Metabolic Consequences of Inhibition of Cerebral Adenosylmethionine Decarboxylase by Methylglyoxal bis(Guanylhydrazone)

Abstract: Effects of intracerebroventricularly injected methylglyoxal bis(guanylhydrazone) on polyamine metabolism in mouse brain were recorded during 180 h after a single dose of 3.4 μmol/kg body weight. Cerebral concentrations of 31 other amino compounds were also asayed during the experiment. The drug caused a significant inhibition of adenosylmethionine decarboxylase that lasted for 50 h, with the maximal decrease, about 70%, occurring between 5 and 10 h after injection. Significant decreases of brain spermidine and spermine concentrations were observed in three phases. Two transient decreases occurred at 10 and at 35 h, and a longer-lasting one between 60 and 100 h. Ornithine decarboxylase was stimulated within 5 h after the injection, reaching a maximal level about 30-fold above normal at 60 h, and returned to control level at 140 h. This stimulation was accompanied by significant accumulation of the reaction product, putrescine, in the brain. It was maximally > 10-fold above normal at 160 h, and was still significantly above control at the end of the observation period. The time course of changes in the parameters of polyamine metabolism was regarded as support of a previously presented hypothesis that limiting putrescine concentration may play a role in the regulation of cerebral polyamine metabolism. In addition, the present results emphasize the possibility that changes in the activities of catabolic reactions may also contribute to the regulation of cerebral polyamine concentrations. Of the 31 amino compounds analyzed, only the concentrations of ornithine, urea, glutamine, and glutamate showed significant changes from normal. Ornithine concentration first was significantly increased at 25 h, whereafter it decreased and was somewhat below normal for most of the period between 60 and 180 h. The urea concentration showed a tendency to increase throughout the experiment, being significantly elevated at the end. These changes were regarded as suggesting that the increased need for ornithine in putrescine synthesis is satisfied mainly by increased arginine uptake and degradation. The magnitude of urea accumulation suggested that metabolism of ornithine to glutamate was also accelerated. An unexpected shift toward glutamine in the glutamine/glutamate relationship was observed during the first 100 h. However, the total concentration of these two compounds was quite constant throughout the experiment. Inhibition of ornithine decarboxylase by intraventricular injection of 2-difluoromethylornithine was tried during the study, but sufficient doses could not be used without induction of acute side effects.     

Glutamic Acid Decarboxylase Activity of the Preoptic Area and Hypothalamus Is Influenced by the Serotonergic System

The effect of the serotonergic system on glutamic acid decarboxylase (GAD) activity of the preoptic area and the hypothalamus was studied in female rats on the day of proestrus. A circadian rhythm of GAD activity was observed with higher values in rats killed at 1130 h than in rats killed at 1500 h. In rats bearing lesions of the median raphe nucleus (MRn), a nucleus that sends 5-hydroxytryptamine nerve terminals to the areas under study decreased GAD activity. On the contrary, electrochemical stimulation of the MRn enhanced GAD activity in intact rats killed at 1500 h, but not in those killed at 1130 h, an effect that was prevented by the injection of the 5-hydroxytryptamine antagonist, methysergide. Furthermore, the injection of 5-hydroxytryptamine into the third ventricle, either in intact rats in the afternoon or in MRn-lesioned rats in the morning, also increased GAD activity. The results of the present study suggest that activation of the serotonergic system increases GAD activity in the preoptic area and hypothalamus.     

Glutamate Decarboxylase Distribution in Discrete Motor Nuclei in the Cat Brain

Abstract: The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising γ-aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro-caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.     

