Metabolism of 6-ketoprostaglandin F1 alpha and prostacyclin to 6,15-diketo-13,14-dihydroprostaglandin F1 alpha-like material in cats and rabbits.

Using previously described radioimmunoassays for 6-ketoprostaglandin F1 alpha and 6,15-diketo-13,14-dihydroprostaglandin F1 alpha, plasma levels of these two products of degradation of prostacyclin (prostaglandin I2) were monitored in cats and rabbits. It was shown that both exogenous prostaglandin I2 and 6-ketoprostaglandin F1 alpha could be rapidly converted to 6,15-diketo-13,14-dihydroprostaglandin F1 alpha immunoreactivity, 6-ketoprostaglandin F1 alpha to an even somewhat larger extent. This difference in conversion could be explained by competing mechanisms which could delay or hinder the access of prostaglandin I2 to the sites of metabolism by 15-hydroxyprostaglandin dehydrogenase. The 6-ketoprostaglandin F1 alpha and 6,15-diketo-13,14-dihydroprostaglandin F1 alpha immunoreactivities were further characterized in two different thin-layer chromatographic systems and were shown to co-chromatograph exclusively with their respective standards. Similar results were obtained when tritium-labeled prostaglandin I2 or 6-ketoprostaglandin F1 alpha were infused into cats. Extracts of plasma samples taken at different time intervals after the infusion were submitted to thin-layer chromatography. The radioscans of the chromatograms showed two consistent peaks, one co-chromatographing with authentic 6-ketoprostaglandin F1 alpha and the other one with 6,15-diketo-13,14-dihydroprostaglandin F1 alpha. After infusion of 6-ketoprostaglandin F1 alpha the conversion to the 6,15-diketo-13,14-dihydro-prostaglandin F1 alpha-like product occurred somewhat faster than after infusion of prostaglandin I2. We conclude that, under in vivo conditions in the two species investigated, 6-ketoprostaglandin F1 alpha can be rapidly and effectively metabolized by 15-hydroxyprostaglandin dehydrogenase.

Adrenocorticotropin and beta-endorphin release from rat adenohypophysis in vitro: inhibition by prostaglandin E2 formed locally in response to vasopressin and corticotropin-releasing factor.

The present study examined the involvement of prostaglandins (PGs) in the mechanisms of ACTH and beta-endorphin release from rat anterior pituitary quarters incubated in vitro. Various cyclooxygenase inhibitors (indomethacin, diclofenac, flurbiprofen) had no effect on basal release of ACTH-like or beta-endorphin-like immunoreactivity (beta-EI), but enhanced ACTH-immunoreactivity/beta-EI release upon stimulation by arginine-vasopressin (AVP) or synthetic ovine corticotropin-releasing factor [CRF-(1-41)]. The lowest effective concentration of indomethacin was just sufficient to prevent PG synthesis. Indomethacin was similarly active after blockade of the phosphodiesterase by 3-isobutyl-1-methylxanthine. When added to the incubation media in concentrations up to 1 microM, PGE2, D2, F2 alpha, or prostacyclin (PGI2) did not alter basal beta-EI release; however, with stimulation by AVP or CRF-(1-41), PGE2 but not PGD2, F2 alpha, or I2 inhibited beta-EI release by about 60%. The concentrations of PGE2 in the incubation media, as measured by RIA, were somewhat higher than those of any other cyclooxygenase product (PGD2, F2 alpha, 6-keto-PGF1 alpha, thromboxane B2). Upon stimulation by AVP or CRF-(1-41), the concentrations of PGE2 increased, whereas those of PGD2 or F2 alpha remained unchanged. The release of beta-EI stimulated by high potassium concentration was not enhanced by indomethacin, although this release was sensitive to inhibition by PGE2. We conclude that PGE2 is formed locally subsequent to binding of the neurohormones and may act as a negative feedback-modulator of vasopressin's and CRF-(1-41)'s activity in the anterior pituitary gland.

Formation of sulphidopeptide-leukotrienes in brain tissue of spontaneously convulsing gerbils.

Five minutes after the onset of seizures high amounts of immunoreactive prostaglandin (PG) F2 alpha and smaller amounts of sulphidopeptide (SP)-leukotriene (LT)-like immunoreactivity could be detected in gerbil brain tissue. Bilateral carotid artery ligation followed by 15 min of reperfusion even more enhanced brain tissue contents of PGF2 alpha and SP-LT-like material. Analysis of the immunoreactive SP-LT-like material by reversed phase high pressure liquid chromatography (h.p.l.c.) revealed immunoreactivity co-eluting with authentic LTC4 and LTD4.

Identification of mechanisms involved in the modulation of release of noradrenaline in the hippocampus of the rabbit in vitro.

