Search

Following transient focal stroke, rapid accumulation and activation of neutrophils in the ischaemic region is deleterious due to release of reactive oxygen species and myeloperoxidase (MPO). The purpose of this study was to examine whether AM-36, both a Na+ channel blocker and an antioxidant, afforded neuroprotection by modulating neutrophil accumulation into brain, following endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAo) in conscious rats.AM-36 was administered at 3 and 24 h after ET-1-induced MCAo. Functional recovery was determined using grid-walking and cylinder tests. Image analysis of brain sections was used to determine infarct volume. The effect of AM-36 on neutrophil infiltration and their interaction with macrophages was examined in rats at 48 h following MCAo by both an MPO assay and double-label immunofluorescence. Blood brain barrier (BBB) breakdown was measured by the area stained by intravenous Evans Blue.AM-36 reduced functional deficits in both tests such that no difference existed from pre-ischaemic values at 48 h. Neutrophil infiltration, assessed by MPO activity, and infarct volume were significantly reduced following AM-36 administration by 54 and 60% respectively. Similarly, immunofluorescence revealed that AM-36 reduced neutrophil infiltration by approximately 50% in selected brain regions, when compared to controls, and also modulated macrophage phagocytosis of neutrophils. Breakdown of the BBB was significantly reduced by 60% following AM-36 treatment.These findings suggest that AM-36 can directly modulate the neutrophil inflammatory response and reduce BBB breakdown following MCAo.

Neuropeptide Y (NPY), one of the most abundant neuropeptides found in the central nervous system (CNS), has been implicated in the regulation of many autonomic functions, including cardiovascular control and the central stress response. The present study represents a detailed investigation of the effects of acute and chronic restraint stress on the expression of the mRNA encoding the NPY precursor, prepro-NPY, in the CNS of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) using in situ hybridization histochemistry. Basal (unstressed) levels of prepro-NPY mRNA expression were found to be significantly increased in the hypothalamic arcuate nucleus of SHR compared to WKY rats, with similar levels of prepro-NPY mRNA expression found in the remaining central nuclei. Following exposure to both acute and chronic restraint, significant changes in prepro-NPY mRNA expression were found in a variety of central regions in both strains, including the arcuate nucleus and hippocampus (both strains), medial amygdala and cortex (WKY only), and dentate gyrus, nucleus of the solitary tract and ventrolateral medulla (SHR only). A comparison of the temporal response to restraint revealed that significant differences between strains existed in regions such as the arcuate nucleus, hippocampus and dentate gyrus, providing further evidence that hypertensive rats apparently have an impaired neural stress response. The present study demonstrates that exposure to restraint results in significant changes in prepro-NPY mRNA expression in specific nuclei of both WKY and SHR that are components of not only the central circuitry regulating the stress response, but also the neural network modulating autonomic function.

The neuropeptide galanin (GAL) has been implicated in the neural response to a number of stressors including restraint; however, the effect of restraint stress on GAL receptor density in the central nervous system (CNS) has not been investigated. Normotensive (Wistar-Kyoto; WKY) and hypertensive (spontaneously hypertensive; SHR) rats were subjected to a daily 60-min restraint stress paradigm for 0 (control), 1, 3, 5 or 10 consecutive days, and the density of [125I]-GAL binding sites following exposure to restraint was compared between strains using quantitative autoradiography. Significant differences in basal (no stress) levels of GAL receptor density between WKY and SHR were detected in regions such as the central nucleus of the amygdala (Ce) and ventromedial hypothalamus (VMH) (P0.05). In WKY, restraint stress (1 day) induced significant decreases in GAL receptor density in forebrain regions such as the Ce (-41%) and medial nucleus of the amygdala (-41%) (P0.05). Chronic restraint (10 days) did not induce significant decreases in these nuclei in WKY, indicating that forebrain neurons containing GAL receptors in WKY possessed a functional ability to adapt to repeated restraint. In addition, restraint stress induced significant decreases in GAL receptor density in SHR in regions such as the lateral parabrachial nucleus (-43%; 5 days of restraint) and hypoglossal nucleus ( approximately -18% for entire restraint period) (P0.05). In conclusion, restraint stress resulted in region- and strain-specific alterations in GAL receptor density, some of which may contribute to the altered stress response previously observed in hypertensive rats. The results clearly support the hypothesis that neuropeptides such as GAL are an integral component of the neural response to psychological stress, although the functional significance of the changes in GAL receptor density described in this study awaits elucidation.

