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Biomedical subjects

D J Reis

Publications and source records attributed to D J Reis.

At least 91 records · Page 5Linked to original sources
PubMed1995

Imidazoline receptors in vascular smooth muscle and endothelial cells.

We sought to determine if smooth muscle and endothelial cells of blood vessels express imidazoline receptors. Membranes of cultured smooth muscle cells specifically bind with high affinity to alpha 2-adrenergic ligands, [3H]p-aminoclonidine, [3H]rauwolscine, and [3H]idazoxan. All of [3H]rauwolscine and [3H]p-aminoclonidine but less than 10% of [3H]idazoxan binding was displaced by 10 microM epinephrine, indicating a nonadrenergic binding site for [3H]idazoxan. [3H]Idazoxan binding was inhibited with a rank order of potency: cirazoline > idazoxan > naphazoline >> guanabenz > amiloride > clonidine = phentolamine. Agmatine, an endogenous ligand for I-receptors, inhibited binding with a Ki of 240 +/- 25 nM. The binding of [3H]idazoxan to membranes of pulmonary artery endothelial cells was to both alpha 2-adrenergic and imidazoline receptors. Cultured smooth muscle cells, as well as rat carotid arterioles, were specifically immunostained by antibodies to an I-receptor-associated protein. We conclude that vascular smooth muscle and endothelial cells express not only alpha 2-adrenergic receptors but also I-receptors of the I2 subclass with high affinity for agmatine. Since serum contains an endogenous ligand for I-receptors, possibly agmatine, the results suggest the presence of a novel receptor mechanism on vascular smooth muscle which may regulate vascular tone.

Animals↗
PubMed1995

Decerebration does not alter hypoxic sympathoexcitatory responses in rats.

In anesthetized, paralyzed and ventilated rats, hypoxia, produced by intratracheal administration of 100% N2 for 20 s, increases sympathetic nerve activity and produces cardiovascular responses. Acute midcollicular decerebration has no effect on these responses in chemo-innervated or chemo-denervated animals. Suprapontine neural structures are, therefore, not required for the rapid sympathetic and cardiovascular responses to acute hypoxia. The results support the view that sympathoexcitatory responses to acute hypoxia depend entirely on the functions of reticulospinal sympathoexcitatory vasomotor neurons of the rostral ventrolateral medulla (RVL).

Anesthesia↗
PubMed1995

GABA-mediated inhibition of pacemaker neurons of rostral ventrolateral medulla by clonidine in vitro.

In vitro, clonidine (1, 10, or 30 microM) dose-dependently and reversibly inhibited tonically active pacemaker neurons that correspond to the relatively fast-conducting reticulospinal vasomotor neurons of the rostral ventrolateral reticular nucleus of the medulla oblongata in rats. The clonidine-induced membrane-hyperpolarizing response of these neurons was abolished by either tetrodotoxin, bicuculline, a GABAA receptor antagonist, or 4,4'-diisothiocyano-1,2'-disulphonic stilbene acid, a Cl- channel blocker. We conclude that the clonidine-induced inhibition of the pacemaker neurons of the rostral ventrolateral reticular nucleus is indirect, mediated by synaptic release of gamma-aminobutyric acid (GABA) or GABA-like substances, which activate Cl- channels of the pacemaker neurons of the rostral ventrolateral reticular nucleus.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Medullary visceral reflex circuits: local afferents to nucleus tractus solitarii synthesize catecholamines and project to thoracic spinal cord.

Visceral feedback circuits in lower brainstem were elucidated with retrograde tracers by mapping neurons that issue local projections to the general visceral afferent division of the nucleus tractus solitarii (NTS) and dorsomotor vagal nucleus (DMX) in adult male rats. In study 1, spinal and intramedullary afferents to the visceral-sensorimotor complex (NTS-X) were traced to contiguous populations of cell bodies arranged in cylindrical segmental organization. NTS-X afferents derive from curvilinear arrays of neurons that parallel the efferent radiations of the solitariotegmental tract. Newly discovered afferents arise from circumscribed cell groups in the dorsal reticular formation and periventricular zone. Another source was traced to a paraambigual cell column in the apex of the rostral ventrolateral reticular nucleus (n.RVL). In study 2, catecholaminergic afferents were initially defined with combined retrograde transport-immunocytochemical methods. Deposits of retrograde tracers into NTS-X transported to neurons containing tyrosine hydroxylase (TH) in the A1, C1, and C3 areas or phenylethanolamine N-methyltransferase (PNMT) in the C1 area of the n.RVL and C3 area. In study 3, it was revealed that NTS-X afferents arise, in part, as collaterals of thoracic reticulospinal neurons. Deposits of the retrograde fluorescent tracer Fluorogold into the upper thoracic cord and rhodamine-labeled microbeads into NTS-X transported to the same neurons within a subambigual locus in n.RVL and parts of nucleus raphe magnus. In study 4, dual retrograde tracer-immunocytochemical analysis demonstrated that catecholamines are synthesized by a subset of neurons in the n.RVL that issue collaterals to the NTS-X and thoracic cord. Double retrogradely labeled TH- or PNMT-immunoreactive cell bodies were restricted to the C1 area within a 450-microns column bordered rostrally by the facial nucleus and ventrally by the medullary subpial surface. We conclude that visceral reflex arcs are reciprocally organized. Targets of NTS projection are also sources of local NTS-X afferent innervation. Catecholaminergic and other local afferents from reticular formation, periventricular, and spinal gray may, via collaterals, simultaneously modulate visceral reflex excitability at the level of NTS and the outflow of autonomic and respiratory motoneurons.

