Clonidine-displacing Substance Research Articles

Agmatine

Agmatine is an endogenous neuromodulator that, based on animal studies, has the potential for new drug development. As an endogenous aminoguanidine compound (1-amino-4-guanidinobutane), it is structurally unique compared with other monoamines. Agmatine was long thought to be synthesised only in lower life forms, until its biosynthetic pathway (decarboxylation of arginine) was described in the mammalian brain in 1994. Human arginine decarboxylase has been cloned and shown to have 48% identity to ornithine decarboxylase. In neurons of the brain and spinal cord, agmatine is packaged into synaptic vesicles and released upon neuronal depolarisation. Other evidence of a neuromodulation role for agmatine is the presence of a specific cellular uptake mechanism and a specific metabolic enzyme (agmatinase; which forms putrescine).Initially, agmatine was conceptualised as an endogenous clonidine-displacing substance of imidazoline receptors; however, it has now been established to have affinity for several transmembrane receptors, such as alpha(2)-adrenergic, imidazoline I(1) and glutamatergic NMDA receptors. In addition to activity at these receptors, agmatine irreversibly inhibits neuronal nitric oxide synthase and downregulates inducible nitric oxide synthase. Endogenous agmatine is induced in response to stress and/or inflammation. Stressful conditions that induce agmatine include hypoxic-ischaemia and cold-restraint stress of ulcerogenic proportion. Induction of agmatine in the brain seems to occur in astrocytes, although neurons also synthesise agmatine. The effects of injected agmatine in animals include anticonvulsant-, antineurotoxic- and antidepressant-like actions. Intraperitoneal or intracerebroventricular injections of agmatine rapidly elicit antidepressant-like behavioural changes in the rodent forced swim test and tail suspension test. Intraperitoneal injections of agmatine into rats and mice also elicit acute anxiolytic-like behavioural changes in the elevated plus-maze stress test. In an animal model of acute stress disorder, intraperitoneal agmatine injections diminish contextual fear learning. Furthermore, intraperitoneal injections of agmatine reduce alcohol and opioid dependence by diminishing behaviour in a rat conditioned place preference paradigm. Based on these findings, agmatine appears to be an endogenous neuromodulator of mental stress. The possible roles and/or beneficial effects of agmatine in stress-related disorders, such as depression, anxiety and post-traumatic stress disorder, merit further investigation.

In vitro and ex vivo distribution of [ 3H]harmane, an endogenous β-carboline, in rat brain

The endogenous β-carboline, harmane, has been shown to bind to monoamine oxidase A (MAO-A) and a separate, high affinity, non-MAO site. Research in our laboratory has shown that harmane is an active component of clonidine-displacing substance (CDS), the proposed endogenous ligand for imidazoline binding sites (IBS). In the present study we have investigated the distribution of [ 3H]harmane in rat brain, and related the binding profile to the distribution of the MAO-A selective ligand [ 3H]Ro41-1049 and the I 2BS ligand [ 3H]2-BFI. The in vivo distribution of [ 3H]harmane following intravenous administration was also investigated. Receptor autoradiography revealed a highly significant correlation for the distribution of [ 3H]harmane and [ 3H]Ro41-1049, and a significant correlation for [ 3H]harmane and the I 2BS ligand [ 3H]2-BFI. The in vivo distribution of [ 3H]harmane suggests that the ligand accumulates in the adrenal gland and throughout the brain with the primary route of excretion occurring via the duodenum. In conclusion, these studies have shown that [ 3H]harmane labels a population of binding sites that reflect the distribution of MAO-A. Further evidence for a non-MAO, IBS [ 3H]harmane population has not been shown but the high level of expression of the MAO-A site is likely to have masked the much smaller population of I 2BS.

Harmane and Harmalan Are Bioactive Components of Classical Clonidine-Displacing Substance

Elucidation of the structure of the endogenous ligand(s) for imidazoline binding sites, clonidine-displacing substance (CDS), has been a major goal for many years. Crude CDS from bovine lung was purified by reverse-phase high-pressure liquid chromatography. Electrospray mass spectrometry (ESMS) and nuclear magnetic resonance ((1)H NMR) analysis revealed the presence of L-tryptophan and 1-carboxy-1-methyltetrahydro-beta-carboline in the active CDS extract. Competition radioligand binding studies, however, failed to show displacement of specific [(3)H]clonidine binding to rat brain membranes for either compound. Further purification of the bovine lung extract allowed the isolation of the beta-carbolines harmane and harmalan as confirmed by ESMS, (1)H NMR, and comparison with synthetic standards. Both compounds exhibited a high (nanomolar) affinity for both type 1 and type 2 imidazoline binding sites, and the synthetic standards were shown to coelute with the active classical CDS extracts. We therefore propose that the beta-carbolines harmane and harmalan represent active components of classical CDS. The identification of these compounds will allow us to establish clear physiological roles for CDS.

