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We sought to characterize the interactions of an endogenous clonidine-displacing substance (CDS) with the specific receptor sites to which clonidine and its analogs bind: (a) the non-adrenergic imidazole binding site, which is present in the ventrolateral medulla (VLM) but not the frontal cortex, (b) high-affinity and (c) low-affinity states of the alpha 2-adrenergic receptor, and (d) the alpha 1-adrenergic receptor. CDS, like clonidine, potently and completely inhibited specific p-[3H]aminoclonidine binding to membranes from the VLM or from the frontal cortex. Both CDS and clonidine bound with highest affinity to imidazole binding sites in the VLM, both were 3-fold selective for high-affinity over low-affinity alpha 2-adrenergic receptors, and both exhibited lowest affinity for alpha 1-adrenergic receptors. Unlike clonidine, CDS exhibited 30-fold selectivity for imidazole over alpha 2-adrenergic receptors but showed only a weak preference for alpha 2- over alpha 1-adrenergic receptors, indicating that CDS and clonidine are not identical. We conclude that CDS is an endogenous clonidine-like substance which may be the natural ligand for imidazole binding sites in the VLM. The receptor-binding properties of CDS are consistent with the view that it is a unique and as yet unrecognized compound.

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 substance in brain, clonidine-displacing substance, binds to the same receptor populations as clonidine and is biologically active. Since receptor binding sites can be modeled by using specific antiligand antibodies, we tested the hypothesis that polyclonal antibodies raised in rat and rabbit against the clonidine analog p-aminoclonidine coupled to hemocyanin would recognize compounds structurally related to clonidine, including clonidine-displacing substance. Binding to anti-p-aminoclonidine antibodies was examined by using a competitive radioimmunoassay with tritiated p-aminoclonidine as the radioligand. Central vasodepressor agents that, like clonidine, are known to bind with high affinity to both imidazole sites and alpha 2-adrenergic receptors in brain inhibited radioligand binding to anti-p-aminoclonidine antibodies. All of these agents contain imidazol(in)e and phenyl ring moieties as part of their chemical structures (e.g., oxymetazoline); a number of other compounds without one or both of these rings failed to cross-react with the antisera. Clonidine-displacing substance, partially purified from bovine brain, also inhibited specific radioligand binding to anti-p-aminoclonidine antibodies. The inhibition was dose dependent and high affinity (IC50, 4 Units). The endogenous substance had no effect on the apparent affinity of the antibodies for the radioligand, but blocked a specific number of binding sites. Immunoprecipitation experiments showed that authentic clonidine-displacing substance, that which displaces tritiated p-aminoclonidine binding to membrane receptors, is recognized by anti-p-aminoclonidine antibodies. We conclude that a unique subset of structural determinants required for ligand interaction with both imidazole and alpha 2-adrenergic receptors is critical for binding to anti-p-aminoclonidine antibodies, and that since clonidine-displacing substance is recognized by highly clonidine-specific antisera, it may also contain these determinants within its structure, namely the imidazol(in)e and phenyl ring systems.

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.

In order to characterize putative high- and low-affinity states of the renal alpha 1-adrenoceptor, binding sites for the selective antagonist radioligand [3H]prazosin were examined in washed membranes prepared from rat renal cortex and medulla. Norepinephrine competition curves at [3H]prazosin sites were biphasic and were best fit by a two-site model. Na+ and GTP selectively decreased the proportion of sites exhibiting a high affinity for norepinephrine. In contrast, Mg2+ facilitated high-affinity interactions of norepinephrine at the renal alpha 1-receptor. Guanine nucleotides and Na+ increased the affinity of some antagonists [( 3H]prazosin, WB-4101), but not others (phentolamine). Mg2+ again had opposite effects. The effects of ions and nucleotides on both agonist and antagonist interactions were concentration-dependent. The order of potencies for monovalent cations (Na+ greater than Li+ much greater than K+), divalent cations (Mn2+ greater than Mg2+) and nucleotides (Gpp (NH)p, GTP much greater than GMP, ATP) were similar to those reported for cyclase-coupled receptor systems. However, unlike other divalent cations Ca2+ decreased both agonist and antagonist binding, possibly due to a Ca2+-sensitive proteinase. Receptor binding properties were similar in renal cortex and medulla. Renal alpha 1-receptor sites appear to display high- and low-affinity states with respect to agonists, and the equilibrium between these states may be modulated by guanine nucleotides and mono- and divalent metal ions. Some antagonists appear to bind preferentially to sites with low agonist affinity, and this effect is probably independent of retained endogenous catecholamines.

