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A Villarroel

Publications and source records attributed to A Villarroel.

At least 19 recordsLinked to original sources
PubMed2004

Intravenous and subcutaneous pharmacokinetics of florfenicol in sheep.

Pharmacokinetic parameters of florfenicol were determined in 10 adult sheep (five wethers and five ewes) after a single 40 mg/kg intravenous (i.v.) dose, and three daily subcutaneous (s.c.) doses of 40 mg/kg of a commercial preparation (Nuflor((R))). The concentration of florfenicol in serum samples was assayed using a proprietary HPLC assay method, and pharmacokinetic parameters derived for individual animal data by each route using compartmental and noncompartmental approaches. Two animals (one male and one female) were excluded due to observed i.v. dosing problems, and a biexponential model was found to fit the i.v. data well for six of the other eight animals. Data from two males showed prolonged low concentrations of florfenicol in serum and were better fit by a three-compartment model. The mean +/- SD for the half-lives of the distribution and elimination phases for the six sheep best fit with a two-compartment model were 0.069 +/- 0.018 and 1.01 +/- 0.09 h respectively, and for the V(d(ss)) and clearances were 0.503 +/- 0.035 L/kg and 366 +/- 53 mL/h/kg respectively. The data collected during the s.c. multiple dose study were analyzed using noncompartmental methods only. The bioavailability (F%) after s.c. dosing was calculated in three ways to compare estimation methods as steady-state had not been reached and single dose s.c. data were not obtained past 24 h. Using the AUC(0--24) and AUC(0--> infinity ) from the first dose, the F% values averaged 27 and 40% respectively. Using the AUC(0--> infinity ) for all doses, the F% was 65%. Calculations of the mean time during which the serum concentration exceeded 0.5 and 1.0 microg/mL were 105 +/- 3.9 and 74.7 +/- 12.2 h respectively.

Animals↗
PubMed2003

Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation.

The KCNQ family of voltage-dependent non-inactivating K+ channels is composed of five members, four of which (KCNQ2-5) are expressed in the CNS and are responsible for the M-current. Mutations in either KCNQ2 or KCNQ3 lead to a hereditary form of dominant generalized epilepsy. Using specific antisera to the KCNQ2, KCNQ3 and KCNQ5 subunits, we found that KCNQ3 co-immunoprecipitated with KCNQ2 and KCNQ5 subunits, but no association was detected between KCNQ2 and KCNQ5. Intense KCNQ5 immunoreactivity was found to be widely distributed throughout the temporal neocortex and the hippocampal formation. In these structures, both pyramidal and non-pyramidal neurons and a population of glial cells in the white matter expressed the KCNQ5 subunit. In the sclerotic areas of the CA fields of epileptic patients, a marked loss of KCNQ5 immunoreactive pyramidal neurons was found in relation with the loss of neurons in these regions. However, in the regions adjacent to the sclerotic areas, the distribution and intensity of KCNQ5 immunostaining was apparently normal. The widespread distribution of KCNQ5 subunits, its persistence in pharmacoresistant epilepsy, along with the significant role of the M-current in the control of neuronal excitability, makes this protein a possible target for the development of anticonvulsant drugs.

Adolescent↗
PubMed2001

Intersubunit cooperativity in the NMDA receptor.

Opening of the NMDA receptor channel requires simultaneous binding of glutamate and glycine. Although the binding sites for each agonist are in different subunits, the presence of one agonist influences the binding of the other. We have localized regions in the S1 binding domain of both subunits required for the transmission of allosteric signals from the glutamate binding NR2A subunit to the glycine binding NR1 subunit. Three-dimensional modeling indicates that these segments are not directly involved in ligand binding, but likely form solvent-accessible loops protruding out of the binding pocket, making them suitable to relay interactions between adjacent subunits. Thus, these segments mediate negative allosteric coupling between the two subunit types that form the NMDA receptor.

Allosteric Regulation↗
PubMed2001

Sex differences in the acetylcholine receptor kinetics of postnatal and denervated rat muscle.

Single-channel recording from visualised endplates in freshly dissociated muscles from postnatal and denervated rat muscle revealed the presence of a low conductance, fetal type of acetylcholine receptor. Kinetic analysis showed a main component in the burst durations with a mean of 10.8 +/- 2.7 ms (n = 29). Receptors from female rats had an additional 27.3 +/- 5.5 ms (n = 5) kinetic component which was found in one-third of the 15 female endplates. Recordings from male and female denervated muscles gave more homogeneous kinetics with single time constants of 7.2 +/- 1.3 and 7.4 +/- 1.3 ms, respectively. It is concluded that the acetylcholine receptor channels present during early development are different from those of denervated muscle.