Relapse after abstinence can be modelled in rats using an alcohol deprivation effect (ADE) of enhanced ethanol consumption after a period of enforced abstinence from ethanol; however, not all rat strains display such an effect. We wanted to examine the effect of naltrexone on ethanol consumption by ethanol-preferring Fawn-Hooded (FH) rats using such a model.FH rats were given continual free-choice access to a 5% ethanol solution or water (4 weeks) followed by 2 weeks of water alone. At the end of this abstinence period, osmotic minipumps were implanted subcutaneously to deliver saline (n = 4) or naltrexone (n = 4; 8.4 mg/kg/day for 4 weeks). After recovery from surgery, the rats were again given access to 5% ethanol under the same free-choice conditions (4 weeks). A third group of age-matched controls drank only water during the behavioral trial. At the end of the behavioral trial, the rats were decapitated and an autoradiographic examination was made of micro-opioid receptor density through the forebrain using the ligand [125I]FK-33824.First, a period of enforced abstinence from ethanol consumption caused a significant (p0.05) and prolonged increase in ethanol preference (+18%) and decrease in water consumption (-53%), although the volume of ethanol consumed (ml/day) did not vary, indicating an atypical ADE in this rat strain. Second, naltrexone significantly (p0.05) decreased ethanol consumption by the FH rats in terms of absolute amount of ethanol consumed and preference for ethanol solution, but this effect of naltrexone diminished over time, concurrent with a robust and significant elevation in micro-opioid receptor density in all brain regions examined (p0.05). Finally, ethanol consumption alone also upregulated micro-opioid receptor density relative to nondrinking controls in a number of brain regions, which included the nucleus accumbens (+29%) and caudate-putamen (+15%,p0.05), but decreased micro-opioid receptor density in other regions including the substantia nigra pars reticulata, which was suggestive of an indirect effect on micro-opioid receptors.The data suggest that continual long-term naltrexone treatment may not be effective in the treatment of alcoholism, possibly because of the induced increase in micro-opioid receptor density.

1. The objective of this study was to determine the influence of gender on the development of renal hypertension in Sprague-Dawley rats using the Goldblatt two-kidney, one-clip (2K1C) model. In addition, this study examined the effect of ovariectomy upon the development of hypertension in female rats.2. At 10 weeks of age, male, intact female and ovariectomized female rats underwent clipping of the right renal artery or sham operation. Tail-cuff plethysmography was used to monitor the systolic blood pressure of all animals for 7 weeks post-clipping or sham operation. Rats were sub-grouped according to whether or not they developed hypertension (systolic blood pressure=150 mmHg).3. Within 2 to 3 weeks of clipping, hypertension was induced in only 53% (n=120) of the intact female 2K1C rats, but in 83% (n=18) of the male and 78% (n=18) of the ovariectomized female rats.4. Seven weeks after right renal artery clipping, plasma renin activity was determined in a subset of each group and was found to be 5-6 fold higher in male (17.29+/-4.04 ng angiotensin I.h-1.ml-1) and ovariectomized female (9.71+/-1.25 ng angiotensin I.h-1.ml-1) hypertensive rats compared with their respective normotensive or sham-operated counterparts (3.39+/-0.58 ng angiotensin I.h-1.ml-1 and 1.60+/-0.41 ng angiotensin I.h-1.ml-1 respectively) (P0.05, analysis of variance). In contrast, the plasma renin activity measured in intact female hypertensive rats was not significantly different from that measured in the corresponding 2K1C normotensive or sham-operated groups.5. These results indicate that the success rate of inducing renal hypertension in Sprague-Dawley rats is higher in males than in intact females. Furthermore, these results suggest that the induction of 2K1C hypertension may be influenced by ovarian hormones.

