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Because of the central role of adrenergic mechanisms in the expression of crucial genes during brown adipocyte differentiation, we examined the activation (phosphorylation) of CREB (cAMP-response-element-binding protein) in mouse brown adipocytes in primary culture. We found that noradrenaline ('norepinephrine') stimulated CREB phosphorylation rapidly (maximum effect in < or =5 min with slow decay) and efficiently (EC(50), 6 nM). The increase in CREB phosphorylation coincided with increased expression of an artificial cAMP-response-element-containing reporter construct. CREB phosphorylation was partly inhibitable, both by the beta-adrenergic antagonist propranolol and by the alpha(1)-adrenergic antagonist prazosin. Adenylate cyclase hyperactivation (by forskolin) could stimulate CREB phosphorylation to the same extent as noradrenaline. The alpha(1)-adrenergic agonist cirazoline also increased CREB phosphorylation. An increase in intracellular [Ca(2+)] had, however, no effect, but protein kinase C activation by PMA was a potent stimulator. The cirazoline-stimulated (alpha(1)-adrenergic) CREB phosphorylation was inhibited by a desensitizing pretreatment with PMA, demonstrating that the alpha(1)-stimulation was mediated via protein kinase C activation; neither Src nor extracellular-signal-regulated kinases 1 and 2 activation was involved in the signalling process. We conclude that CREB phosphorylation in brown adipocytes is mediated not only through the classical beta-adrenergic/cAMP pathway but also through a novel alpha(1)-adrenergic/protein kinase C/CREB pathway, which has not been described previously in any tissue.

The aim of the present study was to classify release-inhibiting receptors on rat pheochromocytoma PC12 cells. Veratridine-evoked [3H]noradrenaline release from PC12 cells was inhibited by micromolar concentrations of the imidazoline and guanidine derivatives cirazoline, clonidine, aganodine, 1,3-di(2-tolyl)guanidine, BDF6143 and agmatine, and of the cannabinoid receptor agonist WIN55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-yl](1-naphthalenyl)methanone mesylate), but not by noradrenaline. The inhibitory effect of clonidine was antagonized by micromolar concentrations of rauwolscine and SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide). The potencies of the agonists and antagonists were compatible with an action at previously characterized presynaptic imidazoline receptors. 1-Oleoyl-lysophosphatidic acid, but not sphingosine-1-phosphate, produced an inhibition of release that was antagonized by 30 microM rauwolscine, 1 microM SR141716A and 10 microM LY320135 as well as by pretreatment of the cells with 100 microM clonidine for 72 h. Polymerase chain reaction (PCR) experiments on cDNA from PC12 mRNA suggest mRNA expression of lysophospholipid receptors encoded by the genes edg2, edg3, edg5 and edg7, but not of receptors encoded by edg1, edg4, edg6 and edg8, and not of alpha(2A(-))nd CB(1) receptors. In conclusion, PC12 cells are not endowed with alpha(2)-adrenoceptors and CB(1) cannabinoid receptors, but with an inhibitory receptor recognizing imidazolines, guanidines and WIN55,212-2 similar to that on sympathetic nerves. The PCR results and the ability of 1-oleoyl-LPA to mimic these drugs (also with respect to their susceptibility to antagonists) suggest that the release-inhibiting receptor may be an edg-encoded lysophospholipid receptor.

The present study was undertaken to investigate and compare the properties of noradrenaline release in the locus coeruleus (LC) and prefrontal cortex (PFC). For that aim the dual-probe microdialysis technique was applied for simultaneous detection of noradrenaline levels in the LC and PFC in conscious rats. Calcium omission in the LC decreased noradrenaline levels in the LC, but increased its levels in the PFC. Novelty increased noradrenaline levels in both structures. Infusion of the az-adrenoceptor agonist clonidine decreased extracellular noradrenaline in the LC as well as in the PFC. Infusion of the alpha (2A)-adrenoceptor antagonist BRL44408, or the alpha (1)-adrenoceptor agonist cirazoline into the LC or PFC caused a similar dose-dependent increase in both structures. When BRL44408 or cirazoline were infused into the LC. few effects were seen in the PFC. Infusion of the 5-HT1A-receptor agonist flesinoxan into the LC or the PFC decreased the release of noradrenaline in both structures. When flesinoxan was infused into the LC. no effects were seen in the PFC. When the GABA(A) antagonist bicuculline was applied to the LC, noradrenaline increased in the LC as well as in the PFC. It is concluded that the release of noradrenaline from somatodendritic sites and nerve terminals responded in a similar manner to presynaptic receptor modulation. The possible existence of dendritic noradrenaline release is discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

Clonidine is a partial agonist at brain alpha(2)-adrenoceptors (alpha(2)AR), but also has high affinity (K(D) = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and alpha(2)AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 microM norepinephrine (NE; alpha(2)AR agonist) or 12.5 microM methoxy-idazoxan (MIDX; selective alpha(2)AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 microM cirazoline. Levels of [3H]-clonidine binding to alpha(2)AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r(2) = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: alpha(2)AR (4:1). Quantitative transepts drawn across hippocampal images revealed alpha(2)AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus-areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I(1) and I(2) subtypes. The rank-order of affinities were cirazolineharmaneBDF6143idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 +/- 12 nM).1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from alpha(2)AR; and 2) the affinities of these sites was distinct from alpha(1)AR, alpha(2)AR, I(1) or I(2) sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.

Intravenous administration of the alpha2-adrenoceptor antagonist, idazoxan, elicits variable cardiovascular effects, depending on experimental conditions. In this study, the effects of idazoxan were investigated in rats with high, low, or no basal sympathetic tone. In a group of conscious Sprague-Dawley rats (n = 9), mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nervous activity (RSNA) were recorded. Idazoxan (250 microg/kg, i.v.) induced a transient decrease in MAP (-12+/-3 mm Hg) that was accompanied by increases in HR (49+/-14 beats/min) and RSNA (53+/-14%). In six of nine rats, a light pentobarbitone anesthesia was given. Basal RSNA was decreased (6.0+/-1.3 microV from 12.8+/-4.1 microV; p

Several analogues of cirazoline (2), a selective alpha1-adrenoreceptor agonist, were prepared and their pharmacological profiles studied. Although at the alpha1-adrenoreceptor all the compounds displayed a significant agonist activity, at the alpha2-adrenoreceptor they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure-activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for alpha1-agonist activity, while the cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead, the same substitution (i.e., isopropoxy for the cyclopropyl ring) at alpha2-adrenoreceptors causes a reversal of activity. On the other hand, the cyclopropyl ring seems to be important for alpha1-selectivity. Compound 20 is the most potent alpha1-agonist of the series, being equiactive with cirazoline on rat vas deferens and in pithed rat.

Biochemical and pharmacologic evidence supports the heterogeneous nature of imidazoline receptors (IRs). However, only monoamine oxidase (MAO) (55- and 61-kD) isozymes have been identified as imidazoline binding site-containing proteins. Idazoxan-binding proteins of approximately 70- and approximately 45-kD of unknown amino acid sequences have been isolated from chromaffin cells and rat brain, respectively. Other proteins of approximately 27-30 to > 80 kD have been visualized by immunologic and photoaffinity labeling techniques in different tissues and species. The specific antiserum that recognizes the approximately 70-, approximately 45-, and approximately 29-kD IR proteins, but not MAO, was used to quantitate these proteins in the rat brain cortex. Treatments (7 days) with the I2-selective imidazoline drugs idazoxan (10 mg/kg), cirazoline (1 mg/kg), and LSL 60101 ([2-(2-benzofuranyl) imidazole; 10 mg/kg]) induced differential changes in these proteins: levels of the approximately 29-kD IR were increased by idazoxan and LSL 60101 (23%), levels of the approximately 45-kD protein only by cirazoline (44%), and those of the approximately 66-kD protein only by idazoxan (50%). These treatments also increased the densities of [3H]-idazoxan (I2) binding sites (32-42%). Chronic treatment with efaroxan, RX821002, and yohimbine (10 mg/kg), which possess very low affinity for I2-IRs, did not alter either their immunoreactivities or the density of I2 sites. Chronic treatment with MAO inhibitors clorgyline and phenelzine (10 mg/kg) and acute treatment with EEDQ (1.6 mg/kg, 6 h) induced decreases in the levels of these IR proteins (17-47%) and I2 sites (31-57%). Significant correlations were found when the mean percentage changes in immunoreactivity of IR proteins were related to the mean percentage changes in the density of I2 sites after treatment with the foregoing drug (r = 0.92, r = 0.69, and r = 0.75 for the approximately 29-, approximately 45-, and approximately 66-kD proteins, respectively). These results indicate that in the rat cerebral cortex, the I2 sites labeled by [3H]idazoxan are heterogeneous and that the related immunoreactive IR proteins contribute differently to the modulation of I2 sites after drug treatment.

