Carmen Estrada | Universidad de Cadiz (original) (raw)

Papers by Carmen Estrada

Journal of Cerebral Blood Flow and Metabolism, Sep 1, 1997

The partIcipation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regul... more The partIcipation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated, Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH diaphorase staining in the adventitial layer of bovine cerebral arteries, NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunola beling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 rnA, 0.2 millisec onds, I to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2ex, produced tetrodo toxin-sensitive relaxations that were completely suppressed by AP-nitro-L-arginine methyl ester (L-NAME) and by the gua nylyl cyclase inhibitor I H-[ I ,2.4Joxadiazolo[ 4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536

Brain Research, Jun 1, 1990

The choline analogue ethylcholine mustard aziridinium ion (AF64A) produces both neuronal and non-... more The choline analogue ethylcholine mustard aziridinium ion (AF64A) produces both neuronal and non-neuronal alterations in the rat retina 9. 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 ceils 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.

PubMed, Mar 1, 1984

The effects of vasopressin on the cerebral circulation were studied in conscious goats and in iso... more The effects of vasopressin on the cerebral circulation were studied in conscious goats and in isolated human and goat cerebral arteries. Infusion of 1 to 12 mU of vasopressin into the internal maxillary artery of unanesthetized goats caused dose-dependent reductions in cerebral blood flow, a decrease of 36 +/- 4.7% (mean +/- S.E.) occurring with the highest dose. Cumulative application of vasopressin (10(-12) to 10(-6) M) markedly constricted human and goat cerebral arteries in vitro, the effect being more prominent in human vessels. (1-Deaminopenicillamine, 4-valine)-8-D-arginine-vasopressin, a competitive antagonist of the pressor effects of vasopressin, partially inhibited the cerebral vasoconstriction produced by vasopressin in vivo and in vitro without affecting the vasoconstrictor responses to norepinephrine, 5-hydroxytryptamine and potassium chloride. The results indicate that low concentrations of vasopressin produce constriction of cerebral vessels by direct excitatory effects on specific receptor sites. This effect should be considered in certain pathophysiological states in which vasopressin is released in amounts that could interfere with the proper blood supply to the brain.

PubMed, Apr 1, 1982

Muscarinic cholinergic receptor sites in cerebral blood vessels were analyzed directly by using r... more Muscarinic cholinergic receptor sites in cerebral blood vessels were analyzed directly by using radioligand binding techniques with the specific muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) as ligand. Specific binding of [3H]QNB to membrane preparations from isolated bovine pia-arachnoid vessels was found to be saturable, of high affinity (Kd = 5.4 X 10(-10) M) and selectively inhibited by muscarinic antagonists (atropine) and agonists (acetylcholine, carbachol and methacholine). Scatchard and Hill plot analyses of the data indicated one class of sites for the antagonists, but two independent classes of sites were revealed with the agonists. Interestingly, the high and low affinities of the agonist sites correlated with the concentrations of agonist which have been reported to produce dilation and contraction, respectively, of isolated pial artery segments. A substantial number of specific [3H]QNB binding sites also were measured in isolated bovine cerebral microvessels providing direct evidence for muscarinic receptors in intracerebral vessels. In contrast, specific [3H]QNB binding was barely detectable in mesenteric blood vessels. Choline acetyltransferase (ChAT) activity was measured in parallel with [3H]QNB binding. The relative distribution of choline acetyltransferase was correlated with that for the binding, with the highest values observed in the intracerebral microvessels. This suggests a functional innervation of the cerebrovascular muscarinic receptors.

Canadian Journal of Physiology and Pharmacology, 1990

Rabbit central ear arteries, with and without endothelium, were perfused at a constant flow rate ... more 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.Key words: adrenergic innervation, endothelium-derived relaxant factor, endothelial receptors, endothelium, vascular innervation.

