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.

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.

Endogenous ligands of imidazoline receptors: classic and immunoreactive clonidine-displacing substance and agmatine.

1. There are several endogenous ligands that bind to I-receptors of both the I1 and I2 subclass. These include: (a) classic CDS, a partially purified entity isolated by the criteria that it displaces binding ligands to alpha 2- and I-receptors; (b) immunoreactive (ir)-CDS, a moiety that binds to antibodies raised against clonidine, para-amino-clonidine, or idazoxan; and (c) agmatine.

Distribution of imidazoline receptor binding protein in the central nervous system.

I-receptors can be localized immunocytochemically in rat nervous system with polyclonal antibodies to an IRBP. I-receptors are cytoplasmic and detected in neuronal perikarya, processes, and glia. Labeled neuronal perikarya in the CNS are uncommon and localized to the mesencephalic trigeminal nucleus.

Studies on neuropeptide Y receptors in a mouse adrenocortical cell line.

The mouse adrenocortical Y-1 cell line has been found to express high affinity binding sites for neuropeptide Y (NPY). Pharmacological studies have shown that these NPY binding sites are of the Y1 type. Reverse transcription-polymerase chain reaction using primers specific for the rat Y1 receptor revealed that the NPY Y1 receptor mRNA is present in Y-1 cells.

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.

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.

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.

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.

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.

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.

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.

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.

Cardiovascular responses to agmatine, a clonidine-displacing substance, in anesthetized rat.

We investigated the cardiovascular responses in anesthetized ventilated rats to agmatine (decarboxylated arginine), an amine which is an endogenous clonidine-displacing substance (CDS) synthesized in brain. Intracisternal agmatine dose-dependently increased sympathetic nerve activity and arterial pressure (at 400 nmol by 8.7 +/- 2.

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.

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.

Sympatho-excitatory neurons of the rostral ventrolateral medulla are oxygen sensors and essential elements in the tonic and reflex control of the systemic and cerebral circulations.

MEDULLARY ROSTRAL VENTROLATERAL RETICULAR NUCLEUS (RVL): Reticulospinal neurons are critical to control of the circulation by the brain. Its actions are implemented by a few reticulospinal neurons, 200 in the rat. These directly innervate and excite preganglionic sympathetic neurons of the spinal cord by releasing L-glutamate.