Effect of neuroleptics and other drugs on monoamine uptake by membranes of adrenal chromaffin granules.

1 The effects have been investigated of various reserpine-like, neuroleptic, antidepressant and other compounds on the adenosine-5'-triphosphate (ATP)-dependent uptake of noradrenaline (NA) (reserpine-sensitive) and tryptamine (reserpine-resistant) by membranes of isolated chromaffin granules of bovine adrenal medulla. 2 Reserpine and Ro 4-1284 (2-hydroxy-2-ethyl-3-isobutyl-9,10-dimethoxy-hexahydro-11bH-benzo(a)quinolizine) as well as neuroleptics (e.g. chlorpromazine and haloperidol) inhibited the NA uptake, but the reserpine-like drugs were more potent. In contrast, Ro 4-1284 showed a considerably weaker effect thatn the neuroleptics in interfering with tryptamine uptake. Chlorpromazine had about the same potency in inhibiting NA and tryptamine uptake, whereas the action of haloperidol was more pronounced on the uptake of NA than of tryptamine. 3 The relative potencies of neuroleptic drugs in inhibiting NA uptake by granule membranes in vitro corresponded only partly to their relative potencies in enhancing dopamine turnover in vivo. 4 The inhibition of NA uptake by chloropromazine and Ro 4-1284 appeared to be of the noncompetitive type. 5 Chlorpromazine did not influence the decrease in ATP induced by granule membranes in the incubation medium. 6 Other basic, but not acidic compounds also inhibited NA uptake by granule membranes; their potency was of the order of that of chlorpromazine (antidepressants) or weaker (e.g. benzodiazepines). 7 In conclusion, the mechanism of action of neuroleptics probably differs from that of reserpine-like drugs in the inhibition of monoamine uptake by membranes of catecholamine storage organelles. While interference with the granular storage of dopamine at the granule membrane level may contribute to the in vivo action of neuroleptics (e.g. in enhancing dopamine turnover), additional effects of these drugs must be involved in vivo, e.g. blockade of pre- and postsynaptic dopamine receptors.     

The outward transport of axoplasmic noradrenaline induced by a rise of the sodium concentration in the adrenergic nerve endings of the rat vas deferens

Summary The adrenergic nerve endings of the rat vas deferens were loaded with ³H-(–)-noradrenaline; COMT was inhibited by the presence of 10 mol/l U-0521, and all experiments were carried out with calcium-free solution. After 100 min of wash-out a neuronal efflux of tritium was obtained which remained constant with time (when expressed as fractional rate of loss; FRL); it contained more DOPEG than noradrenaline.The in vitro administration of reserpine-like drugs (reserpine and Ro 4-1284) increased the FRL of tritium, presumably because of an increase in the leakage of noradrenaline from storage vesicles; the efflux of DOPEG increased more than that of noradrenaline, and the ratio NA/DOPEG declined.Inhibition of the membrane ATPase (by omission of potassium from the medium or by the presence of 3 mmol/l ouabain) increased the FRL of tritium, presumably because of an increase in the net leakage of noradrenaline from the storage vesicles (as a consequence of the fall in the concentration of free axoplasmic noradrenaline; see below).Veratridine also increased the FRL of tritium, partly because of its known reserpine-like effect (Bönisch et al. 1983); in the presence of 1 mol/l veratridine, the efflux of DOPEG increased.Irrespective of the presence or absence of reserpine or Ro 4-1284, inhibition of the membrane ATPase or the presence of veratridine (agents or procedures which increase the axoplasmic sodium concentration) always resulted in a brisk increase of the efflux of noradrenaline that was accompanied by a simultaneous decrease in the efflux of DOPEG (see above for one exception). In all experiments the rise in internal sodium caused the ratio NA/DOPEG to increase.These results indicate that—as long as the sodium gradient is normal—the axonal membrane functions as a barrier that largely prevents any outward movement of axoplasmic noradrenaline. Consequently, the axoplasmic amine is largely deaminated, and the ratio NA/DOPEG is low. However, when the axoplasmic sodium concentration rises, axoplasmic noradrenaline is transported out of the nerve ending at such high rates that the axoplasmic noradrenaline concentration falls; the fall in the efflux of DOPEG is indicative of a fall in the intraneuronal formation of DOPEG. The results show that changes in the efflux of DOPEG (i.e., of a highly lipophilic metabolite that easily leaves adrenergic nerve endings) can serve as an index of changes in axoplasmic noradrenaline levels.Supported by the Deutsche Forschungsgemeinschaft (Tr.96)     