I2-binding sites (I2-BS) are attributed to be a regulative site on monoamine oxidase (MAO). The in vivo and in vitro effects of various imidazoline and guanidine derivatives on MAO activity and on mitochondrial respiration were studied. Substances with high affinity for I2-BS (antazoline, idazoxan, and cirazoline: IC50 = 20.3, 33.8, and 43.4 microM) had a stronger inhibitory effect on MAO activity than did I1-ligands (efaroxan, rilmenidine, clonidine, and moxonidine: IC50 = 277, 801, 1,224, and > 10,000 microM). Substances with the highest inhibitory effects were BDF8082 (IC50 = 1.7 microM) and 2-(2-benzofuranyl)-2-imidazoline (BFI; IC50 = 4.0 microM). The enzyme is inhibited noncompetitively and is reversible, because its activity is completely or partially restored after dialysis. Agmatine, the putative endogenous ligand for IBS, also decreased MAO activity (IC50 = 168 microM), whereas its precursor, L-arginine, and its metabolite, putrescine, had no effects. In vitro inhibition of MAO and mitochondrial respiration by the IBS-ligands tested could not be correlated, suggesting no link between the function of the inner and outer mitochondrial membrane. MAO activity in vivo was significantly reduced only by pargyline (-95%), BDF8082 (-68%), BFI (-43%), and 1-(m-chlorophenyl)-biguanide (-28%). Catecholamine content of livers obtained from animals treated with different IBS-ligands was consequently increased. In conclusion, the strong inhibitory effects of I2 selective imidazoline ligands confirm the existence of I2-BS as a regulatory site on MAO.

Presynaptic, release-inhibiting imidazoline receptors have hitherto been detected mainly under conditions of ongoing alpha 2-autoinhibition. We tried to find them under alpha2-autoinhibition-free conditions, in the majority of experiments in the rabbit pulmonary artery but in a few experiments also in rabbit atria, rabbit brain cortex and rat brain cortex. Tissue segments were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Ten compounds, some thought to inhibit noradrenaline release through activation of presynaptic imidazoline receptors, some thought to act through alpha 2-adrenoceptors, were tested. Rauwolscine and 6-chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine (SKF 86466) were used as antagonists. In rabbit pulmonary artery segments stimulated by trains of 20 pulses/50 Hz (alpha 2-autoinhibition-free conditions), all ten "agonists" [medetomidine, aganodine, 4-chloro-2-guanidyl-isoindoline (BDF 7579), 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143), 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), alpha-methylnoradrenaline, clonidine, moxonidine, cirazoline and idazoxan] reduced the stimulation-evoked overflow of tritium, with potency decreasing in that order and with maximal inhibition by 59% (idazoxan) to 95% (moxonidine). Rauwolscine competitively antagonized the effects of all ten "agonists" with similar potency, the pKd-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pKd 7.6). Cirazoline was also studied in the other three tissues. In rabbit atrial segments stimulated by 20 pulses/50 Hz and rabbit brain cortex slices stimulated by 4 pulses/100 Hz (both autoinhibition-free), cirazoline reduced the evoked overflow of tritium with concentration-inhibition curves similar to the curve in the pulmonary artery. In the brain cortex, the pKd-value of rauwolscine against cirazoline was 7.7 (pulmonary artery: 7.6). In rat brain cortex slices stimulated by 3 pulses/100 Hz (autoinhibition-free), cirazoline failed to change the evoked overflow of tritium but antagonized the inhibitory effect of UK 14304, pKd of cirazoline against UK 14304 6.9. In rat brain cortex slices stimulated by trains of 36 pulses/3 Hz, finally (marked alpha 2-autoinhibition), cirazoline increased the evoked overflow of tritium. In the rabbit pulmonary artery, rauwolscine and SKF 86466 acted with the same potency against typical presynaptic imidazoline receptor agonists (such as clonidine) and typical alpha 2-adrenoceptor agonists (such as moxonidine). Hence, presynaptic imidazoline receptors were not detectable, in a tissue that is prototypical for presynaptic imidazoline receptors, in the absence of alpha 2-autoinhibition, i.e. under conditions usually thought to be optimal for presynaptic receptor characterization. The pKd-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the alpha 2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an alpha 2(A)-autoreceptor agonist in rabbit tissues but as an alpha 2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) alpha 2A-adrenoceptors than the orthologous (rodent) alpha 2D-adrenoceptors.

The role of the alpha-1 adrenoceptor in prepulse inhibition (PPI) was evaluated in rats. The alpha-1 adrenergic agonist cirazoline disrupted PPI; this effect was reversed by the alpha-1 adrenergic antagonist prazosin or the atypical antipsychotic quetiapine. Alpha-1 adrenoceptors may thus be involved in the regulation of PPI and in the psychotherapeutic actions of certain antipsychotics.

The mechanism of alpha1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the alpha1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 microM. The contractile response to a submaximal concentration of cirazoline (0.3 microM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 microM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22+/-0.06 mol of Pi/mol of LC20 (n=3) and reached a maximum of 0.58+/-0.08 mol of Pi/mol of LC20 (n=3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by re-addition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 microM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 microM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 microM cirazoline were partially inhibited by chloroethylclonidine (50 microM), suggesting the involvement of both alpha1A and alpha1B adrenoceptors in these contractile responses.

The alpha-1 adrenergic agonist, cirazoline, was examined for effects on spatial working memory performance in aged rhesus monkeys. Cirazoline has additional high affinity for imidazoline receptors and has good brain penetrance when administered systemically. Spatial working memory was assessed using the variable delayed response task, a test dependent upon prefrontal cortical function in monkeys. Low doses of cirazoline (0.00001-0.001 mg/kg) impaired delayed response performance significantly. This impairment did not appear to result from nonspecific changes in behavior, because cirazoline had no significant effect on performance of control trials where the delay was "0" s, and had no significant effect on behavioral ratings. Impairment was reversed by pretreatment with the alpha-1 adrenergic antagonist, prazosin, consistent with drug actions at alpha-1 adrenergic receptors. In contrast, preliminary data suggest that higher cirazoline doses (0.001-0.01 mg/kg) occasionally produced improved performance that was not reversed by prazosin, but rather, by the imidazoline/alpha-2 adrenergic antagonist, idazoxan. The finding that alpha-1 adrenergic receptor stimulation impairs spatial working memory performance complements previous research demonstrating that alpha-2 adrenergic receptor stimulation improves working memory, and suggests that norepinephrine may have opposing actions at alpha-1 vs. alpha-2 receptors in the prefrontal cortex as it does in the hypothalamus and thalamus.

The isolated perfused rat mesenteric bed releases endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine (ACh) or histamine. I propose that EDHF released in the mesenteric vascular bed is a cytochrome P450 (CYP)-linked, arachidonate metabolite. In the presence of nitro-L-arginine methyl ester (L-NAME) and indomethacin, injections of ACh (0.001 to 10 nmol) or histamine (0.1 to 1,000 nmol) elicited transient, dose-dependent dilation of cirazoline (an alpha1-adrenoceptor selective agonist) preconstricted mesenteric beds. The L-NAME-resistant responses to ACh or histamine were insensitive to tetrodotoxin (1 micromol/L), thus negating its neuronal origin, but were profoundly attenuated by a K+ channel inhibitor tetrabutylammonium (0.5 mmol/L). 7-Ethoxyresorufin (a selective and competitive blocker of CYP 1A isozyme) blunted ACh and histamine mediated EDHF responses but did not alter vasodilation initiated through K+ channel activation by either cromakalim or NS-1619, or through the nitric oxide-cGMP pathway (sodium nitroprusside). Clotrimazole, an imidazole that inhibits CYP by binding to the heme moiety, attenuated ACh, histamine, and cromakalim but not sodium nitroprusside-mediated vasodilator responses. Other CYP isozyme selective inhibitors, such as metyrapone (CYP 2B), 7-pentoxyresorufin (CYP 2B1), sulfaphenazole (CYP 2C/3A), and 17-octadecynoic acid (4A-linked omega-hydroxylase inhibitor), did not alter ACh or histamine-induced EDHF response. The EDHF-mediated dilations initiated by ACh and histamine, as well as K(ATP) activation by cromakalim, were blocked by mepacrine, a nonselective phospholipase A2 inhibitor. Mepacrine did not alter K(Ca) activation by compound NS-1619. I conclude that 1) the isolated perfused rat mesenteric prearteriolar bed releases in response to ACh and histamine, an EDHF that causes vasodilation through K+ channel activation; 2) the EDHF is most likely a CYP-derived arachidonate product; 3) CYP 1A is well suited as the isozyme responsible for EDHF production in this vascular bed; and 4) PLA2 appears to mediate the release of the precursor arachidonic acid.

[ 3 H]RX821002, [ 3 H]clonidine and [ 3 H]idazoxan have previously been shown to selectively label α 2 -adrenergic receptors, I 1 and I 2 imidazoline receptors in the human central nervous system, respectively. Idazoxan shows relatively high affinity for all three receptors. We investigated the possible selectivity of several compounds towards one of those receptors in human striatum. Addition of an alkoxy group at the 2-position of the benzodioxan moiety of idazoxan (ethoxy-idazoxan, methoxy-idazoxan) increases the α 2 -selectivity in human brain. Efaroxan is also α 2 -selective. On the contrary, BU224, BU239, cirazoline and RX801077 display imidazoline receptor selectivity. Our results indicate that for all molecules tested, idazoxan and ‘flat’ analogs possess I 1 I 2 receptor selectivity. A ‘bulky’ substituent at the 2-position of the benzodioxan ring gives rise to α 2 -adrenergic receptor selectivity. Until now, we found no more than 3-fold difference in IC 50 between both imidazoline receptors. Both receptors also display similar stereoselectivity, suggesting that they might be ‘interconnected’ in the human striatum.