Imidazoline Receptors and their Ligands as Potentiators of Nutrient-Induced Insulin Secretion

It is now well established that many tissues express binding sites that are defined pharmacologically as “imidazoline” receptors. These are still undergoing full molecular characterisation but candidate clones have been identified for both the I 1 and I2 subtypes. The physiological roles of these receptors remain to be the subject of continuing investigation although there is strong evidence that I1 sites play a role in the central regulation of blood pressure while I2 sites may control feeding and other aspects of behaviour. In molecular terms, the third imidazoline receptor subtype (I3) is the least well characterised although progress in understanding its functional pharmacology is continuing. This receptor plays a role in the regulation of insulin secretion and mediates glucose-dependent increases in secretion in response to synthetic ligands such as efaroxan and antazoline. These agents were originally developed as selective α2-adrenoceptor antagonists but it is now clear that they also act as I3 agonists. Molecular characterisation of the functional activity of I3 agonists has been complicated by the fact that most of the ligands also interact with other cellular proteins, leading to potential difficulties in the interpretation of experimental data. In particular, it was established more than a decade ago that many I3-agonists induce the closure of ATP-sensitive potassium (KATP) channels and it is now clear that an imidazoline binding site is located within the pore-forming subunit (Kir6.2) of the KATP channel. However, increasing evidence implies that closure of this channel is not primarily responsible for the ability of imidazoline drugs to enhance insulin secretion. In support of this, imidazoline compounds have recently been described that do not close KATP channels but which potentiate insulin secretion very effectively. These probably interact with intracellular targets within pancreatic β- cells and the results suggest that the I3 receptor may be distinct from the imidazoline binding site associated with Kir6.2. Rather, the I3-site appears to control a distal step in the exocytotic pathway. The present review examines recent progress in identifying the I 3 receptor and describes the pharmacology of I3-agonists that regulate insulin secretion. The possibility that these agonists might then serve as prototypes for the development of novel ligands having therapeutic potential is also considered. Keywords: type 2 diabetes, imidazoline receptors, efaroxan, clonidine-displacing substance, islets of langerhans, insulin secretion, glucagon secretion

No evidence for activation of alpha(2)-adrenoceptors by methanolic extracts of bovine brain and lung containing clonidine-displacing substance.

Methanolic extracts of bovine brain and lung are capable of displacing [(3)H]-clonidine from alpha(2)-adrenoceptor binding sites, indicating the presence of a clonidine-displacing substance (CDS). We have examined alpha(2)-adrenergic responses and the extracts in three models: [(3)H]-cyclic AMP accumulation in miniprisms of guinea pig cerebral cortex, isometric tension measurements of isolated segments of the rat vas deferens, and porcine palmar lateral vein. The selective alpha(2)-adrenoceptor agonist, 5-bromo-6-2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304) inhibited forskolin-stimulated [(3)H]-cyclic AMP accumulation in the cerebral cortex and elicited contractions of the porcine isolated palmar lateral vein. Clonidine (0.1-30 nM) inhibited neurogenic contractions of the rat vas deferens. Responses to both agonists were inhibited by the alpha(2)-adrenoceptor antagonists, idazoxan or rauwolscine. Brain CDS (5 units/mL) reduced forskolin-stimulated [(3)H]-cyclic AMP accumulation in the guinea pig cerebral cortex, whereas lung CDS (1 unit/mL) increased the accumulation of the cyclic nucleotide. Neither response to the extracts was inhibited by 1 microM idazoxan. Low concentrations of both extracts (0.05 unit/mL) reduced electrically evoked contractions of the rat vas deferens by approximately 20%, but higher concentrations enhanced neurogenic contractions by approximately 50%. Again, the effect of the brain extract was not altered by 1 microM idazoxan. Lung CDS (0.02-1 unit/mL) induced contractions of the porcine palmar lateral vein that were also insensitive to rauwolscine. The results suggest that brain and lung CDS do not activate either central or peripheral alpha(2)-adrenoceptors.

Comparative effects of efaroxan and beta-carbolines on the secretory activity of rodent and human beta cells.