Stroke, Mar 1, 1981

We studied the in vivo and in vitro effects of diazoxide on the cerebral circulation of 8 normote... more We studied the in vivo and in vitro effects of diazoxide on the cerebral circulation of 8 normotensive (mean arterial pressure = 100 mm Hg) and 5 renal hypertensive (mean arterial pressure = 146 mm Hg) goats. Injections of diazoxide (03-27 mg) into the internal maxillary artery of unanesthetized goats produced dose-dependent increases in cerebral blood flow (electromagnetic flowmeter), this effect being significantly higher in hypertensive goats. Intravenous injection of 5 mg/kg of diazoxide into normotensive goats increased cerebral blood flow 40 ml/mln/100 g and mean arterial pressure dropped 22 mm Hg whereas in hypertensive goats cerebral blood flow was unchanged and mean arterial pressure decreased 50 mm Hg. The increase in heart rate due to intravenous diazoxide was similar in normotensive and hypertensive goats (35 beats/min). Cumulative applications of diazoxide (10** to 10~*M) on isolated middle cerebral arteries produced dilatory responses both under resting conditions and after previous tonic contraction by serotonin. This relaxation was significantly greater in arterial segments from hypertensive goats. The results indicate that diazoxide exerts powerful dilatatory effects on cerebral vessels, both in vivo and in vitro, and that these effects are particularly evident in hypertensive animals.

Journal of Cerebral Blood Flow and Metabolism, Sep 1, 1991

British Journal of Pharmacology, Oct 1, 1996

We investigated the vascular effects of agmatine (decarboxylated arginine), an endogenous ligand ... more We investigated the vascular effects of agmatine (decarboxylated arginine), an endogenous ligand for x2-adrenoceptors and non-adrenoceptor imidazoline (I-) receptors, present in endothelium, smooth muscle and plasma, using the rat tail artery as a model. 2 While by itself agmatine (10 nM-1 mM) was without effect on isolated arterial rings, at the highest concentration used (1 mM) it slightly increased EC50 values for contractions elicited respectively by the xl-and x2-adrenoceptor agonists methoxamine and clonidine. 3 Agmatine (0.03-1 mM) produced a concentration-dependent transient inhibition of the contractions induced by transmural nerve stimulation (TNS; 200 mA, 0.2 ms, 1 Hz, 10 s). This effect was abolished by the CL2-adrenoceptor antagonists, rawolscine and idazoxan. 4 In the presence of rawolscine or idazoxan, agmatine produced a concentration-dependent delayed facilitation of TNS-induced contractions, which was prevented by cocaine. 5 Neither inhibitory nor potentiating actions were produced by agmatine on contractions induced by noradrenaline (NA) administration. 6 Agmatine did not directly affect [3H]-NA uptake in bovine cultured chromaffin cells. 7 Agmatine can regulate vascular function by two opposing actions at sympathetic nerve terminals, with different latencies: a transient inhibition of NA release mediated by prejunctional x2-adrenoceptors and a cocaine-sensitive delayed facilitation the mechanism of which is undetermined at present. 8 The results reveal the existence of a novel endogenous amine modulating NA release in the perivascular sympathetic terminals.

