Carlos Barajas-López

Protease‐activated receptor 2 sensitizes TRPV1 by protein kinase Cɛ‐ and A‐dependent mechanisms in rats and mice

Proteases that are released during inflammation and injury cleave protease‐activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2‐induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cɛ and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCɛ and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cβ, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin‐stimulated increases in [Ca2+]i and whole‐cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non‐algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCɛ and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCɛ and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

Mast cell tryptase and proteinase‐activated receptor 2 induce hyperexcitability of guinea‐pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long‐term hyperexcitability in submucosal neurons from the guinea‐pig ileum by activating proteinase‐activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT‐PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL‐NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min–hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR‐NH2) had no effect on neuronal membrane potential or long‐term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5‐HT, prostaglandins) also caused depolarization, and following washout of antigen, long‐term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long‐term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

Functional interactions between nicotinic and P2X channels in short‐term cultures of guinea‐pig submucosal neurons

1 Functional interactions between nicotinic and P2X receptors in submucosal neurons were investigated. Whole‐cell currents induced by ACh (IACh) and ATP (IATP) were blocked by hexamethonium and PPADS), respectively. Currents induced by simultaneous application of the two transmitters (IACh+ATP) were only as large as the current induced by the most effective of these substances. This current occlusion indicates that activation of nicotinic and P2X channels is not independent. 2 Kinetic parameters of IACh+ATP indicate that they are carried through channels activated by either substance. In agreement with this interpretation, both IACh and IATP amplitudes were decreased when ATP and ACh were applied simultaneously, whereas no cross‐desensitization was observed when nicotinic and P2X receptors were desensitized individually. 3 Current occlusion was observed at membrane potentials of −60 and +10 mV, when IACh and IATP were inward. However, when these currents were outward (at +40 mV), current occlusion was not observed. Current occlusion was still observed at +40 mV in experiments in which the reversal potential of these currents had been adjusted to more positive values. 4 Current occlusion occurred as soon as currents were detected (< 5 ms), was still present in the absence of Ca2+, Na+ or Mg2+, and after adding staurosporine, genistein, K‐252a, or N‐ethylmaleimide to the pipette solution. Similar observations were noted after substituting α,β‐methylene ATP for ATP, or GTP for GTP‐γ‐S in the pipette and in experiments carried out at 36, 23 and 9 °C. 5 We propose that nicotinic and P2X channels are in functional clusters of at least two, and that the influx of ions through one activates (through allosteric interactions) a mechanism that inhibits the other channel.

P2x‐purinoceptors of myenteric neurones from the guinea‐pig ileum and their unusual pharmacological properties

1 Whole‐cell and outside‐out patch clamp recordings were used to characterize the physiological and pharmacological properties of the P2x‐purinoceptors of myenteric neurones from the guinea‐pig ileum. 2 Adenosine 5′‐triphosphate (ATP) and analogues (1–3000μm) evoked a rapid inward current in > 90% of all recorded neurones. The reversal potential of this current was dependent on the extracellular sodium concentration, at +14 ± 1.9, 0 ± 1.6 and −12 ± 1 mV for 166, 83 and 42 mM of sodium, respectively. The fast activation and inactivation of this current occurred even when guanosine 5′‐triphosphate (GTP) was omitted from the pipette solution or substituted with an equimolar concentration of guanosine 5′‐o‐[2‐thiotriphosphate] (GTP‐γ‐S). Single channel currents were observed when these outside‐out membrane patches were exposed to ATP (10–30 μm). These channels have a unitary conductance of about 17 picosiemens. 3 The rank‐order of potency of the agonists used to induce the whole‐cell currents was: ATP‐γ‐S = ATP = 2‐methylthio‐ATP (2‐Me‐S‐ATP) > > α,β‐methylene ATP = β,γ‐methylene ATP; adenosine and uridine 5′‐triphosphate (UTP) (up to 1 mM) were inactive. 4 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (1–30μm) antagonized the effects of ATP (1 mM) with an IC50 of 4 μm. α,β‐Methylene ATP (100 μm) did not affect the ATP (30 μm)‐induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E‐3G had a very weak antagonistic effect (IC50 μ 3 mM). Suramin potentiated the currents induced by ATP through a mechanism that was independent of its inhibitory effect on ectonucleotidase activity, as suramin also potentiated the effect of α,β‐methylene ATP (an ATP analogue that is resistant to nucleotidases). 5 In conclusion, the myenteric P2x‐purinoceptor shares some properties with other purinoceptors in particular with the P2times4‐ and P2X6‐purinoceptors. This receptor has also some unusual pharmacological properties suggesting that myenteric neurones express a novel subtype of P2x‐purinoceptors. The properties of this receptor, however, might be a result of the combination of two or more of the homomeric purinoceptors so far characterized.