The pancreatic beta-cell expresses an imidazoline-binding site that is involved in the regulation of insulin secretion. This site is pharmacologically atypical in comparison with the I(1) and I(2) sites described in other tissues, and it has been classified as I(3). The structural requirements for binding of ligands to the I(3) site have not been fully defined, although a range of synthetic I(3) ligands have been characterized in functional terms. Evidence has been presented that an endogenous I(3) ligand may exist, because extracts of brain contain an active principle that stimulates insulin secretion in a manner consistent with the involvement of I(3) sites. The active component has not been identified but has been equated with the long-sought clonidine displacing substance (CDS) that is proposed as the endogenous ligand for imidazoline-binding sites. Recent evidence has indicated that one active component of CDS may be a beta-carboline, but it is not known whether beta-carbolines can stimulate insulin secretion. Thus, we have studied the effects of beta-carbolines on insulin secretion and cytosolic Ca(2+) levels in rodent and human islet cells. The results reveal that harmane, pinoline, and norharmane cause a dose- and glucose-dependent increase in insulin secretion but show that this response differs in a number of ways from that elicited by the well-characterized I(3)-agonist, efaroxan. Thus, beta-carbolines represent a new class of insulin secretagogues, although it remains unclear whether their action is mediated solely by I(3) sites in the beta cell.

Spinal and supraspinal agmatine activate different receptors to enhance spinal morphine antinociception.

The endogenous clonidine-displacing substance, agmatine, enhances morphine-induced antinociception and inhibits development of tolerance to morphine. Because agmatine binds with relatively high affinity to both alpha(2) adrenergic and imidazoline (I) receptors, the following studies were designed to determine which receptor type is involved in the morphine-enhancing activity. Mice were injected by various routes with agmatine, clonidine or norepinephrine as well as selective antagonists, and tail flick antinociception was measured. Agmatine administered subcutaneously (s.c.), intracerebroventricularly (i.c.v.), or intrathecally (i.t.) had no antinociceptive activity, but enhanced the antinociception produced by i.t. morphine. The morphine-enhancing activity of i.c.v. agmatine (and clonidine) was attenuated by coadministered yohimbine, idazoxan and SK and F 86466 (alpha(2) antagonists). When given i.t., agmatine, yohimbine and SK and F 86466 attenuated the antinociception produced by coadministered i.t. clonidine. In contrast, i.t. yohimbine and SK and F 86466, but not agmatine, attenuated antinociception produced by i.t. norepinephrine. These results suggested that agmatine actions were different at brain and spinal sites. Thus, agmatine may act at both alpha(2) and I receptors in the brain and I receptors in the spinal cord.

Clonidine-Displacing Substance Reduces Glucagon Secretion from Mouse Pancreatic α-Cells by KATP-Channel-Independent Inhibition of Exocytosis

Clonidine-displacing substance (CDS) is a potent stimulator of insulin release from pancreatic β-cells and has been suggested to constitute the endogenous ligand for the islet imidazoline-binding site. Here we have explored the effects of CDS on glucagon release from mouse pancreatic α-cells. CDS (5 U/ml) produced a 35% inhibition (P < 0.05) of glucagon release from intact islets. This effect was dose-dependent and half-maximal inhibition by CDS was observed at 0.03 U/ml. Inhibition of glucagon release was not associated with a change in whole-cell ATP-sensitive K+-channel activity in single α-cells. However, during intracellular application through the recording pipette, CDS produced a 36% (P < 0.05) decrease in the rate of exocytosis, measured as changes in cell capacitance. The inhibitory effect of CDS on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A. These data provide further evidence for a role of CDS as an endogenous ligand controlling islet hormone secretion.

Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells

A crude extract of clonidine-displacing substance (CDS) has previously been extracted from the NG108-15 cell line. This study aimed to purify CDS extracted from this cell line further, by the technique of reverse phase-HPLC (RP-HPLC), and subsequently determine whether this refined CDS bears any similarity to CDS's extracted from other tissues. Crude CDS was extracted from NG108-cells and fractionated by RP-HPLC eluting with a linear gradient of methanol (5–65%; 1 ml min −1 flow rate) over 50 min., and collected at 1 min. intervals. The pharmacological activities of the CDS fractions were determined by their abilities to displace bound [ 3H]clonidine to α 2-adrenoceptors in rat brain membranes. RP-HPLC analysis of CDS revealed a pharmacologically active fraction distinct from agmatine, eluting at 24 min, corresponding to an absorbance peak observed at this time. Collectively, these results confirmed that CDS was present in the NG108-15 cell line. However, the RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDS's extracted from bovine tissues. These results support the notion of the existence of several CDS's.