Liberation of catecholamines from blood platelets

1 Platelet-rich plasma (PRP) of guinea-pigs with or without reserpine was preincubated either with [¹⁴C]-5-hydroxytryptamine ([¹⁴C]-5-HT) plus [³H]-dopamine or with [¹⁴C]-5-HT plus [³H]-noradrenaline ([³H]-NA). After isolation on two successive dextran gradients the double-labelled platelets were incubated in Tris-buffer in the presence or absence of various drugs. The decrease in radioactivity in the platelets was measured in order to determine the amount of the amine that had been liberated.

2 Spontaneous liberation of the labelled amines was more marked in reserpine-treated platelets than in normal ones and somewhat more pronounced for the ³H-catecholamines than for [¹⁴C]-5-HT.

3 The reserpine-like benzoquinolizine, Ro 4-1284, caused liberation of all three labelled amines in normal but not in reserpine-treated platelets. More [³H]-dopamine was liberated than [¹⁴C]-5-HT and less [³H]-NA.

4 The arylalkylamines, tyramine and p-chloromethamphetamine (PCMA), liberated all three labelled amines from normal platelets, and [¹⁴C]-5-HT and [³H]-dopamine, but not [³H]-NA from reserpine-treated ones. In normal platelets dopamine was reduced to a greater extent than [¹⁴C]-5-HT and [³H]-NA to a smaller extent, whereas in reserpine-treated platelets [¹⁴C]-5-HT was more markedly diminished than [³H]-dopamine.

5 The 5-HT uptake inhibitor, imipramine, had little influence on the spontaneous and drug-induced liberation of [¹⁴C]-5-HT and [³H]-dopamine.

6 It is concluded that ³H-catecholamines like [¹⁴C]-5-HT are mostly localized in the granular pool of platelets; the three drugs tested liberate [³H]-dopamine [³H]-NA and [¹⁴C]-5-HT from the granular pool. Ro 4-1284 does not liberate ³H-catecholamines and [¹⁴C]-5-HT from extragranular sites whereas tyramine and PCMA also act on the extragranular pool of [³H]-dopamine and [¹⁴C]-5-HT but not [³H]-NA.

7 The liberation of catecholamines from platelets differs from that of 5-HT in several respects and platelets are only partly comparable to neurones as far as drug-induced liberation of biogenic amines is concerned.

     

Effects of the noradrenaline metabolites on tyrosine hydroxylase activity in guinea-pig atria

Summary The effects of noradrenaline, its five metabolites and metanephrine, were studied on tyrosine hydroxylase activity in guinea-pig atria. The deaminated metabolite, (±)-3,4-dihydroxyphenylglycol (DOPEG), was equipotent with (±)-noradrenaline in its inhibitory action on tyrosine hydroxylase activity in the homogenates of guinea-pig atria. The inhibition by DOPEG was competitive with the cofactor, reduced pteridine. The deaminated acid, 3,4-dihydroxymandelic acid (DOMA) and the O-methylated deaminated acid, 3-methoxy, 4-hydroxymandelic acid (VMA) had 1/50th and 1/30th, respectively, the potency of noradrenaline in inhibiting tyrosine hydroxylase. The rest of the metabolites did not inhibit tyrosine hydroxylase in homogenates in concentrations up to 1.0 mM. In intact guinea-pig atria noradrenaline was considerably more potent than DOPEG in inhibiting tyrosine hydroxylase. Normetanephrine 1.4×10^–4 M inhibited tyrosine hydroxylase in the intact tissue but failed to inhibited the enzyme in the homogenate even in higher concentrations. The effect of normetanephrine in the intact tissue is related to the ability of this compound to release endogenous noradrenaline.A reserpine-like agent, Ro 4-1284, did not inhibit tyrosine hydroxylase activity in the homogenate but in the intact tissue the inhibition was more than 50%. This effect of Ro 4-1284 in the intact tissue appears to be related to the releasing effects of this agent and to an increase in the axoplasmic levels of DOPEG.Since the formation of the deaminated glycol, DOPEG, represents the main metabolic pathway for the neurotransmitter in adrenergic nerve endings, the present results are compatible with the view that, in addition to the pool of extravesicular noradrenaline, the cytoplasmic concentration of DOPEG could also participate in the regulation of the activity of tyrosine hydroxylase.     