Recently, a new imidazoline (I 2 ) ligand, [ 3 H]2-(2-benzofuranyl)-2-imidazoline (BFI) was shown to be more selective for I 2 vs α 2 binding in rodent brain. We characterized [ 3 H]BFI binding in human brain cortex and lateral reticular nucleus (NRL). Membranes were incubated with [ 3 H]BFI at 22 °C in 50 mM Tris, 1.5 mM EDTA at pH 7.5. Saturation experiments with [ 3 H]BFI (0.5–80 nM) were analyzed using non-linear curve fitting. The NRL had 4X more binding sites than cortex with similar affinity (B max = 2085 ± 732 and 471 ± 41 fmol mg protein; K D = 9.3 ± 3.5 and 11.9 ± 2.7 nM, respectively). In competition studies, cortical [ 3 H]BFI binding was displaced in order of decreasing potency by clorgyline > BFI ≥ cirazoline > idazoxan ≥ guanabenz > clonidine > RX821002. The monoamine oxidase (MAO) inhibitor tranylcypromine (TCP) (1 nM-10 μM), markedly enhanced [ 3 H]BFI binding in both NRL and cortex. Enhanced binding was maximal at 300 nM (12 X control) and returned to baseline at 30 μM. Potentiation was not seen with pargyline or clorgyline. TCP did not effect [ 3 H]BFI binding in rat cortex, or [ 3 H]idazoxan binding in human cortex and NRL. In human cortex, inhibition of MAO by preincubation with pargyline (10 μM) abolished the TCP effect. Upon preincubation with TCP, the stimulation of [ 3 H]BFI binding was dose-dependently related to a simultaneous inhibition of MAO. Thus, [ 3 H]BFI labels a site in human NRL and cortex that appears similar to the previously described I 2 site labeled by [ 3 H]idazoxan. However, [ 3 H]BFI binding is dramatically stimulated by TCP in human brain via a mechanism dependent on endogenous MAO activity.

The present study was carried out to clarify whether the imidazolines clonidine, moxonidine and cirazoline as well as the guanidine aganodine inhibit noradrenaline release in the rat and rabbit brain via imidazoline receptors, alpha 2-adrenoceptors and/or histamine H3 receptors. Slices or synaptosomes from the rat or the rabbit brain were incubated with 3H-noradrenaline and exposed to phenoxybenzamine, which irreversibly blocks presynaptic alpha 2-adrenoceptors and, at considerably lower potency, imidazoline receptors. Tritium overflow in the superfused preparations was evoked electrically (3 Hz; slices) or by K+ 15 mmol/l (synaptosomes). Noradrenaline and rauwolscine, which possess low affinity, if any, for imidazoline receptors, were used as reference drugs. The evoked overflow in rat brain cortex slices and synaptosomes and in rat medulla oblongata slices, not exposed to phenoxybenzamine, was inhibited by clonidine, moxonidine and noradrenaline. Phenoxybenzamine markedly attenuated the effect of each drug to about the same extent. In rabbit brain cortex slices, not exposed to phenoxybenzamine, the evoked overflow was inhibited by clonidine, moxonidine, aganodine and noradrenaline, facilitated by BDF 6143 (4-chloro-2-(2-imidazoline-2-yl-amino)-isoindoline), idazoxan and rauwolscine and not affected by cirazoline. In slices exposed to phenoxybenzamine, the inhibitory effects of the imidazolines, of aganodine and of noradrenaline were again attenuated by about the same high degree, the facilitatory effects of BDF 6143, idazoxan and rauwolscine were abolished and cirazoline produced a slight inhibition of the evoked overflow. The latter effect was not affected by high concentrations of rauwolscine and idazoxan (at which these drugs act antagonistic at imidazoline receptors in other models). The specific binding of 3H-N alpha-methylhistamine to H3 receptors in rat brain cortex membranes was displaced only by high concentrations of moxonidine (pKi = 6.16) and at even lower affinity by aganodine, BDF 6143, cirazoline, clonidine and idazoxan (pKi5). Histamine, which was used as a reference drug, proved to be very potent (pKi = 8.20). In conclusion, imidazolines affect noradrenaline release in the rat and rabbit brain cortex and medulla oblongata via alpha 2-adrenoceptors but not via imidazoline receptors resembling the presynaptic imidazoline receptors previously identified in peripheral tissues of the rabbit. In addition, the involvement of I1- or I2-imidazoline binding sites or of H3 receptors is very improbable in view of the low affinity of aganodine, moxonidine and/or clonidine for these recognition sites and/or incompatibility of the rank order of their affinities with the potencies of the drugs in inhibiting noradrenaline release.

Publisher Summary Compounds such as the α 1 -adrenergic receptor (AR) agonist/ α 2 -AR antagonist cirazoline, the α 2 -AR antagonist idazoxan, the α 2 -AR agonist guanabenz, the ion transport inhibitor amiloride, and other structurally related ligands that have an imidazoline or guanidinium moiety elicit a number of pharmacological effects. Although such ligands interact with known receptor systems, some of their functional effects are pharmacologically illdefined, such as their centrally mediated effects on blood pressure and the ability of certain imidazoline derivatives to augment glucose-induced insulin secretion from pancreatic β cells. Indeed several studies indicate that these molecules interact with distinct membrane-bound proteins variously termed imidazoline/ guanidinium receptive sites (IGRS), imidazoline binding proteins, I 1 and I 2 receptors, imidazoline receptors, nonadrenergic imidazoline binding sites, or imidazole receptors. These sites share the common property of not recognizing endogenous agonists for known monoamine receptors, but subgroups of these binding sites differ in their ligand recognition properties in various tissues. The synthetic ligands recognized by imidazoline binding proteins elicit several cell responses; however, the signaling pathways involved and the relative importance of these proteins in these events are unclear.

The observation that all the attempts to characterize imidazoline (I) receptors have been carried out with non-selective or poorly selective ligands prompted us to undertaken research aimed at developing selective ligand(s). In previous work using, as a starting point, cirazoline I, a potent alpha 1-adrenergic receptor agonist that also binds to I receptors, we showed that removal of the cyclopropyl ring (2) retains high affinity for I2 receptors while reducing alpha 1-adrenergic agonist activity. However, it was felt that this residual, albeit modest, alpha 1-adrenergic agonist activity might diminish the usefulness of compound 2, and we now report on our continuing efforts in this field. Starting from compound 2, we first eliminated the alpha 1-agonist component by isosteric replacement and then, by means of conformational restrictions on compound 7, succeeded in discovering tracizoline (9) and benazoline (12). These two new ligands with high affinity (pKi value 8.74 and 9.07, respectively) and unprecedented selectivity with respect to both alpha 2- (I2/alpha 2 7,762 and 18,621) and alpha 1- (I2/alpha 1 2,344 and 2,691) adrenergic receptors, are valuable tools in the study of I receptor structure and function. In addition, the large number of derivatives studied has allowed us to establish congruent qualitative structure-activity relationships and identify some structural elements governing affinity and selectivity.

The lack of an effective analgesic treatment makes pain a clinical challenge and the need of a novel approach to identify new agents is urgent. In this scenario I2-ligands can be considered an alternative strategy in pain therapy. The development of an ex vivo model useful for the evaluation of functional activities at both a2 and I2-IBs (imidazoline binding sites) is an important task in pharmacological sciences since several I2 ligands display activity also towards a receptors. The present study aims to develop an ex vivo model for estimating the activity of I2-IBs ligands in a biological sample where a1 and a2 adrenergic receptors are present. For this purpose the imidalzoline endogenous ligand, harmane, reference compounds, 2BFI and BU224, and imidazoline derivatives 1-3 have been selected taking into account their in vitro activity towards IBs and adrenergic receptors. All compounds have been tested ex vivo in guinea pig-ileum where a2A-ARs are prejunctionally and I2-IBS postjunctionally localized. Adrenergic component has been identified by the studying the interference of compounds on the electrically-evoked contraction while I2-IBs activity by testing the ability of compounds to inhibit the carbachol-evoked contractions in the presence of prazosin to mask the a1 adrenoceptors. Compounds 1 and 2 were found I2-IBs antago nists (pIC50=4.2 and 4.0, respectively) whereas compound 3 was I2-IBs agonist (EC50=0.38 mM); All ligands were a2 adrenergic agonists. This paper suggests guinea-pig ileum as the first ex vivo approach for establishing both the intrinsic activity of I2-IBs ligands and the physiological correlation between IBs and adrenergic system.

