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The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b(5) and b(5) reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes.

In previous studies, it was shown that liver microsomes from rabbit, rat, pig, and human are involved in the reduction of N-hydroxylated amidines, guanidines, and amidinohydrazones of various drugs and model compounds (Drug Metab Rev 34: 565-579). One responsible enzyme system, the microsomal benzamidoxime reductase, consisting of cytochrome b5, its reductase, and a cytochrome P450 isoenzyme, was isolated from pig liver microsomes (J Biol Chem 272:19615-19620). Further investigations followed to establish whether such enzyme systems are also present in microsomes of other organs such as brain, lung, and intestine. In addition, the mitochondrial reduction in human and porcine liver and kidney preparations was studied. The reductase activities were measured by following the reduction of benzamidoxime to benzamidine, guanoxabenz to guanabenz, and Ro 48-3656 ([[1-[(2S)-2-[[4-[(hydroxyamino)iminomethyl]benzoyl]amino]-1-oxopropyl]-4-piperidinyl]oxy]-acetic acid) to Ro 44-3888 ([[1-[(2S)-2-[[4-(aminoiminomethyl)benzoyl]amino]-1-oxopropyl]-4-piperidinyl]oxy]-acetic acid). Interestingly, preparations of all tested organs were capable of reducing the three compounds. The highest specific rates were found in kidney followed by liver, brain, lung, and intestine, and usually the mitochondrial reduction rates were superior. From the determined characteristics, similarities between the enzyme systems in the different organs and organelles were detected. Furthermore, properties of the benzamidoxime reductase located in the outer membrane of pig liver mitochondria were studied. In summary, these results demonstrate that in addition to the microsomal reduction, mitochondria are involved to a great extent in the activation of amidoxime prodrugs. The importance of extrahepatic metabolism in the reduction of N-hydroxylated prodrugs is demonstrated.

The mitochondrial amidoxime reducing component (mARC) is a molybdenum-containing enzyme and capable of reducing N-hydroxylated structures such as amidoxime prodrugs. In this study, we tested the involvement of mARC in the reduction of N-oxides (amitriptyline-N-oxide, nicotinamide-N-oxide), oximes ((E)-/(Z)-2,4,6-trimethylacetophenonoxime) and a N-hydroxyamidinohydrazone (guanoxabenz). All groups are reduced by mARC proteins, and the enzymes are therefore involved in the interconversion of N-oxygenated metabolites originating from cytochrome P450s and flavin-containing monooxygenases. In addition, these structures open up further options for serving as prodrugs. Thus, with respect to these reactions, testing of candidates with N-oxygenated structures should not solely be carried out in microsomal enzyme sources but as well in mitochondria. However, differences in the reduction of oximes and N-oxides between the two isoforms, namely mARC1 and mARC2, were detectable; N-oxides are exclusively reduced by mARC1. We therefore assume differences between the so far unknown 3D structures of the two proteins.

The mechanism for formation of high-affinity binding of 1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine (guanoxabenz) to α 2 -adrenoceptors was studied in particulate fractions from the rat spleen. The proportion of apparent high versus low-affinity α 2 -adrenoceptor binding sites increased with increasing incubation time and was also augmented by Mg 2+ ions. The formation of high-affinity guanoxabenz binding seemed to be inhibited by a series of N -hydroxyguanidine analogs to guanoxabenz, as well as by a series of metabolic inhibitors that included allopurinol, 1-chloro-2,4-dinitrobenzene, 5,5′-dithiobis-(2-nitrobenzoic acid), cibacron blue, phenyl- p -benzoquinone, didox, and trimidox. The formation of guanoxabenz high-affinity binding was also inhibited in a time- and concentration-dependent fashion by preincubating the membranes with the LW03 N -hydroxyguanidine analogue of guanoxabenz. Moreover, when the spleen membranes were extensively washed for 30 min with buffers at 25°, the guanoxabenz high-affinity binding disappeared. However, when these washed membranes were supplemented with xanthine, the apparent affinity of guanoxabenz increased four to five-fold. Taken together, all data were compatible with the theory that the formation of high-affinity binding was dependent on the generation of a guanoxabenz metabolite that showed an approximate 100-fold greater affinity for the α 2 -adrenoceptors than guanoxabenz itself. Because the most potent blocker of the formation of high-affinity binding was allopurinol (apart from some N -hydroxyguanidine analogs to guanoxabenz) and since the activity could be restored with xanthine, a likely candidate responsible for the metabolic activation is xanthine oxidase.

