An acid-sensing ion channel that detects ischemic pain

Ischemic pain occurs when there is insufficient blood flow for the metabolic needs of an organ. The pain of a heart attack is the prototypical example. Multiple compounds released from ischemic muscle likely contribute to this pain by acting on sensory neurons that innervate muscle. One such compound is lactic acid. Here, we show that ASIC3 (acid-sensing ion channel #3) has the appropriate expression pattern and physical properties to be the detector of this lactic acid. In rats, it is expressed only in sensory neurons and then only on a minority (~40%) of these. Nevertheless, it is expressed at extremely high levels on virtually all dorsal root ganglion sensory neurons that innervate the heart. It is extraordinarily sensitive to protons (Hill slope 4, half-activating pH 6.7), allowing it to readily respond to the small changes in extracellular pH (from 7.4 to 7.0) that occur during muscle ischemia. Moreover, both extracellular lactate and extracellular ATP increase the sensitivity of ASIC3 to protons. This final property makes ASIC3 a "coincidence detector" of three molecules that appear during ischemia, thereby allowing it to better detect acidosis caused by ischemia than other forms of systemic acidosis such as hypercapnia.

Phenotypic and genotypic profile of pyrethroid resistance in populations of the mosquito Aedes aegypti from Goiânia, Central West Brazil

ABSTRACTINTRODUCTION:The mosquito Aedes aegypti has evolved resistance to pyrethroid insecticides. The present study evaluated Ae. aegypti from Goiânia for the resistant phenotype and for mutations associated with resistance.METHODS:Insecticide dose-response bioassays were conducted on mosquitoes descended from field-collected eggs, and polymerase chain reaction (PCR) was used to genotype 90 individuals at sites implicated in pyrethroid resistance.RESULTS:All mosquito populations displayed high levels of resistance to deltamethrin, as well as high frequencies of the 1016Ile kdr and 1534Cys kdrmutations.CONCLUSIONS:Aedes aegypti populations in the Goiânia area are highly resistant to deltamethrin, presumably due to high frequencies of kdr(knockdown-resistance) mutations.

Exploration of receptor binding of Bacillus thuringiensis toxins

Wild type and mutant toxins of Bacillus thuringiensis delta-endotoxins were examined for their binding to midgut brush border membrane vesicles (BBMV). CryIAa, CryIAb, and CryIAc were examined for their binding to Gypsy moth (Lymantria dispar) BBMV. The binding of CryIAa and CryIAc was directly correlated with their toxicity, while CryIAb was observed to have lower binding than expected from its toxicity. The latter observation confirms the observation of Wolfersberger (1990). The "rule" of reciprocity of binding and toxicity is apparently obeyed by CryIAa and CryIAc, but broken by CryIAb on L. dispar. Alanine substitutions were made in several positions of the putative loops of CryIAa to test the hypothesis that the loops are intimately involved in binding to the receptor. The mutant toxins showed minor shifts in heterologous binding to Bombyx mori BBMV, but not enough to conclude that the residues chosen play critical roles in receptor binding.

When population and evolutionary genetics met behaviour

In this review, we analyse the impact of a population and evolutionary genetics approach on the study of insect behaviour. Our attention is focused on the model organism Drosophila melanogaster and several other insect species. In particular, we explore the relationship between rhythmic behaviours and the molecular evolution of clock and ion channel genes.

Anestésicos locais: interação com membranas biológicas e com o canal de sódio voltagem-dependente

Many theories about the mechanism of action of local anesthetics (LA) are described in the literature. Two types of theories can be distinguished: those that focus on the direct effects of LA on their target protein in the axon membranes, i.e. the voltage-gated sodium channel and the ones that take into account the interaction of anesthetic molecules with the lipid membrane phase for the reversible nerve blockage. Since there is a direct correlation between LA hydrophobicity and potency, it is crucial to take this physico-chemical property into account to understand the mechanism of action of LA, be it on the sodium channel protein, lipid(s), or on the whole membrane phase.

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (~98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (~76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

Purinergic signalling: past, present and future

The discovery of non-adrenergic, non-cholinergic neurotransmission in the gut and bladder in the early 1960's is described as well as the identification of adenosine 5'-triphosphate (ATP) as a transmitter in these nerves in the early 1970's. The concept of purinergic cotransmission was formulated in 1976 and it is now recognized that ATP is a cotransmitter in all nerves in the peripheral and central nervous systems. Two families of receptors to purines were recognized in 1978, P1 (adenosine) receptors and P2 receptors sensitive to ATP and adenosine diphosphate (ADP). Cloning of these receptors in the early 1990's was a turning point in the acceptance of the purinergic signalling hypothesis and there are currently 4 subtypes of P1 receptors, 7 subtypes of P2X ion channel receptors and 8 subtypes of G protein-coupled receptors. Both short-term purinergic signalling in neurotransmission, neuromodulation and neurosecretion and long-term (trophic) purinergic signalling of cell proliferation, differentiation, motility, death in development and regeneration are recognized. There is now much known about the mechanisms underlying ATP release and extracellular breakdown by ecto-nucleotidases. The recent emphasis on purinergic neuropathology is discussed, including changes in purinergic cotransmission in development and ageing and in bladder diseases and hypertension. The involvement of neuron-glial cell interactions in various diseases of the central nervous system, including neuropathic pain, trauma and ischemia, neurodegenerative diseases, neuropsychiatric disorders and epilepsy are also considered.