1. Discrete, non-adrenergic binding sites for imidazolines have been characterized in the brain and periphery. The I1 clonidine-preferring site is mainly distributed in the brain and brain stem, while the I2 idazoxan-preferring site is more widely distributed. 2. The I1 site appears to be associated with modulation of blood pressure. Imidazolines act within the rostral ventrolateral medulla to produce hypotension. The underlying signal transduction mechanism is poorly understood. 3. The imidazolines clonidine and cirazoline inhibited nicotine-stimulated calcium entry into rat phaeochromocytoma (PC-12) cells by a non-adrenergic mechanism. This effect was not attributable to the stimulation of protein kinases. 4. Similarly, clonidine and cirazoline inhibited nicotine-stimulated inward currents into PC-12 cells. This inhibitory action was not altered by inhibitors of signal transducing G-proteins. 5. Clonidine and cirazoline displaced the ion channel ligand [3H]-phencyclidine from nicotinic acetylcholine receptors, suggesting that these drugs act by direct blockade of the intrinsic ion channel of the nicotinic acetylcholine receptor. 6. This ion channel-blocking activity represents a novel action of these imidazolines and may underlie some of the proposed physiological actions of I1 sites.

alpha 2-Adrenoceptor agonists inhibit the firing of locus coeruleus (LC) neurons. It was recently observed that the alpha-adrenoceptor agonists clonidine, rilmenidine and cirazoline, when injected intravenously in anaesthetized rats pretreated with the irreversible alpha 2-adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), excite the LC. The effect was attributed to activation of I1 imidazoline receptors. The aim of the present experiments was to characterize the direct effect of alpha 2-adrenoceptor and I1 imidazoline receptor agonists on LC neurons. Electrical activity of LC neurons was extracellularly recorded in midpontine slices prepared from the rat brain. Concentration-response curves were obtained for the alpha 2-agonist noradrenaline and the mixed I1/alpha 2-receptor agonists clonidine, rilmenidine and moxonidine in slices without treatment and in slices treated with 6-chloro-N-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF86466) or EEDQ, alpha 2-adrenoceptor antagonists with low affinity for I1 and I2 imidazoline receptors, respectively. All four agonists concentration-dependently reduced the firing rate of the neurons, with full inhibition at higher concentrations. SKF86466 shifted the concentration-response curves of the agonists to the right; the calculated antagonist dissociation constants are compatible with an effect of the agonists on alpha 2-adrenoceptors. EEDQ completely prevented the inhibition by the agonists. Neither in SKF86466- nor in EEDQ-treated slices was an excitation by clonidine, rilmenidine and moxonidine observed. We conclude that the LC neurons do not possess functional I1 (and also no I2) imidazoline receptors. The effects of noradrenaline, clonidine, rilmenidine and moxonidine on the neurons can be fully explained with an interaction with inhibitory alpha 2-adrenoceptors (probably of the alpha 2D subtype). The excitation of the LC by imidazoline receptor agonists under in vivo conditions, hence, is not a direct effect on the neurons of the LC.

OBJECTIVE Both intracellular and extracellular sources of calcium are involved in the activation of contraction in vascular smooth muscle. In the diabetic or hypertensive state, or both, changes induced in calcium handling by various types of agonists may vary considerably. METHODS We investigated in which manner L-type calcium-channel blockade with nifedipine influences the pressor effects of the alpha 1-adrenoceptor agonists cirazoline and ST 587, the alpha 2-adrenoceptor agonist UK 14.304 and angiotensin II, all exerting a differential influence on calcium homeostasis, in pithed spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats rendered diabetic by an injection of 55 mg/kg streptozotocin. RESULTS In diabetic WKY rats and SHR, the maximal pressor response was impaired for all agonists. The hypertensive state enhanced the maximal pressor response to all agonists. No difference was found in the nifedipine-induced depression of the pressor response to cirazoline and angiotensin II in the four groups of rats. The maximal pressor responses to ST 587 and UK 14.304 were more effectively depressed by administration of 0.3 mg/kg nifedipine both in diabetic WKY rats and in diabetic SHR than they were in their non-diabetic controls. CONCLUSIONS Hypertension was associated with enhanced pressor response, whereas the diabetic state counteracted this effect. The pressor responses in pithed diabetic and diabetic hypertensive rats were clearly more dependent on the nifedipine-sensitive calcium influx than were those in their non-diabetic controls.

Hypertension is often present in the diabetic individual and it is known to aggravate the vascular complications associated with diabetes. The pressor responses to two alpha 1-adrenoceptor agonists (ST587 and cirazoline), two alpha 2-adrenoceptor agonists (azepexole dihydrochloride (B-HT933) and UK14.304) and angiotensin II were investigated in pithed spontaneously hypertensive rats (SHR) and in pithed normotensive Wistar Kyoto rats (WKY) made diabetic by a single i.v. injection (55 mg/kg) of streptozotocin (STZ). Two months after diabetes was induced, the effect of the agonists on basal diastolic blood pressure (DBP) was determined. In pithed diabetic WKY and SHR, the maximal pressor response was impaired for all agonists. The dose response curves were shifted to the right when compared with their non-diabetic controls. The hypertensive state enhanced the maximal pressor response to all agonists compared with non-hypertensive animals. Additional diabetes blunted this increase in the effects of ST587, B-HT933 and angiotensin II, but not in those of cirazoline and UK14.304. Hypertension caused a leftward shift of the dose response curve for ST587 when compared with the non-hypertensive state. However, this effect was not observed when diabetes was present as well. In conclusion, hypertension resulted in an enhanced pressor effect, possibly caused by vascular hypertrophy, whereas the diabetic state counteracted this effect.

The profile of the contractile effects of calcium ions, the Ca(2+)-entry promoter Bay K 8644 and two alpha 1-adrenoceptor agonists (cirazoline and ST587) was studied in isolated perfused Langendorff hearts taken from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) with simultaneous diabetes. In both the hypertensive and the diabetic state the isolated hearts showed a tendency to an impaired inotropic response (increase in left ventricular pressure (LVP)) towards an increase of extracellular calcium ions. The impaired inotropic response was most pronounced in hearts of rats with simultaneous diabetes and hypertension. The Ca(2+)-influx promoter Bay K 8644 did not influence the maximally developed LVP in (diabetic) SHR and WKY preparations. The inotropic responses to both cirazoline and ST587 were increased in hearts from diabetic WKY and diabetic SHR, when compared with those from control WKY and SHR. Hypertension, however, blunted the inotropic response to ST587 but not that to cirazoline when compared to hearts from control WKY or diabetic WKY. The present study indicates that the combination of hypertension and diabetes leads to progressive cardiac deterioration, likely to be the result of important changes in calcium homeostasis.

1. To investigate the structure-activity relationships of alpha-adrenoceptor agonists for the alpha 1-adrenoceptor subtypes, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a-, alpha 1b-, and alpha 1d-subtypes). 2. Radioligand binding studies with [125I]-HEAT showed that cirazoline and oxymetazoline had higher affinities at alpha 1a-subtype than at alpha 1b- and alpha 1d-subtypes, while noradrenaline had higher affinity at the alpha 1d-subtype than at alpha 1a- and alpha 1b-subtypes. 3. In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+]i response) in a concentration-dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the alpha 1a-subtype, while it acted as a partial agonist at alpha 1b- and alpha 1d-adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at alpha 1a-adrenoceptors, and has no intrinsic activity at the other subtypes. 4. Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline-induced [Ca2+]i response for alpha 1a- and alpha 1d-subtypes were found to be linear, whereas it was moderately hyperbolic for the alpha 1b-subtype, indicating the absence of receptor reserves in CHO cells expressing alpha 1a- and alpha 1d-subtypes while there exists a small receptor reserve for CHO cells expressing the alpha 1b-subtype. 5 In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of alphal-adrenoceptor.

Clonidine and other imidazoline/oxazoline drugs, such as cirazoline and rilmenidine, have been shown to stimulate the activity of noradrenergic neurones in the locus coeruleus (NA-LC) by an alpha 2-adrenoceptor-independent mechanism through the activation of I-imidazoline receptors. The endogenous modulation of the stimulatory effect of clonidine on NA-LC neurones was further investigated after inactivation of alpha 2-adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). In EEDQ-pretreated rats (6 mg/kg, i.p., 6 h), clonidine caused a rapid and dose-dependent (320-5120 micrograms/kg, i.v.) increase in the firing rate of NA-LC neurones (ED50 = 809 micrograms/kg, Emax = 90%). The stimulatory effect of clonidine on NA-LC neurones was completely blocked by pretreatment of rats with the excitatory amino acid receptor antagonist kynurenic acid (1-3 mumol in 10-30 microliters, i.c.v., 2-5 min before clonidine). In contrast, the stimulatory effect of clonidine on NA-LC neurones was potentiated by pretreatment with reserpine (5 mg/kg, s.c., 18 h) (Emax increased by 63%). Pretreatment with alpha-methyl-p-tyrosine (250 mg/kg, i.p., 24 h) did not alter the stimulatory effect of clonidine, but pretreatment with p-chloro-phenylalanine (400 mg/kg, i.p., 24 h) markedly enhanced the stimulatory effect of clonidine on NA-LC neurones (Emax increased by 139%). The present results indicate that the imidazoline receptor-mediated stimulatory effect of clonidine on NA-LC neurones is an indirect effect dependent on an excitatory amino acid pathway and modulated by an inhibitory serotonin mechanism.