The present study has employed immunocytochemistry on free-floating sections of adult rat medulla oblongata to characterise the distribution of nitric oxide synthase- (NOS), adenosine deaminase- (ADA) and neuropeptide Y- (NPY) immunoreactivity (IR) throughout the entire rostro-caudal axis of the nucleus tractus solitarius (NTS). In addition, unilateral nodose ganglionectomy was performed in a group of rats to determine whether any observed immunoreactivity was associated with central vagal afferent terminals. NOS-IR was found throughout the entire NTS, in cells, and both varicose and non-varicose fibres. Furthermore, unilateral nodose ganglionectomy resulted in a clear reduction in NOS-IR (visualised with diaminobenzidine) in a highly restricted portion of the ipsilateral medial NTS. Similarly, ADA- and NPY-containing cells, fibres and terminals were also found throughout the adult rat NTS. However, following unilateral nodose ganglionectomy, there was no apparent reduction in either ADA-IR or NPY-IR on the denervated side of the NTS. These data indicate a role for nitric oxide, purines and neuropeptide Y as neuromodulators within the rat NTS, although only nitric oxide appears to be primarily associated with vagal afferent input. Adenosine deaminase and neuropeptide Y-containing neurons appear to be predominantly postsynaptic to vagal input, although their possible association with vagal afferents cannot be completely excluded.

CV-11974, a newly developed angiotensin II (AII) subtype 1 (AT1) receptor antagonist, lowers mean arterial pressure (MAP) in various hypertensive animal models. The aim of this study was to examine the regional haemodynamic effects of CV-11974, which contribute to its hypotensive action, in conscious, chronically instrumented spontaneously hypertensive rats (SHR) and renal hypertensive 2-kidney, 1-clip (2K1C) rats, as well as in appropriate normotensive Wistar Kyoto and sham-operated rats. In SHR, CV-11974 (0.1 mg/kg, i.v.) lowered MAP and increased renal blood flow and conductance, indicating renal vasodilatation. However, there were minimal changes in blood flow in the mesenteric or hindquarters vascular beds. By contrast, in 2K1C rats, CV-11974 caused marked hypotension which was associated with substantial vasodilatation in all three vascular beds. The cardiovascular effects of exogenous AII were also blocked by CV-11974. These results suggest that there are subtle differences in the haemodynamic profile of CV-11974 in the two hypertensive rat models, despite similar resting MAP, since this compound caused a relatively selective renal vasodilatation in SHR, but more widespread vasodilatation in 2K1C rats.

The activity of baroreceptor reflexes and cardiopulmonary reflexes was examined in conscious spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats. The baroreceptor heart rate reflex, elicited by phenylephrine- and nitroprusside-induced changes in blood pressure, had a reduced range and lower heart rate plateau in SHR than in WKY rats, which suggests impaired vagal control of the heart rate in SHR. Cardiopulmonary receptor reflex activity was assessed by intravenous injections of phenyldiguanide which evoke the Bezold-Jarisch reflex. Phenyldiguanide elicited dose-dependent bradycardic and hypotensive responses in WKY rats, but these were significantly attenuated in SHR. This is the first demonstration of impaired Bezold-Jarisch responses in conscious SHR and provides evidence of both impaired vagally mediated arterial baroreceptor activity and impaired cardiopulmonary receptor activity in this rat strain.

The sections in this article are: 1 Terminology 2 Historical Developments 2.1 Identification of the Sympathetic Transmitter 2.2 Tissue Receptors 2.3 Autonomic Neuroeffector Junctions 2.4 Biochemical Mechanisms 3 Distribution and Storage of Catecholamines 3.1 Tissue Distribution 3.2 Cellular Distribution of Catecholamines 3.3 Subcellular Distribution 3.4 Quantitative Aspects of Transmitter Distribution 3.5 Summary 4 Enzymes Involved in Catecholamine Metabolism 4.1 Biosynthetic Ezymes 4.2 Degradative Enzymes 4.3 Regulation of Catecholamine Turnover 4.4 Summary 5 Release and Inactivation of Catecholamines 5.1 Quantal Liberation of Transmitter by Exocytosis 5.2 Inactivation of Transmitter 5.3 Circulating Catecholamines and Dopamine β-hydroxy lase 6 Adrenotropic Receptors 6.1 Classification of Adrenotropic Receptors 6.2 Receptor Topography 6.3 Receptor Composition 7 Life Cycle of Noradrenergic Synaptic Vesicles 7.1 Origin of Vesicles Within Noradrenergic Neurons 7.2 Turnover and Fate of Synaptic Vesicles 7.3 Topochemical Model of Noradrenergic Transmission