Benzodiazepine receptor ligand influences on learning: an endogenous modulatory mechanism mediated by benzodiazepines possibly of alimentary origin

In rats pre-but not post-training ip administration of either flumazenil, a central benzodiazepine (BSD) receptor antagonist, or of n-butyl-B-carboline-carboxylate (BCCB), an inverse agonist, enhanced retention of inhibitory avoidance learning. Flumazenil vlocked the enhancing effect of BCCB, and the inhibitory effect of the BZD agonists clonazepam and diazepam also given pre-training. Post-training administration of these drugs had no effects. The peripheral BZD receptor agonist/chloride channel blocker Ro5-4864 had no effect on the inhibitory avoidance task when given ip prior to training, buth it caused enhancement when given immediately post-training either ip or icv. This effect was blocked by PK11195, a competitive antagonist of Ro5-4864. These results suggest that ther is an endogenous mechanism mediated by BZD agonists, which is sensitive to inverse agonists and that normally down-regulates the formation of memories through a mechanism involving GABA-A receptors and the corresponding chloride channels. The most likely agonists for the endogenous mechanism suggested are the diazepam-like BZDs found in brain whose origin is possibly alimentary. Levels of these BZDs in the cortex were found to sharply decrease after inhibitory acoidance training or mere exposure to the training apparatus.

Preconcentration of copper from natural water samples using ligand-less in situ surfactant-based solid phase extraction prior to FAAS determination

In the present work, a simple and rapid ligand-less, in situ, surfactant-based solid phase extraction for the preconcentration of copper in water samples was developed. In this method, a cationic surfactant (n-dodecyltrimethylammonium bromide) was dissolved in an aqueous sample followed by the addition of an appropriate ion-pairing agent (ClO4-). Due to the interaction between the surfactant and ion-pairing agent, solid particles were formed and subsequently used for the adsorption of Cu(OH)2 and CuI. After centrifugation, the sediment was dissolved in 1.0 mL of 1 mol L-1 HNO3 in ethanol and aspirated directly into the flame atomic absorption spectrometer. In order to obtain the optimum conditions, several parameters affecting the performance of the LL-ISS-SPE, including the volumes of DTAB, KClO4, and KI, pH, and potentially interfering ions, were optimized. It was found that KI and phosphate buffer solution (pH = 9) could extract more than 95% of copper ions. The amount of copper ions in the water samples varied from 3.2 to 4.8 ng mL-1, with relative standard deviations of 98.5%-103%. The determination of copper in water samples was linear over a concentration range of 0.5-200.0 ng mL-1. The limit of detection (3Sb/m) was 0.1 ng mL-1 with an enrichment factor of 38.7. The accuracy of the developed method was verified by the determination of copper in two certified reference materials, producing satisfactory results.

ClC-5 chloride channel and kidney stones: what is the link?

Nephrolithiasis is one of the most common diseases in the Western world. The disease manifests itself with intensive pain, sporadic infections, and, sometimes, renal failure. The symptoms are due to the appearance of urinary stones (calculi) which are formed mainly by calcium salts. These calcium salts precipitate in the renal papillae and/or within the collecting ducts. Inherited forms of nephrolithiasis related to chromosome X (X-linked hypercalciuric nephrolithiasis or XLN) have been recently described. Hypercalciuria, nephrocalcinosis, and male predominance are the major characteristics of these diseases. The gene responsible for the XLN forms of kidney stones was cloned and characterized as a chloride channel called ClC-5. The ClC-5 chloride channel belongs to a superfamily of voltage-gated chloride channels, whose physiological roles are not completely understood. The objective of the present review is to identify recent advances in the molecular pathology of nephrolithiasis, with emphasis on XLN. We also try to establish a link between a chloride channel like ClC-5, hypercalciuria, failure in urine acidification and protein endocytosis, which could explain the symptoms exhibited by XLN patients.

Involvement of the TRPV1 channel in the modulation of spontaneous locomotor activity, physical performance and physical exercise-induced physiological responses

Physical exercise triggers coordinated physiological responses to meet the augmented metabolic demand of contracting muscles. To provide adequate responses, the brain must receive sensory information about the physiological status of peripheral tissues and organs, such as changes in osmolality, temperature and pH. Most of the receptors involved in these afferent pathways express ion channels, including transient receptor potential (TRP) channels, which are usually activated by more than one type of stimulus and are therefore considered polymodal receptors. Among these TRP channels, the TRPV1 channel (transient receptor potential vanilloid type 1 or capsaicin receptor) has well-documented functions in the modulation of pain sensation and thermoregulatory responses. However, the TRPV1 channel is also expressed in non-neural tissues, suggesting that this channel may perform a broad range of functions. In this review, we first present a brief overview of the available tools for studying the physiological roles of the TRPV1 channel. Then, we present the relationship between the TRPV1 channel and spontaneous locomotor activity, physical performance, and modulation of several physiological responses, including water and electrolyte balance, muscle hypertrophy, and metabolic, cardiovascular, gastrointestinal, and inflammatory responses. Altogether, the data presented herein indicate that the TPRV1 channel modulates many physiological functions other than nociception and thermoregulation. In addition, these data open new possibilities for investigating the role of this channel in the acute effects induced by a single bout of physical exercise and in the chronic effects induced by physical training.

The FML (Fucose Mannose Ligand) of Leishmania donovani: a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovanii infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0 % in antibody response (p<0.001) and reduction of 85.5 % parasite liver burden (p<0.001) was detected in animals immunized with FML saponin, unrespectivety of the immunization route.