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M Criado

Publications and source records attributed to M Criado.

At least 37 records · Page 2Linked to original sources
PubMed1997

Expression of alpha 7 neuronal nicotinic receptors during postnatal development of the rate cerebellum.

Several lines of evidence suggest that alpha-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors may play a developmental role by modulating plasticity in neuronal circuits. The alpha 7 subunit, a main component of these receptors, is expressed in most regions of the brain, including the cerebellum, where it is present almost exclusively in Purkinje cells and deep cerebellar nuclei. Purkinje cells constitute the only efferent pathway of the cerebellum and their development involves complex interactions, which have been extensively studied. They therefore provide a potentially useful model for analysis of development plasticity which could be influenced by alpha 7 neuronal nicotinic receptors. In the present study a previously characterized monoclonal antibody (mAb 307) has been used to determine the temporal pattern of expression of the alpha 7 subunit in the developing rat cerebellum. No detectable alpha 7 immunoreactivity is found between P0 and P2. Between P3 and P5, however, the Purkinje cell layer shows moderate immunolabeling. alpha 7 expression in this layer increases rapidly between P8 and P15. This increase in alpha 7 staining, which overlaps in time with important developmental and synaptogenic events, is not uniform throughout the cerebellar cortex. Thus, between P3 and P5 all Purkinje cells are weakly labeled, while at later stages (P8-P15) immunolabeling becomes more intense, but at the same time, disappears from Purkinje cells in rostral lobules. In addition, a very well defined pattern for discontinuous or columnar labeling is detected in regions of the Purkinje cell layer where alpha 7 subunits were being expressed. Finally, at P20, alpha 7 subunit labeling is found again in all Purkinje cells, although with lower intensity. These results suggest that alpha 7 receptor expression is developmentally regulated, with a time course that parallels the final differentiation of Purkinje cells. In addition, the heterogeneous spatial distribution of alpha 7-containing nicotinic receptors indicates that, during cerebellar maturation, these cells may receive different signals that modulate receptor gene expression in a very specific way.

Animals↗
PubMed1997

Elevated glomerular and blood mononuclear lymphocyte nitric oxide production in rats with chronic bile duct ligation: role of inducible nitric oxide synthase activation.

Recent work indicates that nitric oxide (NO) plays an important role in the systemic and renal alterations of cirrhosis. In the present study, we have evaluated whether the inducible NO synthase (iNOS) isoform participates in the enhanced renal and systemic NO production of a rat model of cirrhosis. In vitro and in vivo experiments were performed in rats subjected to chronic bile duct ligation (BDL) and in sham-operated (SO) animals. Plasma nitrite (3.1 +/- 0.1 micromol/L in SO and 6.6 +/- 0.2 micromol/L in BDL), glomerular nitrite production (6.4 +/- 0.1 vs. 9.8 +/- 0.1 nmol/24h/7,000 glomeruli, respectively), and mononuclear lymphocyte cells nitrite production (0.3 +/- 0.04 vs. 0.6 +/- 0.12 nmol/10(6) cells, respectively) were all significantly higher in BDL than in SO. Moreover, mononuclear lymphocytes and glomeruli from BDL rats showed an increased expression of macrophage-type iNOS, detected by Western blot. Kidneys from BDL animals also showed an increased calcium-independent NO synthase activity, compared with those from SO rats. Constitutive endothelial-type NO synthase expression in glomeruli or the activity of calcium-dependent NO synthase in whole kidney did not show differences between BDL and SO rats. In cultured mesangial cells from normal rats, the addition of plasma from BDL but not of plasma from SO significantly stimulated (35%) nitrite production and increased the expression of macrophage-type iNOS. In addition, administration of aminoguanidine (AG), a preferential iNOS inhibitor, elevated dose-dependently mean arterial pressure in both groups, but this increase was greater in BDL (26.5 +/- 4.4 mm hg) than in SO (13.6 +/- 2.6). In BDL, AG also increased sodium and water excretion and glomerular filtration rate. In contrast, there were only small nonsignificant changes in SO animals. Therefore, these results indicate that the expression, activity and production of NO in kidneys, glomeruli, and mononuclear lymphocyte cells is elevated in BDL rats, and this is partly because of a plasma-derived substance(s), which stimulates iNOS formation. The amelioration of the arterial hypotension and the associated reduced excretory levels of these cirrhotic animals by aminoguanidine further support the involvement of the inducible NO synthase isoform in the renal alterations observed in BDL animals.

