Polarimetric parameters associated to special microstructured fibers.

The Mueller matrices associated to six homemade special index-guiding microstructured fibers are determined at a transmission wavelength of 1550 nm. Diattenuation, polarizance, and polarization-dependent loss, among other polarimetric parameters, are determined from the Mueller matrix associated to a 2 m length of each fiber studied here. Results show that the fiber fabrication parameters have a clear effect on the output degree of polarization (DoP), providing an inverse relationship between the DoP output and the relative hole diameter, the d/Λ value.

Determination of the Mueller matrix of UV-inscribed long-period fiber grating.

An explicit method for determination of the Mueller matrix elements of a commercial long-period fiber grating inscribed with ultraviolet CW laser irradiation (UV-LPFG) is presented. From the Mueller matrix obtained for such UV-LPFG, the full polarimetric response of the grating was found. Our polarimetric analysis was focused mainly on the polarization-dependent loss and other polarimetric properties, such as the polarizance, the depolarization index, and the diattenuation parameters. The full polarimetric analysis allows us to obtain more complete information than the usually reported ones, in which only two orthogonal linear polarizations are considered; for example, with our analysis, we prove that a small depolarization effect is inherent in UV-LPFG and that attenuation depends on the polarization state. This additional polarimetric information could be useful to control the output LPFG signal, for instance, for the realization of wavelength switchable or Q-switched fiber lasers, among other applications.

Generation of unconventional polarization from light scattered by metallic cylinders under conical incidence.

A simple experimental method to generate unconventional polarized states from the conical scattering of light by thin metallic cylinders is presented. Results show that radial and azimuthal polarizations are easily obtained. This opens up the possibility for a broad range of applications, from surgery to industrial and even to remote sensing. To the best of our knowledge, this is the cheapest and easiest way to generate radial and azimuthal unconventional polarization states.

Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons.

The three most common Cys-loop receptors expressed by myenteric neurons are nACh, 5-HT3 and GABAA . To investigate the function of these proteins researchers have used channel inhibitors such as hexamethonium (antagonist of nACh receptors), ondansetron (antagonist of 5-HT3 receptors), picrotoxin and bicuculline (both antagonists of GABAA receptors). The aim of this study was to investigate the specificity of these inhibitors on Cys-loop receptors of primary cultured neurons obtained from the guinea-pig small intestine. The whole-cell configuration of the patch clamp techniques was used to record membrane currents induced by ACh (IACh ), 5-HT (I5-HT ) and GABA (IGABA ) in the absence and the presence of various concentrations of hexamethonium, ondansetron, picrotoxin or bicuculline. The three Cys-loop receptors present in enteric neurons are expressed independently and they do not cross-desensitized. Hexamethonium inhibited IACh without affecting I5-HT and IGABA . Ondansetron inhibited I5-HT and also IACh but did not affect IGABA . Picrotoxin and bicuculline inhibited I5-HT , IACh and IGABA with different potency, being the lowest potency on 5-HT3 receptors. All these inhibitory effects were concentration dependent and reversible. Our observations showed that except for hexamethonium, all other inhibitors used here show different degrees of selectivity, which has to be considered when these antagonists are used in experimental studies aimed to investigate the functions of these receptors. In particular, in tissues expressing nACh receptors because these are the targets of all other inhibitors used here. The low potency of picrotoxin and bicuculline to inhibit 5-HT3 receptors suggests that these receptors are heteromeric proteins.

Retention of a new-defined intron changes pharmacology and kinetics of the full-length P2X2 receptor found in myenteric neurons of the guinea pig.