In vitro, Mar 1, 1985

Cerebrovascular endothelial cells from adult bovine brain were carried successfully in long-term,... more Cerebrovascular endothelial cells from adult bovine brain were carried successfully in long-term, serial culture. Endothelial cells were obtained from the middle and anterior cerebral arteries and from capillaries isolated from grey matter of the cerebral cortex or caudate nucleus. Capillary ceils were found to grow best in RPMI 1640 with 20% fetal bovine serum. They did not require tumor-conditioned medium or matrix-coated surfaces, although fibronectin was used to enhance the initial plating efficiency of the primary cultures. The same conditions were used to support satisfactory growth of arterial endothelial cells; however they did not grow as rapidly as the capillary cells. Retention of endothelial-specific characteristics were shown for capillary-derived ceils carried up to Passage 28, arterial-derived cells up to Passage 11, and after frozen storage of both types of cultured cells. Cultures of both arterial and capillary cells stained positively for Factor VIII antigen, exhibited a nonthrombogenic surface, and produced prostacyclin in response to arachidonic acid. Arterial endothelial cells produced more prostacyclin than capillary endothelium. The capillary ceils had a unique tendency to assume a ringlike morphology after subculture and sometimes formed capillarylike networks of cell cords in dense cultures. When cultured in a three-dimensional plasma clot, capillary and arterial endothelial cells, but none of the other cell types studied, organized into tubelike structures reminiscent of capillary formation in vivo. The availability of long-term cultures of cerebrovascular endothelial ceils provides an opportunity to compare properties of arterial and capillary endothelium from the same tissue and to investigate such processes as angiogenesis and blood-brain barrier induction.

Brain Research, May 1, 1983

Muscarinic cholinergic receptor sites were detected with [3H]quinuclidinylbenzilate (QNB) binding... more Muscarinic cholinergic receptor sites were detected with [3H]quinuclidinylbenzilate (QNB) binding techniques in two fractions of bovine intracerebral vessels; one of the fractions contained primarily small arteries and veins with some attached capillaries, and the other one was highly enriched in capillaries. The amounts of binding were similar in equivalent vascular fractions isolated from cerebral cortex, caudate nucleus and cerebellar cortex in spite of large differences among the 3 regions in [aH]QNB binding to brain tissue. The different distribution of muscarinic receptors in brain tissue and blood vessels argues against the possibility that the receptors represent a contamination of the vascular fractions by brain parenchyma. Cerebral endothelial cells, which were isolated by treating capillaries with collagenase, bound [ZH]QNB to the same extent as did cerebral capillaries. This is consistent with an endothelial localization of capillary muscarinic receptors. Choline acetyltransferase (CHAT) activity, a marker for cholinergic neurons, also was present in the vascular preparations. Within each brain region, ChAT activities in capillaries and larger vessels were similar, but significant regional differences were found for vascular ChAT activity, with the highest values in the caudate. Isolated endothelial cells contained significantly lower levels of ChAT activity than intact capillaries, suggesting a periendothelial location of the enzyme, as would also be the case for attached nerve terminals. The presence of [ZH]QNB binding sites and ChAT activity in intracerebral blood vessels is consistent with an innervation of the cerebral vasculature by a cholinergic system that may regulate cerebral blood flow and capillary permeability.

Journal of Vascular Research, 1981

The effect of diazoxide on isometric tension of goat middle cerebral arteries was investigated bo... more The effect of diazoxide on isometric tension of goat middle cerebral arteries was investigated both under resting conditions and under contraction produced by 10(-6) M serotonin. In addition, the inhibitory action of diazoxide was tested against contractile effects induced by different experimental interventions such as electrical field stimulation, norepinephrine, tyramine, histamine, and KCl. Diazoxide caused dose-dependent relaxation of cerebral arteries which was more pronounced when the vessels were previously contracted. High doses of diazoxide (10(-3) M) inhibited significantly the contraction induced by electrical field stimulation and all the vasoactive agents used. This inhibitory effect of diazoxide was greater for those drugs that directly or indirectly act through alpha-adrenergic receptor stimulation. Tritium release induced by electrical field stimulation of cerebral arteries previously labelled with 3H-norepinephrine was not affected in the presence of diazoxide. We conclude that diazoxide has a dilatory effect on goat brain vessels due to direct relaxation of smooth muscle together with a possible blockade of the alpha -adrenergic receptors. This effect might explain the maintenance of cerebral blood flow observed in vivo during diazoxide-induced arterial hypotension.

Journal of Neurochemistry, Apr 1, 1993

The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibi... more 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.

