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D J Reis

Publications and source records attributed to D J Reis.

At least 109 records · Page 6Linked to original sources
PubMed1994

Adrenergic and non-adrenergic spinal projections of a cardiovascular-active pressor area of medulla oblongata: quantitative topographic analysis.

A cardiovascular-active pressor area of medullary reticular formation was defined by mapping changes in arterial blood pressure produced by microinjections of the neuroexcitatory amino acid, L-Glutamate (L-Glu). Sites where L-Glu provoked pressor responses larger than 10 mmHg were localized to a rostral longitudinal cell column of the nucleus reticularis rostroventrolateralis (n.RVL) extending 450 microns posteriorly to the facial nucleus. Spinal projections from the ventrolateral medulla were studied with a dual retrograde transport-immunocytochemical method. A striking correspondence was observed between the ventrolateral pressor area (VLPA) of n.RVL and rostrocaudal distribution of a circumscribed population of thoracic reticulospinal neurons containing tyrosine hydroxylase (TH)- or phenylethanolamine N-methyltransferase (PNMT)_immunoreactivity. Quantitative analysis revealed that 72% of the total number of retrogradely labeled neurons within the active area were immunocytochemically positive for TH; 28% of the reticulospinal projection cells were immunonegative. Deposits of L-Glu and dye through the same micropipettes verified a consistent correlation of vasopressor sites and the rostral subset of catecholaminergic neurons. Since comparable numbers of cell bodies in the VLPA contain TH and PNMT all are presumed to be adrenergic. At levels of n.RVL immediately adjacent to the VLPA commencing at a level 450 microns caudal to the facial nucleus, sites were unresponsive to Glu-stimulation or vasodepressor. At these levels, only non-adrenergic reticulospinal neurons project to cervical or thoracic spinal segments. We conclude that the VLPA is highly restricted to a narrow column of n.RVL < 0.5 mm in length and corresponds precisely with a population of predominantly adrenergic thoracic reticulospinal neurons that project exclusively to sympathoadrenal preganglionic motoneurons [cf 46]. These findings corroborate the idea that an adrenergic-spinal pathway may play a role in controlling sympathetic outflow.

Animals↗
PubMed1994

Effects of moxonidine, an imidazoline antihypertensive agent, on second messenger systems in rat brain.

Moxonidine, an imidazoline, binds to alpha 2-adrenoceptors and imidazoline receptors. We sought to determine the effects of moxonidine on accumulation of cAMP, cGMP, and phosphoinositide turnover in minislices of cerebral cortex and brainstem. In cerebral cortex, but not in brainstem, moxonidine inhibited the stimulated production of cAMP, an effect blocked by alpha 2-adrenergic antagonist rauwolscine. It also increased the hydrolysis of phosphoinositide above 100 microM in cerebral cortex and failed to alter accumulation of cGMP in both regions. We conclude that moxonidine is a typical alpha 2-adrenergic agonist inhibiting cAMP production in cerebral cortex, but not in brainstem, its interaction with I1 imidazoline sites in brainstem did not regulate second messenger systems, and moxonidine increased phosphoinositide turnover in cerebral cortex by unknown mechanisms.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1994

Immunodetection of putative imidazoline receptor proteins in the human and rat brain and other tissues.

A specific anti-imidazoline receptor (IR) protein antiserum was used to detect these putative receptors in brain and other tissues. In immunoblotting experiments, various brain areas and tissues expressed a double band of 29-30 kDa, and some of them bands of 47 and 66 kDa. The human medulla oblongata also showed bands of 44 and 50 kDa; and the rat adrenal medulla, bands of 58 and 86 kDa. Total immunoreactivity correlated with the density of IR binding sites in the various tissues. This is first evidence of heterogeneity of immunoreactive IRs in the mammalian brain.

Adrenal Medulla↗
PubMed1994

Hypoxia-activated Ca2+ currents in pacemaker neurones of rat rostral ventrolateral medulla in vitro.