The N-reduction of the centrally acting α2-adrenoreceptor agonist guanoxabenz (Benzerial), an N-hydroxyamidinohydrazone, to the amidinohydrazone guanabenz (Wytensin, Hipten, Rexitene) by microsomal fractions from rabbits, pigs, and humans has been detected in vitro. The conversion rates with rabbit microsomal fractions were markedly slower than those in the cases of fractions from humans and pigs. It was also possible to demonstrate the N-oxidation of guanabenz to guanoxabenz by the use of microsomal fractions from rabbits, pigs, and humans. Furthermore, the oxidation was also observed in reconstituted systems in the presence of enriched cytochrome P450 fractions, purified isoenzyme P450 2C3, and heterologously expressed P450 2C3 of the subforms 6βH and 6βL. The analyses were performed with a newly developed HPLC technique and were confirmed by LC-MS methods. The kinetic parameters determined for the metabolic cycle (bioreversible reactions) are indicative of a predominance of the reduction of guanoxabenz...

Analysis of saturation isotherms of the novel α2-adrenoceptor antagonist radioligand [3H]-MK 912 revealed that the ligand labelled a homogenous population of α2B-adrenoceptors in the neonatal rat lung with a Kd of 0.77 nM and a Bmax of 231 fmol/mg protein. In rat kidney, combined saturation and competition experiments, using [3H]-MK 912 and the α2A-adrenoceptor selective drug guanfacine, revealed that ~ 81% of the sites labelled by [3H]-MK 912 were α2B-adrenoceptors and ~ 19% α2A-adrenoceptors; the Kds of [3H]-MK 912 being. 1.1 and 2.0 nM and the Bmax 134 and 33 fmol/mg protein, respectively. The kidney α2B-adrenoceptors were studied separately by using ~ 1.5 nM [3H]-MK 912 in the presence of 0.32 μM guanfacine, the latter which blocked ligand binding to α2A-adrenoceptors completely. Analysis of drug competition curves obtained during these conditions revealed that 18 out of 20 different agonists and antagonists yielded steep and uniphasic competion curves which modelled best into one site fits. However, both guanoxabenz and LT 11 appeared to inhibit [3H]-MK 912 binding at two sites; the Kds of guanoxabenz differing ~ 120-fold and that of LT 11 differing ~ 35-fold for the two sites. Moreover, the addition of mutual fixed concentrations of either 20 μM guanoxabenz or 20 μM LT 11 completely prevented the binding of the other compound to its high affinity site, indicating that both compounds labelled the same site with the high affinity. The analysis indicated that 29% of the sites were of high and 71% of low affinity. However, in the neonatal rat lung guanoxabenz and LT 11 (as well as 15 other compounds) yielded competition curves which modelled only into one site fits. The Kds obtained in the lung correlated well with the Kds obtained in the kidney for α2B-adrenoceptors; for guanoxabenz and LT 11 the values from the lung were close to those determined in the kidney for the low affinity site for guanoxabenz and LT 11. Moreover, when the rat RNG α2B-adrenoceptor was expressed in COS-7 cells and its binding properties tested using [3H]-MK 912 binding, guanoxabenz, LT 11 as well as a number of other drugs inhibited the ligand binding at a single α2-adrenoceptor site; the drug Kds being practically the same as those found for the neonatal rat lung. It is suggested that rat α2B-adrenoceptors may exist in two forms: α2B1 and α2B2. The α2B1-form, which shows high affinity for guanoxabenz and LT 11, is present in the adult rat kidney. The α2B2-form, which shows low affinity for guanoxabenz and LT 11, is present in the neonatal rat lung as well as in the adult rat kidney and it is encoded by the RNG gene.