—A study has been made of the catechol-O-methyl transferase activity of some peripheral tissues after sympathetic denervation. A fall in catechol-O-methyl transferase activity was found in some organs, e.g. rat and rabbit vas deferens, cat nictitating membrane and rabbit submaxillary gland but not in mouse heart and spleen. It was found that suboptimal concentrations of S-adenosylmethionine did not reveal a significant difference between normal and denervated organs but at optimal concentrations a fall was seen in some organs. Catechol-O-methyl transferase activity was present in bovine splenic nerve and in adrenal medulla. It is suggested that the fall in enzyme activity after denervation indicates a neuronal cellular localization. A kinetic study of catechol-O-methyl transferase from normal and denervated rat vas deferens suggested that the neuronal and extraneuronal catechol-O-methyl transferase had different kinetic properties and an estimation of the kinetic constants of the neuronal enzyme was made.

1. The question of cellular localizations of monoamine oxidase and catechol-o-methyl transferase, enzymes involved in the metabolism of noradrenaline, has been investigated by following the changes in the enzyme activity of the smooth muscle of the cat nictitating membrane after sympathetic denervation. Any falls in enzyme activity coinciding with the time course for degeneration of the sympathetic nerve endings (2-3 days) can reasonably be ascribed to a former localization of the enzyme within the sympathetic nerve.2. Monoamine oxidase activity of the inferior and medial smooth muscle was significantly reduced 3 days after sympathectomy and remained reduced 14 days after sympathectomy.3. A correlation was found between the fall in monoamine oxidase activity and the endogenous noradrenaline concentration of the contralateral normal muscle, indicating that the higher the endogenous noradrenaline concentration, the greater the fall in monoamine oxidase activity after denervation.4. It was concluded that the fall in monoamine oxidase activity after denervation indicated a former localization of the enzyme within the sympathetic nerve endings.5. The fall in monoamine oxidase activity of the denervated muscle was significantly less measured with benzylamine as a substrate than with tyramine. This suggested that the neuronal monoamine oxidase may have different properties than the extraneuronal enzyme.6. A small fall in the catechol-o-methyl transferase activity of denervated smooth muscle was found and a correlation between the fall in enzyme activity and endogenous noradrenaline indicated that in this smooth muscle a proportion of the catechol-o-methyl transferase activity may be of presynaptic origin.

—Monoamine oxidase activity of peripheral organs of various species has been examined after surgical, chemical and immunological sympathectomy to assess the proportion of enzyme activity in adrenergic neurons and in extraneuronal cells. Significant falls in monoamine oxidase activity of vas deferens, submaxillary gland, iris and spleen were seen after sympathetic denervation although not in heart, small intestine and kidney. It was suggested that a correlation exists between the extent of the fall in monoamine oxidase activity after sympathectomy and the density of sympathetic innervation of the control organ. Studies of monoamine oxidase activity in vas deferens after inhibition with clorgyline suggested multiple forms of monoamine oxidase. Differences in inhibitor sensitivity, substrate specificity and thermal inactivation of monoamine oxidase in normal and denervated vas deferens were found and it was suggested that differences exist in the properties of the neuronal and extraneuronal monoamine oxidase.

1. The rate of uptake of (+)- and (-)-noradrenaline was measured in isolated perfused hearts of reserpine treated rats, mice and guinea-pigs by fluorimetric analysis of the removal of catecholamine from the perfusion medium. In rat and mouse heart (-)-noradrenaline was taken up significantly more rapidly than (+)-noradrenaline, but no stereochemical specificity was found for noradrenaline uptake in guinea-pig hearts.2. Using radioactively labelled (+)-(14)C-noradrenaline and (-)-(3)H-noradrenaline, the kinetic constants for uptake into noradrenaline-containing and dopamine-containing synaptosomes from rat brain were determined. The uptake by noradrenaline terminals in the hypothalamus had a higher affinity for (-)-noradrenaline than for the (+)-isomer, but no differences in affinity were found for uptake into dopamine terminals.3. When equal amounts of labelled (+)- and (-)-noradrenaline were injected in vivo in double isotope experiments, the (+)-isomer disappeared more rapidly than the (-)-isomer from rat heart and spleen, but no significant differences were found between the rates of disappearance of the two isomers from rat brain or in the whole mouse.4. Analysis of the radioactive metabolites of the two isomers of noradrenaline after administration of mixed doses of the labelled substances showed that a significantly higher proportion of (+)-noradrenaline was metabolized to normetanephrine than of (-)-noradrenaline, in the whole mouse, rat heart and rat brain.