Clonidine and cirazoline bind with high affinity to a nonadrenergic site in the brain stem, the so-called imidazoline I1 receptor. Our aim was to determine the mechanism by which these receptors act and their possible linkage to signal-transducing heterotrimeric G-proteins. We examined the effects of clonidine and cirazoline on PC-12 cells, a neuronal cell line that is reported to possess the I1 site and have no alpha 2-adrenoceptors. In undifferentiated PC-12 cells loaded with the Ca2+ indicator dye fura-2, clonidine and cirazoline (10-100 microM) inhibited the increase in [Ca2+]i produced by nicotine (10 microM). This inhibition was not reversed by yohimbine (100 microM), and adrenaline and BHT 920 were ineffective at 100 microM. This effect was not inhibited by pretreatment with pertussis toxin (24 hours, 100 ng/ml) and not modulated by pretreatment with IBMX (100 microM). The nicotine-induced increase in [Ca2+]i is apparently due to Ca2+ entering via the intrinsic ion channel of the nicotinic acetylcholine receptor. Clonidine and cirazoline inhibited the inward current produced by nicotine (10 microM) as measured by the whole cell patch-clamp technique in differentiated PC-12 cells, recorded at a holding potential of -60 mV. In agreement with the results found with fura-2, inhibition of inward current was concentration dependent and not blocked by yohimbine (100 microM) or mimicked by adrenaline (100 microM). Pretreatment of PC-12 cells with pertussis toxin or infusion of GDP-beta-S (2 mM) via the patch pipette did not alter the inhibition of the nicotine-induced inward current by clonidine or cirazoline. Clonidine and cirazoline, but not adrenaline, displayed [3H]phencyclidine from Torpedo electroplaque membranes enriched in nicotinic acetylcholine receptors in a concentration-dependent manner (10-100 microM). Taken together, these results suggest that clonidine and cirazoline inhibit Na+ and Ca2+ entry through the nicotinic acetylcholine receptor via a nonadrenergic mechanism that is independent of G-proteins and cyclic nucleotides, presumably by direct blockade of the intrinsic ion channel of the nicotinic acetylcholine receptor.

The effects of the nonpeptide angiotensin II receptor antagonist losartan and the angiotensin-converting enzyme inhibitor captopril on pressor responses to the selective alpha 1-adrenoceptor agonist cirazoline (10 ng/kg-3.0 mg/kg) in the pithed rat were compared. In addition, the effects of losartan and captopril on pressor responses to cirazoline were compared in the presence of the selective irreversible alpha 1-adrenoceptor antagonist SZL-49 (1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo[2,2,2]octa-2,5- dienyl-carbonyl)-piperzine) and/or the Ca2+ channel antagonist nifedipine. Losartan (5.0 mg/kg) and captopril (3.0 mg/kg), as compared to saline, significantly lowered the blood pressure of intact, anaesthetized and pithed rats. Continuous infusion with vasopressin was used to restore the blood pressure of pithed rats pretreated with losartan or captopril to a level comparable to animals that had received saline. Losartan, captopril, nifedipine (1.0 mg/kg), and SZL-49 (10.0 mg/kg) antagonized the pressor actions of cirazoline, which displaced the dose-diastolic blood pressure response curve for the agonist to the right. Moreover, pressor responses to cirazoline were significantly reduced in rats that had received losartan and nifedipine in comparison to nifedipine alone. In contrast, in rats treated with nifedipine, further administration of captopril did not significantly reduce pressor responses to cirazoline as compared to nifedipine alone. Cirazoline-mediated pressor responses at all doses were significantly attenuated in rats treated with SZL-49 and either losartan or nifedipine combined as compared to SZL-49 alone. In contrast, only cirazoline-mediated pressor responses at lower doses were significantly reduced by pretreatment with a combination of SZL-49 and captopril as compared to SZL-49 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

We have identified and characterized non-adrenergic [3H]clonidine binding sites in rat stomach. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 68% specific binding at [3H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to rat stomach membranes was concentration-dependently inhibited by various imidazolines and guanidines including the sigma site ligand 1,2-di-(2-tolyl)guanidine (DTG), by the butyrophenone derivative haloperidol and by the piperidine derivative (+)-3-PPP[(R)-3-(3-hydroxyphenyl)-N-propylpiperidine]; the latter two compounds are also known to exhibit affinity for sigma sites. In contrast, rauwolscine, histamine, ranitidine and the non-hydrolysable GTP-analogue Gpp(NH)p (5′ guanylylimidodiphosphate) did not, or with negligible affinity, inhibit [3H]clonidine binding. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for a single detectable site) was as follows: cirazoline>idazoxan≥DTG>(+)-3-PPP> clonidine>guanabenz>haloperidol. This rank order is not compatible with the pharmacological properties of either I1- or I2-imidazoline binding sites. However, the ability of haloperidol, (+)-3-PPP and DTG to displace [3H]clonidine (the latter two with high affinity) suggests that the [3H] clonidine binding sites in rat stomach may be related to sigma-like sites.

The affinity of AMPI (2-[3-aminophenoxy]methyl imidazoline) for [3H]clonidine and [3H]idazoxan imidazoline binding sites was determined in various rabbit and human tissues. Although cirazoline showed a high affinity (nM range) in all the tested tissues, its derivative, AMPI, had a high affinity (nM range) in rabbit brain and kidney but a low affinity (μM range) in the human brain. These differences in affinities were very similar to those obtained with amiloride. The same results were obtained when considering [3H]clonodine or [3H]idazoxan specific imidazoline binding sites.

Bovine adrenal chromaffin cells, which have been shown to lack alpha 2-adrenoceptors, were used to investigate the pharmacological characteristics of [3H]idazoxan binding sites. The binding of [3H]idazoxan was very rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 50% specific binding at [3H]idazoxan 10 nmol/l), saturable and of high affinity (KD 13nmol/l) and, hence, was compatible with the criteria for the identification of an imidazoline binding site (IBS). Since in competition experiments rauwolscine and (-)-adrenaline showed only negligible affinity for these adrenal medullary binding sites, the lack of alpha 2-adrenoceptors was confirmed. Histamine and amiloride also did not inhibit [3H]idazoxan binding or caused only negligible inhibition. In contrast, the specific binding of [3H]idazoxan was concentration-dependently inhibited by several imidazolines and guanidines with the following rank order of potency which conforms to the characteristics of the previously defined I2-IBS (in parentheses: Ki, nmol/l): idazoxan (4) = cirazoline (4)clonidine (272) = BDF 6143 (299; 4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline)BDF 6100 (563; 2-(2-imidazolin-2-yl-amino)-isoindoline)or = BDF 7579 (868; (4-chloro-2-isoindolinyl)guanidine)phentolamine (1424) = naphazoline (1451). Equilibrium [3H]idazoxan binding was reduced by K+ but not by Na+ or the non-hydrolysable GTP-analogue Gpp(NH)p (5'-guanylylimidodiphosphate; 100 mumol/l). In conclusion, membranes of the bovine adrenal medulla are endowed with non-adrenergic high-affinity [3H]idazoxan sites which exhibit the pharmacological properties of the amiloride-insensitive subtype of I2-IBS and probably are not coupled to a G-protein.

1. The vasoactive mechanisms of bile salts have been investigated in rat isolated portal venous and superior mesenteric arterial rings and perfused mesentery. 2. The isolated perfused mesentery was precontracted with a selective alpha 1-adrenoceptor agonist, cirazoline. Incremental doses of tauroursodeoxycholate (TUDC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC) caused a dose-dependent vasorelaxation. The order of potency of the vasodilator effect was TDC > TCDC > TUDC. 3. The effect of TDC (1.9 x 10(-8)-1.9 x 10(-6) mol) was examined before and after propranolol (3 microM), tetraethylammonium (5 mM), ouabain (10(-5) M), NG-nitro-L-arginine methyl ester (10(-4) M) and capsaicin (50 mg kg-1) to block, respectively, beta-adrenoceptors, K+ -channels, Na+, K+-ATPase, nitric oxide synthase, and primary sensory nerves. The vasodilator effect of TDC was not affected by any of these blocking agents or by denuding vascular endothelium with distilled water. 4. Infusion of TDC (1.9 x 10(-8)-1.9 x 10(-6) mol) with K+-free or high K+ (60 mM) physiological salt solution (PSS) did not affect the vasodilator effect of TDC. 5. Contractions induced by KCl (0.01-1.0 M), arginine vasopressin (AVP, 10(-10)-10(-7) M) or cirazoline (10(-7) x 10(-5) M) were all inhibited by TDC (300 microM). 6. TDC (10(-6) to 10(-3) M) also inhibited the basal tension and the development of spontaneous contractions in the isolated portal vein. 7. TDC (300 microM), however, did not affect noradrenaline-induced phasic contractions elicited in Ca(2+)-free PSS by Ca2+ release from intracellular stores. 8. We conclude that TDC inhibits Ca2+ entry through both voltage-operated and receptor-operated calcium channels, whereas intracellular Ca2+ release is not affected.

