavior and mechanical hypersensitivity without acting on TRPV1 channels.

Phα1ß toxin is a peptide purified from the venom of the armed spider Phoneutria nigriventer, with markedly antinociceptive action in models of acute and persistent pain in rats. Similarly to ziconotide, its analgesic action is related to inhibition of high voltage activated calcium channels with more selectivity for N-type. In this study we evaluated the effect of Phα1ß when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1ß on Ca²âº transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1ß reduced both nocifensive behavior and mechanical hypersensitivity induced by capsaicin similarly to that observed with SB366791, a specific TRPV1 antagonist. Peripheral nifedipine and mibefradil did also decrease nociceptive behavior induced by intraplantar capsaicin. In contrast, ω-conotoxin MVIIA (a selective N-type Ca²âº channel blocker) was effective only when administered intrathecally. Phα1ß, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca²âº transients in DRG neurons. The simultaneous administration of Phα1ß and SB366791 inhibited the capsaicin-induced Ca²âº transients that were additive suggesting that they act through different targets. Moreover, Phα1ß did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1ß may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors.

Electrophysiological effects of tamoxifen: mechanism of protection against reperfusion arrhythmias in isolated rat hearts.

Reperfusion arrhythmias are currently attributed to ionic imbalance and oxidative stress. Tamoxifen is a potent antioxidant that also modulates some ionic transport pathways. In this work, we tried to correlate the electrophysiological effects of 1, 2, and 5 µM of tamoxifen with the incidence and severity of arrhythmias appearing on reperfusion after 10 minutes of coronary occlusion in isolated hearts from female rats. All tamoxifen concentrations inhibited the action potential shortening observed in the control hearts during late ischemia (6-10 minutes), whereas 2 and 5 µM also reduced the resting membrane potential depolarization. The incidence of sustained ventricular tachycardia and/or ventricular fibrillation on reperfusion decreased from 10 of 12 (control group) to 5 of 10 (1 µM, P = 0.1718), 4 of 12 (2 µM, P = 0.0361), and 2 of 10 (5 µM, P = 0.0083). The possible role of chloride currents activated by cell swelling in these effects was explored in hearts submitted to a 10-minute hypotonic challenge, where tamoxifen (5 µM) blocked the action potential shortening and the late resting membrane potential depolarization produced by hypotonicity, mimicking its action in late ischemia. Tamoxifen produced a similar increase of the total antioxidant capacity of myocardial samples at all the concentration tested. In conclusion, our data strongly suggest that the antiarrhythmic action of this agent is mediated by its electrophysiological effect derived from modulation of chloride currents activated by cell swelling.

Targeting mitochondria for cardiac protection.

The critical role of mitochondria in cardiomyocyte survival and death has become an exciting field of research in cardiac biology. Indeed, it is accepted that mitochondrial dysfunction plays a crucial role in the pathogenesis of multiple cardiac diseases. Besides the obvious relevance of mitochondria in energy production, calcium homeostasis, and reactive oxygen species (ROS) production, new processes like mitochondrial fusion/fission, phosphorylation and nitrosylation modifications in mitochondrial proteins have been suggested to form part of a cast of key players in cardiac disease. This review describes currently studied drugs and compounds that target mitochondria in the scenario of cardiovascular diseases.

Transporte eletrônico em biofilmes nanoestruturados para biossensores a base de enzimas / Electronic transport in nanostructures films for biosensors based in enzymes

Os biossensores são dispositivos empregados para a detecção de um analito específico, podendo assim ser no controle de qualidade nos alimentos para determinar a presença de micro-organismos, toxinas ou metabólitos. O presente estudo objetiva desenvolver um biossensor condutométrico, baseado na imobilização de peroxidasse em membranas de quitosana e quitosana com nanopartículas de ouro (AuNP) para a detecção de peroxido de hidrogênio. O trabalho foi dividido em três etapas. Na primeira etapa foi estudada a obtenção de AuNP empregando agentes redutores biológicos, sendo avaliados três monossacarídeos (glicose, frutose e galactose), três dissacarídeos (sacarose, maltose e lactose), dois biopolímeros (amido e quitosana), assim como os extratos obtidos a partir das folhas de hortelã (Mentha piperita) e cascas de furtas de abacaxi (Ananas comosus), banana (Musa sp. ), maracujá (Passiflora edulis), tangerina (Citrus reticulata). A quitosana mostrou-se como o melhor agente redutor na síntese das AuNP, as quais foram empregadas na segunda etapa para a produção de membranas. Três tipos de membranas foram processadas, membranas de quitosana sem AuNP e membranas de quitosana com AuNP com concentrações de 8 e 11mM., as quais foram caraterizadas morfológica e eletricamente. Finalmente foi avaliada a imobilização da peroxidasse usando quatro tratamentos diferentes, sendo a dispersão da peroxidasse nas soluções filmogênicas precursoras das membranas a mais eficiente. A resposta elétrica destas membranas é dependente da concentração de AuNP e da presença de enzimas, e também foi alterada quando as mesmas foram expostas a soluções de tampão fosfato com diferentes concentrações de peroxido de hidrogênio. Isto constitui o principio de operação dos biossensores condutométricos desenvolvidos neste trabalho.