The α2-adrenoceptor antagonists idazoxan and RX811059 induced reciprocal forepaw treading, a component of the 5-HT-behavioural syndrome in rats. This response is independent of 'non-α2-adrenoceptor idazoxan binding sites' (NAIBS) at which RX811059 is inactive. Idazoxan pre-treatment, in rats, enhanced forepaw treading, head weaving and tremor induced by the 5-HT agonist 5-methoxy-N,N dimethyltryptamine (5-MeODMT), increased head twitches (but decreased hindlimb abduction) induced by the 5-HT releaser p- chloroamphetamine (pCA), but did not clearly alter head twitches induced by the 5-HT precursor L-5-hydroxytryptophan in mice. The α1-antagonist prazosin did not alter behaviour induced by either 5-MeODMT or pCA in rats. The α 2-agonist, guanoxabenz, did not alter 5-MeODMT-induced behaviour in rats. St587, an α1-agonist, selectively potentiated tremor induced by 5-MeODMT, but no other behaviour. A possible mechanism for these interactions could be through enhanced, α2-adrenoceptor-mediated, 5-HT release in specific brain areas. Other possibilities, e.g. direct action at subtypes of 5-HT receptors and the importance of these NA-5-HT interactions in the treatment of resistant depression, are discussed.

Primäre Hepatozytenkulturen stellen ein geeignetes in vitro-Modell für Biotransformationsstudien und zur Aufklärung potenzieller Interaktionen von Xenobiotika dar. Im Gegensatz zu den häufig eingesetzten Leberparenchymzellen der Ratte, die jedoch große Speziesunterschiede zum Metabolismus des Menschen aufweist, gelten Hepatozyten porcinen Ursprungs als mögliche Alternative für das begrenzt verfügbare humane Lebermaterial. In dieser Arbeit wurden deshalb vergleichende Phase I- und Phase II-Studien anhand bestimmter stickstoffhaltiger Modellverbindungen in porcinen und humanen Hepatozytenkulturen sowie Studien zur Cytochrom P450-Induktion mit Schweinehepatozyten durchgeführt. Beide Spezies wiesen bezüglich der untersuchten Reaktionen große Übereinstimmungen auf. Aufgrund des Einsatzes zahlreicher N-hydroxylierter Verbindungen als Prodrugs war neben der N-reduktiven Aktivierung insbesondere die Untersuchung von Phase II-Metaboliten der Modellsubstrate Benzamidoxim (Amidoxim), N-Hydroxydebrisoquin (N-Hydroxyguanidin) und Guanoxabenz (N-Hydroxyamidinohydrazon) von großer Bedeutung. LC/MS-Studien lieferten Hinweise auf die Bildung mehrerer Konjugate sowohl in Hepatozytenkulturen als auch mit UDP-Glucuronyltransferasen. Hierbei ermöglichten synthetische Referenzsubstanzen die eindeutige Identifizierung der O-Glucuronide von N-Hydroxydebrisoquin und Benzamidoxim.

Die vorliegende Arbeit befasst sich mit der enzymatischen Reduktion von N-hydroxylierten Verbindungen im Rahmen des extrahepatischen Metabolismus. Zu Beginn der vorliegenden Arbeit wurden zunächst Mikrosomen aus Leber, Niere, Gehirn, Lunge und Darm vom Schwein und Leber, Niere und Darm vom Menschen gewonnen. Daneben wurden auch Blut und Blutbestandteile von Schwein und Mensch beschafft, präpariert und in die Untersuchungen einbezogen. In den Studien wurden die Mikrosomen aus den verschiedenen Organen mit drei Substraten umgesetzt, um das Vorhandensein eines reduktiven Enzymsystems, das in der Leber aus Cytochrom b5, der NADH-Cytochrom b5 Reduktase und einem P450 Isoenzym besteht, zu belegen. Die in dieser Arbeit untersuchten N-hydroxylierten Verbindungen Benzamidoxim, Guanoxabenz und Ro 48-3656 waren von ihrer chemischen Struktur her entweder Amidoxime oder N-Hydroxyamidinohydrazone, die durch reduktive Enzymsysteme dehydroxyliert werden können, sodass als Metaboliten Amidine bzw. Amidinohydrazone entstehen. In allen Mikrosomen, sowohl vom Schwein als auch vom Menschen, wurde eine eindeutige Umsetzung der eingesetzten Amidoxime (bzw. des N-Hydroxyamidinohydrazons) zu den korrespondierenden Amidinen (zum Amidinohydrazon) nachgewiesen, sodass die N-Dehydroxylierung für die drei verwendeten Substrate in den in dieser Arbeit eingesetzten Organen belegt werden konnte. Die Reduktionen zeigten mit Ausnahme des Darms alle die Bevorzugung eines leicht sauren Reaktionsmilieus und in Übereinstimmung mit bisherigen Studien eine Präferenz von NADH gegenüber NADPH.

Guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) and guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine) are both known as centrally active antihypertensive drugs. We have previously shown that enzymatic activity in the rat spleen can induce N-reduction of guanoxabenz, leading to high affinity alpha 2-adrenoceptor binding, due to the formation of the alpha 2-adrenoceptor active drug, guanabenz. The spleen activity appears to reside in xanthine oxidase as it is activated by xanthine and blocked by allopurinol. We report that high affinity guanoxabenz binding is also induced in rat brain membranes after addition of NADH or NADPH cofactors. However, the brain process was clearly different from that of the spleen, as the formation of high affinity binding in the brain was not blocked by allopurinol. Moreover the NADH/NADPH activated mechanism of the brain membranes was not blocked by carbon monoxide and SKF525A, thus the activity appears not to reside in cytochrome P450 enzymes. Instead the activity was blocked by menadione and dicumarol. We conclude that the rat cerebral cortex contains an enzymatic activity that may activate guanoxabenz leading to formation of a metabolite showing high affinity for alpha 2-adrenoceptors. We also conclude that the rat brain activity is clearly distinct from that of the rat spleen.

The subtype determination of auto- and hetero-alpha 2-adrenoceptors modulating the synthesis of noradrenaline (NA) and serotonin (5-HT), respectively, was assessed using the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition as a measure of the rate of tyrosine and tryptophan hydroxylation in the rat brain in vivo. In the cerebral cortex and hippocampus, Org 3770 (non-selective alpha 2-adrenoceptor antagonist, 0.5-10 mg/kg, i.p.) increased (43%-58%) and clonidine (non-selective alpha 2-adrenoceptor agonist, 1 mg/kg) decreased (37%-49%) the synthesis of dopa. Also the antagonist ARC 239 (alpha 2B/C selective, 5-40 mg/kg) increased the synthesis of dopa in cortex (39%-46%) and hippocampus (17%-85%). In contrast, the antagonist BRL 44408 (alpha 2D selective, 1-10 mg/kg) did not increase the synthesis of dopa in cortex, and increased it modestly in hippocampus only. The agonist guanoxabenz (alpha 2B/C selective, 0.03-3 mg/kg) decreased the synthesis of dopa in both brain regions (20%-65%), whereas the agonist oxymetazoline (alpha 2D selective, 0.1-3 mg/kg) failed to do so. These results indicated that the alpha 2-autoreceptors that modulate the synthesis of dopa/NA are probably associated with the alpha 2B/C-subtypes. In cortex and hippocampus, clonidine decreased (35%-53%) the synthesis of 5-HTP but Org 3770 failed to induce the opposite effect (except the 2 mg/kg dose in cortex). BRL 44408 markedly increased the synthesis of 5-HTP in cortex (113%-148%) but not in hippocampus. Similarly, also ARC239 increased the formation of 5-HTP in cortex (36%-48%) but not in hippocampus, where it was decreased (30%-55%). Oxymetazoline decreased the synthesis of 5-HTP in hippocampus (28%-30%) but failed to do so in cortex. Guanoxabenz in the low dose range (0.03-0.3 mg/kg) did not decrease the synthesis of 5-HTP in any brain region. These results indicated that the alpha 2-heteroreceptors that modulate the synthesis of 5-HTP/5-HT may well be different from the proposed alpha 2B/C-autoreceptors modulating the synthesis of dopa/NA. These alpha 2-heteroreceptors appear to be associated with the alpha 2D-subtype.