Animals↗
PubMed1995

An antibody to agmatine localizes the amine in bovine adrenal chromaffin cells.

Agmatine, a newly identified amine in mammalian brain, is an endogenous ligand for imidazoline and alpha 2-adrenergic receptors. We sought to develop a polyclonal antibody to agmatine suitable for immunocytochemistry. Agmatine was conjugated to keyhole limpet hemocyanin and injected into rabbits. The polyclonal antiserum so generated dose-dependently recognized the agmatine conjugate but not carrier protein by dot blot. Its reaction with the conjugate was selectively antagonized by agmatine but not related compounds. The antiserum, but not pre-immune or pre-adsorbed antiserum, selectively stained cultured adrenal chromaffin cells. Our results indicate that agmatine immunoreactivity is contained in a sub-population of adrenal chromaffin cells and, thus, these antibodies are useful for immunocytochemical localization of the amine in mammalian tissues.

Adrenal Medulla↗
PubMed1995

NMDA receptor-mediated sympathetic chemoreflex excitation of RVL-spinal vasomotor neurones in rats.

1. We investigated the role of N-methyl-D-aspartic acid (NMDA) receptors in mediating hypoxic excitation of the reticulospinal vasomotor neurones of the rostroventrolateral reticular nucleus (RVL) of the medulla oblongata in paralysed ventilated rats. 2. Unilateral close arterial injection of sodium cyanide (100 nmol) into the carotid sinus region or ventilation with 100% N2 for 12 s rapidly, reversibly and reproducibly excited the RVL-spinal vasomotor neurones, followed about 1-2 s later by increases in sympathetic nerve activity and arterial pressure, effects abolished by denervation of the ipsilateral carotid sinus nerve. 3. Ionophoresis onto the RVL-spinal vasomotor neurones of kynurenate (a wide-spectrum antagonist of the excitatory amino acid receptors) or of 2-amino-5-monophosphovaleric acid (APV; a selective NMDA receptor antagonist), but not of xanthurenate (an inactive analogue of kynurenate), blocked the excitation elicited by intracarotid cyanide or 12 s of hypoxia. Kynurenate completely and APV partially blocked the excitatory responses to ionophoretically applied L-glutamate. APV, however, did not alter the excitatory responses of the vasomotor neurones to ionophoreses of kainate and quisqualate. 4. Bilateral microinjection of kynurenate (10 nmol, 50 nl per site) or APV (5 nmol, 50 nl per site) into the RVL blocked the increases in arterial pressure elicited by intracarotid cyanide or 12 s of 100% N2 ventilation. 5. Twenty seconds of intratracheal administration of 100% N2 resulted in complex and prolonged elevations of arterial pressure, the late component of which was affected neither by sinus denervation nor by microinjections of kynurenate or APV into the RVL. 6. We conclude that the sympathetic and cardiovascular responses to stimulation of arterial chemoreceptors result from excitation of RVL-spinal vasomotor neurones via activation of the NMDA subtypes of the excitatory amino acid receptors of the neurones. In contrast, the failure of these antagonists to influence the delayed excitation of the RVL-spinal vasomotor neurons by more prolonged exposure to N2 inhalation further supports the view that these neurones are directly stimulated by hypoxia.

Adrenergic Fibers↗
PubMed1995

Agmatine, the bacterial amine, is widely distributed in mammalian tissues.