Extraction of active clonidine-displacing substance from bovine lung and comparison with clonidine-displacing substance extracted from other tissues

Crude methanolic clonidine-displacing substance (CDS) extracted from bovine lung competed for radioligand binding from α 2-adrenoceptors and I 2-sites present in rat brain membranes, and from I 1-sites present in rat brain and kidney membranes. There was no difference in the competition of [ 3 H ]clonidine binding to α 2-adrenoceptors present in either rat or rabbit brain membranes by the crude CDS extract and therefore either tissue could be used to estimate the number of units of CDS present in extracts. Further purification by reverse phase high performance liquid chromatography (RP-HPLC), with UV detection, of extracts obtained from bovine lung, brain and rat brain exhibited similar three-peak profiles, previously reported. Corresponding fractions competed for radioligand binding to α 2-adrenoceptors present in rat brain membranes, eluting between 19 and 23 min, which corresponded with the middle peak of the three-peaks. Therefore, we propose the CDS-like material eluting from all these tissues to be similar. Interestingly, CDS extracted from bovine adrenal glands under the same conditions showed a similar three-peak profile, but did not repeat the displacement of binding just at 19–23 min, but at every time point after 4 min. This suggests this tissue could represent a source of CDS in this species.

Isolation and partial structure determination of a clonidine-displacing substance from bovine lung and brain

A large scale extraction and isolation method was developed for the purification of clonidine-displacing substance (CDS) activity from bovine lung or brain. This optimised method used direct freeze drying of tissue, hexane removal of lipids, and methanol extraction of CDS activity. Using a bioassay directed isolation strategy a new CDS compound was purified from an extract of bovine lung. The isolation strategy involved subsequent steps of flash C-18 chromatography, ion exchange, size exclusion, and C-18 HPLC. An HPLC detection method was developed and applied to show that the new CDS is present in both lung and brain tissue. Spectroscopic data for this new CDS indicates that it is related to guanosine, but is not noradrenaline, adrenaline, histamine, agmatine, guanosine, GMP, GDP or GTP.

'Seeing through a glass darkly': casting light on imidazoline 'I' sites.

Although imidazoline sites have been the subject of research for several years, there is still controversy about their structure, diversity and physiology. The I1 site is thought to exist principally as a binding site and is widely purported to play a role in controlling systemic blood pressure, although this is still unclear. The majority of I2 sites are widely accepted as being allosteric sites on monoamine oxidase; however, even with selective ligands, their exact function remains to be determined. A putative I3 site modulates insulin secretion and could represent the first functional site to be pharmacologically defined with selective agonists and antagonists. The structure and relevance of the proposed endogenous ligand 'clonidine-displacing substance' remains elusive. A potential candidate for this substance is agmatine; however, although it is capable of displacing bound clonidine from imidazoline sites, it lacks the functionality ascribed to the clonidine-displacing substance. In this review, Richard M. Eglen and colleagues assess our knowledge of imidazoline sites in the light of recent data.

Do alpha 2-adrenoceptors and imidazoline binding sites coexist in the human term placenta? Evidence from direct binding studies.

Alpha2-Adrenergic receptors and non-adrenergic imidazoline binding sites (IBS) in human placental membranes were investigated by means of the radioligands [3H]-RX 821002 and [3H]-RX 781094 (idazoxan) respectively. Human term placentae (38-40 weeks) were obtained immediately after vaginal delivery. The specific binding of the alpha2-subtype-selective [3H]-RX 821002 confirms the presence of alpha2-adrenoceptors in the human placenta, while [3H]-idazoxan binds to non-adrenergic IBS. The sites were characterized by displacement analyses with various imidazoline and non-imidazoline drugs. The presence of an endogenous ligand for IBS has not yet been demonstrated. Clonidine displacing substance (CDS) was recently identified as agmatine; it recognizes both alpha2 and imidazoline receptors. This phenomenon was studied in crude placental membranes. The studies revealed that: (i) alpha2-adrenoceptors coexist with non-adrenergic IBS in human placental membranes; (ii) there is a strong probability that alpha2-adrenoceptors and IBS are pharmacologically distinct; and (iii) agmatine binds to placental alpha2 and imidazoline receptors with different affinities.

The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans.