Biosensors are devices used for detecting a specific analyte, and thus can be used in quality control of food for determining the presence of micro-organisms, toxins or metabolites. The present study aims to develop a conductometric biosensor based on the immobilization of peroxidase in membranes of chitosan and chitosan with gold nanoparticles (AuNP) for the detection of hydrogen peroxide. The work was divided into three stages. In the first stage, methods for obtaining AuNP employing biological reducing agents were studied, evaluating three monosaccharides (glucose, fructose and galactose), three disaccharides (sucrose, maltose and lactose), two biopolymers (starch and chitosan), as well as the extracts obtained from the leaves of mint (Mentha piperita) and husks dost thou pineapple (Ananas comosus), banana (Musa sp), passion fruit (Passiflora edulis), mandarin (Citrus reticulata). Chitosan exhibited the best behavior as reducing agent for the synthesis of AuNP, which were employed in the second step for the production of membranes. Three types of membranes were processed, chitosan membranes without AuNP and chitosan membranes with AuNP with concentrations of 8 and 11mM, which were morphologically and electrically characterized. Finally the peroxidase immobilization was evaluated using four different procedures, being the dispersion of the peroxidase in filmogenic solutions precursor of membranes the more efficient. The electrical response of these membranes, depends on the AuNP concentration and the presence of enzymes, and was also altered when they were exposed to hydrogen peroxide containing phosphate buffer solutions. This constitutes the principle of operation of the conductometric biosensor developed in this work.

Pharmacological properties of a pore induced by raising intracellular Ca2+.

Recent studies on the P2X(7) receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca(2+) concentration induces a pore opening similar to P2X(7) receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca(2+) concentration is associated to P2X(7) receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X(7) receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 microM) increased intracellular free Ca(2+) concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t(1/2)) for ethidium bromide uptake of 118 +/- 17 s, and t(1/2) for Lucifer yellow of 122 +/- 11 s. P2X(7) receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca(2+) concentration. However, 5-(N,N-hexamethylene)-amiloride, a P2X(7) receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca(2+) at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca(+2) induces a novel membrane pore pharmacologically different from the P2X(7) associated pore and hemigap-junction pore.

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation.

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.

Aortas isolated from sinoaortic-denervated rats exhibit rhythmic contractions that are regulated by pharmacologically distinct calcium sources.

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca(2+) sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca(2+) released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca(2+) entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca(2+)-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca(2+) release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca(2+) entry through L-type Ca(2+).

Monoamine transporter inhibitors and norepinephrine reduce dopamine-dependent iron toxicity in cells derived from the substantia nigra.

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean +/- SD uptake of 100 microm 59FeCl3 in RCSN-3 cells was 25 +/- 4 pmol per min per mg, which increased to 28 +/- 8 pmol per min per mg when complexed with dopamine (Fe(III)-dopamine). This uptake was inhibited by 2 microm nomifensine (43%p < 0.05), 100 microm imipramine (62%p < 0.01), 30 microm reboxetine (71%p < 0.01) and 2 mm dopamine (84%p < 0.01). The uptake of 59Fe-dopamine complex was Na+, Cl- and temperature dependent. No toxic effects in RCSN-3 cells were observed when the cells were incubated with 100 microm FeCl3 alone or complexed with dopamine. However, 100 microm Fe(III)-dopamine in the presence of 100 microm dicoumarol, an inhibitor of DT-diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 microm nomifensine, 30 microm reboxetine and 2 mm norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe3+, (2) the uptake of Fe-norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe-norepinephrine complex even when DT-diaphorase is inhibited. These results support the proposed neuroprotective role of DT-diaphorase and norepinephrine.