The frequency and duration of rapid eye movement (REM)-sleep episodes were examined in normotensive rats during and after cessation of continuous infusion of either clonidine (10 micrograms/kg/h) or the oxazoline derivative S3341 (50 micrograms/kg/h) for 10 days. Both compounds abolished REM-sleep during the infusion. On cessation of the clonidine infusion there was a rebound increase in the frequency and duration of REM-sleep episodes. These episodes were associated with markedly exaggerated blood pressure fluctuations. On cessation of the S3341 infusion, the frequency and duration of REM-sleep episodes returned to control levels. Blood pressure responses were not different from controls. These post-infusion differences may be related to the greater degree of selectivity of S3341 for the alpha 2-adrenoceptor rather than the alpha 1-adrenoceptor subtype.

The disposition of the potent antihypertensive drug clonidine has been poorly understood through the lack of a convenient and sensitive assay. A radioimmunoassay for clonidine has been developed and is capable of detecting as little as 10 pg of clonidine. 2,6-Dichlorophenyl-guanidine, a known metabolite of clonidine, did not cross-react with the antiserum whereas another metabolite, 4-hydroxyclonidine, was as potent as clonidine in displacing labeled clonidine from the antibody. However, a simple solvent extraction step before the radioimmunoassay selectively extracted clonidine from a mixture of clonidine and 4-hydroxyclonidine in alkaline plasma and this procedure permitted a specific assay for clonidine. The plasma levels of clonidine in rats after the administration of a hypotensive dose (100 microgram/kg i.v.) were determined by radioimmunoassay and these data indicated that the disposition of clonidine conforms to an open two-compartment, pharmacokinetic model. Clonidine rapidly accumulated in the brain as shown by the attainment of peak concentrations within 2 min of i.v. injection.

The intracerebroventricular injection of the mast cell degranulator, compound 48/80 (C48/80, 10 micrograms/kg), produced a marked behavioural syndrome in rats which included head and body shakes, paw tremor, excessive grooming, unusual posture and gait, mild diarrhoea, piloerection, extreme agitation and irritability to touch, sedation and catatonia. Fifteen minutes after C48/80, the histamine concentrations were decreased significantly in all brain regions examined, i.e. the cortex, cerebellum, midbrain, medulla oblongata-pons (MO-P) and hypothalamus. The noradrenaline (NA) concentrations were decreased in the cerebellum, hypothalamus and MO-P, whereas the dopamine (DA) content was decreased in the MO-P only. The concentrations of serotonin were not affected. As such, the behaviours following the acute degranulation of brain mast cells by C48/80 may result predominantly from the release of histamine and possibly NA and DA.

Neuropeptide (NPY) and norepinephrine (NE) concentrations were measured in the right ventricle (RV) of rats with cardiac failure resulting from coronary artery ligation. RV NE concentrations fell significantly in rats with infarcts while RV NPY concentrations were unaltered.

The presence of a methyltransferase enzyme in human red blood cells (RBCs) capable of S-methylation of captopril is described. The apparent Michaelis-Menten (Km) constant for captopril was 0.5 mM and the maximum velocity (Vmax) was 0.391 pmoles S-methylcaptopril (mg protein)-1 min-1. There is some evidence presented to show that S-methylcaptopril inhibited its own formation with a ki value of 5.81 mM. Captopril thiol methyltransferase activity was also examined in rat tissues and was found to be present in all tissue studied. Subcellular localisation studies in rat liver suggest that the enzyme was microsomal in origin. The order of activity was liver greater than heart greater than spleen greater than lung greater than kidney much greater than RBC (rat). This tissue distribution was quite different from previous studies using other thiol substrates and is consistent with more than one form of thiol methyltransferase enzyme in tissues.

The effects of continuous clonidine infusion (10 micrograms/kg/h for 10 days) and the cessation of this infusion on the circadian body temperature rhythm of the spontaneously hypertensive (SH) rat were examined. This circadian rhythm was blunted significantly during the infusion of clonidine. The fall in body temperature which normally occurs at the onset of each light phase was attenuated during the clonidine infusion, and as such, these rats displayed a relative hyperthermia over the light but not the dark phases. On cessation of infusion (24:00 h), a distinct hyperthermia occurred within the immediate dark phase and the subsequent light phase. The results demonstrate that the circadian control of body temperature is disturbed both during and after continuous clonidine infusion.