Animals↗
PubMed1997

Neuronal nicotinic acetylcholine receptors on bovine chromaffin cells: cloning, expression, and genomic organization of receptor subunits.

Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the alpha 3 subunit, which we have previously cloned, the presence of alpha 5 and beta 4 but not of beta 2 subunits was detected by reverse transcription-PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that alpha 3, alpha 5, and beta 4 subunits are coexpressed in all chromaffin cells. The primary structure of alpha 5 and beta 4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other beta 4-containing nicotinic receptors, the ones formed by the bovine beta 4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of alpha 3 and alpha 5 subunits, which together with the beta 4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the alpha 3 and alpha 5 genes overlap at their 3' ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.

Animals↗
PubMed1996

Acetylcholine receptor subunit homomer formation requires compatibility between amino acid residues of the M1 and M2 transmembrane segments.

The neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha3 and alpha7 have different assembly behavior when expressed in heterologous expression systems: alpha3 subunits require other subunits to assemble functional nAChRs, whereas alpha7 subunits can produce homomeric nAChRs. A previous analysis of alpha7/alpha3 chimeric constructs identified a domain comprising the first putative membrane-spanning segment, M1, as essential to homomeric assembly. The present study dissected further this domain, identifying three amino acid residues, which are located at the most intracellular third of the M1 transmembrane segment, as important in the assembly of homomers. Moreover, formation of homooligomeric complexes seems to require a compatible accommodation between this region and certain residues of the second transmembrane segment, M2. Thus, compatibility between defined domains of the M1 and M2 transmembrane segments appears as a determinant factor governing homomer association of nAChR subunits.

Amino Acid Sequence↗
PubMed1996

A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspartate residue therein, Asp-266 in the alpha7 subunit, was identified by site-directed mutagenesis as crucial, since mutants at this position exhibited very poor functional responses to three different nicotinic agonists. We have extended this investigation to another neuronal nicotinic receptor (alpha3/beta4), and found that a homologous residue in the beta4 subunit, Asp-268, played a similar role in coupling. These findings are consistent with a hypothesis that the aspartate residue in the M2-M3 loop, which is conserved in all homomer-forming alpha-type subunits and all neuronal beta-type subunits that combine to form functional receptors, is a major determinant of information transmission from binding site to channel gate in all neuronal nicotinic receptors.

Amino Acid Sequence↗
PubMed1995

alpha-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: molecular cloning, functional expression and alternative splicing of the alpha 7 subunit.

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine alpha 7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat alpha 7 and 89% to chicken alpha 7 subunits. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors; the alpha 7 receptor has a pentameric structure. Upon activation with nicotinic agonists the alpha 7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription-polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer expression when coinjected with the undeleted isoform.

Alternative Splicing↗
PubMed1995

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel.

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.

Amino Acid Sequence↗
PubMed1994

Role of two acetylcholine receptor subunit domains in homomer formation and intersubunit recognition, as revealed by alpha 3 and alpha 7 subunit chimeras.

Differential expression of subunit genes from the nicotinic acetylcholine receptor (AChR) superfamily yields distinct receptor subtypes. As each AChR subtype has a specific subunit composition and many subunit combinations appear not to be expressed, each subunit must contain some information leading to proper assembly. The neuronal AChR subunits alpha 3 and alpha 7 are expressed in bovine chromaffin cells, probably as constituents of two different AChR subtypes. These subunits have different assembly behavior when expressed in heterologous expression systems: alpha 7 subunits are able to produce homomeric AChRs, whereas alpha 3 subunits require other "structural" subunits for functional expression of AChRs. This feature allows the dissection of the requirements for subunit interactions during AChR formation. Analysis of alpha 7/alpha 3 chimeric constructs identified two regions essential to homomeric assembly and intersubunit recognition: an N-terminal extracellular region, controlling the initial association between subunits, and a second domain within a region comprising the first putative transmembrane segment, M1, and the cytoplasmic loop coupling it to the pore-forming segment, M2, involved in the subsequent interaction and stabilization of the oligomeric complex.