Comparative Characterization of Glutamate Decarboxylase in Crude Homogenates of Oviduct, Ovary, and Hypothalamus

Some biochemical characteristics of L-glutamate decarboxylase (GAD) were compared using crude homogenates of the rat oviduct, ovary, and hypothalamus. As estimated by the measurement of CO2 production, the specific activities of oviductal and ovarian GAD were about 10 and 3% of the hypothalamic value, respectively. Stoichiometric formation of gamma-aminobutyric acid (GABA) and CO2 from L-glutamate could be observed in oviduct and hypothalamus, while in ovarian homogenates the production of CO2 was more than nine times that of GABA. Hypothalamic and tubal GAD showed similar time course, temperature dependence, and pH dependence. The dependence on protein concentration and on exogenous cofactor supply was also similar in these two tissues. The kinetic constant for L-glutamate as a substrate was nearly the same in oviduct (1.30 mM) and hypothalamus (1.64 mM). The responsiveness of tubal and hypothalamic GAD to various inhibitors was also similar. The present findings indicate that the oviductal and the hypothalamic GAD may be identical, and they suggest a possible GABAergic innervation of the Fallopian tube.     

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Abstract: In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (～ 15%) of collagen-tailed forms (mostly A₁₂ and A₈), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m -NaCl, 5 mm -EDTA, 1% Triton X-100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G₄ and the monomeric G₁ forms, with a smaller proportion of the dimeric G₂ form. (3) The tetrameric enzyme exists as a minor soluble component (G^S₄) that does not interact with Triton X-100, and a major hydrophobic component (G^H₄) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G₁ form presents a marked hydrophobic character, as indicated by its interaction with Triton X-100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G₄) that does not interact with Triton X-100, and a major hydrophobic component (G^H₄, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent-free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris-HCl buffer produce a larger overall yield of G₁ form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G₁ form apparently derives in part from a pool of G^H₄ form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G₄) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex-tracts, in a manner that depends on the ionic conditions. In Tris-HCl buffer, G₁ is converted into a well-defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm -Ca_2- to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X-100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.     

Enkephalinergic Markers in Substantia Nigra and Caudate Nucleus from Parkinsonian Subjects

Marked reductions in opiate receptor binding (-42%), "enkephalinase" activity (-39%), and Met5-enkephalin levels (-72%) accompanied the well-established dopamine depletion in the substantia nigra pars compacta of Parkinsonian subjects. In contrast, enkephalinergic markers were not significantly modified in caudate nucleus.     

Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus

The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time-dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier-mediated transport system. The second component, which accounted for about 10-30% of the DA uptake at 2 microM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier-mediated system. The saturable, Na-dependent component had an apparent Km(DA) of about 0.5 microM. The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier-mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.     

A Cyclic AMP Mechanism Mediates the Serotonin-Induced Increase in Glutamic Acid Decarboxylase Activity in the Preoptic Area and Hypothalamus

Previous studies have shown that the injection of 5-hydroxytryptamine (5-HT) into the third ventricle of rats on the afternoon of proestrus increases glutamic acid decarboxylase (GAD) activity in the preoptic area and the hypothalamus. In the present report we examine the adenylate cyclase-cyclic AMP (cAMP) system as mediator of that effect. The increase in GAD activity induced by intraventricular injection of 5-HT was completely blocked by injecting an antiserum against cAMP into the third ventricle 30 min earlier, whereas an injection of serum from normal rabbits was ineffective. On the contrary, activation of adenylate cyclase activity by intraventricular injection of forskolin increased GAD activity, an effect that was also blocked by anti-cAMP serum. Anti-cAMP serum also lowered GAD activity in the preoptic area and hypothalamus when injected on the morning of proestrus but not when injected in the afternoon, when the values of GAD activity were already low. The results suggest that a cAMP mechanism may be involved in the changes in preoptic-area and hypothalamic GAD activity such as the rise in enzyme activity induced by intraventricular injection of 5-HT.     