Animals↗
PubMed2000

Four residues of the extracellular N-terminal domain of the NR2A subunit control high-affinity Zn2+ binding to NMDA receptors.

NMDA receptors are allosterically inhibited by Zn2+ ions in a voltage-independent manner. The apparent affinity for Zn2+ of the heteromeric NMDA receptors is determined by the subtype of NR2 subunit expressed, with NR2A-containing receptors being the most sensitive (IC50, approximately 20 nM) and NR2C-containing receptors being the least sensitive (IC50, approximately 30 microM). Using chimeras constructed from these two NR2 subtypes, we show that the N-terminal LIVBP-like domain of the NR2A subunit controls the high-affinity Zn2+ inhibition. Mutations at four residues in this domain markedly reduce Zn2+ affinity (by up to >500-fold) without affecting either receptor activation by glutamate and glycine or inhibition by extracellular protons and Ni2+ ions, indicating that these residues most likely participate in high-affinity Zn2+ binding.

Allosteric Site↗
PubMed1999

Alternative splicing generates multiple mRNA forms of the acetylcholine receptor gamma-subunit in rat muscle.

The fetal type acetylcholine receptor, composed of the alphabeta gammadelta subunits, has shown a highly variable channel kinetics during postnatal development. We examine the hypothesis whether such a variability could result from multiple channel forms, differing in the N-terminus of the gamma-subunit. RT-PCR revealed, in addition to the full-length mRNA, three new forms lacking exon 4. One of them in addition lacks 19 nucleotides from exon 5, predicting a complete subunit, with a 43 residues shorter N-terminus. A third one lacking the complete exon 5 predicts a subunit without transmembrane segments. These forms, generated by alternative splicing, may account for the kinetic variability of the acetylcholine receptor channel.

Alternative Splicing↗
PubMed1999

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

Agmatine↗
PubMed1998

Glycine-independent NMDA receptor desensitization: localization of structural determinants.

In studying chimeras of NR2A and NR2C subunits of the NMDA receptor, we have found that glycine-independent desensitization depends on two regions of the extracellular N-terminal domain. One corresponds to a stretch of approximately 190 amino acids preceding the glutamate-binding domain S1. The other localizes at the interface between the N-terminal segment and the first transmembrane domain of NR2A subunits and involves A555 and S556. Both regions support desensitization in the absence of the other with different time courses. Desensitization did not develop with time in receptors containing the entire N-terminal region of NR2C. The introduction of A555 into the corresponding position of NR2C subunits enabled the receptors to manifest time-dependent increase in desensitization. Thus, this determinant behaves as an allosteric effector for glycine-independent desensitization.

Allosteric Regulation↗
PubMed1997

Nonstationary noise analysis of M currents simulated and recorded in PC12 cells.

M current relaxations recorded in PC12 cells were subjected to nonstationary noise analysis (NSNA) to obtain estimates of single-channel current (i), channel number (N), and open probability (Po) for the channels responsible for M current. The analysis was constrained such that N and single-channel conductance were the same at two potentials. The relation between variance and current indicated that the fraction of channels open was 0.58 +/- 0.06 (mean +/- SD) and 0.05 +/- 0.04 (mean +/- SD: n = 9) at -33 and -63 mV, respectively. The single M channel conductance was 4.0 pS, and a density of 1 functional M channel per 4 microm2 was estimated. Monte Carlo simulations of a two-state model of M channels were used to obtain sets of simulated macroscopic M currents that were subjected to the same NSNA procedure so as to evaluate the accuracy of M channel parameters obtained with this method. The influence of current rundown and filter frequency on estimates of i, N, and Po were evaluated. The single-channel parameters estimated from the simulations differed by < 10% from actual values at any level of current rundown, N, or Po. The dispersion in the estimation of N and Po increased as Po decreased. Decreasing filter frequency caused an underestimation of i, paralleled by an overestimation of N. The estimation of Po was relatively immune to the filter frequency, especially for data simulated with Po = 0.77.