P2X2 plays an important role in ATP signaling in guinea pig myenteric plexus. Here, we cloned and characterized three P2X2 isoforms expressed in myenteric neurons. RT/PCR was used to amplify the cDNA of P2X2 variants. These were expressed in Xenopus oocytes, and nucleotide-induced membrane currents were recorded with the two-electrode voltage clamp technique. Three P2X2 cDNAs were identified in myenteric single neurons, named P2X2-1, P2X2-2 and P2X2-4. Based on the analysis of the structural organization of these variants we predicted that P2X2-2 is the fully processed variant, which lead us to propose a new exon-intron arrangement of P2X2 receptor gene with 12 exons and 11 introns. In agreement with this new model, the intron 11 is retained in P2X2-1 and P2X2-4 variants by alternative splicing. Expression of P2X2-1, P2X2-2 and P2X2-4 were found in 92, 42 and 37%, respectively, out of 40 analyzed single neurons. P2X2-4 does not form functional channels, and homomeric channels formed by P2X2-1 and P2X2-2 have different pharmacological profile. Thus, the former receptor is more sensitive to ATP, BzATP, and PPADS, whereas, suramin inhibited both receptors in a biphasic- and monophasic-manner, respectively. α,ß-meATP has very low efficacy on either channel. Furthermore, ionic currents mediated by P2X2-1 have slower desensitization than P2X2-2. These results indicate that P2X2-1 was the most common P2X2 transcript in myenteric neurons and displays significant phenotypical changes implicating that retention of the intron 11 plays a major role in ATP signaling in the intestinal myenteric plexus.

5-Hydroxytryptamine and atropine inhibit nicotinic receptors in submucosal neurons.

The whole-cell recording technique was used to investigate the pharmacological properties of acetylcholine-activated ion channels of cultured submucosal neurons from guinea-pig small intestine. Acetylcholine induced whole-cell membrane currents (I(ACh)) in a concentration-dependent manner (EC(50)=79 microM). I(ACh) exhibited strong inward rectification, had a reversal potential of +19+/-2 mV (Na(+) outside, Cs(+) inside), was reversibly inhibited in a concentration-dependent manner by hexamethonium (EC(50)=5 microM) and atropine (EC(50)=1.6 microM), and was unaffected by alpha-bungarotoxin (30 nM). Atropine was less potent in inhibiting the currents induced by 30 microM acetylcholine than those induced by 1 mM acetylcholine. I(ACh) was mimicked by the current induced by nicotine (I(Nic); EC(50)=52 microM). I(Nic) was also blocked by atropine (EC(50)=1.7 microM) and hexamethonium (EC(50)=3.6 microM). 5-Hydroxytryptamine (5-HT) also inhibited I(ACh) in a concentration-dependent manner (EC(50)=180 microM) in the experiments carried out in the presence of a 5-HT(3) receptor antagonist. 5-HT had a similar inhibitory effect after the desensitization of 5-HT(3) receptors or in neurons with relative small 5-HT(3)-mediated currents. The inhibitory actions of hexamethonium, atropine, and 5-HT on I(ACh) were voltage-dependent. Thus, inhibition was significantly smaller for outward currents (recorded at +40 mV) than for inward currents (recorded at -60 mV). Our observations indicate that the I(ACh) of submucosal neurons are mediated by activation of nicotinic channels, which are blocked by atropine, 5-HT, and hexamethonium. The possibility that one of the 5-HT roles in the gastrointestinal tract might be to directly modulate nicotinic channels is discussed.

Changes in intracellular Ca2+ by activation of P2 receptors in submucosal neurons in short-term cultures.

Electrophysiological and Ca2+ microfluorimetric techniques were used to characterize the pharmacological profile of the P2 receptors expressed in submucosal neurons and the changes in intracellular Ca2+ associated with activation of these receptors. ATP caused a fast and slow membrane depolarizations during intracellular recordings. ATP induced a rapid inward current during whole-cell experiments. Receptors mediating the inward current and fast depolarization have the same pharmacological profile and these ATP responses were more sensitive to pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid than Basilen BlueE-3G, and potentiated by suramin. The slow depolarization was not blocked by these P2 receptor antagonists, pertussis toxin, or KT5720 (protein kinase A inhibitor). N-ethylmaleimide or protein kinase C inhibitors (staurosporine and calphostin) blocked this depolarization. ATP induced complex multi-phasic Ca2+ transients in most neurons, classified as fast, slow, or mixed fast/slow responses. In conclusion, the fast and slow Ca2+ responses were mediated by respective activation of P2X and P2Y receptors and were associated with fast and slow depolarizations, respectively.

Slow wave and spike action potentials recorded in cell cultures from the muscularis externa of the guinea pig small intestine.