Hypericum perforatum L. extract does not inhibit 5-HT transporter in rat brain cortex

The hydroalcoholic extract of Hypericum perforatum L. is an effective antidepressant, although its mechanism of action is still unknown. It inhibits the synaptosomal uptake of serotonin (5-HT), dopamine and noradrenaline, suggesting a biochemical mechanism similar to the synthetic standard antidepressants. In the present study, further investigating this hypothesis, we confirmed that a hydromethanolic extract of H. perforatum inhibited [3H]5-HT accumulation in rat brain cortical synaptosomes with an IC50 value of 7.9 microg/ml. The IC50 of pure hyperforin was 1.8 microg/ml, so the activity of the total extract is not related only to its hyperforin content (<5%). This inhibitory effect, however, is not due to a direct interaction with, and blockade of, the 5-HT transporters since the extract, like hyperforin, did not inhibit [3H]citalopram binding (IC50 > 100 microg/ml and 10 microg/ml, respectively). We also found that 3-10 microg/ml of the extract, or 0.3-1 microg/ml hyperforin, induced marked tritium release from superfused synaptosomes previously loaded with [3H]5-HT. The releasing effect of the extract resembles the releasing effect of a reserpine-like compound (Ro 04-1284), i.e. it was slightly delayed and was 5-HT carrier- and calcium-independent. These data suggest that the hydromethanolic extract of H. peforatum, similarly to Ro 04-1284, rapidly depletes storage vesicles, raising the cytoplasmic concentration of 5-HT, and this increase is presumably responsible for the apparent inhibition of [3H]5-HT uptake. Therefore, our in vitro data do not confirm that the hydromethanolic extract of H. perforatum acts as a classical 5-HT uptake inhibitor but indicate reserpine-like properties. However, the concentrations of the active component(s) effective in vitro as reserpine-like agent(s) (i.e. corresponding to > or =3 microg/ml of the hydromethanolic extract) do not seem to be achieved in the brain after pharmacologically effective doses of the extract, as indicated by the finding that there were no significant changes of rat brain 5-HT and 5-hydroxyindoleacetic acid levels after a schedule of treatment (3 x 300 mg/kgday, orally) active in an animal model predictive of antidepressant-like activity. These data also suggest that the antidepressant effect of H. perforatum extracts is unlikely to be associated with interaction with GABA, benzodiazepine and 5-HT1 receptors since, in receptor binding studies, we found IC50 values higher than 5 microg/ml. Therefore other, still unknown, mechanisms are possibly involved in H. perforatum antidepressant effects.     

Nonexocytotic noradrenaline release induced by pharmacological agents or anoxia in human cardiac tissue

In acute myocardial ischemia, noradrenaline is released locally from sympathetic varicosities by a Ca²⁺-independent nonexocytotic release mechanism that is effectively suppressed by inhibitors of the neuronal noradrenaline carrier (uptake₁). The purpose of the present study was to elucidate the significance of free axoplasmic amine concentration and disturbed neuronal sodium homeostasis for nonexocytotic noradrenaline release in the human heart by comparing the release induced by anoxia with that induced by reserpine, tyramine, or veratridine. The overflow of endogenous noradrenaline and dihydroxyphenylethyleneglycol was assessed in human atrial tissue incubated in calcium-free Krebs-Henseleit-solution to prevent interferences by exocytotic release. The overflow of dihydroxy-phenylethyleneglycol served as indicator of the free axoplasmic noradrenaline concentration.When vesicular uptake was blocked by the reserpine-like agent Ro 4-1284, high dihydroxyphenylethyleneglycol overflow was observed without concomitant noradrenaline overflow. If, however, Ro 4-1284 was combined with sodium pump inhibition (by omission of extracellular potassium) or with alteration of the transmembrane sodium gradient (by lowering the extracellular sodium concentration), both dihydroxyphenylethyleneglycol and noradrenaline were released. The indirectly acting sympathomimetic tyramine induced a marked increase in noradrenaline overflow which was accompanied by overflow of high amounts of dihydroxyphenylethyleneglycol, indicating interference of the drug with both vesicular catecholamine transport and amine transport via uptake₁. Likewise, veratridine induced an overflow of noradrenaline (which was prevented by blockade of uptake₁) and dihydroxyphenylethyleneglycol indicating a reserpine-like action of the drug. A disturbed energy status of the sympathetic neuron induced by cyanide intoxication or anoxia caused noradrenaline overflow which was suppressed by uptake, blockade. Blockade of sodium channels by tetrodotoxin effectively reduced noradrenaline overflow during cyanide intoxication but not during anoxia. Anoxia-induced noradrenaline release, however, was markedly suppressed by inhibition of Na⁺/H⁺ exchange with ethylisopropylamiloride, indicating the Na⁺/H⁺ exchange as the predominant pathway for sodium entry into the sympathetic neuron during anoxia.The results demonstrate that disturbed neuronal sodium homoeostasis and impaired vesicular storage function are critical conditions, causing nonexocytotic noradrenaline release in anoxic human cardiac tissue.     