We sought to determine whether agmatine (decarboxylated arginine), a bacterial product recently discovered for the first time in mammalian brain, was contained in other organs. A method was developed for isolation of agmatine from tissue and detection by RP-HPLC following solid-liquid extraction and derivatization with o-phthalaldehyde and mercaptoethanol. Recovery was about 80% and the limit of fluorometric detection was about 10 pg on column. In male Sprague-Dawley rats agmatine was unevenly and widely distributed in many tissues confirming its presence in mammals. The highest concentration (approximately 71 ng/mg net weight) was found in stomach, with aorta and small intestine next, followed by smaller levels in spleen, adrenal, aorta, and skeletal muscle and brain. Serum concentrations were high. Agmatine in male Long Evans rats of 3, 12, and 24 months of age demonstrated similar but not identical tissue distribution without any effect of aging. Since agmatine binds to alpha 2-adrenergic and imidazoline receptors, is bioactive in a number of tissues, is contained in neurons and is found in serum and tissues, the findings are consistent with a potential role for agmatine as a neurotransmitter and/or hormone. It also raises the possibility that agmatine may, as in bacteria, serve as a polyamine precursor along metabolic pathways previously not detected in mammals.

Agmatine↗
PubMed1995

Supercomputer algorithms for efficient linear octree encoding of three-dimensional brain images.

We designed and implemented algorithms for three-dimensional (3-D) reconstruction of brain images from serial sections using two important supercomputer architectures, vector and parallel. These architectures were represented by the Cray YMP and Connection Machine CM-2, respectively. The programs operated on linear octree representations of the brain data sets, and achieved 500-800 times acceleration when compared with a conventional laboratory workstation. As the need for higher resolution data sets increases, supercomputer algorithms may offer a means of performing 3-D reconstruction well above current experimental limits.

Algorithms↗
PubMed1995

Molecular characterization and isolation of a 45-kilodalton imidazoline receptor protein from the rat brain.

Imidazoli(di)nes bind to molecular entities different from alpha 2-adrenoceptors: the so-called imidazoline receptors (IRs). Two main types of IRs have been described, the clonidine- and the idazoxan-preferring types, as well as other IRs whose pharmacological properties do not fit either type, but little is known about the molecular features of these receptors. In this study, IR proteins have been solubilized from the rat brain, using the zwitterionic detergent CHAPS, and analyzed by pharmacological and immunological means two of the four peak discriminated by gel filtration chromatography using [3H]idazoxan binding and a specific antibody. The IR eluted in the first peak accounted for 80% of the specific binding of [3H]idazoxan to solubilized brain membranes, and its pharmacological features corresponded to the non-adrenoceptor component of [3H]idazoxan binding in rat brain native membranes. The elution volume of this peak corresponded to a 130-140-kDa protein, but immunoblot analysis with a specific anti-IR antiserum showed the presence of a approximate 45-kDa IR protein, suggesting that this receptor is either an oligomeric protein complex or that it is associated with other proteins. This result was in agreement with the isolation and immunodetection of a 45-kDa peptide by affinity chromatography, which supported the relationship between this protein and a rat brain imidazoline binding site. The second peak, accounting for 15% of the specific binding of [3H]idazoxan to solubilized membranes, had a Mr of approximately 65-70,000, as determined by gel filtration chromatography and immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed1995

Neurons in the barosensory area of the caudal ventrolateral medulla project monosynaptically on to sympathoexcitatory bulbospinal neurons in the rostral ventrolateral medulla.

Neurons in the caudal ventrolateral medulla may function as interneurons in the baroreceptor reflex are by inhibiting sympathoexcitatory bulbospinal neurons in rostral ventrolateral medulla. While some caudal ventrolateral medullary neurons are excited orthodromically by baroreceptors and antidromically from the rostral ventrolateral medulla, there is no anatomical evidence to prove that these barosensory neurons of the caudal ventrolateral medulla monosynaptically innervate the bulbospinal neurons in the rostral ventrolateral medulla. To establish the presence of such a direct projection, barosensory neurons were identified in the rostral caudal ventrolateral medulla of anesthetized rats by criteria that they spontaneously discharged with a cardiac rhythm and were excited by baroreceptor stimulation. The anterograde tracer biocytin was iontophoresed onto these neurons and, in the same animal, the retrograde tracer wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles was injected by micropressure into the ipsilateral spinal (thoracic level 3) intermediolateral cell column to label bulbospinal neurons. After 18-24 h, rats were killed and sections through the rostral ventrolateral medulla were processed for both markers. By light microscopy, numerous biocytin-labeled varicose processes overlapped rostral ventrolateral medullary neurons containing wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles. By electron microscopy, biocytin was found in axons and terminals. The terminals (n = 76) were large (0.6-1.2 microns in diameter), contained numerous small, clear vesicles and formed primarily symmetric synapses on perikarya and large (1.5-4.5 microns) dendrites within the rostral ventrolateral medulla. Some of these target neurons contained wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles associated with lysosomes and multivesicular bodies in the cytoplasm. The results indicate that: (i) neurons in the barosensory sympathoinhibitory region of the caudal ventrolateral medulla directly synapse on bulbospinal neurons in the rostral ventrolateral medulla; and (ii) the synaptic profile (symmetric synapse) and location (perikarya and large dendrites) is consistent with the conclusion that baroreceptor neurons of the caudal ventrolateral medulla potently and monosynaptically inhibit sympathoexcitatory neurons of the rostral ventrolateral medulla. The findings support the hypothesis that the barosensory region of the rostral caudal ventrolateral medulla is an intermediate relay in the baroreceptor reflex are.