Intracellular recordings were obtained to investigate whether slow wave and spike type action potentials are present in cell cultures of the muscularis externa from the guinea pig small intestine. The muscularis externa of the small intestine was dissociated by using specific purified enzymes and gentle mechanical dissociation. Cells were plated on cover slips and maintained in culture for up to 4 weeks. Dissociated cells obtained in this way reorganized themselves in a few days to form small cell clumps showing spontaneous movements. Intracellular recordings of these clumps displayed both spike and slow wave type action potentials. Spikes were observed on top of some slow waves and were abolished by the addition of nifedipine or the removal of extracellular calcium. Slow waves, however, were nifedipine insensitive and temperature sensitive, and were abolished by octanol (a gap junction blocker) and forskolin (an adenyl cyclase activator). Slow waves were never observed in small clumps (<50 microm), suggesting that a critical mass of cells might be required for their generation. These observations demonstrated for the first time the presence of nifedipine-insensitive slow waves in cell cultures of the muscularis externa from the guinea pig small intestine. Cell cultures allow rigorous control of the immediate environment for the cells and this should facilitate future studies on the molecular and cellular mechanisms responsible for the slow waves in the gastrointestinal tract.

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 alpha, beta-methylene ATP for ATP, or GTP for GTP-gamma-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 (1-3000 microM) 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-gamma-S). Single channel currents were observed when these outside-out membrane patches were exposed to ATP (10-30 microM). 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-gamma-S = ATP = 2-methylthio-ATP (2-Me-S-ATP) > > alpha, beta-methylene ATP = beta, gamma-methylene ATP; adenosine and uridine 5'-triphosphate (UTP) (up to 1 mM) were inactive. 4. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1-30 microM) antagonized the effects of ATP (1 mM) with an IC50 of 4 microM. alpha, beta-Methylene ATP (100 microM) did not affect the ATP (30 microM)-induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E-3G had a very weak antagonistic effect (IC50 > or = 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 alpha, beta-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 P2x4- 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.

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.

Cellular mechanisms underlying adenosine actions on cholinergic transmission in enteric neurons.

Whole cell recordings were used to investigate the effects of adenosine and several of its analogues on voltage-activated calcium currents (VACC) of myenteric and submucosal neurons. Electrophysiological and pharmacological properties of the soma VACC recorded in myenteric neurons indicate that they are carried through N-type calcium channels, similar to those of the submucosal neurons and to those of the calcium conductance that mediates acetylcholine release at the submucosal ganglia. Adenosinergic compounds inhibited, in a concentration-response and in a voltage-dependent manner, VACC in neurons from both enteric plexuses. The pharmacological profile of the receptors that mediate this effect was similar to that of the receptors involved in presynaptic inhibition in enteric neurons and likely of the A1 subtype. The effects of 2-chloroadenosine (CADO) on VACC were prevented by pretreatment with pertussis toxin (PTX), became irreversible with guanosine 5'-O-(3-thiotriphosphate) (inside the pipette), and were abolished with N-ethylmaleimide (NEM; known to uncouple receptors from G protein complexes). Intracellular recordings were used to further evaluate presynaptic effects of adenosine at the submucosal plexus. Adenosinergic compounds reduced the amplitude of fast excitatory postsynaptic potentials (EPSPs) by acting at nerve terminals. This effect was insensitive to PTX and staurosporine (a protein kinase inhibitor) but was abolished by NEM. CADO effects on EPSPs were not reversed by increasing the extracellular calcium concentration. In conclusion, activation of A1 adenosine receptors inhibits VACC via PTX-sensitive G proteins in myenteric and submucosal neurons. Reduction of cholinergic transmission also involves A1 adenosine receptors and appears to involve the activation of PTX-insensitive G proteins.

ATP inhibits the synaptic release of acetylcholine in submucosal neurons.