Release studies with rat brain cortical synaptosomes indicate that tramadol is a 5-hydroxytryptamine uptake blocker and not a 5-hydroxytryptamine releaser.

Tramadol is a centrally acting opioid analgesic whose mechanism of action could also involve an increase in central serotoninergic transmission. Thus, tramadol inhibits synaptosomal serotonin (5-hydroxytryptamine, 5-HT) reuptake and induces tritium release from [3H]5-HT-preloaded slices. We investigated the effect of (+/-)-tramadol in release studies with superfused rat brain cortex synaptosomes preloaded with [3H]5-HT. Tramadol had no releasing effect up to 30 microM, whereas at 10 microM tramadol significantly inhibited by 45% D-fenfluramine-induced [3H]5-HT release. At 100 microM, tramadol showed a slight releasing effect in the absence or in the presence of pargyline, which was not augmented in synaptosomes pre-exposed to Ro 04-1284 (2-ethyl-1,3,4,6,7,11b-hexahydro-3-isobutyl-9,10-dimethoxy-2H-benzo [a]quinolizin-2-ol hydrochloride), a reserpine-like compound that enhances cytoplasmic 5-HT levels. In summary, (+/-)-tramadol behaved as a classical 5-HT uptake blocker (like citalopram) and not as a substrate of the 5-HT carrier with indirect 5-HT mimetic properties (like D-fenfluramine).     

CXCR4 and NMDA Receptors Are Functionally Coupled in Rat Hippocampal Noradrenergic and Glutamatergic Nerve Endings

Previous studies had shown that the HIV-1 capsidic glycoprotein gp120 (strain IIIB) modulates presynaptic release-regulating NMDA receptors on noradrenergic and glutamatergic terminals. This study aims to assess whether the chemokine CXC4 receptors (CXCR4s) has a role in the gp120-mediated effects. The effect of CXCL12, the endogenous ligand at CXCR4, on the NMDA-mediated releasing activity was therefore investigated. Rat hippocampal synaptosomes were preloaded with [³H]noradrenaline ([³H]NA) or [³H]D-aspartate ([³H]D-Asp) and acutely exposed to CXCL12, to NMDA or to both agonists. CXCL12, inactive on its own, facilitated the NMDA-evoked tritium release. The NMDA antagonist MK-801 abolished the NMDA/CXCL12-evoked tritium release of both radiolabelled tracers, while the CXCR4 antagonist AMD 3100 halved it, suggesting that rat hippocampal nerve endings possess presynaptic release-regulating CXCR4 receptors colocalized with NMDA receptors. Accordingly, Western blot analysis confirmed the presence of CXCR4 proteins in synaptosomal plasmamembranes. In both synaptosomal preparations, CXCL12-induced facilitation of NMDA-mediated release was dependent upon PLC-mediated src-induced events leading to mobilization of Ca²⁺ from intraterminal IP₃-sensitive stores Finally, the gp120-induced facilitation of NMDA-mediated release of [³H]NA and [³H]D-Asp was prevented by AMD 3100. We propose that CXCR4s are functionally coupled to NMDA receptors in rat hippocampal noradrenergic and glutamatergic terminals and account for the gp120-induced modulation of the NMDA-mediated central effects. The NMDA/CXCR4 cross-talk could have a role in the neuropsychiatric symptoms often observed in HIV-1 positive patients.     

