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C Estrada

Publications and source records attributed to C Estrada.

At least 37 records · Page 2Linked to original sources
PubMed1994

Effects of retinoic acid on NB 69 human neuroblastoma cells and fetal rat mid brain neurons.

Retinoids are chemical compounds which play important roles in ontogenetic development and cranio-caudal differentiation in animals, but their effect on phenotypic expression of neurotransmitters are unknown. We studied the pharmacological and morphological effects of retinoic acid (RA) on two types of immature vertebrate neurons, the human derived neuroblastoma cells, NB69, and fetal rat mid brain neurons in culture. The pharmacological effects of RA on the cultures and their relation to catecholamine and acetylcholine neurotransmission were evaluated according the levels of catecholamines, tyrosine hydroxylase (TH) activity, TH immunostaining, and choline acetyltransferase (CAT) activity, respectively. RA reduces catecholamine levels and TH activity in NB69 cells and the number of dopamine neurons in cultures derived from rat fetal mid brain. The detrimental effect of RA on mid brain neurons is dose- dependent; limited to TH+ cells at low concentrations (100 to 500 nM) and toxic for all types of cells at high concentrations (1 to 2 microM). RA increases CAT activity in NB 69 cells and produces phenotypic differentiation of these to a more mature neuronal phenotype with more prolonged neurite extensions. Therefore, RA may play a trophic positive role in the differentiation of immature cells to cholinergic neurons; this contrasts with the detrimental effects of RA on catecholamine neurons.

Animals↗
PubMed1993

Local NADPH-diaphorase neurons innervate pial arteries and lie close or project to intracerebral blood vessels: a possible role for nitric oxide in the regulation of cerebral blood flow.

Electrical stimulation of perivascular nerves induced a relaxation of endothelium-denuded cat pial arteries that was significantly reduced by nitric oxide (NO) synthase inhibition, indicating that NO was involved in the neurogenic relaxation of these vessels. Histochemical staining of the pial arteries for NADPH-diaphorase (NADPH-d), used as a marker for NO synthase, showed positive nerve fibers in the adventitial layer. Interestingly, in some restricted areas stained neuronal cell bodies were also observed. These neurons were scattered or distributed in small groups in a ganglion-like manner, and they sent fibers to the vessel wall. No NADPH-d-positive nerve fibers or cell bodies were detected in forelimb, pulmonary, or coronary arteries. Within the brain parenchyma, blood vessels also showed positive fibers around their walls. These fibers were organized in a branching pattern and presented varicosities. NADPH-d-positive neurons were found in the proximity of the intracerebral vascular profiles, sending processes to the vessels and/or being directly apposed to their wall. The neurovascular contacts were preferentially located close to the interface between the cerebral cortex and white matter. The anatomical relationship between NADPH-d-positive neurons and fibers and the cerebral blood vessels, together with the participation of NO in the neurogenic relaxation of pial arteries, suggests that NO is involved in the regulation of cerebral blood flow.

Animals↗
PubMed1993

Direct cytotoxicity of ethylcholine mustard aziridinium in cerebral microvascular endothelial cells.

The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibitor of choline uptake in brain synaptosomes and is used as a neurotoxin to produce animal models of cholinergic hypofunction. However, previous studies have shown that intraocular administration of AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of lactate dehydrogenase activity in the culture medium, AF64A produced similar concentration-dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human cholinergic neuroblastoma cell line SK-N-MC, but not in bovine cerebral smooth muscle cells. The toxic effect of AF64A correlated well with the affinity of the choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell choline carrier, as demonstrated by the following observations: (a) AF64A inhibited [3H]choline uptake in a concentration-dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of choline or hemicholinium-3 to the culture medium prevented the AF64A-induced toxicity in endothelial cell cultures.

Animals↗
PubMed1992

Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.

The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and gamma-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock.

Acetylcholine↗
PubMed1992

Nitric oxide mediates tumor necrosis factor-alpha cytotoxicity in endothelial cells.

Tumor necrosis factor alpha (TNF-alpha) exerts multiple actions on endothelial cells including among others the expression of pro-coagulant activity and adhesion molecules, and secretion of cytokines. We now show that TNF-alpha induces a time- and dose-dependent cytotoxic effect on cultured bovine aortic endothelial cells. This TNF-induced cytotoxicity, which is preceded by increased production of nitric oxide (NO), is significantly decreased by the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-alpha-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-alpha on endothelial cells.

Amino Acid Oxidoreductases↗
PubMed1992

Ouabain-sensitive choline transport system in capillaries isolated from bovine brain.