A major problem in the treatment of clinical epilepsy with benzodiazepines is the development of tolerance. This is most marked with the 1,5-benzodiazepine, clobazam, but is also noted with the 1,4-benzodiazepine, clonazepam. This study examined the effects of chronic treatment with clobazam, clonazepam and the non-benzodiazepine sodium valproate on the amygdaloid kindled rat with a view to developing an animal model of tolerance. Twice daily intraperitoneal injections of vehicle (1 mL/kg), clonazepam (0.3 mg/kg), clobazam (4.0-8.0 mg/kg) or sodium valproate (200 mg/kg) were given to fully kindled rats for 12 days, in the case of clonazepam and valproate, and for 16 days for clobazam. All rats were stimulated 30-60 minutes (depending on the drug) after the morning dose. The amygdala after-discharge duration (recorded on the EEG) and the seizure stage were determined. While all three drugs initially blocked the kindled seizure, tolerance developed to clonazepam and clobazam but not to valproate. Statistical analysis of the results showed a significant linear trend in the development of tolerance to clonazepam after day 5, but tolerance to clobazam appeared in a more abrupt manner after day 2. The clobazam group were then given a further 2 days of treatment with clonazepam (0.3-0.6 mg/kg) and this blocked the kindled seizures. The amygdaloid kindled rat is a suitable model for studying tolerance to the anticonvulsant effects of benzodiazepines. Further experiments are currently in progress to examine the effectiveness of change-over therapy between clobazam and clonazepam as a means of overcoming tolerance.

Studies using a sensitive radioenzymatic assay for plasma noradrenaline suggest there is a selective overactivity of the sympathetic nervous system in essential hypertension. Methodology which allows the study of local sympathetic turnover in CNS nuclei and peripheral blood vessels is described. This approach has been used to study the non-innervated sympathetic turnover phaeochromocytoma. It is suggested that studies of local regulatory mechanism in neurotransmitter release are required to give a greater understanding of the central and peripheral role of the sympathetic nervous system in the pathogenesis of hypertension.

In rats, the existence of circadian rhythms in many physiological, biochemical and behavioural systems has been well established (Sheving et al, 1968; Rusak and Zucker, 1975; Rosenfeld and Rice, 1979). However, controversy exists as to whether or not such rhythms exist for arterial blood pressure. Although Friberg et al (1982) reported that the blood pressure of normotensive rats was higher during the dark phase than the light phase, Norman et al (1980) did not find evidence for a circadian rhythm in this parameter. Nevertheless, it appears that the cardiovascular effects of drugs could be complicated by the presence of circadian rhythms in vagal and sympathetic activity, hormone release and the responsiveness of the heart and blood vessels (Rusak and Zuker, 1979; Hossman et al., 1980). For example, Brown and Lever (1982) demonstrated that the pressor effects of a continuous infusion of angiotensin II in normotensive rats were more dramatic during the dark phases of a four day infusion period.

The interaction between some examples of mononuclear and binuclear DNA-intercalating antitumor agents and alpha- and beta-adrenoceptors has been studied using radioligand-binding assays. Competition for 125I-BE 2254, [3H]rauwolscine, and (-)-[3H]dihydroalprenolol binding was used to assess affinity for alpha 1-, alpha 2-, and beta-adrenoceptor-binding sites, respectively. Two homologous series of alkyl-linked diacridines and diquinolines were found to interact poorly with beta-adrenoceptors, with only the largest members having appreciable affinity. By contrast, these compounds bind strongly and in a complex manner to alpha 1- and alpha 2-adrenoceptors. The affinity of diacridines for both alpha-adrenoceptor classes has a parabolic dependence on alkyl chain length with the hexyl and pentyl derivatives being the most potent at the alpha 1- (Ki = 11.5 +/- 2.3 nM) and alpha 2- (Ki = 143 +/- 26 nM) binding sites, respectively. The dependence of inhibition constants on linker chain length for the diquinolines is more complicated, with the ethyl- and heptyl-linked dimers having the greatest affinity for each alpha subclass. There is a nadir in affinity for the pentyl and butyl ligands and an increase in dissociation constant for octyl and longer homologues. Thus, the ethyl diquinoline has Ki values of 6.6 +/- 1.2 and 110 +/- 14 nM for the alpha 1- and alpha 2- adrenoceptors, respectively, and, correspondingly, the heptyl derivative has values of 39 +/- 4 and 51 +/- 1 nM. These findings are discussed with respect to a model of the alpha-adrenoceptor in which the radioligand-binding site is situated in a trench or cleft, surrounded by a flat surface bounded by walls. Daunomycin was found to have no affinity for adrenoceptors of any type and mitoxantrone similarly fails to interact with alpha 2- and beta-adrenoceptors, but binds to the alpha 1 subclass with an inhibition constant (Ki) of 3930 +/- 420 nM. Bisantrene also has no affinity for beta-adrenoceptors but binds to alpha 1- and alpha 2- adrenoceptors with Ki values of 145 +/- 24 and 2310 +/- 430 nM, respectively. Among the mononuclear acridine drugs studied, only nitracrine shows detectable interaction with beta-adrenoceptors (Ki = 760 +/- 50 nM). This compound, like bisantrene, has high affinity for the alpha 1-adrenoceptor (Ki = 131 +/- 17 nM) and moderate affinity for the alpha 2 subclass (Ki = 2180 +/- 500 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