Competition [3H]RX821002 ([3H]2-methoxyidazoxan) binding experiments with alpha 2-adrenoceptor subtype-specific antagonists--BRL 44408 (alpha 2A selectively), ARC 239 (alpha 2B selective), and others--were performed to delineate through rigorous computer modeling receptor subtypes in the postmortem human brain. In the hippocampus, hypothalamus, cerebellum, and brainstem the whole population of alpha 2-adrenoceptors appears to belong to the alpha 2A subtype (100%; Bmax = 34-90 fmol/mg of protein). In the frontal cortex, the predominant receptor was the alpha 2A subtype (87%; Bmax = 53 fmol/mg of protein), although a small population of the alpha 2B/C subtype (13%; Bmax = 8 fmol/mg of protein) was also detected. In the caudate nucleus, a mixed population of alpha 2A (64%; Bmax = 9 fmol/mg of protein) and alpha 2B/C (36%; Bmax = 5 fmol/mg of protein) subtypes was detected. In the cortex and caudate and in the presence of ARC 239 (to mask the alpha 2B/C-adrenoceptors), competition experiments with the agonist guanoxabenz clearly modeled the high- and low-affinity states of the alpha 2A subtype. In the presence of ARC 239 and the GTP analogue guanylyl-5'-imidodiphosphate together with NaCl and EDTA (to eliminate the high-affinity alpha 2A-adrenoceptor) guanoxabenz only recognized the low-affinity alpha 2A-adrenoceptor. The results indicate that in the human brain the predominant alpha 2-adrenoceptor is of the alpha 2A subtype and that this functionally relevant receptor subtypes is not heterogeneous in nature.

Summary The α2A-adrenoceptors in rat spleen, kidney, spinal cord and cerebral cortex were studied using [3H]-RX821002 radioligand binding. In the spleen, spinal cord and cerebral cortex, the ligand bound to saturable sites with a K d of about 1 nmol/l and capacities of 134, 240 and 290 fmol/mg protein, respectively. Computer modelling competition curves for 39 drugs, including those for α2A-, α2B- or α2C-adrenoceptor selective drugs, indicated that the sites labelled by [3H]-RX821002 in the spleen consisted of a single population of α2A-adrenoceptors. However, the competition curves for guanoxabenz were definitely biphasic and resolved into two site fits, indicating that guanoxabenz was binding to both high affinity (K d = 35 nmol/1) and low affinity (K d = 8900 nmol/1) α2A-adrenoceptor sites in the proportions 57% and 43%, respectively. The K d Sfor a number of α2-adrenoceptor subtype selective drugs, measured in competition with [3H]-RX821002 in cerebral cortex and spinal cord, were highly correlated with those obtained in the spleen indicating their α2A-adrenoceptor nature. However, by contrast to the results with the spleen, the guanoxabenz competition curves for the spinal cord and cerebral cortex were monophasic and resolved only into one site fits, the K d of guanoxabenz being about 4000 nmol/l for both tissues. Drug K d Sfor kidney α2A-adrenoceptors were also determined using [3H]-RX821002. For nearly all drugs tested, the K d Swere highly correlated with those found for the α2A-adrenoceptors in the other rat tissues. However, for guanoxabenz, the data indicated that it competed with [3H]-RX821002 at a single α2A-adrenoceptor site with a K d of 39 nmol/1. When the rat α2A-adrenoceptor gene RG20 was transiently expressed in COS-7 cells and its ligand binding properties probed using [3H]-RX821002, the drug K d Sobtained were also highly correlated with those found for the α2A-adrenoceptors in the spleen, cerebral cortex, spinal cord and kidney of the rat. For the RG20 encoded receptor, the guanoxabenz competition curves were steep and monophasic and modelled best into one site fits, with the Kd of guanoxabenz being 5200 nmol/1. It is suggested that guanoxabenz can differentiate between two forms of α2A-adrenoceptors in the rat: α2A1 and α2A2. The α2A1-form is present in the spleen and kidney where it shows a high apparent affinity for guanoxabenz. The α2A2-form shows a low apparent affinity for guanoxabenz and is present in the spleen, cerebal cortex and spinal cord. The α2A2-form of the rat α2-adrenoceptor appears to be encoded by the RG20 gene. The α2A, and α2A2-adrenoceptor forms do not represent high and low affinity receptor forms for agonists because assays included EDTA, Gpp(NH)p and Na+, which eliminated the high affinity receptors for agonists.

1. Simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked [3H]-RX821002 saturation curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves revealed that the drugs bound to three alpha 2-adrenoceptor subtypes in the rat kidney with grossly differing selectivities. These alpha 2-adrenoceptor subtypes were termed alpha 2 A, alpha 2B1 and alpha 2B2. The order of affinities for [3H]-RX821002 for the adrenoceptor sites was alpha 2A greater than alpha 2B1 greater than alpha 2B2, the KdS being 0.62 +/- 0.05, 2.52 +/- 0.11 and 6.74 +/- 1.21 nM, respectively. The order of affinities for ARC 239 was alpha 2B1 greater than alpha 2B2 much greater than alpha 2A with KdS 4.78 +/- 1.04, 28.8 +/- 4.1 and 1460 +/- 270 nM, respectively. For guanoxabenz the order of affinities was alpha 2A greater than alpha 2B1 much greater than alpha 2B2 with KdS 99.7 +/- 15.1, 508 +/- 135 and 25,400 +/- 2400 nM, respectively. 2. Binding constants for 14 compounds for the three rat kidney alpha 2-adrenoceptor subtypes were determined by the simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked drug competition curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves. Of the 14 compounds tested, oxymetazoline and guanfacine were found to bind with low affinities to both of the alpha 2B1- and alpha 2B2-adrenoceptor but with high affinity to the alpha 2A-adrenoceptor. 3. (-)-Adrenaline and (-)-noradrenaline showed dissimilar order of affinities for the three alpha2-adrenoceptors. For (-)-adrenaline the order of affinities was alpha2Bl >- alpha2A> alpha2B2 and for (-)-noradrenaline alpha2B2 > alpha2Bl > alpha2A. All three alpha2-adrenoceptors showed the expected stereoselective binding for adrenaline enantiomers, the (+)-form being 7-10 fold less potent than the (-)form. 4. [3H]-yohimbine was also used as radioligand. The data with this ligand were fully compatible with the [3H]-RX821002 data. However, [3H]-yohimbine appeared to label only alpha2Bl- and alpha2B2-adrenoceptors presumably because it had too low an affinity for alpha2A-adrenoceptors. 5. We conclude that three pharmacological subtypes of alpha2-adrenoceptors are labelled by [3H]-RX821002 in the rat kidney. Guanoxabenz and ARC 239 may be used in competition studies to delineate between these three alpha2-adrenoceptor subtypes. Moreoever, we here present a method allowing the determination of binding constants for an arbitrary drug to the three alpha2-adrenoceptor subtypes.