3H]5-hydroxytryptamine uptake and release in cat cerebral arteries

1. Field electrical stimulation induced tritium release from cat cerebral arteries preincubated with [3H]serotonin (5-HT). 2. This release was markedly reduced by tetrodotoxin (0.8 microM), B-HT 920 (1 microM), denervation with 6-OH-dopamine (6-OHDA) and OCa2+, and increased by phentolamine (1 microM) and phorbol 12,13-dibutyrate (1 and 3 microM). 3. 5-HT (10 and 100 microM) and NA (0.1, 1 and 10 microM) caused concentration-dependent tritium release in control arteries, but not in those denervated with 6-OHDA. 4. [3H]5-HT uptake was greatly reduced by preincubation of arteries with cocaine (10 microM), ouabain (100 microM) or denervation with 6-OHDA. 5. 5-HT did not amplify contractions elicited by noradrenaline (NA) in middle cerebral arteries. 6. These data indicate: (1) 5-HT is mainly accumulated in adrenergic nerve endings; (2) 5-HT release is modulated by presynaptic alpha 2-adrenoceptors; (3) protein kinase C of perivascular adrenergic nerve endings participates in 5-HT release, and (4) 5-HT did not amplify NA responses.     

3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampus: role of Na+ entry on Ca2+ pools and of protein kinase C.

Slices of rat hippocampus, preincubated with [3H]noradrenaline [(3H]NA), were superfused continuously and stimulated by addition of 3,4-diaminopyridine (3,4-DAP; 100 microM) for 10 min to the superfusion medium. An overflow of 3H evoked by 3,4-DAP (representing [3H]NA release) was measurable not only in the presence but also in the absence of extracellular Ca2+. Both the protein kinase C (PKC) activator 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) and the PKC inhibitor polymyxin B, affected mainly the evoked release in the absence of extracellular Ca2+ in a facilitatory or inhibitory manner, respectively. Moreover, in the absence of extracellular Ca2+, both the 3,4-DAP-evoked [3H]NA release and the facilitatory effect of 4 beta-PDB were abolished in the presence of tetrodotoxin or in the absence of Na+ in the superfusion medium. Ruthenium red, a blocker of mitochondrial Ca2+ reuptake, potently increased 3,4-DAP-evoked [3H]NA release in Ca(2+)-free EGTA-containing medium. The facilitatory effects of ruthenium red and 4 beta-PDB were additive. From these and earlier observations we conclude (1) that the mechanism of 3,4-DAP-evoked [3H]NA release involves both Ca2+ influx into the nerve terminals and mobilization of intraneuronal Ca2+ pools. Most probably Ca2+ release from cytoplasmic Ca2+ stores (e.g. endoplasmic reticular pools or mitochondria) is induced by Na+ ions entering the nerve endings during 3,4-DAP-evoked repetitive action potentials. (2) The facilitatory effect of phorbol ester on 3,4-DAP-evoked NA release appears to be mediated not by changes in Ca2+ influx, but by enhancement of intraneuronal events distal to Na+ ion entry and increased intracellular Ca2+ availability.     

Effects of lithium chloride administered in combination with methamphetamine or a reserpine- like drug on brain norepinephrine and dopamine metabolism in rats]

Lithium chloride (LiCl) was injected acutely (2.4 mEq/kg, i.p.) or subacutely (2.5 mEq/kg X 2/day for 4.5 days, i.p.) in rats treated concurrently with methamphetamine (MAPT) or a reserpine-like drug (Ro4-1284), and effects of LiCl on behavior as well as metabolism of brain norepinephrine (NE) and dopamine (DA) were investigated. Acute administration of LiCl suppressed the MAPT-induced hyper-locomotor activity but did not affect the Ro4-1284-induced behavioral depression. LiCl administered subacutely increased the MAPT-induced stereotyped behavior, and it reduced the Ro4-1284-induced initial transient excitation in spontaneous locomotor activity and tended to facilitate the recovery from behavioral depression. As for NE and DA metabolism, LiCl administered subacutely inhibited the MAPT-induced decrease of the deaminated NE metabolites and Ro4-1284-induced decrease of DA metabolism and the Ro4-1284-induced alterations in NE metabolism. From these results, it is likely that the inhibitory effect of LiCl on the MAPT and Ro-1284-induced changes in NE and DA metabolism may to some extent be involved in the therapeutic effects on manic-depressive illness.     