BACTERIAL CELL PRODUCTS SIGNAL TO MOUSE COLONIC NOCICEPTIVE DORSAL ROOT GANGLIA NEURONS

This study examined whether bacterial cell products that might gain access to the intestinal interstitium could activate mouse colonic nociceptive dorsal root ganglion (DRG) neurons using molecular and electrophysiological recording techniques. Colonic projecting neurons were identified by using the retrograde tracer fast blue and Toll-like receptor (TLR) 1, 2, 3, 4, 5, 6, 9, adapter proteins Md-1 and Md-2, and MYD88 mRNA expression was observed in laser-captured fast blue-labeled neurons. Ultrapure LPS 1 microg/ml phosphorylated p65 NF-kappaB subunits increased transcript for TNF-alpha and IL-1beta and stimulated secretion of TNF-alpha from acutely dissociated DRG neurons. In current-clamp recordings from colonic DRG neurons, chronic incubation (24 h) of ultrapure LPS significantly increased neuronal excitability. In acute studies, 3-min superfusion of standard-grade LPS (3-30 microg/ml) reduced the rheobase by up to 40% and doubled action potential discharge rate. The LPS effects were not significantly different in TLR4 knockout mice compared with wild-type mice. In contrast to standard-grade LPS, acute application of ultrapure LPS did not increase neuronal excitability in whole cell recordings or afferent nerve recordings from colonic mesenteric nerves. However, acute application of bacterial lysate (Escherichia coli NLM28) increased action potential discharge over 60% compared with control medium. Moreover, lysate also activated afferent discharge from colonic mesenteric nerves, and this was significantly increased in chronic dextran sulfate sodium salt mice. These data demonstrate that bacterial cell products can directly activate colonic DRG neurons leading to production of inflammatory cytokines by neurons and increased excitability. Standard-grade LPS may also have actions independent of TLR signaling.

Interferon-α inhibits long-term potentiation and unmasks a long-term depression in the rat hippocampus

Interferons (IFN) appear to have various neuromodulatory actions. Here, we characterized the actions of IFN-alpha on the electrophysiological properties of CA1 hippocampal neurons using intracellular recordings. Superfusion of this cytokine did not alter the resting membrane potential, cell input resistance, action potentials, nor GABA-mediated fast synaptic potentials. IFN-alpha inhibited glutamate-mediated excitatory postsynaptic potentials (gEPSPs) and reversed or prevented long-term potentiation (LTP) induced by high-frequency tetanic stimulation. IFN-alpha reduced gEPSP amplitude far below its control value. Only a short-term potentiation (STP) was observed when either IFN-alpha or D-2-amino-5-phosphonovalerato (APV; NMDA receptor antagonist) were present during tetanic stimulation. After this STP in presence of APV, IFN-alpha had no effect on gEPSPs. APV had no effect on LTP when applied after tetanic stimulation and did also not prevent IFN-alpha effect on LTP. Genistein (a tyrosine kinase inhibitor) or heat inactivation prevented IFN-alpha effects. IFN-alpha also decreased the depolarization induced by local application of glutamate but did not modify those induced by NMDA. Similarly, IFN-alpha reversed the potentiation (induced by tetanic stimulation) of glutamate-induced depolarizations. IFN-alpha did not affect long-term depression (LTD) induced by low-frequency tetanic stimulation. In conclusion, IFN-alpha-induced inhibition of LTP is, at least in part, mediated by a postsynaptic effect, by tyrosine kinase activity, and by non-NMDA glutamate receptors. Inhibition of LTP by IFN-alpha unmasks LTD which is induced by the same high-frequency tetanic stimulation.

Melatonin modulates cholinergic transmission by blocking nicotinic channels in the guinea-pig submucous plexus

Melatonin, a hormone produced and released by the pineal gland is also synthesized by cells of the gastrointestinal wall, where it might be a local regulator of gut functions. In this study, we investigated the possible role of melatonin as a modulator of the enteric nervous system. Intracellular recordings were made in neurons of the submucosal plexus from the guinea-pig ileum to measure the melatonin effects on their electrophysiological properties. Melatonin did not alter the membrane potential, the membrane resistance and the noradrenergic inhibitory postsynaptic potentials. However, melatonin (30-3000 microM) reversibly decreased the amplitude of nicotinic excitatory postynaptic potentials (EPSPs) in a concentration-dependent manner (IC50 = 247 microM). These actions of melatonin were not modified by the presence of idazoxan and atropine indicating that they are not mediated by endogenous release of acetylcholine, noradrenaline, or by direct activation of alpha 2-adrenoceptors or muscarinic receptors. The superfusion of melatonin also blocked the nicotinic depolarizations induced by locally applied acetylcholine, indicating that at least part of its effects are postsynaptic. In voltage-clamp experiments, using the whole-cell configuration, melatonin also inhibited the nicotinic inward currents induced by acetylcholine (IACh) in a concentration-dependent manner (IC50 = 257 microM). Melatonin decreased the maximal IACh but did not affect the potency of acetylcholine to induce this current, indicating a noncompetitive antagonism. This effect was voltage-dependent. Our observations indicate that melatonin inhibits the fast EPSPs by directly and specifically blocking the nicotinic channels. The relative high concentrations of melatonin required to produce such an effect rules this out as one of its humoral actions. Such an effect, however, might be of physiological significance close to the cells that release melatonin in the gastrointestinal wall or in other organs.