Use of [3H] Spiperone for Labelling Dopaminergic and Serotonergic Receptors in Bovine Caudate Nucleus

Abstract— [³H]Spiperone binding has been used to study neurotransmitter receptors in bovine caudate nucleus in displacement and saturation binding experiments. Displacement curves for several antagonists are biphasic and can be analysed into contributions from dopaminergic and serotonergic sites. Antagonist binding at each class of sites follows the simple mass action equations for binding at a homogeneous set of sites (slope factors close to unity). Agonist displacement curves also indicate complex behaviour, but agonist binding to the dopaminergic sites alone exhibits heterogeneous properties (slope factors less than unity). Saturation binding experiments have been conducted on each class of site, defining dopaminergic binding of [³H]spiperone as that binding displaced by 0.1 m m -dopamine and serotonergic binding as that displaced by 0.3 μ m -mianserin. In each case, a single class of binding sites was detected: the binding parameters derived in this way have been used to calculate the proportions of the two classes of binding site observed in displacement experiments. Good agreement was obtained between calculated and observed values.     

Effects of Amphetamine on Carrier-Mediated and Electrically Stimulated Dopamine Release in Slices of Rat Caudate Putamen and Nucleus Accumbens

Abstract: The effects of (+)-amphetamine on carrier-mediated and electrically stimulated dopamine release were investigated using fast cyclic voltammetry in rat brain slices incorporating the nucleus accumbens, and in the caudate putamen. In the caudate putamen, dopamine release either increased with increasing frequency of local electrical stimulation (hot spots) or did not increase significantly (cold spots); dopamine release increased with increasing frequency of electrical stimulation in the nucleus accumbens. Local pressure application of (+)-amphetamine from a micropipette caused dopamine efflux at all sites examined, and this was not affected by sulpiride, indicating that efflux of dopamine caused by (+)-amphetamine is not regulated by dopamine D₂ autoreceptors. (+)-Amphetamine reduced single-pulse electrically stimulated dopamine release at all sites; sulpiride reversed this decrease, indicating that endogenous dopamine released by (+)-amphetamine activates dopamine D₂ autoreceptors. In nucleus accumbens and hot spots, (+)-amphetamine did not affect 20-pulse 50-Hz-stimulated dopamine release, whereas in cold spots it potentiated 20-pulse 50-Hz-stimulated dopamine release. We conclude that (+)-amphetamine modifies electrically stimulated dopamine release by uptake inhibition or by indirect activation of D₂ autoreceptors; the precise mechanism is determined by site and duration of electrical stimulation.     

Effect of Cholecystokinin Octapeptide on Endogenous Amino Acid Release from the Rat Ventromedial Nucleus of the Hypothalamus and Striatum

The sulphated octapeptide of cholecystokinin (CCK-8S) was found to cause a dose-dependent increase in the basal release of aspartate, glycine, and gamma-aminobutyric acid from the striatum and the ventromedial nucleus of the hypothalamus (VMH). No effect on amino acid release was observed after electrical (VMH) or potassium (striatum) stimulation. Experiments performed using the CCKB-selective antagonist L-365,260 and the CCKA-selective antagonist L-364,718 suggested that this action of CCK-8S was mediated via the CCKB receptor. The ability of CCK-8S to evoke amino acid release was not dependent on the presence of extracellular calcium, though the effect was abolished by tetrodotoxin. Inhibition of protein kinase activity by staurosporine prevented the excitatory effects of CCK-8S on amino acid release.     

Iron Deficiency Alters Discrete Proteins in Rat Caudate Nucleus and Nucleus Accumbens

Young rats (21 days old) made nutritionally iron deficient, by feeding them a semisynthetic diet containing skimmed milk for 5 weeks, had significantly lowered hemoglobin levels (5.2 +/- 4 g/100 ml). The nonheme iron content in caudate nucleus was decreased by 47%. The behavioral response of iron-deficient rats to apomorphine (2 mg/kg) and the density of 3,4-dihydroxyphenylethylamine (dopamine) D2 receptors, as measured by [3H]spiperone binding in caudate nucleus, were significantly reduced by 70 and 53%, respectively. The possibility that nutritional iron deficiency may affect protein content in brain was investigated by measuring the apparent concentration of proteins in caudate nucleus and nucleus accumbens from iron-deficient and control animals using two-dimensional gel electrophoresis. The data indicate that iron deficiency can affect content in these two brain regions. Significant changes in the content of 10 proteins were noted in the caudate nucleus and nucleus accumbens in iron-deficient rats. The albumin level was significantly increased in both regions studied, whereas the neuron-specific enolase level was increased in the nucleus accumbens and the glial fibrillary acidic protein level was reduced in the caudate nucleus. The significance of these protein content changes, as well as a reduction in content of a 94-kilodalton protein (a molecular size similar to that of the D2 dopamine receptor), remains to be established.     