Amino Acid Sequence↗
PubMed1994

Immunocytochemical localization of the alpha 7 subunit of the nicotinic acetylcholine receptor in the rat central nervous system.

Previous molecular cloning studies have revealed that alpha-bungarotoxin binding proteins present in the brain are members of the neuronal nicotinic acetylcholine receptor gene family. The alpha 7 subunit is structurally related to the agonist binding subunits present in the central and peripheral nervous systems and, when expressed in Xenopus oocytes, forms functional channels blockable by alpha-bungarotoxin. In the present study, three different monoclonal antibodies raised against the alpha 7 subunit were used to map its distribution throughout the central nervous system of the rat. Immunohistochemical localization revealed that the alpha 7 subunit is expressed in most regions of the brain, being, overall, well correlated with previous "in situ" localization of alpha 7 transcripts and alpha-bungarotoxin autoradiographic binding studies. Particularly strong immunoreactivity was observed in several sensory and motor nuclei of the brainstem as well as the red nucleus. At the cellular level, alpha 7 immunostaining was usually found both in somata and dendrites, whereas axonal and terminal labeling was not observed. The widespread distribution of the alpha 7 subunit polypeptide is consistent with immunoprecipitation data demonstrating that it is a component of the predominant subtype of brain alpha-bungarotoxin-sensitive nicotinic receptors.

Amino Acid Sequence↗
PubMed1994

A delayed rectifier potassium channel cloned from bovine adrenal medulla. Functional analysis after expression in Xenopus oocytes and in a neuroblastoma cell line.

Using a cDNA library from bovine adrenal medulla, and, subsequently, a bovine genomic library, we have isolated the gene coding for a non inactivating potassium channel. This gene encodes a 597-amino acid protein which we have called BAK5 as its sequence is very similar to members of Kv1.5 potassium channel family. Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK5 DNA. Protein expression was under the control of a heat-shock promoter. Transfected cells showed a current highly selective for potassium, insensitive to tetraethylammonium but reversibly blocked by 4-aminopyridine. Oocytes injected with BAK5 mRNA also expressed a potassium current with the same characteristics.

4-Aminopyridine↗
PubMed1992

Molecular cloning and permanent expression in a neuroblastoma cell line of a fast inactivating potassium channel from bovine adrenal medulla.

Using a cDNA library from bovine adrenal medulla, we have isolated cDNAs coding for a potassium channel. These cDNAs encode a 660-amino acid protein that has a molecular weight of 73,288 kDa and no amino-terminal signal peptide. We have called it BAK4. Analysis of its sequence reveals close similarity (94% homology) with a recently described potassium channel from rat brain (RCK4) and heart (RHK1). Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK4 DNA. Expression of the DNA was under the control of a heat-shock promoter. Several clones, that were isolated by neomycin resistance selection, had integrated the plasmid DNA in a stable form. Upon heat induction, these cells produced BAK4 RNA and a potassium outward current, not present in control non-transfected cells. The current, which was transient and decayed markedly during the duration of 200 ms-pulses, can be described as a Ik(A) potassium current. The expression of these types of channels in brain (RCK4,RHK1), heart (RHK1) and adrenal medulla (BAK4) suggest their possible implication in important functions for the cell.

Adrenal Medulla↗
PubMed1992

Primary structure of an agonist binding subunit of the nicotinic acetylcholine receptor from bovine adrenal chromaffin cells.