Uptake of 5-hydroxytryptamine in blood platelets and its inhibition by drugs: role of plasma membrane and granular storage

The initial uptake of ³H-5-hydroxytryptamine (³H-5-HT) showed linearity for short time intervals in normal and reserpinized blood platelets of guinea-pigs, but was lower in reserpinized platelets. The K_m values for the ³H-5-HT uptake were virtually identical in normal and reserpinized platelets, whereas Vmax was lower in the latter. Imipramine and chlorpromazine caused the same percentage inhibition of ³H-5-HT uptake in normal and reserpinized platelets; the reserpine-like compound Ro 4–1284 inhibited the uptake of ³H-5-HT in the normal, but not markedly in the reserpinized platelets. Haloperidol, prenylamine and Ro 4–9040 were more potent inhibitors in normal than in reserpinized platelets. It is concluded that (a) the K_m of the initial uptake of 5-HT by platelets is probably determined by the mechanism at the plasma membrane, whereas V_max may be codetermined by the intracellular storage capacity, (b) platelets are models for differentiating the site of action (plasma membrane or storage organelles) of drugs interfering with 5-HT uptake, and (c) neuroleptics- and reserpine-like compounds may either act selectively on the plasma membrane or on the intracellular storage organelles, or affect both of these subcellular sites.     

Effect of peptide and nonpeptide antagonists of angiotensin II receptors on noradrenaline release in hypothalamus of rats with angiotensin II-induced increase of water intake

Angiotensin II (Ang II) administered intracerebroventriculary (icv) at a dose that induces drinking behavior in rats significantly increased K⁺-stimulated release of [³H] noradrenaline (NA) in hypothalamus without affecting basal [³H] NA release. The observed difference between the effects of Ang II on basal and K⁺-stimulated [³H]NA release may possibly be due to the fact that peptides are released after increased neuronal activity. It can be suggested that Ang II is important primarily in pathological states and that NA plays a substantial role in the brain Ang II-induced drinking response. The imidazolic nonpeptidic compound 2-n-butyl-4-chloro-5-hydroxymethyl-1-{[2-(1H-tetrazol-5-yl)-biphenyl-4-yl]methyl}imidazole potassium salt (DuP 753, losartan), its active metabolite 2-n-butyl-4-chloro-1-{[2-(1H-tetrazol-5-yl)-biphenyl-4-yl]methyl}imidazole-5-carboxylic acid (EXP 3174) and peptide Ang II analogue, sarmesin, antagonized the Ang II-induced effect on [³H]NA release, in spite of the differences in their chemical structures. Thus, the drugs tested inhibited K⁺-stimulated [³H]NA release in hypothalamus, acting via the angiotensin (AT) 1 receptor subtype. We could not reject the possibility of a non-receptor mechanism of action for DuP 753, EXP 3174 and sarmesin. This research allows us to suggest a neurochemical mechanism for the modulatory role of these drugs on the NA-ergic system. The Ang II receptor antagonists studied may become important therapeutic agents, which act preferentially on pathologically activated systems. These agents may be of use for the prevention of excessive ingestion of water in some neuropsychotic diseases.     

Possible physiological significance of the initial step in the catabolism of noradrenaline in the central nervous system of the rat