Changes in Nine Enzyme Markers for Neurons, Glia, and Endothelial Cells in Agonal State and Huntington''s Disease Caudate Nucleus

Enzymes considered to be markers for neurons (angiotensin converting enzyme, thermolysin-like metalloendopeptidase, alanine aminopeptidase, and glutamate-oxaloacetate transaminase), glia (glutamine synthetase, pyruvate carboxylase, and beta-glucuronidase), and endothelial cells (alkaline phosphatase and plasminogen activator) were measured in caudate nucleus from 10 sudden death controls, eight agonal state controls, and 16 Huntington's disease patients. Glutamate-oxaloacetate transaminase was slightly reduced by agonal state. The four enzymes with a neuronal distribution were all correlatively reduced in Huntington's disease caudate nucleus. Glutamine synthetase activity was reduced and beta-glucuronidase mean activity increased over twofold in Huntington's disease caudate nucleus, with the two enzyme activities being inversely related. Pyruvate carboxylase was markedly affected by agonal state and was very variable in Huntington's disease caudate nucleus. The two endothelial enzymes were unaltered in Huntington's disease caudate nucleus. The findings are indicative of neuronal loss, an increased proportion of altered glia, and also of maintained vasculature in Huntington's disease caudate nucleus. Measurement of enzyme activities can help to delineate the types of cell altered in Huntington's disease.     

Distribution of Putrescine in Rat Brain Measured by Gas Chromatography-Mass Spectrometry

We measured putrescine levels in minute sites of single rat brains using a sensitive, specific assay involving gas chromatography-mass spectrometry. The putrescine level was measured in 20 sites of single rat brains: three sites in the cerebral cortex, six sites in the hypothalamus, three sites in the basal ganglia, three sites in the thalamus, three sites in the limbic system, and two sites in the cerebellum. The level of putrescine was very high in the hypothalamus, high in the basal ganglia and limbic system, and low in the thalamus, cerebellum, and two of the three sites in the cerebral cortex. The highest levels were in the anterior hypothalamic area and the lateral hypothalamic area, and the lowest levels were in the vermis and the lobe of the cerebellum.     

The Effect of Mesencephalic Lesions on Tyramine and Dopamine in the Caudate Nucleus of the Rat

Abstract: The dopamine (DA)-containing nerve terminals in the caudate nucleus arise from cell bodies located in the substantia nigra (pars compacta), and it is possible that p-tyramine- and m-tyramine-containing neurons may also exist in this nucleus. We have studied the effects of unilateral electrolytic lesions of the pars compacta in rat on levels of DA, p-tyramine, m-tyramine, and homovanillic acid in the caudate nucleus after various survival times. At 12 and 24 h following lesioning the ipsilateral level of p-tyramine was significantly reduced compared with the contralateral side, whereas the concentrations of m-tyramine, DA, and homovanillic acid were significantly increased. Thus, in the short term, the lesion results in an increase in DA turnover, which is accompanied by an increase in m-tyramine levels and a decrease in p-tyramine levels. Similar changes occur following pharmacological treatments (chlorpromazine, d-amphetamine, l-DOPA) that increase DA turnover. At survival times of 2, 11, and 25 days, the ipsilateral concentrations of m-tyramine, DA, and homovanillic acid were reduced along with p-tyramine. These longer-term alterations in amine levels are most likely a consequence of degeneration of nigro-striatal axons. Placement of a lesion 1 mm dorsal to the usual position centering on the pars compacta produced different biochemical changes from those seen after the pars compacta lesion. One day following this lesion the concentration of p-tyramine was slightly reduced; DA was unaffected, but the concentration of m-tyramine was profoundly increased, even more so than after the pars compacta lesion. This could indicate the existence of specific m-tyramine-containing cell bodies located dorsal to the substantia nigra. The results suggest that p- and m-tyramine in the caudate nucleus originate from neurons in or close to the substantia nigra. The results in the short term following the lesion support the observation that there is an inverse relationship between p-tyramine concentration and DA turnover in the caudate nucleus.     