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 alpha 2-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 alpha 2-receptor mediates the clonidine hypotensive response, a series of clonidine analogues were tested (1) in radioligand binding assays of their affinity at imidazole and alpha 2-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 alpha 2-adrenergic affinity (r = 0.36). An imidazole receptor in the rostral ventrolateral medulla may participate in the hypotensive action of clonidine.

Clonidine-displacing substance (CDS) is biologically active in the brain, as well as the gastric fundus, platelets and vas deferens. We sought to determine whether CDS is contained within peripheral tissues in the rat. Using competitive radioimmunoassay with a clonidine-specific antiserum and 3H-p-aminoclonidine rat adrenal gland and gastric fundus were shown to contain significantly greater amounts of CDS-like radioimmunoactivity than the brain; intermediate-to-low activity was present in the heart, small intestine, serum, kidney and liver. Lung and skeletal muscle exhibited near-background levels. CDS may not be unique to the brain, but also may be synthesized and stored in peripheral organs.

A polyclonal antiserum was raised in rabbit against the clonidine analog p-aminoclonidine (PAC) coupled to hemocyanin. The antiserum (anti-PAC3) exhibited high affinity for unconjugated [3H]PAC (Kd 0.32 +/- 0.07 nM) in a rapid-filtration radioimmunoassay. Competition experiments showed that PAC, clonidine, and naphazoline cross-reacted with the anti-PAC3 antiserum, whereas a number of other structurally related compounds did not. An endogenous clonidine-displacing substance (CDS) partially purified from bovine brain also inhibited specific [3H]PAC binding to anti-PAC3 in a dose-dependent manner. Thus, (a) anti-PAC3 antiserum is specific for clonidine and closely related compounds, and (b) CDS may structurally resemble clonidine since it is recognized by this highly specific antiserum.

In order to assess the relationship of behavior to blood pressure and salt intake, open-field behavior was studied in 123 rats of the spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), Sprague-Dawley (SD), and Dahl resistant (DR) and sensitive (DS) strains. DS rats become hypertensive upon exposure to either high dietary salt or psychogenic stress, while DR rats remain normotensive. A circular photobeam open field was used under full room illumination. Three 5-min trials were given on consecutive days. Rats were tested with or without 5 days of an 8% NaCl diet. We found that DS rats were less active than DR rats, regardless of diet. In contrast, SHR rats were more active than WKY rats. However, DS and SHR rats were equally active. Thus, behavioral differences between these two models of hypertension are expressed by the normotensive control strains. The relative ranking of activity levels between strains was DR = SD greater than SHR = DS greater than WKY. High-salt-enhanced intertrial habituation, defined as the decrease in activity across trials (DS, 100%; SD, 82%; SHR, 90%; WKY, 1350%; but DR, -50%) as well as intratrial habituation, defined as the decrease from the first to the second half of the trial (14%, all subjects). Defecation was increased with high salt (DS, 975%; SD, 59%; SHR, 267%; WKY, 89%; but DR, 40%). These effects of high salt may reflect an increase in an emotionality factor. DR rats were largely resistant to the behavioral effects of salt. Total activity was positively correlated with blood pressure in hypertensive rats, r(42) = 0.33, p less than .01, but negatively correlated in normotensive rats, r(81) = -0.34, p less than .01. The proportion of total activity occurring in the first half of the trial for the initial test day was correlated with blood pressure in normotensive rats r(81) = 0.44, p less than .01. Dietary salt excess has behavioral as well as cardiovascular consequences.

Clonidine-displacing substance (CDS) isolated from bovine brain potently inhibits clonidine binding and elicits contraction of gastric smooth muscle. We sought to determine if CDS was contained in neuron-like clonal cells (neuroblastoma X glioma hybrid NG108-15). Extracts were prepared from osmotically shocked P2 fractions of NG108-15 cells. One unit of CDS, as defined by a [3H]p-aminoclonidine radioreceptor assay using bovine frontal cortex membranes, was obtained from each 1.3 million cells processed. CDS isolated from NG108-15 cells was biologically active on gastric smooth muscle. NG108-15 cells may serve as a model system for the study of this endogenous clonidine-like ligand.

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.