Summary The hypothalamus, the cerebral cortex and the cerebellar cortex of the rat were labelled in vitro with ³H-noradrenaline (³H-NA) and the metabolism of the tritiated transmitter was studied during spontaneous outflow and under conditions of release elicited by exposure to 20 mM K⁺.In the three areas of the central nervous system of the rat, ³H-NA accounted for approximately 40% of the total radioactivity in spontaneous outflow while the ³H-O-methylated deaminated fraction (³H-OMDA) and ³H-3,4-dihydroxyphenylglycol (³H-DOPEG) were the main metabolites. Exposure to the reserpine-like agent, Ro 4-1284 induced a selective increase in the spontaneous outflow of ³H-DOPEG, while the contribution of the ³H-OMDA metabolites to the release induced by Ro 4-1284 was very small.During ³H-transmitter release elicited by exposure to 20 mM K⁺, approximately 80% of the radioactivity was collected as unmetabolized ³H-NA, while ³H-DOPEG was the main metabolite formed under these experimental conditions. Exposure to cocaine prevented ³H-DOPEG formation from ³H-NA released by K⁺, indicating that ³H-DOPEG was formed after neuronal reuptake of the transmitter released by K⁺.After in vitro labelling with ³H-NA, the unmetabolized transmitter represented approximately 70% of the total radioactivity retained in the tissue. However, when ³H-NA was administered in vivo, by intraventricular injection, only 30% of the total radioactivity retained by the tissue was accounted for by ³H-NA, and 60% of the radioactivity corresponded to the ³H-OMDA fraction, most of which was retained as ³H-MOPEG sulfate.When the rats were pretreated with pyrogallol, free ³H-DOPEG accounted for nearly 50% of the radioactivity retained in the three areas of the central nervous system after in vivo labelling with ³H-NA. When monoamine oxidase was inhibited by pargyline and ³H-NA was administered by intraventricular injection, ³H-NMN accounted for approximately 50% of the total radioactivity retained in the three areas of the central nervous system of the rat.The results obtained are compatible with the view that formation of the deamined glycol is the first step in the metabolism of ³H-NA in the rat central nervous system. In addition, it is concluded that the determination of the levels of some NA metabolites retained in the central nervous system does not necessarily represent an accurate reflection of the degree of central noradrenergic activity or of selective metabolic pathways. Consequently, in studies on the metabolism of NA it is important to take into account not only the transmitter and its metabolites in the tissue but also in the outflow from the structures studied either under in vivo or in vitro conditions.     

Flunitrazepam photoaffinity labeling of the GABAA receptor reduces inhibition of [3H]Ro15-4513 binding by GABA

The benzodiazepine drugs modulate γ-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA_A receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [³H]Ro15-4513, [³H]flumazenil (Ro15-1788), and [³H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [³H]flunitrazepam and [³H]flumazenil, but increased the binding of [³H]Ro15-4513. The increase in [³H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent K_d, with no change in B_max. Following photoaffinity labeling, negative modulation of [³H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [³H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [³H]Ro15-4513.     

Noradrenergic vasoconstriction of pig prostatic small arteries

The current study investigated the distribution of adrenergic nerves and the action induced by noradrenaline (NA) in pig prostatic small arteries. Noradrenergic innervation was visualized using an antibody against dopamine-beta-hydroxylase (DBH), and the NA effect was studied in small arterial rings mounted in microvascular myographs for isometric force recordings. DBH-immunoreactive nerve fibers were located at the adventitia and the adventitia-media border of the vascular wall. Electrical field stimulation (EFS, 1-32 Hz) evoked frequency-dependent contractions that were reduced by guanethidine and prazosin (adrenergic neurotransmission and α₁-adrenoceptors blockers, respectively) and by the α₂-adrenoceptor agonist UK 14,304. The α₂-adrenoceptor antagonist rauwolscine reversed the UK 14,304-produced inhibition. NA produced endothelium-independent contractions that were antagonized with low estimated affinities and Schild slopes different from unity by prazosin and the α_1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α-α-dimethyl-1H-indole-3-ethanamine (RS 17053). The α_1A-adrenoceptor antagonist 5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione (RS 100329), which also displays high affinity for α_1L-adrenoceptors, and the α_1L-adrenoceptor antagonist tamsulosin, which also has high affinity for α_1A- and α_1D-adrenoceptors, induced rightward shifts with high affinity of the contraction-response curve to NA. The α_1D-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) failed to modify the NA contractions that were inhibited by extracellular Ca²⁺ removal and by voltage-activated (L-type) Ca²⁺ channel blockade. These data suggest that pig prostatic resistance arteries have a rich noradrenergic innervation; and NA, whose release is modulated by prejunctional α₂-adrenoceptors, evokes contraction mainly through activation of muscle α_1L-adrenoceptors coupled to extracellular Ca²⁺ entry via voltage (L-type)- and non-voltage-activated Ca²⁺ channels.     

Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release

Muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors (nAChRs) regulating GABA release from striatal nerve endings were studied by monitoring release of previously accumulated [³H]GABA or endogenous GABA from superfused mouse striatal synaptosomes. Oxotremorine inhibited the release of [³H]GABA elicited by depolarization with 4-aminopyridine (4-AP), an effect antagonized by atropine. Agonists at nAChRs, including the α₄β₂^∗ subunit-selective RJR2403, provoked the release of [³H]GABA as well as of the endogenous transmitter; these effects also were prevented by oxotremorine and pilocarpine suggesting coexpression of functional mAChRs and α₄β₂^∗ nAChRs on GABAergic nerve endings. The inhibitory effects of oxotremorine on the release of [³H]GABA evoked by 4-AP or by RJR2403 were: (i) prevented by the M₂/M₄ mAChR antagonist himbacine; (ii) insensitive to the M2 antagonist AFDX116; (iii) blocked by the selective M₄ mAChR antagonists MT3, thus indicating the involvement of receptors of the M₄ subtype. In conclusion, in the corpus striatum, acetylcholine released from cholinergic interneurons can activate α₄β₂^∗ nAChRs mediating release of GABA; this evoked release can be negatively modulated by M₄ mAChRs coexpressed on the same GABAergic terminals.     

Pharmacological and kinetic properties of uptake of [H]-norepinephrine by superior cervical ganglia of rats in organ culture

The increase in uptake of [³H]-norepinephrine ([³H]-NE) by superior cervical ganglia grown in organ culture was used as an index of the uptake of norepinephrine (NE) by axonal sprouts formed during organ culture. The kinetic and pharmacologie properties of NE uptake by such axonal sprouts were examined. The K_m (1.9 × 10⁻⁶M) for [³H]-dl-NE uptake by axonal sprouts was similar to that found in sympathetic nerve endings; the K_m in fresh ganglia was 8.0 × 10⁻⁶M. The intensity of inhibition by cocaine, metaraminol and phenoxybenzamine of the [³H]-NE uptake into axonal sprouts also resembled that found for nerve endings and was significantly different from that of fresh ganglion. Our observations indicate that the uptake mechanism for NE into axonal sprouts is pharmacologically and kinetically similar to the uptake mechanism in sympathetic nerve endings.     

Brain microdialysis in rats: a technique to reveal competition in vivo between endogenous dopamine and moclobemide, a RIMA antidepressant

In this study, the effect of the interaction between the short-acting reserpine-like dopamine (DA) releaser Ro 4-1284 (1 mg/kg IP) and the reversible inhibitors of monoamine oxidase type A (MAO-A), moclobemide (Aurorix®) and Ro 41-1049 (20 mg/kg IP, each), on the outflow of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) was investigated by rat transstriatal microdialysis. The injection of Ro 4-1284 after MAO-A inhibitors produced a marked increase of DA concentrations corresponding to a bell-shaped change in DOPAC outflow. This effect was more pronounced in rats treated with moclobemide than with Ro 41-1049. These data support the view that the increment of the endogenous substrate DA might displace moclobemide more rapidly than Ro 41-1049 from MAO-A active sites. The microdialysis method is proposed as a more reliable in vivo technique to investigate the degree of reversibility of the reversible MAO-A inhibitors.     

The effect of the methionine antagonist l-2-amino-4-methoxy-trans-3-butenoic acid on the growth and metabolism of walker carcinosarcoma in vitro

l-2-Amino-4-methoxy-trans-3-butenoic acid (Ro07–7957) is a structural analogue ot methionine with a potent tumour growth inhibitory activity in vitro. This agent is transported by the methionine carrier system in Walker carcinoma and causes an initial dose-related depression of the acid-soluble pool of methionine. The depression of the incorporation of l-[methyl-³H]methionine into acid-insoluble material in the presence of Ro07–7957 is greater than that of l-[2-³H]methionine and l-[4,5(n)-³H]lysine, suggesting an inhibition of the methylation of macromolecules as well as an inhibition of protein synthesis. There is no effect of the drug on the incorporation of [³H]thymidine into acid-insoluble material during the first 24 hr after treatment, while the incorporation of [5-³H]uridine is stimulated 40 per cent. The ratio of incorporation of l-[2-³H]methionine to l-[methyl-¹⁴C]methionine into proteins increases with increasing drug concentration, suggesting an inhibition of protein methylation. This effect is more prevalent at 24 hr than after 8 hr of treatment. The specific activity of tRNA methylase using E. coli MRE 600 tRNA as substrate is elevated more than two-fold within 24 hr after treatment, as is also the intracellular level of S-adenosyl-l-methionine (SAM). The effects of this agent on macromolecular metabolism is in some respects similar to that observed with the carcinogen ethionine, and suggests the initial formation of an inhibitor of methylation, which is followed by a later attempt by the cells to maintain homeostasis by production of increased amounts of tRNA methylating enzymes.