We examined the effects of brief periods of hypoxia or application of cyanide on the discharge and membrane properties of medullary pacemaker neurones in slices of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata of rats. Stable intracellular recordings were obtained from seventy-nine neurones within the RVL which exhibited spontaneous rhythmic discharge in the absence of excitatory postsynaptic potentials (EPSPs). The membrane potential cycles of these neurones could be reset with an evoked spike without eliciting EPSPs or inhibitory postsynaptic potentials and hence met criteria of RVL pacemaker neurones. Hypoxia, produced by reducing O2 from 95 to 20% for 40 s or exposure to cyanide (30-300 microM for 40 s), reversibly increased neuronal discharge 1.6-fold (20% O2) or 2.6-fold (300 microM cyanide), respectively, in association with membrane depolarization and a significant fall in membrane resistance. The membrane responses to hypoxia and cyanide were observed in the presence of tetrodotoxin (TTX) at a concentration (10 microM) which eliminated spontaneous spikes or spikes evoked by intracellular depolarization. When recorded at a holding potential of -70 mV by single-electrode voltage clamp, hypoxia or cyanide (300 microM) elicited inward currents of 0.44 +/- 0.06 and 0.58 +/- 0.08 nA, respectively, which are attenuated by reducing the concentration of extracellular Ca2+ ions, and abolished by 2 mM CoCl2 and 100 microM NiCl2, but not affected by 50 microM CdCl2, replacement of 83% extracellular Na+, or adenosine deaminase (2U ml-1). We conclude that hypoxia and cyanide directly excite RVL pacemaker neurones in vitro by a common mechanism: activation of Ca2+ channel conductance.

Action Potentials↗
PubMed1994

Agmatine: an endogenous clonidine-displacing substance in the brain.

Clonidine, an antihypertensive drug, binds to alpha 2-adrenergic and imidazoline receptors. The endogenous ligand for imidazoline receptors may be a clonidine-displacing substance, a small molecule isolated from bovine brain. This clonidine-displacing substance was purified and determined by mass spectroscopy to be agmatine (decarboxylated arginine), heretofore not detected in brain. Agmatine binds to alpha 2-adrenergic and imidazoline receptors and stimulates release of catecholamines from adrenal chromaffin cells. Its biosynthetic enzyme, arginine decarboxylase, is present in brain. Agmatine, locally synthesized, is an endogenous agonist at imidazoline receptors, a noncatecholamine ligand at alpha 2-adrenergic receptors and may act as a neurotransmitter.

Adrenal Medulla↗
PubMed1994

Cloning and expression of inducible nitric oxide synthase from rat astrocytes.

In primary cultures of rat astroglial cells exposure to bacterial endotoxin lipopolysaccharide (LPS) causes induction of a Ca(2+)-independent form of the nitric oxide synthase (iNOS) enzyme. We have now cloned the mRNA encoding astroglial iNOS using a combination of cDNA library screening and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides directed against conserved regions of all NOS enzymes. The sequence of astroglial iNOS cDNA is highly similar to the mouse macrophage sequence, having an overall homology of 92% at the DNA level and 93% at the protein level. As in other NOSs, canonical binding sites for redox cofactors are present. The 3'-untranslated region displays 4 consensus AU-pentamers, 2 polyadenylation sites, and terminates in a stretch of 17 adenosine residues. In situ hybridization studies with LPS-treated astrocyte cultures demonstrated the presence of iNOS mRNA in the majority of astroglial cells, identified by antibody staining to the glial fibrillary acidic protein (GFAP). PCR analysis showed that LPS stimulated synthesis of astrocyte iNOS mRNA, which was detected as early as 2 hr after exposure to LPS, peaked at 4 hr, and slowly declined over the next 20 hr. These results confirm that astrocytes can express iNOS and provide tools for the subsequent analysis of iNOS gene expression in rodent brain.

Amino Acid Oxidoreductases↗
PubMed1994

Inhibition of protein kinase C differentially affects baroreflex inhibition and hypoxic excitation of medullary vasomotor neurons in rats.

We examined whether rapid sympathoexcitatory responses to hypoxia in rats resulting from excitation of reticulospinal vasomotor neurons of rostral ventrolateral medulla (RVL) results from activation of protein kinase C. In peripherally chemodenervated rats, the specific phosphokinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) administered intracisternally (i.c., 1 mumol), abolished responses of medullary vasomotor neurons and sympathetic nerves to baroreceptor activation, but did not attenuate excitation of RVL or sympathetic neurons to systemic hypoxia (100% N2 for 20 s). Responses of the vasomotor neurons to iontophoretically applied GABA were not attenuated by H-7. In slices, arachidonic acid (100 microM, 40 s) did not change membrane currents (VH = -70 mV) of RVL neurons in the presence tetrodotoxin (10 microM). The inward membrane currents (0.49 nA) induced by cyanide (300 microM, 40 s) were not reduced by H-7 (100 microM, 20 min). The results indicate that activation of protein kinase C does not underlie mechanisms involved in rapid hypoxic excitation of reticulospinal vasomotor neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Lesions of the rostral ventrolateral medulla reduce the cerebrovascular response to hypoxia.