In physiological conditions, there is a net transport of choline from brain to blood, despite the fact that the choline concentration is higher in plasma than in CSF. Because of the blood-brain barrier characteristics, such passage against the concentration gradient takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, [3H]choline uptake properties have been analyzed in capillaries isolated from bovine brain. [3H]Choline uptake was linear with time for up to 1 h. Nonlinear regression analysis of the uptake rates at different substrate concentrations gave the best fit to a system of two components, one of which was saturable (Km = 17.8 +/- 4.8 microM; Vmax = 11.3 +/- 3.4 pmol/min/mg of protein) and the other of which was nonsaturable at concentrations up to 200 microM. The [3H]choline transport was significantly reduced in the absence of sodium and after incubation with 10(-4) M ouabain for 30 min. Ouabain also inhibited choline uptake in purified cerebral endothelial cells, but not in the endothelium isolated from bovine aorta. Accordingly, cerebral endothelial cells were able to concentrate [3H]choline, with this effect being abolished by ouabain, whereas in aortic endothelial cells the [3H]choline intracellular concentration was never higher than that of the incubation medium. These results suggest that the blood-brain barrier endothelium is specifically provided with an energy-dependent choline transport system, which may explain the choline efflux from the brain and the maintenance of a low choline concentration in the cerebral extracellular space.

3-O-Methylglucose↗
PubMed1991

Periendothelial acetylcholine synthesis and release in bovine cerebral cortex capillaries.

Choline acetyltransferase (ChAT) activity is present in isolated cerebral capillaries, where it has been considered to be a marker for perivascular cholinergic nerve terminals. However, ChAT-like immunoreactivity has been visualized in endothelial cells. This finding raised the possibility that at least part of the biochemically detected ChAT has a nonneuronal origin. To evaluate the relative contribution of endothelial cells and nerve fibers to the total acetylcholine (ACh)-synthesizing capacity of cerebral capillaries, ChAT activity and ACh release were measured in capillaries and in purified endothelial cells isolated from bovine cerebral cortex. Isolated capillaries showed ChAT activity, which was inhibited by 2-benzoylethyl trimethylammonium to the same extent as cerebral ChAT. When preincubated with [3H]choline, these capillaries presented a calcium-dependent enhancement in tritium release upon electrical field stimulation. Purified endothelial cells had minor ChAT activity and lacked the ability to release tritium in response to electrical stimulation, although the endothelial markers alkaline phosphatase, gamma-glutamyltranspeptidase, and 1,1'-dioctadecyl-1,3,3',3'-tetramethyl-iodocarbocyanide perchlorate-labeled acetylated low-density lipoprotein uptake were fully preserved. These data indicate that, within isolated cerebral capillaries, ACh is synthesized and released by a periendothelial structure. The fact that ACh release is provoked by electrical stimulation and by a calcium-dependent mechanism strongly suggests that cerebrovascular ACh has a neuronal origin.

Animals↗
PubMed1991

Choline acetyltransferase activity associated with cerebral cortical microvessels does not originate in basal forebrain neurons.

Cerebral cortical microvessels are innervated by cholinergic fibers that are probably involved in the regulation of local cerebral blood flow and blood-brain barrier permeability. The possibility exists that the cholinergic terminals associated with the cortical microvasculature belong to neurons from the nucleus basalis magnocellularis (NBM), where 70% of the cortical cholinergic projections originate. To test this hypothesis, ibotenic acid (25 nmol) was injected unilaterally in the NBM in rats, and 14 days later, choline acetyltransferase (ChAT) activity was measured in the frontoparietal cortex and in a blood vessel fraction isolated from this region. Lesions of the NBM resulted in a 50% decrease of cortical ChAT as compared with control or sham-operated hemispheres; however, no changes were observed in the ChAT activity associated with cortical microvessels. These results indicate that, in rat cerebral cortex, the perivascular cholinergic terminals do not originate in the basal forebrain.

Animals↗
PubMed1991

Nitric oxide mediates the neurogenic vasodilation of bovine cerebral arteries.