The effects of α1-and α2-adrenoceptor agonists on smooth muscle tone and on cholinergic excitatory and non-adrenergic, non-cholinergic inhibitory responses to field stimulation have been investigated in the rat gastric fundus in-vitro. None of the α-adrenoceptor agonists tested, noradrenaline, phenylephrine, cirazoline, guanoxabenz or UK-14,304 showed any contractile effects at concentrations up to 30 μM. In preparations where tone was raised by barium (0.5-2 mM), the mixed α1-and α2-adrenoceptor agonist noradrenaline (0.01–10 μM), and the selective α1-adrenoceptor agonists cirazoline (0.01–10 μM) and phenylephrine (0.01–10 μM) produced concentration-dependent relaxations which were antagonized by the α1-adrenoceptor antagonist prazosin (0.01-1.0 μM). The selective α2-adrenoceptor agonists UK-14,304 (0.03–30 μM) and guanoxabenz (0.03–30 μM), had no relaxant effects in raised tone. UK-14,304 (0.03-1.0 μM) produced a concentration-dependent inhibition of cholinergic nerve-induced responses which was antagonized by the α2-adrenoceptor antagonist idazoxan (0.03-1.0 μM) but not by prazosin (0.03-1.0 μM). Noradrenaline (0.03-1.0 μM) also produced an inhibition of cholinergic nerve-induced responses which was antagonized by idazoxan (0.03–10 μM). A small component of the noradrenaline inhibitory effects was antagonized by prazosin (10%). Cirazoline (0.03-1.0 μM) produced a small inhibition of cholinergic nerve-induced responses which was antagonized by prazosin (0.03-1.0 μM). The prazosin-sensitive components of the inhibitory effects of noradrenaline and cirazoline occurred at concentrations which also produced post-junctional relaxation. UK-14,304 (0.03-1.0 μM) also attenuated non-adrenergic, non-cholinergic (NANC) inhibitory nerve-induced responses and this effect was antagonized by idazoxan (0.03-1.0 μM). The effects of α1-adrenoceptor agonists on NANC nerve-induced responses proved difficult to assess since these agonists caused post-junctional relaxation which itself reduced the size of the inhibitory nerve-induced responses. It is concluded that α-adrenoceptor mediated responses in the rat gastric fundus in-vitro consist of post-junctional α1-adrenoceptor-mediated relaxation of the smooth muscle and pre-junctional α2-adrenoceptor-mediated inhibition of both cholinergic excitatory and non-adrenergic, non-cholinergic inhibitory nerve-induced responses.

The porphyrin precursor, delta-aminolaevulinic acid (ALA) at concentrations of 0.23-7.6 mM caused dose-dependent inhibition of spontaneous contractions in isolated preparations of rabbit small intestine, suspended in Ringer-Locke solution. Contractions which returned after inhibition from 3.8 and 7.6mM ALA showed increased amplitude and from 7.6 mM ALA a reduction in rate. Pretreatment with prazosin (10(-7) M) significantly reduced the duration of inhibition exerted by 3.8 and 7.6 mM ALA and also the amplitude of contractions returning after this inhibition. Pretreatment with yohimbine (10(-6) M) and propranolol (7 X 10(-6) M) had no significant effects. Blocking of the release of noradrenaline in preparations by incubation with 6-hydroxydopamine (10(-3) M) or guanethidine (1.5 X 10(-5) M) did not prevent the inhibitory effects of ALA. The alpha 1 agonist, cirazoline (10(-5) M), inhibited contractile activity. This effect was blocked by prazosin (10(-7) M). The alpha 2 agonist, guanoxabenz (10(-5) M) had no detectable effect, in the presence of prazosin, to block any residual alpha 1 actions. It is concluded that alpha 1 receptors mediate inhibitory effects in this preparation and that ALA appears to have direct effects upon these receptors.

Several neuropharmacological effects of RX 781094, a new selective α 2 -adrenoceptor antagonist, have been investigated in rodents. In rats, RX 781094 (0.1–1.0 mg kg −1 , i.v.) produced a rapid dose-related reversal of cortical EEG synchronisation and behavioural sedation, induced by clonidine or the more selective α 2 -adrenoceptor agonist, guanoxabenz. The α 2 -adrenoceptor antagonists yohimbine and mianserin were also effective in blocking guanoxabenz-induced EEG synchronisation but had a lower potency than did RX 781094. In specificity experiments, RX 781094 (1.0 mg kg −1 , i.v.) failed to antagonise the EEG synchronisation and pronounced behavioural sedation induced by the CNS depressant sodium pentobarbitone (15 mg kg −1 , i.v.). In mice, pretreatment (i.v. or p.o.) with RX 781094 inhibited in a dose-dependent way both guanoxabenz-induced behavioural hypoactivity and clonidine-induced hypothermia. By itself, RX 781094 had no effect on the temperature of normal mice. In sleep-waking studies in rats, RX 781094 (0.1 and 1.0 mg kg −1 , i.v.) had no measurable stimulant or depressant effect on the CNS, in contrast to (+)-amphetamine (1.0 mg kg −1 , i.v.) which elicited marked CNS stimulation. These results support the conclusion that RX 781094 is a potent antagonist at central α 2 -adrenoceptors.