Animals↗
PubMed1995

Regulation of phenylethanolamine N-methyltransferase gene expression by imidazoline receptors in adrenal chromaffin cells.

As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of alpha 2-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 microM. Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I1-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 microM clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.

Adrenal Medulla↗
PubMed1995

Transient expression of calcium-independent nitric oxide synthase in blood vessels during brain development.

The calcium-independent nitric oxide synthase, known as NOS-II or inducible NOS, is only present in adult brain during pathologies that involve inflammatory events. We sought to establish whether NOS-II was also expressed in the course of normal brain development. NOS-II mRNA, measured by quantitative polymerase chain reaction, appeared during the perinatal period in different brain areas. It was detected on embryonic day 14, the earliest time analyzed, and then levels decayed until the first postnatal week when a second, more robust phase of expression arose to peak around postnatal day 7. Expression then declined to negligible levels in adulthood. Immunohistology revealed translation of the NOS-II protein in both embryonic and postnatal animals, localized to parenchymal blood vessels, whereas no vessel staining was detected in adults. Furthermore, NOS-II mRNA and enzymatic activities were present in isolated brain microvessels from developing animals but not from adults, and P7 vessels contained a 125 kDa protein band detected with a monoclonal antibody raised against rat NOS-II protein. These results indicate that the calcium-independent NOS mRNA and functional protein are transiently expressed in vessels throughout the brain in the course of normal development.

Animals↗
PubMed1995

Vasodilation evoked from medulla and cerebellum is coupled to bursts of cortical EEG activity in rats.

Cerebral blood flow (rCBF), measured by laser-Doppler flowmetry, spontaneously fluctuates at approximately 6 events/min in the anesthetized rat. These cerebrovascular waves (CWs) are preceded by simultaneous and synchronous bursts of electrocorticographic activity similar to burst-suppression/spindle-burst electroencephalogram patterns. Identical burst-CW complexes are evoked by single electrical pulses of specific sites in the cerebellar fastigial nucleus or rostral ventrolateral medulla. These consist, sequentially, of a constant initial triphasic (positive-negative-positive) potential reversing polarity in lamina V, variable afterbursts, and transient elevations of rCBF appearing approximately 1.2 s after burst onset. Evoked bursts are occluded by spontaneous bursts appearing < 50 s earlier. Procainization of the cortex reversibly blocks burst-CW complexes. Gradually increasing stimulus frequency proportionally increases the numbers of burst-CW complexes before rCBF rises. We conclude that spontaneous and evoked burst-CW complexes result from excitation of common neurons in lamina V. These intracortical "vasodilator" neurons are spontaneously excited by thalamocortical afferents generating burst-suppression electroencephalogram (EEG) patterns and excited reflexively by afferent signals from the fastigial nucleus or rostral ventrolateral medulla and couple intrinsic neuronal activity to local vascular mechanisms generating vasodilation.

Animals↗
PubMed1994

Protection of focal ischemic infarction by rilmenidine in the animal: evidence that interactions with central imidazoline receptors may be neuroprotective.

Rilmenidine and idazoxan reduce the volume of focal ischemic infarctions produced by occlusion of the middle cerebral artery in the rat by 33% and 29%, respectively, by preserving neurons within the ischemic penumbra. In contrast, the alpha 2-selective antagonist SKF-86466 is without effect. The neuroprotective action of rilmenidine is dose dependent and parallels its antihypertensive actions. Neuroprotection cannot be attributed to changes in cerebral blood flow. We conclude that the neuroprotection produced by rilmenidine is attributable to an interaction with imidazoline receptors (IRs). However, the mechanism of action is not obvious. If it results from an action within the penumbra (direct), it is mediated by mitochondrial I-2 receptors on astrocytes, since cortical neurons are devoid of IRs. Neuroprotection might occur by selectively stimulating Ca2+ uptake into astrocytes, and thereby reducing Ca2+ uptake into neurons. Alternatively, rilmenidine may act indirectly to activate pathways in the brain that are neuroprotective. Neuroprotection may be a therapeutic target for rilmenidine and allied agents that act at central IRs.

Adrenergic alpha-Agonists↗