Nitric oxide (NO) is a mediator of the vasodilation induced by a variety of physiological and pharmacological stimuli. The possible role of NO in the relaxation elicited in cerebral arteries by perivascular nerve stimulation has been investigated. Electrical field stimulation of precontracted bovine cerebral arteries induced a relaxation that was blocked by tetrodotoxin, but not by adrenergic or muscarinic receptor antagonists, suggesting the existence of noradrenergic, noncholinergic dilator nerves, as has been shown in other species. The relaxation was significantly reduced by the inhibitors of NO synthesis, NG-monomethyl-L-arginine and nitro-L-arginine methyl ester, but not by the enantiomer, NG-monomethyl-D-arginine. Such a reduction was reversed by L-arginine. In addition, transmural nerve stimulation (TNS)-induced relaxation was potentiated by superoxide dismutase. No response to TNS was observed in arteries without endothelium. These results suggested that neurogenic relaxation of bovine cerebral arteries is mediated by endothelium-derived NO.

Animals↗
PubMed1990

Neuronal and microvascular alterations induced by the cholinergic toxin AF64A in the rat retina.

The choline analogue ethylcholine mustard aziridinium ion (AF64A) produces both neuronal and non-neuronal alterations in the rat retina. The possible involvement of the retinal capillaries in the origin of the apparently non-specific lesions has been investigated. Two hours after a single intraocular injection of 5 nmol AF64A, ultrastructural alterations were observed in neurons of the inner nuclear layer and the ganglion cell layer, where cholinergic cells are located. One week later, the number of cholinergic neurons, identified by choline acetyltransferase immunohistochemistry, was decreased to 65% of control, the neurons located in the inner nuclear layer being more sensitive than those in the ganglion cell layer. The same dose of AF64A also induced ultrastructural changes in retinal capillaries, which showed a significant increase in the number of pinocytotic vesicles and microvilli in the endothelial cells, 2-5 h after the toxin administration. One day later, arterioles and capillaries presented contracted profiles and the lumen was occasionally lost. The sensitivity of endothelial cells to the toxic effects of AF64A may be explained by the presence in the cerebral endothelium of a choline transport mechanism with an affinity close to that of cerebral synaptosomes. In vitro, both neuronal and endothelial choline uptake systems were equally sensitive to the toxin inhibitory effect. The early and severe vascular alterations induced in the retinal microvessels by AF64A may produce changes in blood perfusion and capillary permeability that could account for the apparently non-specific histological damage.

Animals↗
PubMed1990

Astrocyte growth stimulation by a soluble factor produced by cerebral endothelial cells in vitro.

Conditioned medium from isolated cerebral capillary endothelial cells (ECCM) was found to promote DNA synthesis in astrocytes and pericytes, but not in oligodendrocytes or endothelial cells (EC) in vitro. The astrocyte was the cell of primary interest and the cell tested in the following experiments. The effect of ECCM on astrocytes was concentration and time dependent. The growth factor was released by EC into the medium in a cumulative manner for up to 72 hours. This release was not the result of a nonspecific leakage of an internal store, since the DNA synthetic activity of cell lysates was negligible. The growth factor secretion per cell was higher in sparse than in confluent EC cultures and was partially inhibited by preincubation of EC with interleukin-1. The DNA synthetic activity was due to a peptide, different from basic fibroblast growth factor, transferrin, bovine fibronectin and platelet derived growth factor, with a molecular weight greater than 50,000. The peptide derived from the cerebral capillary EC could be involved in the local signaling between cell types that control new vessel formation in development, in regeneration after brain tissue injury, or in tumor formation.

Animals↗
PubMed1990

Choline uptake by cerebral capillary endothelial cells in culture.

A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood-brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier-mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed a Km of 7.59 +/- 0.8 microM and a maximum capacity of 142.7 +/- 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1-100 microM). When cells were preincubated in the presence of choline, a single saturable component was observed with a Km of 18.5 +/- 0.6 microM and a maximum capacity of 452.4 +/- 42 pmol/2 min/mg of protein. [3H]Choline uptake by endothelial cells was temperature dependent and was inhibited by the choline analogs hemicholinium-3, deanol, and AF64A. The presence of ouabain or 2,4-dinitrophenol did not affect the [3H]choline transport capacity of endothelial cells. Replacement of sodium by lithium and cell depolarization by potassium partially inhibited choline uptake. When cells had been preincubated without choline, recently transported [3H]choline was readily phosphorylated and incorporated into cytidine-5'-diphosphocholine and phospholipids; however, under steady-state conditions most (63%) accumulated [3H]choline was not metabolized within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Endothelial cells inhibit the vascular response to adrenergic nerve stimulation by a receptor-mediated mechanism.

