Paul Ernsberger

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.

Rilmenidine lowers arterial pressure via imidazole receptors in brainstem C1 area

We sought to determine the site of action and receptor type responsible for the antihypertensive actions of rilmenidine, an oxazoline analogue of clonidine. In anesthetized paralyzed rats decerebration did not alter the dose dependent reductions in arterial pressure and heart rate elicited by i.v. drug. Rilmenidine microinjected bilaterally into the C1 area of the rostral ventrolateral medulla (RVL), but not nucleus tractus solitarii (NTS) nor caudal ventrolateral medulla (CVL), elicited dose-dependent falls in arterial pressure and heart rate at doses an order of magnitude less than required systemically. Prior microinjection into the C1 area of the selective alpha 2-adrenoceptor antagonist SKF-86466, even at high doses, failed to modify the hypotension to i.v. rilmenidine. However, microinjection of 3- to 10-fold lower doses of idazoxan, a ligand for imidazole as well as alpha 2-adrenoceptors, blocked the effects. Rilmenidine also competed with the clonidine analogue [3H]p-aminoclonidine ([3H]PAC) at specific binding sites in membranes of bovine ventrolateral medulla and frontal cortex. In RVL rilmenidine competed with binding to imidazole and alpha 2-adrenergic binding sites with a 30-fold selectivity for the imidazole binding sites. In frontal cortex binding was of lower affinity and restricted to alpha 2-adrenergic sites. We conclude that rilmenidine, like clonidine, acts to lower arterial pressure by an action on imidazole receptors in the C1 area of RVL. The higher selectivity of rilmenidine for imidazole to alpha 2-adrenoceptors as compared to clonidine may explain the lower sedative effects of rilmenidine.

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.

The I1-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease

Objective To review previous work and present additional evidence characterizing the I1-imidazoline receptor and its role in cellular signaling, central cardiovascular control, and the treatment of metabolic syndromes. Second-generation centrally-acting antihypertensives inhibit sympathetic activity mainly via imidazoline receptors, whereas first-generation agents act via α2-adrenergic receptors. The I1subtype of imidazoline receptor resides in the plasma membrane and binds central antihypertensives with high affinity. Methods and results Radioligand binding assays have characterized I1-imidazoline sites in the brainstem site of action for these agents in the rostral ventrolateral medulla. Binding affinity at I1-imidazoline sites, but not at other classes of imidazoline binding sites, correlates closely with the potency of central antihypertensive agents in animals and in human clinical trials. The antihypertensive action of systemic moxonidine is eliminated by the I1α2-antagonist efaroxan, but not by selective blockade of α2-adrenergic receptors. Until now, the cell signaling pathway coupled to I1-imidazoline receptors was unknown. Using a model system lacking α2-adrenergic receptors (PC12 pheochromocytoma cells) we have found that moxonidine acts as an agonist at the cell level and I1-imidazoline receptor activation leads to the production of the second messenger diacylglycerol, most likely through direct activation of phosphatidylcholine-selective phospholipase C. The obese spontaneously hypertensive rat (SHR; SHROB strain) shows many of the abnormalities that cluster in human syndrome X, including elevations in blood pressure, serum lipids and insulin. SHROB and their lean SHR littermates were treated with moxonidine at 8 mg/kg per day. SHROB and SHR treated with moxonidine showed not only lowered blood pressure but also improved glucose tolerance and facilitated insulin secretion in response to a glucose load. Because α2-adrenergic agonists impair glucose tolerance, I1-imidazoline receptors may contribute to the multiple beneficial effects of moxonidine treatment. Conclusion The I1-imidazoline receptor is a specific high- affinity binding site corresponding to a functional cell-sur-face receptor mediating the antihypertensive actions of moxonidine and other second-generation centrally-acting agents, and may play a role in countering insulin resistance in an animal model of metabolic syndrome X.

A glutamate mechanism in the intermediolateral nucleus mediates sympathoexcitatory responses to stimulation of the rostral ventrolateral medulla

Publisher Summary The rostral ventrolateral medulla (RVL) contains a population of reticulospinal, sympathoexcitatory neurons. The regulation of the discharge of RVL-spinal vasomotor neurons by afferent pathways to the RVL underlies many reflex adjustments of circulation in response to peripheral and central stimuli. While vasomotor neurons of the RVL are presumed to exert their sympathoexcitatory effects over pathways terminating in the intermediolateral nucleus (IML) of the thoracolumbar spinal cord, little is known of the role played by specific spinal transmitter systems in mediating the increases in sympathetic nerve activity evoked by RVL neuronal discharge. This chapter discusses the combination of electrophysiological, immunocytochemical, and receptor autoradiographic techniques to test the hypothesis that an excitatory amino acid is the fast transmitter implicated in RVL-evoked excitation of sympathetic preganglionic neurons (SPNs). Results suggest that RVL stimulation causes the release of glutamate from nerve terminals in the IML that interacts with glutamate receptors of the kainate subtype to excite SPNs, thereby, producing an increase in sympathetic nerve activity and arterial pressure.