This study examined the mechanism underlying the influence of clozapine upon serotoninergic transmission in the rat. In vitro, clozapine manifested weak affinity at 5-HT1A receptors (pKi = 6.5) as compared to the agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (9.0), but high affinity at alpha 1-adrenoceptors (8.2) as compared to the alpha 1-adrenoceptor antagonist, prazosin (9.7). Ex vivo, clozapine (inhibitory dose (ID)50 = 0.7 mg/kg s.c.) mimicked prazosin (0.5) in potently occupying central alpha 1-adrenoceptors whereas, as compared to 8-OH-DPAT (0.2), it failed to occupy 5-HT1A receptors (> 10.0). The firing of serotoninergic neurones in the dorsal raphe nucleus was abolished by 8-OH-DPAT, clozapine and prazosin with ID50 values of 0.006, 0.09 and 0.07 mg/kg i.v., respectively. At comparable doses, they reduced striatal turnover of 5-HT. While the 5-HT1A receptors antagonists, (-)-tertatolol (2.0 mg/kg i.v.) and spiperone (0.63 mg/kg i.v.), blocked the action of 8-OH-DPAT upon dorsal raphe nucleus firing, they failed to modify the effect of clozapine and prazosin. In contrast, the alpha 1-adrenoceptor agonist, cirazoline (0.005 mg/kg i.v.) prevented the actions of clozapine and prazosin, but not that of 8-OH-DPAT. It is concluded that clozapine only weakly interacts with 5-HT1A receptors and that its potent alpha 1-adrenoceptor antagonist properties underlie inhibition of serotoninergic transmission.

IBI [2-(4'-isothiocyanatobenzyl)imidazoline, 3] has been shown to cause slow-onset, long-lasting contractions of rat thoracic aorta through a non-alpha-adrenergic receptor (non-alpha-AR) mediated mechanism. A series of IBI-related anlogs 7-14 and 16 was prepared to determine the structural requirements for the interaction with non-alpha-AR in rat aortic strips. All IBI analogs produced concentration-dependent contractile responses on rat thoracic aorta. Whereas the actions of analogs 7, 14, and 16 were partly mediated by alpha-ARs, the stimulatory activities of the remaining IBI analogs were unaffected by phenoxybenzamine pretreatment, suggesting that a non-alpha-adrenergic mechanism is involved. We have shown that the contractile actions of IBI and analogs 10-13 were not blocked with the imidazoline/guanidinium receptive site (IGRS) ligands idazoxan, cirazoline, or clonidine. However, the calcium channel blockers nifedipine or verapamil shifted the concentration-response curve of IBI and its analogs 10-13 to the right and reduced the maximal contractile responses. The action of IBI on rat thoracic aorta was reduced by the omission of extracellular calcium in the medium. These results suggest that the stimulatory activities of IBI and analogs 10-13 are not related to the activation of alpha-AR or IGRS receptors and are likely coupled to the voltage-dependent Ca2+ channels.

1. The purpose of this study was to characterize the effect of NG-nitro-L-arginine methyl ester (L-NAME) on the perfusion rate/pressure relations, and on the pressor responses induced to cirazoline and KCl in isolated, perfused mesenteric arterial beds from normotensive and spontaneously hypertensive rats. 2. The basal perfusion pressure of arterial beds perfused with either physiological salt solution (PSS) or PSS containing 1% polyvinylpyrrolidone increased as the perfusion rate increased. L-NAME, in concentrations up to 100 microM, failed to alter the basal pressure regardless of the perfusion rate and viscosity; however, at 5 microM, it potentiated cirazoline-induced vasoconstriction at each of the perfusion rates. 3. L-NAME but not D-NAME caused a leftward shift of cirazoline concentration-response curves with a marked increase in the maximal response. The potentiating action of L-NAME was abolished in arterial beds perfused with a Ca(2+)-free physiological salt solution and also in beds denuded of endothelium by an infusion of distilled water for 5 min. 4. In endothelium-intact and -denuded preparations, L-NAME potentiated KCl pressor responses; the endothelium-independent potentiation of KCl pressor activity was stereospecific, time-independent and was not prevented by the presence of dexamethasone (0.5 microM) in the perfusion medium. However, L-NAME failed to potentiate vasoconstriction obtained to KCl in arterial beds denervated by cold storage (4-5 degrees C) for 2 days. 5. The absence of K+ in the perfusate did not inhibit the ability of L-NAME to potentiate alpha-adrenoceptor-mediated pressor responses, and nor did L-NAME inhibit KCl-induced vasodilatation in preconstricted arteries. It was thus concluded that L-NAME does not affect Na+/K(+)-ATPase activity. 6. No differences in the potentiating ability of L-NAME on either cirazoline- or KCl-mediated pressor responses were apparent between normotensive Sprague Dawley (SD), Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats.7. Our data thus provide evidence that: the presence of a vasoconstrictor is required for basal nitricoxide (NO) release in the mesenteric arterial bed from either normotensive or spontaneously hypertensive rats; L-NAME causes potentiation of cirazoline- and KCl-induced vasoconstriction respectively by inhibiting endothelial and neuronal NO synthase(s). Furthermore, our data indicate that NO synthase activity is not impaired in the mesenteric arterial bed of spontaneously hypertensive rats.

We investigated the influence of high-glucose levels (30.5 mmol/L) and/or hyperosmolarity on pharmacological responses in aortic ring preparations taken from nondiabetic rats. Moderate changes were observed for the concentration-response curves of noradrenaline and phenylephrine, but not for methoxamine and cirazoline. Maximal active forces of the concentration-response curves of potassium chloride and calcium chloride were significantly reduced by the elevated glucose levels per se , but neither the slopes of the curves nor the -logEC 50 values were affected. Concentration-response curves ofserotonin and U 46619 were not affected. For angiotensin II, the −logEC 50 values and maximal active forces of the concentration-response curves were significantly lower under both hyperglycemic and hyperosmolar conditions. The present study suggests that potential changes in contractile behavior in isolated vessels from diabetic animals cannot be attributed to high glucose levels and/or hyperosmolarity as such, but indeed reflect vascular changes associated with the diabetic state. An exception has to be made for the depolarization of aortic ring preparations of nondiabetic rat; the elevated glucose level leads to an impaired calcium influx via the (slow) L-type calcium channels.

The α1- and α2-adrenoceptor-stimulated contractile responses of rat tail artery rings were compared in Sprague–Dawley (SD), spontaneously hypertensive (SHR), and Wistar–Kyoto (WKY) rats that were untreated, treated with pertussis toxin, or treated with cholera toxin. The maximal responses, expressed as milligrams of tension, induced by clonidine (an α2-adrenoceptor agonist) and cirazoline (a selective (α1-adrenoceptor agonist) were significantly greater in SHR than in SD or WKY, and the tissues were more sensitive to the agonists in SHR or SD than in WKY. Yohimbine (0.1 μM), a selective α2-adrenoceptor antagonist, shifted the dose–response curves for clonidine to the right. The effects of yohimbine were greater in SD than in WKY or SHR, but not different between WKY and SHR. Prazosin (0.05 μM), a selective α1-adrenoceptor antagonist, shifted the dose–response curves of cirazoline to the right, but the effects of prazosin were not different among these three strains of rats. Nifedipine (0.05 μM) completely blocked the response to clonidine in SD and WKY; however, in SHR, approximately one-third of the response to clonidine was resistant to nifedipine. Nifedipine, at 0.05 μM, only partially inhibited responses to cirazoline in SD, SHR, and WKY, and no differences were noted between the strains. Pertussis toxin pretreatment (50 μg/kg, 3 days before experiment) almost completely blocked the responses to clonidine, but only partially inhibited those to cirazoline. After pertussis toxin pretreatment, the responses (maximal effects and EC50s) to clonidine and cirazoline were not significantly different in arteries from the three strains of rats. A combination of pertussis toxin and nifedipine resulted in an additive inhibition of the responses induced by cirazoline. cholera toxin pretreatment (0.3 mg/kg, 3 days before experiment), however, had no effects on the contractile responses induced by either clonidine or cirazoline, or on the inhibitory effects of nifedipine in SHR, SD, and WKY. These results indicate that (i) the maximal responses to α1- and α2-adrenoceptor agonists are enhanced in rat tail artery rings from SHR; (ii) tissues from SH and SD rats are also more sensitive to cirazoline and clonidine than are tissues from WKY; (iii) responses to clonidine, but not cirazoline, in tissues from the SHR are less sensitive to nifedipine than tissues from SD and WKY; (iv) a G-protein sensitive to pertussis but not cholera toxin is involved in the regulation of both α1 and α2-adrenoceptor signal transduction processes in rat tail artery smooth muscle; and (v) pretreatment with pertussis toxin reduces the enhanced response levels of SHR tissues so that the maximal contractile responses to both α1- and α2-adrenoceptor agonists are equivalent in arteries from the three strains of rats.Key words: pertussis toxin, cholera toxin, α1-adrenoceptor, α2-adrenoceptor, rat tail artery ring.

