Mary P. Meeley

Clonidine binds to imidazole binding sites as well as α2-adrenoceptors in the ventrolateral medulla

Binding sites labeled by [3H]p-aminoclonidine ([3H]PAC) were characterized in bovine brain membranes prepared from the ventrolateral medulla, the probable site of the antihypertensive action of clonidine and analogs. Comparison was made with [3H]PAC binding to membranes prepared from frontal cortex, which has been studied extensively. Saturation binding isotherms for [3H]PAC were similar in the two regions, although Bmax values were approximately two-fold lower in ventrolateral medulla relative to frontal cortex. Norepinephrine and other phenylethylamines displaced [3H]PAC from a maximum of 70% of the total sites in the ventrolateral medulla. The remaining 30% were norepinephrine-insensitive, non-adrenoceptor sites which displayed high affinity for imidazole compounds. Ligand selectivity differed markedly between ventrolateral medulla and frontal cortex, since some imidazole compounds which potently inhibited [3H]PAC binding in the ventrolateral medulla had no effect in frontal cortex. Imidazole binding sites may mediate, in part, the hypotensive action of clonidine and other imidazole compounds in the ventrolateral medulla. These sites may also participate in the functions of a putative endogenous clonidine-like substance.

AN ENDOGENOUS CLONIDINE-DISPLACING SUBSTANCE FROM BOVINE BRAIN: RECEPTOR BINDING AND HYPOTENSIVE ACTIONS IN THE VENTROLATERAL MEDULLA

A substance has been isolated from bovine brain which displaces 3H-clonidine binding to rat brain membranes (clonidine-displacing substance; CDS). To determine whether CDS is similar to the antihypertensive agent clonidine, the in vitro binding properties of partially-purified CDS and its physiological action in the rostral ventrolateral medulla were examined. Like clonidine, CDS potently inhibited 3H-para-aminoclonidine binding to receptors in bovine ventrolateral medulla membranes (clonidine, IC50 = 24 +/- 8nM; CDS, IC50 = 0.30 +/- .10 Units), with highest affinity for non-adrenergic sites (clonidine, IC50 = 6 +/- 1nM; CDS, IC50 = 0.12 +/- .07 Units). CDS had no effect at beta-adrenergic or muscarinic cholinergic receptors. Like clonidine, CDS elicited a potent, reversible (less than 10 min) dose-dependent fall in arterial pressure (AP) and heart rate when microinjected specifically into the C1 area of the rostral ventrolateral medulla in the rat (maximum delta AP, -65 +/- 7 mm Hg). CDS represents an as-yet-uncharacterized endogenous, physiologically-active agent in brain which may participate in cardiovascular control via non-adrenergic receptors in the rostral ventrolateral medulla.

Intrinsic γ-aminobutyric acid neurons in the nucleus of the solitary tract and the rostral ventrolateral medulla of the rat: An immunocytochemical and biochemical study

Abstract Sympathoexcitatory neurons in the C1 adrenergic area of the rostral ventrolateral medulla (RVL) are tonically inhibited by γ-aminobutyric acid (GABA). To identify the source of this GABAergic input, the distribution of neurons containing glutamate decarboxylase (GAD) was determined immunocytochemically in rats treated with colchicine. Numerous GAD-stained neurons were located in the nucleus of the solitary tract (NTS) and in RVL. Unilateral lesions in NTS did not alter GABA content or GAD activity in RVL, indicating that the afferent projection from NTS to RVL is not GABAergic. Intrinsic GABAergic neurons in RVL may provide tonic inhibition of vasomotor neurons in the C1 area.

Content and in vitro release of endogenous amino acids in the area of the nucleus of the solitary tract of the rat