The His155Tyr (489C>T) single nucleotide polymorphism of P2RX7 gene confers an enhanced function of P2X7 receptor in immune cells from patients with rheumatoid arthritis

We assessed the possible association between several single nucleotide polymorphisms (SNP) of P2RX7 gene with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We determined the function of P2X7 receptor and the frequency of the 489C>T, 1096C>G, and 1513A>C SNP of P2RX7 gene in 111 and 122 patients with SLE and RA, and 98 healthy subjects. We found no significant association between the SNPs studied and SLE or RA. We also detected that lymphocytes from SLE and RA patients with the 489C>T SNP showed a higher ethidium bromide uptake in response to ATP than wild type or 1096C>G/1513A>C subjects. In addition, cells from RA patients and the 489C>T genotype, showed higher [Ca(2+)]i responses to ATP. Our data indicate that the 489C>T SNP of P2RX7 gene confers an enhanced function of this receptor in patients with RA, which may contribute to the pathogenesis of this condition.

Cross-inhibitory interactions between GABAA and P2X channels in myenteric neurones

Inhibitory interactions between GABAA[induced by γ‐aminobutyric acid (GABA)] and P2X [activated by adenosine 5′‐triphosphate (ATP)] receptors of myenteric neurones from the guinea pig small intestine were characterized using whole‐cell recordings. Currents induced by GABA (IGABA) or ATP (IATP) were inhibited by picrotoxin or pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, respectively. Currents induced by GABA + ATP (IGABA+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. This occlusion requires maximal activation of at least one of these receptors. Sequential applications of neurotransmitters, and kinetic and pharmacological properties of IGABA+ATP indicate that they are carried through both GABAA and P2X channels. ATP did not affect IGABA in neurones: (i) in which P2X channels were not present; (ii) after inhibiting P2X channels with Ca2+ (iii) in the presence of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, a P2X receptor antagonist; (iv) after P2X receptor desensitization or (v) at IATP reversal potential. Similarly, GABA did not affect P2X‐mediated currents in neurones: (i) in which GABAA channels were not present; (ii) in the presence of picrotoxin, a GABAA channel blocker; (iii) after GABAA receptor desensitization or (iv) at the IGABA reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11 °C, after adding to the pipette solution a cocktail of protein kinase inhibitors (staurosporine + genistein + K‐252a), after substituting the GTP in the pipette with GDP‐β‐S and after treating the cells with N‐ethylmaleimide. Taken together, all of these results are consistent with a model of cross‐inhibition between GABAA and P2X.

Ionic basis of pacemaker generation in dog colonic smooth muscle.

1. The ionic basis of the slow waves in the circular muscle of the dog colon, in particular the ionic conductances involved in their initiation, were investigated by measuring intracellular electrical activity in the Abe‐Tomita‐type chamber for voltage control. 2. The depolarization that initiates the slow wave activity could be evoked by an increase in inward current and/or by a block of outward current. According to previous work, inward current could be carried by Na+, Cl‐, and Ca2+ ions; K+ ions would carry outward current. 3. The Na+ channel blocker tetrodotoxin (5 x 10(‐7) M) did not affect the slow wave amplitude nor its rate of rise. After omission of Na+, by replacing Na+ with N‐methyl‐D‐glucamine, large slow waves continued to develop although some changes in slow wave characteristics occurred. 4. Replacement of 91% of the Cl‐ by isethionate decreased the slow wave frequency and increased the slow wave amplitude. However, NaCl substitution by sucrose increased the slow wave frequency and decreased the slow wave amplitude. 5. Slow wave activity continued to develop after blockade of Ca2+ influx by D600 (10(‐6) M) or CoCl2 (1‐3 mM). D600 and Co2+ did not affect the membrane potential but reduced the slow wave amplitude and abolished the plateau potential. Slow waves were abolished after omission of extracellular Ca2+ (plus 1 mM‐EGTA). This suggests that Ca2+ influx is probably not necessary but extracellular presence of Ca2+ ions is indispensible for the slow wave generation. 6. The combination of 0 Na+, Li+ HEPES solution, by replacing Na+ with Li+, plus D600 depolarized the cells (up to approximately ‐40 mV) and abolished slow wave activity. This effect was voltage dependent since repolarization caused slow waves to return. 7. Abolition of the slow wave activity was also obtained by current‐induced depolarization to approximately ‐40 mV. However, during high‐K+‐induced depolarization (to approximately ‐40 mV) high amplitude (16 mV) slow waves were still present, slowing that the voltage dependence of the slow waves was shifted positively. This effect probably occurs due to modification by extracellular K+ of a voltage‐dependent K+ conductance, which would suggest that a K+ conductance is involved in slow wave generation. 8. In conclusion, slow waves are generated by cyclic membrane conductance changes, which are dependent on the presence of extracellular Ca2+ ions and on the membrane potential. Our data are consistent with the hypothesis that slow waves are initiated by the blockade of a K+ conductance.