Clonidine and related drugs not only interact with alpha 2-adrenoceptors but also recognise non-adrenoceptor sites in the brain. The involvement of these imidazoline-preferring receptors in the regulation of the activity of locus coeruleus noradrenergic neurones (NA-LC) was investigated after inactivation of alpha 2-adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). In EEDQ-pretreated rats (6 mg/kg, i.p., 6 h), the characteristic inhibitory effect of low doses of clonidine on these neurones was abolished and a paradoxical, dose-dependent increase in firing rate was observed at higher doses (640-5120 micrograms/kg, i.v.) (ED50 = 702 micrograms/kg, Emax = 83%, n = 14). Guanfacine (0.3-20 mg/kg) did not modify neuronal activity but antagonised the stimulatory effect of clonidine. Cirazoline (80-640 micrograms/kg) and rilmenidine (0.3-10 mg/kg) also stimulated neuronal activity (ED50 = 192 micrograms/kg, Emax = 102%, n = 5; ED50 = 1563 micrograms/kg, Emax = 70%, n = 1-5, respectively) by an alpha 2-adrenoceptor-independent mechanism. The results suggest that these drugs can modulate the activity of locus coeruleus noradrenergic neurones through the activation of I1-imidazoline-preferring receptors.

The muscarinic receptor mediating vasodilation of resistance vessels in the rat isolated, constant-pressure perfused kidney (preconstriction by 10(-7) M cirazoline) was characterized by subtype-preferring agonists and selective antagonists. The agonists produced vasodilation with the following rank order of potency: arecaidine propargyl ester (APE)5-methylfurtrethonium = methacholine = oxotremorine(S)-aceclidinearecaidine 2-butyne-1,4-diyl bisester4-Cl-McN-A-343 = (R)-nipecotic acid ethyl ester = N-ethyl-guvacine propargyl ester approximately (R)-aceclidine = (S)-nipecotic acid ethyl esterMcN-A-343. Agonist-induced vasodilation disappeared after destruction of the endothelium with detergent. Highly significant correlations of agonist potencies for vasodilation were found between rat kidney and guinea-pig ileum submucosal arterioles as well as agonist potencies at smooth muscle muscarinic M3 receptors of the guinea-pig ileum. The rank order of antagonist potencies (4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP)(R)-hexahydro-difenidol approximately hexahydro-sila-difenidolpirenzepine approximately p-fluoro-hexahydro-sila-difenidol approximately himbacine approximately AF-DX 384 approximately AQ-RA 741(S)-hexahydro-difenidol) to attenuate vasodilation to APE in rat kidney, correlated significantly with affinities at M3 receptors in submucosal arterioles and in smooth muscle of the guinea-pig ileum, but differed from those at M1 and M2 receptors in rabbit vas deferens. The agonist and antagonist potencies suggest that vasodilation elicited by muscarinic stimuli in endothelium-intact rat renal vasculature is mediated by functional muscarinic M3 receptors.

1. We have used the imidazoline derivative [3H]-idazoxan to define alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in cerebral cortex membranes of calf, mouse, rat, guinea-pig and man. 2. Competition experiments using the selective alpha-adrenoceptor drugs, rauwolscine and corynanthine, indicated that [3H]-idazoxan bound to a single population of sites in the calf and mouse membranes. However, [3H]-idazoxan also labelled non-adrenoceptor, imidazoline binding sites in the rat (15%), guinea-pig (30%) and human (40%) cerebral cortex membranes. 3. Competition experiments with adrenaline and cirazoline in the guinea-pig cortex, verified [3H]-idazoxan binding to both alpha 2-adrenoceptors and to non-adrenoceptor, imidazoline binding sites. 4. It has been postulated by several groups that [3H]-idazoxan may possess partial agonist activity. To investigate this further, saturation experiments were performed in the cerebral cortex membranes of all five species in the absence and presence of 300 microM guanosine triphosphate (GTP). GTP had no effect on [3H]-idazoxan binding in guinea-pig cerebral cortex; in both rat and mouse membranes 300 microM GTP increased the dissociation constant for [3H]-idazoxan by 2-3 fold without significantly affecting the Bmax. GTP reduced the Bmax by approximately 30% and 60% in calf and human cerebral cortex membranes, respectively, without significantly altering the Kd. 5. Saturation experiments were performed in the calf cerebral cortex membranes in the absence and presence of 300 microM GTP with the selective alpha 2-adrenoceptor agonist [3H]-clonidine and the selective muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB). GTP reduced the Bmax for [3H]-clonidine without altering the Kd, but failed to affect either the Bmax or the Kd for [3H]-QNB.6. Saturation experiments were performed in human cerebral cortex membranes in the presence of alpha2-adrenoceptor blockade with and without GTP. GTP 300 microM reduced the Bmax for [3H]-idazoxan at the non-adrenoceptor, imidazoline binding sites, without affecting the Kd. GTP did not affect [3H]-QNB binding to muscarinic sites.7. Thus, there is a need to investigate further the pharmacological actions of [3H]-idazoxan in view of its ability to recognise both alpha2-adrenoceptors and non-adrenoceptor, imidazoline binding sites and because it might possess agonist activity at some of these sites.

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.

Clonidine and related imidazoline agents, beside binding to α2-adrenergic receptors, have been shown to bind to a non-adrenergic site (imidazoline sites) in brain and peripheral tissues. However, which cell types in brain, namely neurons or glia, express this binding site and the cellular effects of activation of this site are not known. We investigated the cellular localization of imidazoline binding sites in cultured rat cortical astrocytes and neurons. Membranes prepared from cultured astrocytes showed specific, high affinity binding (KD: 4 nM) for 3H-idazoxan with about tenfold higher number of binding sites than α2-adrenergic sites (Bmax: 220 vs. 20 fmol/mg protein). Displacement studies exhibited the rank order of potency: cirazoline > idazoxan > amiloride > clonidine >>> epinephrine = ruawolscine defining this site as I-2a subtype of imidazoline binding sites. Moreover, the binding was inhibited by K+ but not by Na+, another characteristic of imidazoline binding sites. In contrast, membranes prepared from cultured neurons showed fewer binding sites for 3H-idazoxan that were completely displaced by adrenergic agents. Incubation of astrocytes with idazoxan, but not rauwolscine, resulted in a concentration-dependent increase in the levels of mRNA for the astrocyte specific molecule glial fibrillary acidic protein. We conclude that (a) the non-adrenergic imidazoline binding sites are expressed in astrocytes but not in neurons in rat cerebral cortex and (b) these “receptors” may influence astrocyte physiology by regulating the levels of GFAP. © 1993 Wiley-Liss, Inc.

The aim of the present study was to verify whether [3H]idazoxan can be considered as a highly selective ligand for imidazoline preferring receptors (IPR). In human frontal cortex membrane preparations [3H]idazoxan at a low concentration (2 nM) only labelled imidazoline sensitive, catecholamine insensitive sites. Binding was of high affinity, saturable and stereospecific. The rank order of potency of different compounds able to inhibit this binding was cirazoline > (+/-)-idazoxan > guanoxan > (-)-idazoxan > tolazoline > UK-14304 > clonidine. Amiloride, imidazol-4-acetic acid and histamine had no significant affinity for IPR labelled by [3H]idazoxan. [3H]idazoxan bound to 2 different sites (KD1 = 1 nM and KD2 = 16.4 nM). Clonidine behaved as a non competitive, non allosteric inhibitor of [3H]idazoxan binding. Both [3H]idazoxan binding sites were equally affected by clonidine. In membrane preparations obtained from the Nucleus Reticularis Lateralis region (NRL) of the brainstem, [3H]idazoxan binding was similar to that in cortical membranes, particularly with regard to specificity and kinetics. However, in the NRL region binding sites were 4-5 times more numerous than in the frontal cortex. Non linear analyses of saturation data obtained with NRL membrane preparations were compatible with both a one site and a two sites model. No significant effects of 1 mM MgCl2 alone or with 100 microM Gpp(NH)p were observed on either [3H]idazoxan binding or the competition with clonidine or rilmenidine. As in the cortical membrane, clonidine was a non competitive inhibitor of [3H]idazoxan binding to membranes from the NRL region. In conclusion, we show that when a low concentration is used, [3H]idazoxan binding to human brain involves sites almost completely insensitive to catecholamines and specific for imidazolines or related compounds. This binding involves two distinct sites. We also report that [3H]idazoxan imidazoline binding sites are not coupled with a G protein. Because of the non competitive interaction between clonidine and [3H]idazoxan for the binding sites of the latter, we are unable to conclude that the binding sites of the two drugs are identical. However, the non competitive, non allosteric interaction suggests a complex model of multiple binding sites.