The I1-imidazoline-binding site is a functional receptor mediating vasodepression via the ventral medulla

I1-imidazoline-binding sites fulfill all essential criteria for identification as receptors, including specificity of binding, association with physiological functions, appropriate anatomic and cellular and subcellular localization, and specific cell signaling pathways. Moreover, binding affinities correlate with functional drug responses. The evidence linking I1 receptors to vasodepression includes expression in RVLM and consistent correlations between vasodepressor potency in humans and animals and I1 binding affinity. Some I1 agonists are antagonists at α2-adrenergic receptors (α2AR), and these elicit vasodepression in RVLM. Potent α2-agonists with phenylethylamine or guanidine structures are inactive in RVLM, yet highly effective in nucleus of the solitary tract, a region with well-defined α2-mediated vasodepressor responses. Selective I1 agonists are used clinically to lower blood pressure with minimal α2-mediated sedation. Moreover, when microinjected into the RVLM only antagonists active at I1 receptors can block the vasodepressor action of either local or systemic imidazolines. RVLM α2-blockade has no effect. Some reports appear to conflict with the I1 receptor hypothesis; but these reports often make incorrect assumptions regarding drug specificity, overlook systemic effects of α2-antagonists, or inappropriately analyze data. Blockade of γ-aminobutyric acid (GABA) receptors blocks the vasodepressor action of imidazolines, implying a multisynaptic pathway. Thus imidazolines act via I1receptors in RVLM to lower blood pressure, although α2AR are also important, especially in NTS.

Hypotensive action of clonidine analogues correlates with binding affinity at imidazole and not alpha-2-adrenergic receptors in the rostral ventrolateral medulla.

Clonidine acts within the rostral ventrolateral medulla to lower arterial pressure. The receptor mechanism for this action is unknown. In the rostral ventrolateral medulla clonidine binds not only to the ap2-adrenergic receptor but also to a novel class of sites which are specific for imidazolines and imidazoles and are distinct from adrenergic or histaminergic receptors. In order to distinguish whether a putative imidazole receptor or the aP2-receptor mediates the clonidine hypotensive response, a series of clonidine analogues were tested (1) in radioligand binding assays of their affinity at imidazole and aP2-adrenergic receptors, and (2) by microinjection into the rostral ventrolateral medulla of anaesthetized rats to measure their capacity to lower arterial pressure. The hypotensive response elicited by the test agents was strongly correlated with affinity at imidazole sites (r = 0.92) but not with aP2-adrenergic affinity (r = 0.36). An imidazole receptor in the rostral ventrolateral medulla may participate in the hypotensive action of clonidine.

Astrocytes cultured from specific brain regions differ in their expression of adrenergic binding sites

We sought to characterize regional heterogeneity of astrocytes using adrenergic receptor sites as cellular markers. Primary cultures made from 6 regions of neonatal rat brain consisted almost exclusively of astrocytes. Membranes from astrocytes cultured 1-3 weeks were prepared for radioligand binding assays of beta- and alpha 2-adrenergic sites using the ligands [3H]dihydroalprenolol and [3H]p-aminoclonidine, respectively. Receptor expression was not affected by time in culture. Astrocytes from different brain regions varied up to 3-fold with respect to number but not affinity for both classes of adrenergic binding site with a rank order of cerebral cortex = superior colliculus greater than hippocampus = ventral midbrain greater than or equal to caudate nucleus greater than or equal to hypothalamus. Binding to beta- and alpha 2-adrenergic receptors was positively correlated across brain regions. Astrocytic receptor binding in each region did not correspond to total receptor levels assessed by quantitative autoradiography. We conclude that: (a) astrocytes are markedly heterogeneous between major brain regions with respect to expression of adrenergic binding sites; (b) regional variations in the density of adrenergic binding sites in brain reflect, in part, local specialization of astrocytes; and (c) a substantial proportion of the adrenergic binding sites in some brain regions may be on astrocytes.

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.

A high density of muscarinic receptors in the rostral ventrolateral medulla of the rat is revealed by correction for autoradiographic efficiency

Previous reports of a low density of muscarinic receptors in the vicinity of the rostral ventrolateral reticular nucleus (RVL) conflict with the presence of a muscarinic cholinergic mechanism regulating arterial pressure, and may reflect low autoradiographic efficiency due to quenching of tritium emissions by white matter. Regional autoradiographic efficiency was determined using brains uniformly labeled with O-[3H]methylglucose. Within the RVL, autoradiographic efficiency was only 50 +/- 2% relative to gray matter. The uncorrected density of muscarinic receptors (M2) labeled by [3H]quinuclidinyl benzilate in the RVL appeared to be low relative to cranial nerve nuclei, but the corrected muscarinic receptor density approximated that within the nucleus tractus solitarii. We conclude that a high density of M2 muscarinic acetylcholine receptors is present in the RVL.