Abstract: We sought to identify amino acid neurotransmitter candidates within the nucleus of the solitary tract in rats. Twenty endogenous amino acids were quantified by reverse‐phase HPLC with fluorescence detection (30‐fmol limit). Mi‐cropunches (1 mm) of the intermediate area of the solitary nucleus were prepared, and the amino acid content determined. Of all the components measured, the putative transmitters Glu, Gly, γ‐aminobutyric acid, taurine, Asp, and Ala appeared in greatest concentrations. Bilateral micropunches superfused in vitro with buffered medium containing 56 mM potassium released Glu, γ‐aminobutyric acid, and Gly in a significant manner (p < 0.05) compared with basal levels. With Glu, 78% was calcium‐dependent and, therefore, presumably from nerve endings; 99% of γ‐aminobutyric acid and 42% of Gly were dependent on calcium. After removal of the nodose ganglion, a bilateral decrease in the calcium‐dependent release of Glu and γ‐aminobutyric acid, but not Gly, was observed; decreases were significant ipsilateral to the site of ablation. We conclude that (a) Glu is a transmitter of primary afferents in the nucleus of the solitary tract; (b) glutamatergic afferents may interact with γ‐aminobutyric acid system(s) in this region; (c) Gly also may participate in the mediation and/or modulation of cardiovascular or other visceral reflexes; and (d) amino acid neurotransmission may play an integral role in the neurogenic control of arterial pressure

A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase (PNMT) but not tyrosine hydroxylase

Intraventricular injection of colchicine in rat results in the appearance within hypothalamus of numerous neurons containing the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase, but not the other catecholamine biosynthetic enzymes. Increased PNMT staining in hypothalamus was paralleled by an increase in PNMT activity measured in micropunch preparations. Immunotitration demonstrated that this increase was due to accumulation of specific enzyme protein. The finding that hypothalamic neurons express PNMT without tyrosine hydroxylase suggests that such neurons may produce methylated amines other than adrenaline.

Effects of clonidine and other imidazole-receptor binding agents on second messenger systems and calcium influx in bovine adrenal chromaffin cells

Clonidine and related imidazoline compounds bind to alpha 2-adrenergic as well as to newly described non-adrenergic imidazole/imidazoline receptors in brain and peripheral tissues. The present study was undertaken to identify the signal transduction mechanism coupled to this new class of receptors (imidazole receptors) using bovine adrenal chromaffin cells. Clonidine did not modify the basal or forskolin-stimulated production of cyclic AMP (cAMP), suggesting the absence of functionally active alpha 2-adrenergic receptors in adrenal chromaffin cells. Clonidine also failed to modify the basal and GTP gamma S- or carbachol-stimulated increase in phosphoinositide hydrolysis. However, clonidine increased significantly the production of cyclic GMP (cGMP) as well as the uptake of 45Ca2+. The cGMP response to clonidine was slower (peak at 15 min) and smaller (only about 50% over control) than the response to acetylcholine and was not shared by other agents that bind to imidazole receptors. In contrast, all agents that bind to imidazole receptors increased the influx of 45Ca2+ into chromaffin cells. It is concluded that (a) alpha 2-adrenergic and imidazole receptors are functionally distinct and linked to different signal transduction mechanisms; (b) the classical G-protein coupled soluble second messenger systems are not coupled to imidazole receptors; (c) clonidine may increase cGMP by a non-receptor-mediated intracellular action; and (d) imidazole receptors may regulate intracellular calcium levels through an ion regulating system that may be different from calcium channels.

Lesions of the basal forebrain in rat selectively impair the cortical vasodilation elicited from cerebellar fastigial nucleus

We sought to determine in rat, whether interruption of the major extrathalamic projections to the cerebral cortex originating in and projecting through the basal forebrain (BF), will impair the increase in regional cerebral blood flow (rCBF), but not metabolism, elicited in the cerebral cortex by electrical stimulation of the cerebellar fastigial nucleus (FN). Studies were conducted in anesthetized, paralyzed, ventilated rats, with blood gases controlled and AP maintained in the autoregulated range. Electrolytic lesions were placed unilaterally in the BF at the level of the lateral preoptic region lying in rostral portions of the medial forebrain bundle and resulted in a reduction of up to 47% of the choline acetyltransferase activity in the ipsilateral cerebral cortex. rCBF was measured in homogenates of 9 paired brain regions by the 14C-iodoantipyrine technique. In unlesioned rats, FN stimulation symmetrically and significantly (P less than 0.05) increased rCBF in all brain regions with the greatest increase (to 180%) in the frontal cortex. Two days following a unilateral BF lesion, FN stimulation failed to increase rCBF in the ipsilateral cerebral cortex distal to the BF lesion. In contrast, rCBF was increased to an almost comparable degree in the remainder of the brain. BF lesions alone resulted in a 18-23% reduction in cortical rCBF ipsilaterally (P less than 0.025). BF lesions did not alter the cerebrovascular vasodilation elicited by CO2 nor perturb autoregulation. The cortical vasodilation elicited by FN stimulation is mediated by intrinsic neuronal pathways and depends upon the integrity of neurons, possibly cholinergic, originating in, or passing through, the BF.