1. RS-15385-197 ((8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro- 3-methoxy-12-(methylsulphonyl)-6H-isoquino [2,1-g][1,6]-naphthyridine) was evaluated in a series of in vitro and in vivo tests as an antagonist at alpha 2-adrenoceptors. 2. RS-15385-197 had a pKi of 9.45 for alpha 2-adrenoceptors in the rat cortex (pA2 in the guinea-pig ileum of 9.72), whereas the 8aS, 12aR, 13aR enantiomer, RS-15385-198, had a pKi of only 6.32 (pA2 6.47) indicating a high degree of stereoselectivity. The racemate RS-15385-196 had a pKi of 9.18. 3. RS-15385-197 showed unprecedented alpha 2 vs. alpha 1 adrenoceptor selectivity in vitro. In the rat cortex, RS-15385-197 had a pKi of 9.45 in displacing [3H]-yohimbine and 5.29 in displacing [3H]-prazosin (alpha 2/alpha 1 selectivity ratio in binding experiments > 14000). The compound had a pA2 of 9.72 as a competitive antagonist of the inhibitory effects of UK-14,304 in transmurally-stimulated guinea-pig ileum and 10.0 against BHT-920-induced contractions in dog saphenous vein (DSV); this latter value was unaltered by phenoxybenzamine. An apparent pKB of 5.9 was obtained against cirazoline-induced contractions in DSV, whilst a pA2 of 6.05 was obtained against phenylephrine-induced contractions in the rabbit aorta (alpha 2/alpha 1 selectivity ratio in functional experiments > 4000). 4. RS-15385-197 was highly selective for alpha 2-adrenoceptors over other receptors: the compound showed low affinity for 5-HT1A (pKi 6.50) and 5-HT1D (pKi 7.00) receptor subtypes, and even lower affinity (pKi < or = 5) for other 5-HT receptor subtypes, dopamine receptors, muscarinic cholinoceptors, beta-adrenoceptors and dihydropyridine binding sites. RS-15385-197 was devoid of affinity for the non-adrenoceptor imidazoline binding site, labelled by [3H]-idazoxan, which provides further evidence that these sites are not related to alpha 2-adrenoceptors. In the DSV, contractile responses to 5-hydroxytryptamine (5-HT) were unaffected by a concentration of 1 microM RS-15385-197. 5. RS-15385-197 was non-selective for the alpha 2A- and alpha 2B-adrenoceptor subtypes in that the pKi for the alpha 2A-adrenoceptor in human platelets was 9.90 and the pKi for the alpha 2B-adrenoceptor in rat neonate lung was 9.70. However, RS-15385-197 showed lower affinity for the alpha 2-adrenoceptor subtype in hamster adipocytes (pKi 8.38). 6. In anaesthetized rats, RS-15385-197 was a potent antagonist of the mydriasis response induced by UK-14,304 or clonidine (AD50 5 and 7 microg kg-1, i.v., respectively; 96 microg kg-1, p.o.) and of UK-14,304-induced pressor responses in pithed rats (AD50 7 microg kg-1, i.v.); the compound therefore is both centrally and orally active. Even at a high dose (10 mg kg-1, i.v.), RS-15385-197 did not antagonize pressor responses to cirazoline in pithed rats, indicating that the selectivity for alpha2 vs. alpha1-adrenoceptors was maintained in vivo.8 RS-15385-197 is therefore a very potent, selective, competitive alpha2-adrenoceptor antagonist, both in vitro and in vivo, is orally active and readily penetrates the brain. It will thus be a powerful pharmacological tool for exploring the various physiological roles of alpha2-adrenoceptors.

Noradrenergic receptor populations within the paraventricular hypothalamus (PVN) modulate feeding. Satiated rats exhibit enhanced feeding subsequent to activation of alpha 2-adrenergic receptors within the PVN induced by exogenous infusion of either norepinephrine (NE) or clonidine (CLON). The feeding-stimulatory effect of alpha 2-adrenergic agents presumably reflects an inhibitory action on receptors located on medial hypothalamic "satiety" cells. Adrenergic receptors of the alpha 1-subclass have been identified within the PVN which are excitatory and which may function to suppress food intake. Microinjection into rat PVN of various alpha 1-adrenergic agonists including cirazoline, methoxamine, phenylpropanolamine and phenylephrine suppress feeding; an effect that is reversed by pretreatment with alpha 1-adrenergic receptor antagonists. The present review argues that alpha 1- and alpha 2-adrenoceptors within brain and specifically within the PVN are organized in an antagonistic fashion and that the effects of various adrenergic agonists on feeding may reflect the degree to which these agonists act at alpha 1- and alpha 2-adrenoceptors as well the relative balance of these receptors and their activity within the PVN.

Activation of alpha-1 adrenoceptors, via systemic injection of agonists such as cirazoline and phenylpropanolamine (PPA), reliably suppresses food intake in rats. These effects are thought to result from stimulation of central alpha 1-adrenoceptors within the rat paraventricular hypothalamic nucleus (PVN), based on studies in which direct injections of cirazoline, methoxamine, phenylephrine and PPA into PVN suppress food intake. Because relatively few alpha 1-agonists have been tested to date using the systemic route of exposure, the present study examined the effects of the alpha 1-adrenoceptor agonists amidephrine and SKF-89748 on food and water intake. Adult male rats received systemic injections (IP) of either amidephrine (0.025, 0.05, 0.01 mg/kg) or of SK&F 89748 (0.01, 0.02, and 0.04 mg/kg). Amidephrine markedly suppressed food intake (ED50 = 0.49 mg/kg) and water intake (ED50 = 0.50 mg/kg), while SK&F 89748 marginally suppressed food intake (ED50 = 0.37 mg/kg) and was less potent in suppressing water intake (ED50 = 0.76 mg/kg). These results document that systemic injection of the alpha 1-adrenoceptor agonists amidephrine and SK&F 89748 induces anorexia with amidephrine exerting greater potency than SK&F 89748. These results further support the hypothesis that stimulation of alpha 1-adrenoceptors suppresses food intake.

1. In the present study, we tested the hypothesis that in the pithed rat preparation two subtypes of the α1-adrenoceptor are linked to two different signal transduction mechanisms, both of which contribute to vasoconstriction, one facilitating Ca2+-entry from the extracellular fluid (α(1A)) and one promoting the release of Ca2+from intracellular sources (α(1B)). 2. The selective α(1A)-adrenoceptor antagonist, 5-methyl-urapidil, and the selective α(1B)-adrenoceptor antagonist, chloroethylclonidine, were unable to discriminate between α1-adrenoceptor-mediated pressor responses, which relied on an entry of extracellular Ca2+sensitive to nifedipine and an intracellular release of Ca2+insensitive to nifedipine, respectively. 3. Chloroethylclonidine, 12.5 and 25 mg kg-1i.v., were equieffective, and had only minor effects on α1-adrenoceptor-mediated increases in diastolic blood pressure. This could be associated with a small decrease in the receptor-reserve of the pithed rat preparation due to irreversible receptor blockade by this antagonist. These data indicate that chloroethylclonidine-sensitive α1-adrenoceptors constitute only a minor fraction of the total α1-adrenoceptor population on rat arterial resistance vessels. 4. Chloroethylclonidine behaved as a partial agonist eliciting a small increase in baseline diastolic blood pressure which could be inhibited by Ca2+-entry blockade with nifedipine. 5. Chloroethylclonidine potentiated the pressor responses elicited by the α2-adrenoceptor agonists UK-14,304 and azepexole (B-HT 933). 6. No evidence was found in the pithed rat that α1-adrenoceptor-mediated Ca2+-entry from the extracellular fluid and Ca2+-release from intracellular stores are mediated by α(1A) and α(1B)-adrenoceptors, respectively.

The effects of efaroxan (RX 821037A; 2-[2-(2-ethyl-2,3-dihydrobenzofuranyl)]-2-imidazoline HCl) at alpha 1- and alpha 2-adrenoceptors were investigated in isolated tissues, pithed rats and conscious rats. In isolated tissues, efaroxan competitively antagonised the inhibitory effects of p-aminoclonidine in the electrically stimulated (0.1 Hz) rat vas deferens, (pA2 = 8.89) and the contractile effects of phenylephrine on the rat anococcygeus muscle (pA2 = 6.03). Efaroxan had a selectivity ratio (alpha 2/alpha 1) of 724 compared to a value of 182 for idazoxan. In pithed rats, the i.v. doses of efaroxan (mumol/kg) producing 2-fold shifts in dose-response curves for UK-14,304 at prejunctional cardiac alpha 2-adrenoceptors and postjunctional vascular alpha 2-adrenoceptors, and for cirazoline at postjunctional vascular alpha 1-adrenoceptors, were 0.05, 0.13 and 2.96, respectively. In conscious fasted rats, prazosin (5 mg/kg p.o.) increased resting glucose levels and exacerbated the hyperglycaemic effects of UK-14,304 and adrenaline. In contrast, efaroxan (1-5 mg/kg p.o.) had little effect on resting plasma glucose but markedly antagonised the hyperglycaemic actions of UK-14,304 and adrenaline. Efaroxan increased resting plasma insulin levels and markedly potentiated the rise in insulin levels produced by adrenaline; this latter effect was prevented by the co-administration of propranolol. These results demonstrate that efaroxan is a potent and selective alpha 2-adrenoceptor antagonist and provide further support for the involvement of alpha 2-adrenoceptors in glucose homeostasis.