Journal of Neurochemistry, Mar 1, 1985

The effects of kainic acid (KA), quisqualic acid (QA), and ibotenic acid (IBO) on histology of th... more The effects of kainic acid (KA), quisqualic acid (QA), and ibotenic acid (IBO) on histology of the retina and on the retinal choline acetyltransferase (ChAT) activity were studied in the rat. KA produced the highest number of altered cells in the ganglion cell layer (GCL) and in the inner nuclear layer (INL), with an almost complete depletion of ChAT activity. QA was less effective than KA in terms of both the number of altered cells and in ChAT depletion. In contrast, retinas injected with IBO showed the mildest morphological lesions together with the highest reduction in the enzyme activity. These re

Journal of Cerebral Blood Flow and Metabolism, Nov 1, 1995

The effects of tumor necrosis factor-a (TNF ex) on the production of the vasoactive substances ni... more The effects of tumor necrosis factor-a (TNF ex) on the production of the vasoactive substances nitric oxide (NO) and endothelin-l (ET-l) were investigated in cerebrovascular cells in culture. Bovine cerebral endo thelial cells (BCEC) stained positively for NADPH diaphorase/NO synthase activity and spontaneously pro duced nitrite, a stable NO oxidation product, which ac cumulated in the culture medium in a linear way for 48 h. Low concentrations of TNF-a (0.5-2 ng/ml) significantly enhanced nitrite production after a 24-h incubation. Higher concentrations or longer exposure times resulted in a cytotoxic effect that altered cell morphology, re leased lactate dehydrogenase (LDH) to the culture me dium, and reduced the protein content. Dexamethasone, but not the NO synthase inhibitor N-iminoethyl-L ornithine (L-NIO), prevented the cytotoxic effect of TNF-a in BCEC. TNF-a also significantly enhanced ni

Biochemical and Biophysical Research Communications, Jul 1, 1992

Tumor necrosis factor alpha (TNF-oJ exerts multiple actions on endothelial cells including among ... more Tumor necrosis factor alpha (TNF-oJ 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-o~ 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-iminoethyI-Lornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-c~-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-c~ on endothelial cells.

Brain Research, 1988

Intraocular injections of ethylcholine mustard aziridinium ion (AF64A) in the rat depressed retin... more 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.

Stroke, 1988

The presence and distribution of a cerebrovascular cholinergic system were studied in goats. Regi... more 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 pres...

Biochemical and Biophysical Research Communications, 1992

The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of norad... more 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-monomethyI-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 7-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. ® 1992 Academic Pr .... Inc. Vascular tone is controlled by the simultaneous influence of neurogenic mechanisms (1) and the release of nitric oxide (NO) by the endothelial cell layer (2). Circulating factors can also affect vascular resistance, either by acting directly on smooth muscle cells or by modulating NO release (3). Although in some vascular beds, such as cerebral (4) or mesenteric vessels (5), both constrictor and dilator perivascular nerves have been described, most vessels are innervated only by

European journal of biochemistry, Apr 1, 2003

Nitric oxide (NO •) strongly inhibits the proliferation of human A431 tumour cells. It also inhib... more Nitric oxide (NO •) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170-kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58-kDa protein which we have denoted NOIPP-58 (nitric oxide-induced 58-kDa phosphoprotein). The NO •-induced phosphorylation of NOIPP-58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP-58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP-independent processes. We also demonstrate that the p38 mitogen-activated protein kinase (p38MAPK) pathway is activated by NO • in the absence and presence of EGF, whereas the activity of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and the c-Jun N-terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate-sensitive dephosphorylation of prephosphorylated EGFR and NOIPP-58. We propose that the dephosphorylation of both NOIPP-58 and EGFR are mediated by a p38MAPKcontrolled phosphotyrosine-protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP-58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.

Biochemical Journal, Sep 1, 1997

Although it has been demonstrated that NO inhibits the proliferation of different cell types, the... more Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its antimitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-Nacetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-o4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butylqpropane-1,3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-$H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the