1. The effects of a rat brain extract containing clonidine-displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated. 2. CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mM glucose, in a dose-dependent manner. The magnitude of this effect was similar to that in response to the well-characterized imidazoline secretagogue, efaroxan. 3. CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose-induced insulin release, in both rat and human islets. 4. These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline-mediated actions of CDS can be blocked by specific imidazoline antagonists. 5. The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri-filtration through a 3,000 Da cut-off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide. 6. Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor.

An antiserum to idazoxan recognizes an immunoreactive substance in human serum and cerebral spinal fluid which is not agmatine

A polyclonal antibody was generated in rabbits to an idazoxan-albumin antigen. The anti-idazoxan antiserum had high affinity for unconjugated 3H-idazoxan ( K d of 19.8 nM) in a radio-immunoassay (RIA). Of various drugs and native molecules only idazoxan potently ( K i of 24 nM) inhibited 3H-idazoxan binding to the anti-idazoxan antibody. A few drugs weakly inhibited 3H-idazoxan binding (IC50 > 605 μM) with rank order of UK 14304 > guanabenz > cirazoline > amiloride > naphazoline. Neither agmatine, an endogenous clonidine displacing substance (CDS), catecholamines or imidazoles inhibited the binding of 3H-idazoxan to the anti-idazoxan antibody. The anti-idazoxan RIA was 4–6 fold more sensitive than an antibody to para-amino clonidine. The CDS detected by ligand displacement from bovine brain dose-dependently inhibited 3H-idazoxan binding. This immunoreactive (ir-) CDS activity was present in human (0.9–4.1 U/ml) and rat sera (1–2 U/ml) and in the cerebro-spinal fluid of eight patients with serious disease of the central nervous system, but not in controls. We conclude: (1) an anti-idazoxan RIA is a sensitive, selective and clinically applicable RIA for measuring ir-CDS; (2) ir-CDS is not agmatine; (3) CDS represents a family of endogenous ligands for imidazoline receptors including ir-CDS and agmatine.

Central cardiovascular actions of agmatine, a putative clonidine-displacing substance, in conscious rabbits

Agmatine, an endogenous clonidine-displacing substance, has been shown to have an affinity for both α 2-adrenoceptors and imidazoline receptors (IR). In conscious rabbits, we have examined the cardiovascular effects of agmatine and its interaction with clonidine, a presumed agonist and 2-methoxy-idazoxan, an antagonist at α 2-adrenoceptors. We have also examined the effect of agmatine on agents having high affinity for I 1-imidazoline receptors namely moxonidine (agonist) and efaroxan (antagonist). Initial dose-response studies showed that agmatine administered in low doses (0.01–10 μg/kg) into the fourth ventricle did not change mean arterial pressure but did produce a dose-dependent bradycardia (maximum −16 ± 3 beats/min). A higher dose of 100 μg/kg produced an adverse reaction in the conscious animals accompanied by a marked increase in mean arterial pressure and a reversal of the bradycardia. This is in contrast to the effects of fourth ventricular clonidine and moxonidine, which caused a dose-dependent fall in both mean arterial pressure and heart rate. Agmatine when administered at the highest well-tolerated dose of 10μg/kg did not further alter the clonidine-induced hypotension but produced a greater bradycardia (−12 ± 4 beats/min clonidine; −29 ± 4 beats/min clonidine plus agmatine; p < 0.05). Similarly, the hypotension induced by moxonidine was not altered by agmatine but heart rate was reduced after the addition of agmatine ( p < 0.01). Efaroxan and 2-methoxy-idazoxan, at doses which produced no effects when given alone, similarly reversed the fall in heart rate elicited by agmatine and caused a small but significant rise in mean arterial pressure. We have previously shown that the doses of these antagonists used in this study produce an equal reversal of the bradycardia induced by fourth ventricular α-methyldopa ( α 2-adrenoceptor agonist) and clonidine and hence have similar α 2-adrenoceptor blocking effects. Our results show that agmatine produces bradycardia as does moxonidine and clonidine but does not mimic or block the hypotensive responses to these agents. These findings do not support the hypothesis that agmatine is an endogenous ligand for IR. However, the bradycardia induced by agmatine may be mediated via α 2-adrenoceptors since it was equally blocked by efaroxan and 2-methoxy-idazoxan. Thus while α 2-adrenoceptor actions of agmatine on heart rate are evident at relatively low doses, the reason for the lack of α 2-adrenoceptor mediated hypotension is not known.