Rabbit central ear arteries, with and without endothelium, were perfused at a constant flow rate and the perfusion pressure was measured as an index of the vessel resistance. Transmural nerve stimulation (TNS) induced a frequency-dependent increase in perfusion pressure in all vessels that was blocked by tetrodotoxin, phentolamine, and prazosin. Removal of endothelium significantly enhanced contractions induced by TNS. The inhibitory effect of endothelium was not modified by indomethacin but was abolished by hemoglobin, indicating that endothelium-derived relaxant factor (EDRF) was the vasodilator involved. The endothelium-dependent inhibitory effect (rubbed vessel minus control vessel contractions) increased with time during the first 10-20 s after the beginning of TNS, and was frequency dependent and inhibited by low doses of phentolamine, which suggest a receptor-mediated mechanism. To analyze whether amine neurotransmitters are able to permeate the artery wall and contact the endothelial cell membrane, the passage of [3H]acetylcholine from the abluminal side to the lumen was studied in intact vessels. [3H]acetylcholine readily permeated the vessel wall, as assessed by radioautography, and appeared in the perfusion fluid at a concentration that explains the relaxation induced by perivascular acetylcholine. These data suggest that endothelial cells modulate the effect of perivascular neurotransmitters by a receptor-mediated mechanism. In the case of the sympathetic innervation, such modulation would be more relevant at low levels of transmitter release and would be minimized during intense sympathetic stimulation.

Acetylcholine↗
PubMed1988

Acetylcholinesterase-containing fibers and choline acetyltransferase activity in isolated cerebral microvessels from goats.

Microvessels have been isolated from goat cerebral cortex and caudate nucleus. The purity of the preparations was assessed by light microscopy and by the high enrichment in the marker enzymes alkaline phosphatase and gamma-glutamyltransferase. Choline acetyltransferase activity was detected in the vascular fractions, being significantly higher in capillaries than in larger vessels. Acetylcholinesterase (AChE)-containing fibers were visualized in vessels of different caliber. Vessels with diameters larger than 70-90 microns showed a network pattern of fibers similar to that of pial arteries. In small vessels (10-70 microns) longitudinal or helical fibers were observed with occasional side-branches that surround the vessel. No AChE staining was visualized in isolated capillaries under light microscopy. This study shows that isolated intracerebral microvessels are suitable preparations for histochemical studies of perivascular nerves. Taken together, the biochemical and histological results are in accordance with a cholinergic innervation of the goat intracerebral vasculature.

Acetylcholinesterase↗
PubMed1988

Biochemical and histological modifications of the rat retina induced by the cholinergic neurotoxin AF64A.

Intraocular injections of ethylcholine mustard aziridinium ion (AF64A) in the rat depressed retinal choline acetyltransferase (ChAT) activity in a dose-dependent manner without any significant change in the content of amino acid neurotransmitters GABA, glycine, aspartate and glutamate. ChAT reduction was already detected 24 h after the injection and persisted for at least one month. In vitro AF64A also inhibited retinal ChAT activity. No changes in muscarinic receptor sites were detected. The histological study showed light cells, characterized by cytoplasmic swelling in the innermost part of the inner nuclear layer and in the ganglion cell layer. We suggest that these light cells are the cholinergic retinal neurons affected by the toxin. In addition, dark cells in the inner nuclear layer, large empty spaces in the outer nuclear layer, inflammatory infiltrate and vascular alterations were also observed in treated retinas. Choline uptake systems in photoreceptors and in endothelial cells or cholinergic perivascular nerve endings may explain the lesions observed in the outer nuclear layer and the vascular alterations.

Amino Acids↗
PubMed1988

Regional differences in cerebrovascular cholinergic innervation in goats.

The presence and distribution of a cerebrovascular cholinergic system were studied in goats. Regional cerebral blood flow was measured in the parietal cerebral cortex, caudate nucleus, and white matter by the hydrogen clearance technique in unanesthetized goats. Intravenous low doses of physostigmine, but not of neostigmine, significantly increased regional blood flow without changing mean arterial blood pressure or behavior. Increases of blood flow were greater in cerebral cortex and caudate nucleus than in white matter although the vasodilation induced by hypercapnia was similar in the three regions. Intracerebral microvessels were isolated from cerebral cortex, caudate nucleus, and white matter to evaluate choline acetyltransferase activity as a marker for perivascular cholinergic nerves. The enzyme level was higher in vessels from cerebral cortex and caudate nucleus than in vessels from white matter, which is in accordance with the functional data. These results suggest the presence of a cholinergic perivascular innervation system in intracerebral microvessels. Such innervation has a nonhomogeneous distribution throughout the brain and might be implicated in the local regulation of cerebral blood flow.

Animals↗