Activation by acetylcholine of a nicotinic acetylcholine receptor on the membrane of bovine chromaffin cells leads to membrane depolarization and to the subsequent triggering of catecholamine secretion. It is evident that acetylcholine receptors play a central role in the initial phase of the secretion process and, therefore, an extensive characterization of their molecular components and properties is of fundamental interest. With this intention, we have screened bovine adrenal medullary cDNA libraries with a probe coding for a fragment of the rat muscle acetylcholine receptor alpha subunit. Several cDNA clones were isolated. The longest cDNA had an open reading frame encoding a 495-amino acid protein with a molecular weight of 56,911. The deduced primary structure contains features that indicate that the encoded protein is an alpha or acetylcholine binding subunit, and, in fact, it manifests significant sequence similarity to previously cloned alpha subunits. Sequence identity is particularly high with the alpha 3 subunit, which is expressed in the rat pheochromocytoma PC12 cell line and in several brain areas, and, consequently, it is considered a component of a neuronal acetylcholine receptor. Accordingly, the present results suggest that the agonist binding subunit of the nicotinic acetylcholine receptor from bovine chromaffin cells is an alpha 3-type subunit, corroborating previous immunological and pharmacological evidence for the presence of a neuronal nicotinic receptor in chromaffin cells.

Acetylcholine↗
PubMed1992

Muscarinic receptor subtypes in bovine adrenal medulla.

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of different selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60-80%) correspond to M2 muscarinic subtypes, and the rest is constituted by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.

Adrenal Medulla↗
PubMed1990

Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle.

The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-, delta- and epsilon-subunit precursors of the rat muscle acetylcholine receptor (AChR) are described. The deduced amino acid sequences indicate that these polypeptides contain 457-519 amino acids and reveal the structural characteristics common to subunits of ligand-gated ion channels. The pattern of subunit-specific mRNA levels in rat muscle shows characteristic changes during development and following denervation, suggesting that innervation of muscle reduces the expression of the alpha-, beta- and delta-subunit mRNAs, suppresses the expression of the gamma-subunit mRNA, and induces expression of epsilon-subunit mRNA. Subunit-specific cRNAs generated in vitro were injected into Xenopus laevis oocytes, resulting in the assembly of two functionally different AChR channel subtypes. The AChR gamma, composed of the alpha-, beta-, gamma- and delta-subunits, has functional properties similar to those of the native AChRs in fetal muscle. The AChR epsilon, composed of alpha-, beta-, delta- and epsilon-subunits, corresponds to the end-plate channel of the adult muscle. Thus in rat skeletal muscle the motor nerve regulates the expression of two functionally different AChR subtypes with different molecular composition by the differential expression of subunit-specific mRNAs.

Amino Acid Sequence↗
PubMed1990

Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle epsilon-subunit DNA.

The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of alpha-, beta-, gamma- and delta-subunits. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle. To investigate the role of the epsilon-subunit, which is believed to replace the gamma-subunit in forming the adult AChR subtype, BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR epsilon-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced epsilon-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant epsilon-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle epsilon-subunit may assemble with mouse muscle alpha-, beta- and delta-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in epsilon-subunit mRNA. Thus, the notion that replacement of the gamma-subunit by the epsilon-subunit during development is the result of the postnatal rise in the level of epsilon-subunit specific mRNA is further supported.

Animals↗
PubMed1989

Developmental regulation of five subunit specific mRNAs encoding acetylcholine receptor subtypes in rat muscle.

The muscular content of the mRNAs encoding the five subunits of the nicotonic acetylcholine receptor was measured during postnatal development in the rat. Subunit specific mRNAs show differential regulation. The levels of the alpha-, gamma- and delta-subunit specific mRNAs decrease steadily after birth, while the beta- and epsilon-subunit mRNAs increase transiently and then decrease. The adult pattern of subunit specific mRNA levels is reached at 4-6 weeks postnatally. The content of gamma- and epsilon-subunit mRNA changes in a reciprocal fashion during the first 2 postnatal weeks, supporting the view that differential regulation of gamma- and epsilon-subunit mRNA during development is one mechanism mediating the appearance of the adult, epsilon-subunit containing, subtype of end-plate channel. Denervation of neonatal muscle increases the levels of all subunit-specific mRNAs during further development. It prevents the postnatal decrease in gamma-subunit mRNA and enhances the initial increase in epsilon-subunit mRNA. This makes it appear that the epsilon-subunit gene is less sensitive to regulation by the nerve in the postnatal period than the gamma-subunit gene.

Actins↗