Animals↗
PubMed1996

An amino acid exchange in the second transmembrane segment of a neuronal nicotinic receptor causes partial epilepsy by altering its desensitization kinetics.

The alpha4 subunit of the neuronal nicotinic acetylcholine receptor is the first gene shown to be involved in a human idiopathic epileptic disease. A missense mutation, leading to the replacement of serine 248 by phenylalanine in the second transmembrane segment, had been detected in patients with autosomal dominant nocturnal frontal lobe epilepsy. The properties of the wild type receptor composed of alpha4 and beta2 subunits and the mutant receptor where alpha4 subunits carried the mutation at serine 248 were compared by means of cDNA manipulation and expression in Xenopus oocytes. The mutant receptor exhibited faster desensitization upon activation by acetylcholine and recovery from the desensitized state was much slower than in the wild type receptor. We conclude that the reported mutation causes seizures via a diminution of the activity of the alpha4beta2 neuronal nicotinic acetylcholine receptor.

Acetylcholine↗
PubMed1996

M-current suppression in PC12 cells by bradykinin is mediated by a pertussis toxin-insensitive G-protein and modulated by intracellular calcium.

Inhibition of the M-current in PC12 cells by bradykinin has been studied under whole-cell recording conditions. In cells dialyzed with GTP-gamma-S, bradykinin produced a total and irreversible M-current suppression, while the inhibition was attenuated in cells dialyzed with GDP-beta-S. Inhibition occurred in cells pretreated with pertussis toxin, while this treatment prevented the modulation of Ca2+ currents by muscarine. The rate and extent of inhibition increased with the level of intracellular Ca2+ from 0 to 70 nM. These results indicate that a pertussis toxin insensitive G-protein mediates the action of bradykinin on the M-current, and some steps in the second messenger cascade are modulated by Ca2+.

Animals↗
PubMed1996

Acetylcholine receptor epsilon-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice.

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the gamma-subunit of the fetal acetylcholine receptor (AChR) by the epsilon-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the epsilon-subunit gene caused in homozygous mutant mice the persistence of gamma-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of epsilon-subunit containing AChRs is essential for normal development of skeletal muscle.

Animals↗
PubMed1996

Calcium permeability increase of endplate channels in rat muscle during postnatal development.

1. Patches of endplate membrane were isolated from rat flexor digitorum brevis muscle at different postnatal stages to measure the time course of development changes in conductance, deactivation time constant and relative Ca2+ permeability of endplate channels. 2. The predominant channel conductance was 40 +/- 1 pS (n = 9) at postnatal day 9 (P9) or younger whereas it was 59 +/- 3 pS (n = 5) at P21 or in older muscle. The deactivation time constant of ensemble patch currents evoked by brief ACh application, decreased from 8 +/- 3 ms (n = 45) at P5-9 to 2.3 +/- 0.3 ms (n = 5) in P21-28 muscle. 3. The relative Ca2+ permeability, measured by the shift of biionic (Ca2+/Cs+) reversal potential of ensemble patch currents upon the replacement of high [Cs+] by high [Ca2+] extracellular solution and with Cs+ as internal reference ion, increased during postnatal development. THe biionic reversal potential shift changed from -21 +/- 1 mV (n = 8) at P5 to -8 +/- 1 mV (n = 10) in P15 or older muscle. 4. Recombinant gamma-AChR channels expressed in Xenopus laevis oocytes had a biionic (Ca2+/Cs+) reversal potential shift of -24.9 +/- 2 mV (n = 14) comparable to that of neonatal endplate channels whereas the reversal potential shift for recombinant epsilon-AChR channels was -7.6 +/- 0.9 mV (n = 13), comparable to that of endplate channels in adult muscle. 5. It is concluded that an approximately 3-fold increase in Ca2+ current through endplate channels during postnatal development is caused by replacement of the fetal gamma-subunit by the epsilon-subunit in juvenile and adult muscle.

Acetylcholinesterase↗
PubMed1996

Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.

1. Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits (GluR-A or GluR-B) and kainate receptor (KAR) subunit (GluR-6) in their unedited (Q)- and edited (R)-forms were expressed in HEK 293 cells. To estimate the dimensions of the narrow portion of these channels, biionic reversal potentials for organic cations of different mean diameters were determined with Cs+ as the internal reference ion. 2. Homomeric channels assembled from Q-form subunits were cation selective. The relation between the relative permeability and the mean size of different organic cations suggests that the diameter of the narrow portion of Q-form channels is approximately 0.78 nm for AMPAR and 0.75 nm for KAR channels. 3. Homomeric channels assembled from R-form subunits were permeant for anions and cations. When probed with CsC1 gradients the relative chloride permeability (PC1/PCs) was estimated as 0.14 for GluR-B(R) and 0.74 for GluR-6(R)-subunit channels. The permeability versus mean size relation for large cations measured with the weakly permeant F- as anion, indicates that for the R-form KAR channels the apparent pore diameter is close to 0.76 nm. 4. Heteromeric AMPAR and KAR channels co-assembled from Q- and R-form subunits were cation selective. The diameter of the narrow portion of these channels is estimated to be in the range between 0.70 and 0.74 nm. 5. The results indicated that the diameters of the narrow portion of AMPAR and KAR channels of different subunit composition and of widely different ion selectivity are comparable. Therefore, the differences in the anion versus cation selectivity, in Ca2+ permeability and in channel conductance are likely to be determined by the difference in charge density of the channel.

Calcium Channels↗
PubMed1996

Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors.

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the gamma-subunit of the fetal muscle acetylcholine receptor (gamma-AChR) is replaced by the epsilon-subunit in the adult muscle receptor (epsilon-AChR), was investigated by analysing the structure of gamma- and epsilon-subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single-channel conductance. 2. The gamma- and epsilon-subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilon(gamma)- or gamma(epsilon)-subunits were constructed from the parental epsilon- and gamma-subunits, respectively. Exchanging the four hydrophobic segments (M1-M4) of the gamma-subunit for those of the epsilon-subunit and vice versa completely reversed the difference in conductance between gamma-AChR and epsilon-AChR channels. 4. Effects of single- and multiple-point mutations in M1-M4 segments of gamma- and epsilon-subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (gamma-subunit) for serine/isoleucine (epsilon-subunit) in M2, and the lysine (gamma-subunit) for glutamine (epsilon-subunit) exchanges in the regions flanking the M2 segment.

Age Factors↗
PubMed1996

Inhibition of the Kv4 (Shal) family of transient K+ currents by arachidonic acid.

We have found that transient A-type currents expressed in Xenopus oocytes from members of the Kv4 family are suppressed by arachidonic acid. Currents from members of the Kv1, Kv2, and Kv3 families showed little or no inhibition by fatty acids in this expression system, although Shaker currents showed a modest increase in peak amplitude. The inhibition of Kv4 channels was not prevented by cyclo-oxygenase, lipoxygenase, or cytochrome P-450 inhibitors and was mimicked by 5,8,11,14-eicosatetraynoic acid, an arachidonic acid analog that is not metabolized by these pathways. Other unsaturated cis fatty acids with more than two double bonds produced a similar effect. In inside-out macropatches, the current was reversibly reduced >50% by 2 microM arachidonic acid, and the inhibition developed in <40 sec. These results suggest that, at concentrations that are likely to be physiologically relevant, arachidonic acid interacts directly with the channel or with a closely associated component. Preliminary mutagenesis of Kv4.2 channels indicates that the N terminal is not required for arachidonic acid action but that the S4-S5 loop may influence the effect.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1996

Inhibition of the Kv4 (Shal) family of transient K+ currents by arachidonic acid.

We have found that transient A-type currents expressed in Xenopus oocytes from members of the Kv4 family are suppressed by arachidonic acid. Currents from members of the Kv1, Kv2, and Kv3 families showed little or no inhibition by fatty acids in this expression system, although Shaker currents showed a modest increase in peak amplitude. The inhibition of Kv4 channels was not prevented by cyclo-oxygenase, lipoxygenase, or cytochrome P-450 inhibitors and was mimicked by 5,8,11,14-eicosatetraynoic acid, an arachidonic acid analog that is not metabolized by these pathways. Other unsaturated cis fatty acids with more than two double bonds produced a similar effect. In inside-out macropatches, the current was reversibly reduced > 50% by 2 mM arachidonic acid, and the inhibition developed in < 40 sec. These results suggest that, at concentrations that are likely to be physiologically relevant, arachidonic acid interacts directly with the channel or with a closely associated component. Preliminary mutagenesis of Kv4.2 channels indicates that the N terminal is not required for arachidonic acid action but that the S4-S5 loop may influence the effect.

5,8,11,14-Eicosatetraynoic Acid↗