Sympathoexcitatory neurons of the rostral ventrolateral medulla are tonically active and required for maintenance of resting levels of arterial pressure. They are also selectively excited by hypoxia and responsible for the associated sympathoexcitation. Since electrical or chemical stimulation of RVL will increase regional cerebral blood flow (rCBF) independently of changes in regional cerebral glucose utilization (rCGU) we investigated whether the RVL was also required to maintain resting levels of rCBF and also participated in the cerebrovascular vasodilation elicited by hypoxia. Rats were anesthetized (chloralose; 40 mg/kg, s.c.), paralyzed (tubocurarine) and ventilated (100% O2). rCBF was measured in 10 dissected brain regions using [14C]iodoantipyrine; rCGU was measured by 2-deoxy-D-[14C]glucose. In controls (n = 6) rCBF ranged from 56 +/- 5 in corpus callosum to 101 +/- 6 ml/min x 100 g in inferior colliculus. Hypoxic-hypoxia (PaO2 = 36 +/- 1 mmHg, n = 6) increased rCBF in all structures maximally, at 204% of control, in occipital cortex. Hypercapnia (PaCO2 = 63.5 +/- 0.9, n = 5) also increased rCBF (P < 0.01) maximally to 299% of control in superior colliculus. Spinal cord transection with maintenance of arterial pressure did not affect resting rCBF and increased the vasodilation to hypoxia (PaO2 = 39 +/- 1 mmHg, n = 5) from 2- to 3-fold in all structures (P < 0.01). Bilateral lesions within the RVL had no effect on resting rCBF or rCGU. However, they significantly reduced, in all areas by 50-69% (P < 0.01, n = 5), the cerebrovascular dilation elicited by hypoxia but not hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-labeled antisense oligodeoxynucleotides in the rat.

1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments. 2. The most intense labeling was observed near the injection site, in periventricular areas, and in perivascular regions. Many of the labeled cells appeared to be neurons, and both the cytoplasm and the nuclei were stained. The labeled cells were detected 15 min after icv injection, demonstrating that the antisense ODNs were taken up rapidly by cells in the parenchyma. The digoxigeninated antisense ODNs were presented in both the cytoplasmic and the nuclear fractions of rat brain extracts, however, the levels appeared to be much lower in the nuclear fractions. 3. Antisense ODNs injected into the lateral ventricle seemed to follow the bulk flow of cerebrospinal fluid (CSF), i.e., from the injection site in the lateral ventricle, through the ventricular system, to the subarachnoid spaces and the perivascular spaces. From the ventricular and perivascular spaces, the antisense ODNs diffused into the extracellular space and were taken up by cells. The full-length digoxigeninated antisense ODNs were detectable within cells after only 15 min, indicating their rapid uptake. In addition, the antisense ODNs appeared to be relatively stable in the brain since the full-length digoxigeninated ODNs were still detectable after 4 hr.

Animals↗
PubMed1994

Intrathecal kynurenate but not benextramine blocks hypoxic sympathoexcitation in chemodenervated anesthetized rats.

In peripherally chemodenervated anesthetized and ventilated rats, inhalation of 100% N2 for 20 s rapidly excites reticulospinal vasomotor neurons of the rostroventrolateral reticular nucleus (RVL) and sympathetic nerves. After an initial fall, arterial pressure is also elevated. The sympathoexcitatory response and secondary increases in arterial pressure were abolished by microinjections of muscimol (250 pmol) into the RVL or by intrathecal administration of kynurenate (500 nmol), a broad-spectrum antagonist of excitatory amino acid receptors, but not by benextramine, an alpha-adrenoceptor antagonist. The results indicate that excitation of pre-ganglionic sympathetic neurons in the spinal cord by hypoxia results from excitation of RVL-spinal vasomotor neurons, probably the glutamatergic neurons.

Adrenergic alpha-Antagonists↗
PubMed1994

Anatomical characterization of a novel reticulospinal vasodepressor area in the rat medulla oblongata.