Previously, we have shown that adenosine inhibits release of acetylcholine (ACh) by acting at A1 presynaptic receptors in guinea pig submucosal synapses. In this study, intracellular recordings were made to investigate the actions of ATP and some analogs on the synaptic release of ACh. Superfusion of these substances decreased the amplitude and duration of electrically induced fast excitatory postsynaptic potentials (EP-SPs) in about 90% of the tested neurons. ATP (0.1-30 microM) effects were concentration dependent with an EC50 of 1.4 microM. ADP, AMP and ATP-gamma-S mimicked ATP inhibitory effects and were equally potent and efficacious. beta,gamma-Methylene-ATP seemed to act as a partial agonist, causing less than 50% of the inhibition obtained with ATP. 2-Methyl-thio-ATP was only active at the highest concentration tested whereas alpha,beta-methylene-ATP and UTP were inactive (0.3-30 microM). ATP-gamma-S did not alter depolarizations induced by exogenous application of ACh, indicating that ATP analogs inhibit EPSPs by acting at a presynaptic site. Although the EC50 values were similar for ATP and adenosine, the maximum responses (76 +/- 4.5% and 40 +/- 1.6%) were different. Adenosine deaminase (which inactivates adenosine) and alpha,beta-methylene-ADP (an ecto-5'-nucleotidase inhibitor) did not alter ATP-induced inhibition of these EPSPs. Inhibition of EPSPs by 30 microM adenosine (maximal concentration) and 1 microM ATP (submaximal concentration) were additive. Suramin or reactive blue 2 (30 microM), antagonists of ATP actions in several tissues, did not modify the effects of ATP on the fast EPSPs. 8-Cyclopentyltheophylline inhibited, in a competitive manner, these ATP inhibitory effects. In conclusion, ATP inhibits synaptic release of ACh by acting at receptors similar to those previously identified as P3-purinoceptors.

ATP closes a potassium and opens a cationic conductance through different receptors in neurons of guinea pig submucous plexus.

Intracellular recordings were made to study the actions of ATP and related nucleotides on neurons from the guinea pig submucous plexus. Local application of ATP, by pressure, induced a depolarization in most AH-type neurons, which had a latency of several milliseconds, lasted for about 5 sec, appeared to reverse at about +4 mV and occurred concomitantly with a reduction in input resistance. Pressure application of ATP also depolarized the S-type neurons. In most of these cells the depolarization had two phases: the first component resembled the depolarization observed in AH cells and the second component was much slower in onset and was longer lasting (30-90 sec). The slower component was associated with an increase in input resistance, reversed polarity near the potassium equilibrium potential and was observed in isolation in 30% of S neurons. Superfusion of ATP or other analogs (0.03-10 microns) induced a slow depolarization in most of S neurons with the following rank order of potency: 2-methylthio-ATP > ATP > adenosine-5'-o-3-thiotriphosphate = ADP; alpha, beta-methylene ATP and beta, gamma-methylene ATP were inactive (10-100 microM). When whole-cell recordings were used, fast superfusion with ATP or other analogs (3-1000 microM) evoked, at negative membrane potentials, a rapidly desensitizing inward current. This current reversed polarity at about 0 mV and was much reduced in low extracellular sodium concentration. The rank order of potency of the used agonists was: ATP = adenosine-5'-o-3-thiotriphosphate = 2-methylthio-ATP > > alpha,beta-methylene ATP = beta,gamma-methylene ATP; adenosine, AMP or ADP (1 mM) were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Suramin increases the efficacy of ATP to activate an inward current in myenteric neurons from guinea-pig ileum.

Previous reports have indicated that suramin antagonizes, in a competitive manner, the actions of adenosine 5'-triphosphate (ATP) mediated by P2 purinoceptors. In neurons from the myenteric plexus, however, suramin has different effects on an ATP-activated conductance. During whole-cell recordings, ATP (3-1000 microM) evoked a rapidly desensitizing inward current in a concentration-dependent manner. The amplitude of the ATP-activated current was increased, in a reversible manner, when suramin was applied previous to or during ATP stimulation. This effect was increased by raising the suramin concentration. Higher concentrations of suramin were required for higher concentrations of ATP; the EC50 values of suramin were 5 +/- 1.1 microM and 143 +/- 48 nM when ATP concentrations of 30 and 10 microM were used. No change in the holding current was observed with suramin alone. These observations suggest that a different subtype of P2 purinoceptors could be expressed in the myenteric neurons.