1 The effects of intravenous administration of the selective alpha 2-adrenoceptor agonists clonidine, UK 14,304 and guanoxabenz on rat pupil diameter were investigated. 2 In rats anaesthetized with pentobarbitone, each agonist produced a marked dose-related increase in pupil diameter; the rank order of potency was: clonidine greater than UK 14,304 greater than guanoxabenz. 3 Pretreatment with the selective alpha 2-adrenoceptor antagonist, RX 781094 (0.5 mg/kg, i.v.), produced a parallel 30-40 fold shift to the right of the dose-pupil dilator response curves for the three agonists. Yohimbine (1.5 mg/kg, i.v.) produced about a 10 fold rightward shift of the dose-response curve for guanoxabenz. In contrast, the alpha 1-selective antagonist, prazosin (0.5 mg/kg, i.v.), failed to affect the dose-response relation for guanoxabenz. 4 Several antagonists of varying selectivities towards alpha 1- and alpha 2-adrenoceptors were tested for their ability to reverse the maximal mydriasis induced by guanoxabenz (0.3 mg/kg, i.v.). The rank order of potency of the antagonists producing a 50% reversal of this effect was: RX 781094 greater than yohimbine greater than piperoxan = rauwolscine greater than mianserin greater than RS 21361. Neither corynanthine nor prazosin reversed the guanoxabenz-induced mydriasis. 5 Topical application of RX 781094 (0.1 to 3% w/v solutions) onto one eye produced a slow reversal of guanoxabenz-induced mydriasis; the time course and degree of reversal were virtually the same in both eyes. 6 Intracerebroventricular administration of RX 781094 (1.25-15 micrograms total dose) caused a rapid dose-related reversal of the maximal mydriasis induced by guanoxabenz (0.3 mg/kg, i.v.). 7 Guanoxabenz (0.3 and 1.0 mg/kg, i.v.) did not produce any dilation of the physostigmine-constricted undamaged pupil of the pithed rat. Intravenous adrenaline was found to produce a small mydriatic effect, while atropine completely antagonized the effects of physostigmine in this preparation. 8 These results indicate that alpha 2-adrenoceptor agonists induce mydriasis in the rat through a central alpha 2-adrenoceptor mechanism. However, the site of action within the central nervous system remains to be determined.

1 The selectivity of a number of agonists for peripheral alpha 1- and alpha 2- adrenoreceptors was determined and related to their cardiovascular effects in pentobarbitone-anaesthetized rats. 2 In isolated tissues, presynaptic alpha 2- and postsynaptic alpha 1-adrenoreceptor activity was determined on the rat vas deferens and anococcygeus muscle respectively. Xylazine, guanabenz, guanoxabenz and guanfacin were more selective, whilst lofexidine, tiamenidine, naphazoline, oxymetazoline and St 91 were less selective than clonidine for presynaptic alpha 2-adrenoreceptors. 3 The anococcygeus muscle of the pithed rat was used to determine the alpha-adrenoreceptor selectivity of the compounds in vivo. The selectivities were similar to those obtained in vitro. 4 Following intravenous administration to pentobarbitone-anaesthetized rats guanabenz and guanfacin caused small transient pressor responses and a prolonged secondary hypotension. In contrast oxymetazoline and St 91 produced marked initial pressor responses and the secondary reduction in blood pressure was absent. Clonidine and tiamenidine produced marked initial pressor responses followed by secondary hypotensive effects. All compounds reduced heart rate. 5 Intracerebroventricular (i.c.v.) administration of clonidine, guanabenz and guanfacin elicited falls in blood pressure and heart rate. In contrast blood pressure was either unaffected or elevated following i.c.v. administration of St 91, oxymetazoline and tiamenidine. 6 It is concluded that in the normotensive rat, central alpha 2-adrenoreceptors play a major role in the hypotensive effects of clonidine and related compounds.