Microinjection of L-glutamate into a subregion of the gigantocellular nucleus of the rat medulla oblongata significantly lowers arterial pressure. This vasodepressor area, the gigantocellular depressor area, is topographically distinct from other vasoactive areas of the medulla. We sought to determine the efferent projections of the gigantocellular depressor area and compare these to the efferent projections of sympathoexcitatory neurons within the rostral ventrolateral medulla. The anterograde tracer Phaseolus vulgaris-leucoagglutinin was deposited into sites in the gigantocellular depressor area or rostral ventrolateral medulla (pressor area) functionally defined as vasodepressor or vasopressor by microinjections of L-glutamate. Following Phaseolus vulgaris-leucoagglutinin injections into the gigantocellular depressor area, labeled punctuate fibers were seen bilaterally within distinct areas of a number of autonomic regions including the nuclei of the solitary tract, subcoeruleus area, parabrachial complex, the medial medullary reticular formation of the medulla and pons, and laminae 7 and 10 of the thoracic spinal cord. Following deposits into the rostral ventrolateral medulla (pressor area), labeled fibers were seen in many of these same autonomic nuclei; however, efferents from the gigantocellular depressor area to the nucleus of the solitary tract, the parabrachial complex and the reticular formation were medial to rostral ventrolateral medulla (pressor area) efferents to these same areas. These data indicate that neurons within the gigantocellular depressor area and the rostral ventrolateral medulla (pressor area) project to autonomic nuclei throughout the central nervous system and further suggest a heterogeneity of function with regard to autonomic control both within the reticular formation and its efferent targets. In addition, these data support the view that the gigantocellular depressor area may be a novel reticulospinal sympathoinhibitory area.

Animals↗
PubMed1994

Nitric oxide and prostanoids participate in cerebral vasodilation elicited by electrical stimulation of the rostral ventrolateral medulla.

We investigated, using laser-Doppler flowmetry, whether nitric oxide (NO)- and/or indomethacin (IND)-sensitive mechanisms mediate the elevations of regional cerebral blood flow (rCBF) elicited by electrical stimulation of the rostral ventrolateral medulla (RVL) in the anesthetized spinalized rat. Stimulation of the RVL for 10 s caused increased rCBF in the frontal cortex by 31% (n = 46), peaking at 22 s and persisting for up to 8 min. Intravenous L-nitro-NG-arginine (NNA) dose dependently and reversibly increased arterial pressure and reduced basal and evoked rCBF to 74 and 54% of the control, respectively (p < 0.05; n = 7). Superfused over the cortex, NNA dose dependently reduced only the evoked elevations of rCBF, to 39% of the control (p < 0.05; n = 6). Intravenous IND decreased the basal rCBF dose dependently and decreased the elevations evoked from the RVL by 38% (p < 0.05), but IND was without effect when superfused. Combined, the effects of intravenous NNA and IND summated, reducing rCBF by 70%. However, when NNA and IND were superfused together, the inhibition of the evoked vasodilation was comparable to that elicited by NNA alone. We conclude that the elevation in rCBF elicited from the RVL is partially mediated by (a) NO synthesized locally in the cortex in response to an afferent neural signal and (b) an IND-sensitive mechanism, probably a product of cyclooxygenase, located in larger cerebral arteries, in response to a retrograde vascular signal resulting from increased blood flow within the brain.

Animals↗
PubMed1994

Induction of nitric oxide synthase in rat C6 glioma cells.

We have examined the induction of nitric oxide synthase (NOS) activity in the rat astrocyte-derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 microgram/ml for 24 h) did not stimulate NO2 production. However, addition of either tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC50 values of 39 ng/ml of TNF-alpha and 9.4 U/ml of IFN-gamma), and the effect of TNF-alpha could be further potentiated (twofold) by the presence of interleukin-1 beta. The simultaneous presence of TNF-alpha and IFN-gamma yielded a greater response than either cytokine alone; however, the respective EC50 values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca(2+)-independent conversion of L-arginine to L-citrulline, with an apparent Km of 51.2 microM, and this activity could be blocked by L-arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.

Amino Acid Oxidoreductases↗
PubMed1994

Nitric oxide synthase expression in glial cells: suppression by tyrosine kinase inhibitors.

Rat brain glial cells have the capacity to express a calcium-independent form of nitric oxide synthase (iNOS). To test if iNOS induction required tyrosine kinase activity, we made use of genistein, a selective inhibitor of tyrosine kinases. In both primary astrocyte cultures and C6 glioma cells, the presence of genistein prevented both lipopolysaccharide- and cytokine-induced NOS activity in a dose-dependent manner. The presence of tyrphostin-25 (10 microM), which is highly specific for tyrosine kinases, also blocked iNOS induction. Additional characterization showed that genistein blocked iNOS induction in a dose-dependent manner (IC50 of approximately 40 microM), that the continuous presence of genistein was not necessary to observe inhibition, and that preincubation with genistein led to higher levels of inhibition than the simultaneous addition of genistein and inducers. The decrease in iNOS activity due to genistein was accompanied by a decrease in iNOS mRNA level as detected by a specific PCR assay. These results indicate that induction of astroglial iNOS expression requires tyrosine kinase activity.