alpha-Adrenoceptors in brain can be studied readily by radioligand binding techniques. This provides valuable information not only on the distribution of receptors in brain regions, but also on the regulation of receptors. The usefulness of this technique is dependent in part on a radioligand with high specificity for the receptor under study. Our studies have shown that 3H-clonidine does not bind exclusively to alpha 2-adrenoceptor subtypes, but also interacts with alpha 1-adrenoceptors. In contrast, 3H-guanfacine labels a high affinity alpha 2 subtype with good selectivity, but 3H-lofexidine probably labels with both alpha 2 and alpha 1-adrenoceptor binding sites.

Tritium-labeled preparations of the selective alpha 2-adrenoceptor agonist, clonidine, and the selective alpha 1-adrenoceptor antagonists, prazosin and WB 4101, were examined for their suitability as ligands for receptor assay. In the rat brain, 3H-clonidine bound specifically with high affinity to membrane sites, and drug displacement studies indicated that these were alpha 2-adrenoceptors. The structural requirements for this receptor were defined by examining a range of clonidine analogues. 3H-Clonidine binding to peripheral tissues of the rat was very low, although peripheral tissues from guinea pig exhibited higher binding. Specific 3H-clinidine binding was not reduced after chemical sympathectomy, suggesting that the binding site, although having characteristics of an alpha 2-adrenoceptor, was not located presynaptically. Studies with 3H-WB 4101 in rat cerebral cortex indicated that this ligand bound to an alpha 1-adrenoceptor which was independent of the 3H-clonidine-binding site. 3H-Prazosin was a ligand for alpha 1-adrenoceptors in guinea pig brain and also in membranes from seminal vesicle. However, in the latter tissue, characterization of the binding site was hampered by a high degree of nonspecific binding (50% of total binding).

1. The combined use of alpha-methyldopa and L-alpha-methyldopa hydrazine (a peripheral decarboxylase inhibitor) has been studied, in a double-blind cross-over comparison, with alpha-methyldopa and L-alpha-methyldopa hydrazine placebo in the treatment of eight patients with essential hypertension. 2. L-alpha-methyldopa hydrazine did not enhance the antihypertensive effect of alpha-methyldopa. This suggests that because methyldopa can inhibit its own decarboxylation, peripheral decarboxylation is not an important metabolic pathway for methyldopa and elevated brain levels of methyldopa do not necessarily result in elevated brain levels of methyldopamine.

—A surgical technique for sympathetically denervating the vas deferens has been evaluated biochemically. A slight fall in soluble muscle protein content and no significant change in DNA content of the operated vas deferens were found. This indicates that the surgical procedure causes only a slight degree of tissue damage and may be useful for investigating the cellular localization and properties of noradrenaline metabolizing enzymes. In three species examined (rat, guinea pig and rabbit), monoamine oxidase activity of the vas deferens fell by approximately 50 per cent after denervation. The time course of the fall in monoamine oxidase activity of rat vas deferens was parallel to that of the disappearance of noradrenaline suggesting that this proportion of the total enzyme activity had a neuronal localization. The remaining enzyme activity is presumably located extraneuronally. Significant falls in catechol-O-methyl transferase activity were found in rat and rabbit vas deferens after denervation but not in guinea pig. The rabbit and rat vas deferens had respectively approximately 60 and 30 per cent of the catechol-O-methyl transferase activity associated with the sympathetic nerves. A complete loss of DOPA decarboxylase and tyrosine hydroxylase activities occurred in rat vas deferens after denervation, suggesting that these noradrenaline synthesizing enzymes have an entirely neuronal localization.