[3H]Imipramine Is Accumulated but Not Released from Slices of the Rabbit Caudate and Hypothalamus

Abstract: Slices of rabbit caudate and hypothalamus take up and accumulate [³H]imipramine. In superfused slices of both structures electrical stimulation or exposure to tyramine failed to release recently taken up [³H]imipramine. De-polarization by exposure to 30–60 mm-potassium caused only a small release of [³H]imipramine that was not concentration-dependent. The release of [³H]imipramine by high potassium was independent of the presence of calcium ions in the superfusion medium. These results contrasted with those obtained for the release of [³H]dopamine from the caudate and [³H]noradrenaline from the hypothalamus, where tyramine, electrical stimulation, and high potassium caused a significant release of the labeled neurotransmitters. The release of [³H]dopamine from the caudate and [³H]noradrenaline from the hypothalamus elicited by electrical stimulation or high potassium was entirely calcium-dependent. It is concluded that [³H]imipramine is taken up into the two brain regions and is accumulated in a nonvesicular site from which it is not released by calcium-dependent depolarizing stimuli.     

Effect of Moxibustion on Dopaminergic and Serotonergic Systems of Rat Nucleus Accumbens

Acupuncture and moxibustion are traditional medical treatments that have come to play important roles in complementary and alternative medicines. Moxibustion also has a long history as a folk remedy in Japan, particularly due to the technical simplicity and selective efficacy on certain types of disease and distress. This study examined the effects of moxibustion focusing on the brain reward system, particularly in the nucleus accumbens. The effects of moxibustion stimulation at various sites and frequencies on monoamine levels of adult male Sprague-Dawley rats were examined using high-preformance liquid chromatography of dissected nucleus accumbens tissues. The rats weighing 290–310 g were divided into 3 groups according to the moxibustion point used: hindlimb, lumbar or parietal points. Each group was further divided into 3 subgroups, with stimulation for 10 consecutive days, for 1 day, or sham treatment (control). On each day of stimulation, 5 moxibustion cones with a peak temperature of 200°C were applied consecutively. Stimulation of any point on 1 day only did not change dopamine or serotonin levels, but lumbar stimulation significantly increased the metabolic turnover of dopamine. Conversely, stimulation for 10 consecutive days resulted in significantly decreased serotonin levels for hindlimb and parietal stimulations, and significantly increased 5-hydroxyindolacetic acid/serotonin ratio for hindlimb stimulation. These results suggest that the metabolic turnover of serotonin release may be accentuated by moxibustion in a reward-related brain area. Moxibustion over consecutive days, especially that to peripheral regions, appears most efficient to influence on monoamine levels in the nucleus accumbens. Special issue dedicated to Dr. Simo S. Oja     

[3H]GBR-12935 Binding to the Dopamine Transporter Is Decreased in the Caudate Nucleus in Parkinson''s Disease

The specific binding of [3H]GBR-12935 to membranes prepared from human caudate nucleus is saturable (Bmax 1.36 +/- 0.18 pmol/mg protein), sodium dependent and of high affinity (KD 2.34 +/- 0.18 nM). Freezing of tissue from rat brain, or refrigeration followed by freezing, results in a small but significant (less than or equal to 20%) decrease in specific [3H]GBR-12935 binding when compared to the binding observed in fresh (nonfrozen) tissue, and this decrease may account, in part, for the differences in specific binding between rat and human brain membranes. Despite small differences in binding site density between fresh and frozen tissue there is a good correlation (r = 0.98; p less than 0.01) between the potencies of a series of drugs in displacing specific [3H]GBR-12935 binding to human caudate membranes and rat striatum as well as in inhibiting dopamine uptake in rat striatal synaptosomes (r = 0.96; p less than 0.01). The specific binding of [3H]GBR-12935 to membranes prepared from the caudate nuclei of patients with Parkinson's disease is decreased compared to membranes prepared from age- and sex-matched controls. These data suggest that [3H]GBR-12935 binds in a sodium-dependent fashion to the dopamine transport complex in human brain and that specific binding is decreased by a pathological degeneration of dopaminergic neurons to the caudate nucleus.     