Amino Acid Oxidoreductases↗
PubMed1994

Spontaneous waves of cerebral blood flow associated with a pattern of electrocortical activity.

We examined the relationship between spontaneous changes in regional cerebral blood flow and electrocortical (ECoG) activity in spinalized rats anesthetized with 1.5% isoflurane. Regional cerebral blood flow, measured by laser-Doppler flowmetry, and ECoG activity were measured bilaterally in frontal and parietal cortex. Spontaneous cerebrovascular waves (SCWs) were seen in all (n = 80) rats and consisted of sawtoothed waves with an average amplitude of 20.1 +/- 0.78%, a duration of 11.7 +/- 0.6 s, and a frequency of 6.3 +/- 0.2 min-1. SCWs were always preceded by a high-amplitude burst of ECoG activity (averaging 752.0 +/- 41.8 microV at 5.6 +/- 0.2 Hz) and comparable to the well-recognized burst-suppression/barbiturate-spindle patterns of ECoG activity. The latency between bursts and SCWs averaged 1.71 +/- 0.05 s. The frequency of bursts and SCWs was highly correlated within and between cortical areas bilaterally (r > 0.9) and appeared synchronously across brain. Deepening anesthesia (to 1.75% isoflurane) reduced the frequency of bursts and SCWs by > 30% but not their correlation (r > 0.9) and minimally increased burst-SCW latency. SCWs differed from an uncommon sinusoidal oscillation regional cerebral blood flow triggered by changes in arterial pressure and independent of the ECoG. Bursts and SCWs were not affected by inhibition of nitric oxide synthase. The results indicate that a population of local cortical neurons, probably driven from subcortical pacemakers, when excited, elicits local cerebrovascular vasodilation.

Anesthesia↗
PubMed1994

Hypoxia selectively excites vasomotor neurons of rostral ventrolateral medulla in rats.

Systemic hypoxia [PaO2 27.3 +/- 1.8 (SE) mmHg] in anesthetized paralyzed rats reversibly increased within seconds the arterial pressure and activities of the sympathetic nerves and the reticulospinal vasomotor neurons of the rostral ventrolateral medulla (RVL). After peripheral chemodenervation, hypoxia also increased activity of the sympathetic nerves and doubled discharges of the vasomotor neurons while inhibiting a majority of the RVL respiratory neurons. Systemic hypercapnia was not effective in eliciting sympathoexcitatory responses. Iontophoresis of sodium cyanide stimulated the vasomotor and inhibited the respiratory neurons. In contrast, iontophoreses of H+, HCO3-, and lactate were without effects on activity of the vasomotor neurons. We conclude 1) hypoxia excites the vasomotor neurons by activating the arterial chemoreceptors and by activating intrinsic cellular mechanisms probably unrelated to accumulation of metabolic byproducts; 2) hypoxia may be the adequate stimulus exciting the RVL-spinal vasomotor and inhibiting the respiratory neurons during the cerebral ischemic response; and 3) these vasomotor neurons may be central oxygen detectors.

Animals↗
PubMed1994

Dopamine or transmitter release from rat carotid body may not be essential to hypoxic chemoreception.

In anesthetized, paralyzed, and ventilated rats, hypoxia or intracarotid cyanide excited the carotid chemoafferents, whereas intracarotid dopamine and tyramine inhibited the chemoafferent discharges. The inhibition was abolished by chlorpromazine without attenuating the hypoxic excitation. Comparably, the hypoxic excitation was not attenuated by the following: 1) inhibition of nitric oxide synthase with NG-nitro-L-arginine; 2) inhibition of heme oxygenase with zinc protoporphyrin IX; 3) antagonism of ATP receptors with reactive blue 2; 4) antagonism of cholinergic receptors with atropine or trimethaphan; 5) inactivation of adenosine with adenosine deaminase; and 6) blockade of glutamate receptors with kynurenate. Systemic administration of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid, in doses reversibly blocking sympathetic ganglionic transmission, was also without effect. Cyanide microinjection (0.05-0.5 nmol) into the petrosal but not nodose ganglion elicited a rapid dose-dependent elevation of arterial pressure. We conclude that excitation of the chemoreceptor afferents by hypoxia/cyanide cannot be attributed to release of these agents nor to others by Ca(2+)-dependent mechanisms. The results suggest that the afferent nerves themselves might function as oxygen detectors.

Afferent Pathways↗