Nω-Hydroxyagmatine: A Novel Substance Causing Endothelium-Dependent Vasorelaxation

Agmatine (Agm), a decarboxylated L-arginine, has been suggested to be an endogenous clonidine-displacing substance in the brain. We hypothesed that Agm enzymatically produces nitric oxide (NO) through N ω-hydroxyagmatine (OHAgm) similar to the well-known endogenous NO generation from L-arginine through N ω-hydroxy-L-arginine, because Agm possesses a guanidyl function in its molecule. OHAgm was originally synthesized from δ-aminopentanoic acid in 36% overall yield. Agm and the synthetic OHAgm were examined using rat aortic rings whether they could cause endothelium-dependent vasorelaxation or not. These substances equally elicited vasorelaxations. The relaxations were completely abolished by a NO synthase inhibitor, N ω-nitro-L-arginine methyl ester, or endothelium denudation. These results suggested that Agm and OHAgm are alternative precursors for NO generated by NOS and that OHAgm may be an endogenous substance distributable in the brain.

Molecular and physiological properties of clonidine-displacing substance.

An endogenous small molecular mass compound, termed clonidine-displacing substance (CDS), has been isolated and purified from bovine brain. The estimated level of CDS in bovine brain is 400-1,000 units/wet brain, with 1 unit of activity calculated to be approximately 1-2 ng. It is present in human serum, urine, and cerebrospinal fluid. The isolation procedure consists of initial aqueous and methanolic extractions followed by a series of HPLC chromatography steps (reverse phase and TSK sizing columns). The reverse-phase chromatography of CDS extracted under identical conditions from bovine brain and human serum show similar retention times. The final chromatography step gives a single active peak with a distinct ultraviolet spectrum, a single molecular peak m/z 587.8 +/- 2 in plasma desorption mass spectrometry (PDMS), and a unique pharmacological and physiological profile. Clonidine-displacing substance does not partition into organic solvents and it is ninhydrin and fluorescamine negative. All of these molecular properties clearly distinguish CDS from agmatine, an endogenous 130-dalton compound of far greater abundance which displays lower affinity for p-aminoclonidine-labeled sites in rat brain membranes. The ultraviolet spectrum of CDS consists of two aromatic peaks at 224 and 276 nm, whereas agmatine is an aliphatic substance with no ultraviolet absorbance. Like many antihypertensive drugs of the guanidine and imidazoline family of compounds, CDS recognizes alpha 2-adrenergic receptors, clonidine sites (IR-I1), and imidazoline sites (IR-I2). A good correlation exists between the affinities of various imidazoline/guanidine type ligands for IR-I2 in both human placenta and rat liver membranes which can be accurately determined because both tissues lack IR-I1 and alpha 2-adrenergic receptors. There is no correlation in the affinities of these ligands for IR-I2 of human-placental versus alpha 2-adrenergic receptors of human platelets. By uncovering the role of CDS in the central nervous system we will be able to understand the coupling of IRs to neurotransmission and, in turn, to changes in arterial pressure.

Agmatine: A novel endogenous vasodilator substance

The purpose of the study was to investigate the effects of agmatine, an endogenous clonidine-displacing substance (CDS), on systemic hemodynamics in the anesthetized rat. Bolus intravenous (i.v.) injections of agmatine decreased systemic arterial pressure (SAP) and systemic vascular resistance in a dose-dependent manner. The development of acute tachyphylaxis to the systemic vasodepressor response to agmatine did not induce cross-tachyphylaxis to the systemic vasodepressor responses to bradykinin, isoproterenol and nitroglycerin. The present data demonstrate agmatine, as a CDS and agonist for imidazoline (I) receptors, possesses marked systemic vasodilator activity in the rat. The present data suggest that activation of I receptors may represent a novel mechanism of vasodilation in vivo.

Influence of Imidazolines on Catecholamine Release in Pithed Spontaneously Hypertensive Rats

The actions of the imidazoline derivatives clonidine, moxonidine, and rilmenidine and of the recently discovered clonidine-displacing substance agmatine on stimulation-induced norepinephrine overflow and epinephrine release were studied in pithed spontaneously hypertensive rats. All three imidazolines dose-dependently decreased norepinephrine overflow and led to an increase in epinephrine release when the highest dose of each compound was injected. The alpha 2-adrenoceptor antagonist rauwolscine shifted the dose-response curves of plasma norepinephrine concentrations to higher levels. Agmatine did not change norepinephrine overflow but increased epinephrine release into plasma after the highest dose administered. It is concluded that the investigated imidazolines decrease norepinephrine overflow via presynaptic alpha 2-adrenoceptors, whereas epinephrine release is mediated through putative imidazoline receptors on the adrenal medulla.