Clonidine displacing substance is biologically active on smooth muscle

A substance has been isolated from brain which potently inhibits the binding of clonidine to brain membranes (clonidine displacing substance, CDS). We sought to determine if CDS is biologically active on smooth muscle. CDS had no effect on vascular smooth muscle. In contrast, CDS potently contracted rat gastric fundus strips in a dose dependent manner. The contractile effect of CDS was not blocked by antagonists selective for biologically active substances known to contract the fundus strip. These results demonstrate that CDS has a unique and potent ability to selectively contract smooth muscle.

Imidazoline receptors in the nervous system

A. lmldarollne receptors In brain. The original insights of Karppanen (1) and Bousquet (2) that the central antihypertensive actions of the imidazoline clonidine could not be attributed to interactions with q-adrenergic receptor (AAR) but related to the imidazoline structure of the drug gave birth to the concept of a novel receptor referred to here as the imidazoline receptor (IR). The original concept also indicated per force that IRs were localized in brain. More direct evidence for the existence of IRs in brain (3) and other kidney (4) was subsequently established by ligand binding studies which indicated that the binding of 3H-para-aminocbnidine (3H-PAC) to membranes of ventral medulla, the site of the antihypertensive actions of clonidine (5) and rilmenidine (6) was fully displaceable by clonidine yet only partially by adrenergic ligands. Non-adrenergic binding was displaceable by agents which were imidazoles, imidazolines (e.g. imidazole 4-acetic acid, cimetidine, cirazoline), some guanidines (e.g. guanabenz, amiloride) and oxazoles (rilmenidine). Thus, these agents bound to both AARs and another binding site considered to be the IR. That IRs in the CNS are functional has, however, largely been indirect. That IRs mediate the hypotensive action of clonidine and rilmenidine in the ventral medulla has been assumed based on evidence that the reduction in arterial pressure elicited by the microinjection of agents into the rostra1 ventrolateral medulla (RVL) correlates with binding at IRs and not AARs and that the microinjection of idazoxan but not of selective AAR antagonists into RVL will block the systemic fall of AP elicited by intravenous clonidine or rilmenidine. There is also indirect evidence that the neuroprotective actions of idazoxan and rilmenidine can be attributed to actions at nonadrenergic receptors presumably representing IRs (7). On the other hand several of the central actions of clonidine including analgesia and sedation appear to represent the actions of the agents at AARs.

Expression of non-adrenergic imidazoline sites in chromaffin cells and mitochondrial membranes of bovine adrenal medulla

We sought to characterize the non-adrenergic binding site for imidazolines, the imidazoline receptor in whole membranes and subcellular compartments of chromaffin cells of bovine adrenal medulla. [3H]Idazoxan exhibited saturable and high affinity (KD = 5 nM) binding to chromaffin cell membranes fully displaceable by idazoxan and cirazoline but not by epinephrine or rauwolscine. Binding sites were highly enriched in mitochondrial but not plasma membranes and absent from nuclear fractions. The rank order of potency for displacement of [3H]idazoxan from mitochondrial membranes was: cirazoline > idazoxan > naphazoline > amiloride > detomedine > clonidine >> phentolamine > cimetidine = imidazole 4-acetic acid > p-iodoclonidine = epinephrine = norepinephrine = rauwolscine. Binding was also inhibited with high affinity by the purported endogenous ligand clonidine-displacing substance and by K+ and the K(+)-channel antagonists 4-aminopyridine and tetraethylammonium bromide but not Na+. We conclude that: (a) adrenal chromaffin cells express imidazoline receptors but not alpha 2-adrenergic receptors; (b) these sites are predominantly localized to adrenal medullary mitochondria; and (c) imidazoline receptors conform to an idazoxan preferring (I-2) rather than the clonidine preferring (I-1) subclass and are amiloride sensitive. The data support the view that alpha 2-adrenergic and imidazoline receptors are distinct receptor species and that adrenal chromaffin cells would be a useful cultured cell system, expressing only imidazoline receptors, for further molecular and functional studies of the receptors.