Effect of Oxotremorine on Ornithine Decarboxylase Activity of the Adrenal Gland in Rat

Abstract: In this work we have studied the mechanism for the increase of adrenal ODC (ornithine decarboxylase, EC 4.1.1.17) activity provoked by oxotremorine, a muscarinic agonist. 1. Oxotremorine increased medullary ODC activity maximally at 2 h. Cortical enzyme responded much more slowly. 2. Blockade of peripheral muscarinic receptors with methylatropine partially reduced the response to oxotremorine in the medulla, but not cortex. 3. Hy-pophysectomy abolished the cortical, but not the medullary, responses to oxotremorine. Methylatropine reduced the effect of oxotremorine on medullary ODC in hypophysectomized rats. 4. In unilaterally splanchnicotomized rats oxotremorine caused an increase of ODC activity of the denervated adrenal gland relative to control value; activities in both medulla and cortex were significantly lower than those observed in the innervated gland. Evidence was obtained for a compensatory increase of ODC activity of the adrenal cortex (but not medulla) on the intact side of unilaterally operated rats. 5. Surgical intervention, in the form of a sham operation for transection of the spinal cord, leads to an increase of ODC activity in both parts of the adrenal gland. Transection of the cord attenuates these increases. 6. The additional increase of medullary ODC activity owing to the administration of oxotremorine to sham-operated rats is partially reduced in the adrenal medulla by muscarinic blockade, and completely in the cortex. This effect of methylatropine in regard to cortical ODC activity was not apparent in the other experiments with intact or unilaterally splanchnicotomized (unoperated side) rats. The results with unilaterally splanchnicotomized rats and those with transected spinal cord suggest that oxotremorine-induced modifications of adrenal ODC activity are centrally mediated, above the level of origin of the splanchnic nerves in the spinal cord (T_8–10). Experiments with hypophysectomized rats show that the response of the adrenal cortex to oxotremorine is entirely mediated by the hypophysis.     

Characterization of Muscarinic Autoreceptors in the Rabbit Hippocampus and Caudate Nucleus

Oxotremorine-induced inhibition of electrically evoked release of ³H-acetylcholine from brain slices preincubated with ³H-choline was used to characterize muscarinic autoreceptors in rabbit hippocampus and caudate nucleus. From the shifts to the right of the concentration-response curves of oxotremorine in the presence of muscarinic receptor antagonists, the following pKB values [95% C.I.] were determined in the hippocampus: tripinamide: 8.7 [8.5, 8.8]; himbacine: 8.4 [8.3, 8.5]; AQ-RA 741: 8.3 [8.2, 8.5]; 4-DAMP: 8.2 [8.0, 8.3]; hexahydrosiladifenidol: 7.4 [7.2, 7.5]; AF-DX 116: 7.3 [7.1, 7.4]; pirenzepine: 6.8 [6.6, 7.0]; and PD102807: 6.3 [6.0, 6.5]. In the caudate nucleus: tripinamide: 9.1 [8.9, 9.2]; 4-DAMP: 8.3 [8.2, 8.5]; himbacine: 8.1 [8.0, 8.2]; AQ-RA 741: 8.1 [8.0, 8.3]; hexahydrosiladifenidol: 7.3 [7.2, 7.4]; AF-DX 116: 7.1 [7.0, 7.2]; pirenzepine: 6.7 [6.6, 6.8]; and PD102807: 6.5 [6.2, 6.8]. These pK_B values fit best to literature values for M₂ receptors, suggesting that the muscarinic autoreceptor of the rabbit hippocampus and caudate nucleus is the m₂ gene product.