Jordi Aleu

Microneurographic identification of spontaneous activity in C-nociceptors in neuropathic pain states in humans and rats.

Summary Microneurography can detect spontaneous activity in C‐nociceptors in neuropathic pain states in humans and in rats. ABSTRACT C‐nociceptors do not normally fire action potentials unless challenged by adequate noxious stimuli. However, in pathological states nociceptors may become hyperexcitable and may generate spontaneous ectopic discharges. The aim of this study was to compare rat neuropathic pain models and to assess their suitability to model the spontaneous C‐nociceptor activity found in neuropathic pain patients. Studies were performed in normal rats (n = 40), healthy human subjects (n = 15), peripheral neuropathic pain patients (n = 20), and in five rat neuropathic pain models: nerve crush (n = 24), suture (n = 14), chronic constriction injury (n = 12), STZ‐induced diabetic neuropathy (n = 56), and ddC‐induced neuropathy (n = 15). Microneurographic recordings were combined with electrical stimulation to monitor activity in multiple C fibers. Stimulation at 0.25 Hz allowed spontaneous impulses to be identified by fluctuations in baseline latency. Abnormal latency fluctuations could be produced by several mechanisms, and spontaneous activity was most reliably identified by the presence of unexplained latency increases corresponding to two or more additional action potentials. Spontaneous activity was present in a proportion of mechano‐insensitive C‐nociceptors in the patients and all rat models. The three focal traumatic nerve injury models provided the highest proportion (59.5%), whereas the two polyneuropathy models had fewer (18.6%), and the patients had an intermediate proportion (33.3%). Spontaneously active mechano‐sensitive C‐nociceptors were not recorded. Microneurographic recordings of spontaneous activity in diseased C‐nociceptors may be useful for both short‐ and long‐term drug studies, both in animals and in humans.

Control of neurotransmitter release by an internal gel matrix in synaptic vesicles

Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Abstract.Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present.

ATP Crossing the Cell Plasma Membrane Generates an Ionic Current in Xenopus Oocytes

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we usedXenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 ± 0.3 pmol/oocyte) generated a slow inward current (2.3 ± 0.1 μA). During hyperpolarizing pulses, the permeability for ATP4– was more than 4000 times higher than that for Cl–. The sensitivity to GdCl3 (0.2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.

Pharmacological characterization of purinergic inhibitory neuromuscular transmission in the human colon

Background  In the present study, we further characterize the purinergic receptors mediating the inhibitory junction potential (IJP) and smooth muscle relaxation in the human colon using a new, potent and selective agonist (MRS2365), and antagonists (MR2279 and MRS2500) of the P2Y1 receptor. The P2Y12 antagonist AR‐C66096 was tested as well. Using this pharmacological approach, we tested whether β‐nicotinamide adenine dinucleotide (β‐NAD) fulfilled the criteria to be considered an inhibitory neurotransmitter in the human colon.

Endogenous hemichannels play a role in the release of ATP from Xenopus oocytes

ATP is an electrically charged molecule that functions both in the supply of energy necessary for cellular activity and as an intercellular signaling molecule. Although controlled ATP secretion occurs via exocytosis of granules and vesicles, in some cells, and under certain conditions, other mechanisms control ATP release. Gap junctions, intercellular channels formed by connexins that link the cytoplasm of two adjacent cells, control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexons, and it has been suggested that these may represent an alternative pathway for ATP release. We have investigated the release of ATP through hemichannels from Xenopus oocytes that are formed by Connexin 38 (Cx38), an endogenous, specific type of connexin. These hemichannels generate an inward current that is reversibly activated by calcium‐free solution and inhibited by octanol and flufenamic acid. This calcium‐sensitive current depends on Cx38 expression: it is decreased in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of these endogenous connexons also allows transfer of Lucifer Yellow. We have found that the release of ATP is coincident with the opening of hemichannels: it is calcium‐sensitive, is inhibited by octanol and flufenamic acid, is inhibited in ASCx38 injected oocytes, and is increased by overexpression of Cx38. Taken together, our results suggest that ATP is released through activated hemichannels in Xenopus oocytes.

Double and triple spikes in C-nociceptors in neuropathic pain states: An additional peripheral mechanism of hyperalgesia

&NA; It was previously reported that in 5 patients with small‐fiber neuropathy, neuropathic pain, and hyperalgesia, application of a single, brief electrical stimulus to the skin could give rise to 2 afferent impulses in a C‐nociceptor fiber. These double spikes, which are attributed to unidirectional conduction failure at branch points in the terminal arborisation, provide a possible mechanism for hyperalgesia. We here report that similar multiple spikes are regularly observed in 3 rat models of neuropathic pain: nerve crush, nerve suture, and chronic constriction injury. The proportion of nociceptor fibers exhibiting multiple spikes was similar (10.1–18.5%) in the 3 models, and significantly greater than the proportion in control (unoperated) animals (1.2%). As in the human patients, multiple spikes in the rat models were often provoked by increasing the stimulation rate from 0.25 to 2 Hz, but provocation by warming was less consistent. Multiple spiking was also directly dependent on stimulus intensity, consistent with a mechanism that depends on activation of multiple branches. Whereas only double spikes had previously been described in patients, in these more extensive recordings from rats we found that triple spikes could also be observed after a single electrical stimulus. The results strengthen the suggestion that multiple spiking, because of impaired conduction in the terminal branches of nociceptors, may contribute to hyperalgesia in patients with neuropathic pain. Double and triple spikes in c‐nociceptors, caused by impaired conduction in terminal branches, may be an important cause of hyperalgesia in patients with neuropathic pain.

Effect of galantamine on the human α7 neuronal nicotinic acetylcholine receptor, the Torpedo nicotinic acetylcholine receptor and spontaneous cholinergic synaptic activity

1 Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimers disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human α4β2 neuronal nicotinic receptor activity. We explored its effect on the human α7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. 2 Galantamine, at a concentration of 0.1 μM, increased the amplitude of acetylcholine (ACh)‐induced ion currents in the human α7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh‐induced currents, was observed at a concentration of 250 μM Ach. 3 The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 μM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh‐induced currents, occurred at a concentration of 50 μM ACh. 4 Galantamine affects not only the activity of post‐synaptic receptors but also the activity of nerve terminals. At a concentration of 1 μM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 μM. 5 In conclusion, our results show that galantamine enhances human α7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects.

Release of ATP induced by hypertonic solutions in Xenopus oocytes

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non‐secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mm). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic‐activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto‐enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium‐dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best‐characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.

Effect of otilonium bromide on contractile patterns in the human sigmoid colon.

Background  The mechanism of action of the spasmolytic compound otilonium bromide (OB) on human colonic motility is not understood. The aim of our study was to characterize the pharmacological effects of OB on contractile patterns in the human sigmoid colon.