After differential centrifugation 24 per cent of the total MAO activity of rat liver homogenates and 42 per cent of that in vas deferens was recovered in a microsomal fraction, the remainder being recovered in a mitochondrial pellet. The distribution of enzyme activities known to be associated with the outer membranes of mitochondria (kynurenine hydroxylase and NADH 2 -cytochrome c reductase) was similar to that of MAO, but succinic dehydrogenase activity was almost entirely mitochondrial. When vas deferens homogenates were subjected to density gradient centrifugation, a small particle fraction containing MAO and NADH 2 -cytochrome c reductase activities could be clearly separated from a fraction containing noradrenaline storage particles. It is concluded that MAO activity is not specifically associated with noradrenaline storage particles, and that the MAO activity recovered in microsomal fractions may arise as an artefact when mitochondria are disrupted during tissue homogenization.

1. The uptake of (+/-)-(3)H-noradrenaline was studied in isolated perfused hearts of rat, mouse, guinea-pig, pigeon and toad (Bufo marinus), and the IC50 (concentration causing 50% inhibition) values for inhibition of uptake of (+/-)-(3)H-noradrenaline by (-)-noradrenaline were calculated. IC50 values ranging from 0.28 muM (rat heart) to 2.34 muM (toad heart) were found.2. In all species except the toad, (-)-noradrenaline showed a higher affinity than (-)-adrenaline for the uptake process, but the reverse was found for the toad heart.3. Mouse and pigeon hearts contained increasing amounts of metabolites of noradrenaline with increasing perfusion concentrations of noradrenaline, but the guinea-pig and toad hearts did not. The in vitro activities of noradrenaline catabolizing enzymes in heart homogenates were measured but did not explain the differences in the pattern of catabolism of noradrenaline found in the intact hearts of the different species.4. In all hearts except the toad, cocaine was an effective blocking agent for the uptake of (+/-)-(3)H-noradrenaline and led to an increase in (3)H-normetanephrine in these hearts. In the pigeon heart, cocaine plus phenoxybenzamine in the perfusate resulted in an inhibition of both (3)H-noradrenaline uptake and (3)H-normetanephrine formation.5. In guinea-pig and pigeon perfused hearts, the uptake of (3)H-noradrenaline into atria and ventricles reflected the relative concentrations of endogenous catecholamines in these regions, but this was not found for rat, mouse and toad hearts.6. It was concluded that species differences exist for both the accumulation and metabolism of catecholamines in isolated perfused hearts.

1. The question of cellular localizations of monoamine oxidase and catechol-o-methyl transferase, enzymes involved in the metabolism of noradrenaline, has been investigated by following the changes in the enzyme activity of the smooth muscle of the cat nictitating membrane after sympathetic denervation. Any falls in enzyme activity coinciding with the time course for degeneration of the sympathetic nerve endings (2-3 days) can reasonably be ascribed to a former localization of the enzyme within the sympathetic nerve.2. Monoamine oxidase activity of the inferior and medial smooth muscle was significantly reduced 3 days after sympathectomy and remained reduced 14 days after sympathectomy.3. A correlation was found between the fall in monoamine oxidase activity and the endogenous noradrenaline concentration of the contralateral normal muscle, indicating that the higher the endogenous noradrenaline concentration, the greater the fall in monoamine oxidase activity after denervation.4. It was concluded that the fall in monoamine oxidase activity after denervation indicated a former localization of the enzyme within the sympathetic nerve endings.5. The fall in monoamine oxidase activity of the denervated muscle was significantly less measured with benzylamine as a substrate than with tyramine. This suggested that the neuronal monoamine oxidase may have different properties than the extraneuronal enzyme.6. A small fall in the catechol-o-methyl transferase activity of denervated smooth muscle was found and a correlation between the fall in enzyme activity and endogenous noradrenaline indicated that in this smooth muscle a proportion of the catechol-o-methyl transferase activity may be of presynaptic origin.