The modulation of the electrically-evoked release of noradrenaline by various possible neurotransmitters or neuromodulators in the hippocampus was studied in the dorsal part of the hippocampus of the rabbit. Slices of this tissue were preincubated with [3H]noradrenaline and superfused with a medium containing 30 microM cocaine. The evoked overflow of tritium was calcium-dependent, tetrodotoxin-sensitive and subject to modulation by presynaptic alpha 2-autoreceptors. Drugs with affinity for beta-adrenoceptors (up to 1 microM), muscarinic (up to 10 microM), nicotinic (up to 100 microM), GABA- (up to 1000 microM), glutamate- (up to 100 microM) and prostaglandin-receptors (up to 1 microM) did not show any modulatory influence on the evoked release of noradrenaline. In contrast, morphine (1 microM) and fentanyl (1 microM) significantly reduced the evoked overflow; this effect was antagonized by naloxone (10 microM), which, given alone, was ineffective. Apomorphine (1 microM) reduced the release of noradrenaline in the absence, and increased it in the presence, of 0.1 microM haloperidol; haloperidol (0.1 microM), given alone was ineffective. From these results it is concluded that, in addition to the well-known alpha 2-autoreceptor mechanism, presynaptic opiate-, D2- and probably D1-receptors might modulate the release of noradrenaline in the hippocampus.

Adenosine: an endogenous modulator of hippocampal noradrenaline release.

In slices of hippocampus from the rabbit, preincubated with [3H]noradrenaline and then continuously superfused, the modulation of the release of noradrenaline by adenosine receptors was studied. Electrical field stimulation of the slices elicited a release of [3H]noradrenaline which was inhibited in a concentration-dependent manner by various adenosine receptor agonists. From the order of potency: cyclohexyladenosine greater than (-)phenylisopropyladenosine [(-)PIA] greater than 5'-N-ethylcarboxamide-adenosine (NECA) greater than 2-chloro-adenosine greater than adenosine (+)phenylisopropyladenosine greater than ATP, the inhibitory adenosine receptor was classified as A1- (Ri-) receptor. The effect of the agonist was strongly reduced by adenosine receptor antagonists, the methylxanthines. A role for endogenous adenosine in the modulation of hippocampal noradrenaline release is supported by these findings: (1) that blockade of adenosine receptors by methylxanthines, especially by 8-phenyltheophylline, increased, whereas (2) inhibition of the uptake of adenosine decreased the evoked release of noradrenaline and (3) that deamination of endogenous extracellular adenosine by addition of adenosine deaminase to the medium enhanced the evoked transmitter release. Inhibitors of endogenous adenosine deaminase and 5'-nucleotidase were without effect. It is concluded that release of noradrenaline in the hippocampus is inhibited at the level of the noradrenergic nerve terminals by endogenous adenosine via A1 (or Ri) receptors.

Quantitative evaluation of the autoinhibitory feedback of release of 5-HT in the caudate nucleus of the rabbit where an endogenous tone on alpha 2-adrenoceptors does not exist.

Slices of caudate nucleus of the rabbit were preincubated with [3H]serotonin [( 3H]5-HT) in the presence of nomifensine, then superfused and twice stimulated electrically. The stimulation-evoked overflow of tritium, representing action potential-induced, exocytotic release of 5-HT, was inhibited concentration-dependently by 5-HT receptor ligands, the potencies of which were compatible with the assumption of a 5-HT1D-like autoreceptor. The inhibitor of the uptake of 5-HT, 6-nitroquipazine, markedly changed the shape of the concentration-response curve of the 5-HT autoreceptor agonist, 5-carboxamido-tryptamine (5-COHT). The maximum effects of the concentration-response curves of 5-COHT and of exogenous 5-HT became more pronounced in the additional presence of the 5-HT autoreceptor antagonists, metitepin or metergoline. Nonlinear regression analysis of these curves was used to estimate the pKd-value of endogenous 5-HT and the 5-HT biophase concentration at the autoreceptor, in the absence and in the presence of 6-nitroquipazine and in the additional presence of metitepin or metergoline. This revealed a highly operative autoinhibitory feedback, via a 5-HT1D type autoreceptor of release of 5-HT in tissue from the caudate nucleus. Also the inhibition by the alpha 2-adrenoceptor agonists, clonidine and UK-14,304, of release of 5-HT was concentration-dependent. There was neither an enhancement of release by rauwolscine, being a 5-HT autoreceptor agonist and alpha 2-adrenoceptor antagonist, in the presence of metitepin, or by the alpha-adrenoceptor antagonist, phentolamine (10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

The serotonin (5-HT) autoreceptor in the hippocampus of the rabbit: role of 5-HT biophase concentration.

Slices of hippocampus from the rabbit were preincubated with [3H]5-HT), then superfused continuously and twice stimulated electrically. The stimulation-evoked overflow of tritium was inhibited by the 5-HT autoreceptor ligands 5-carboxamido-tryptamine (5-COHT), 5-HT, 5-methoxy-N,N-dimethyl-tryptamine (5-MeOMT), (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), methysergide and (+/-)-cyanopindolol in a concentration-dependent manner. These effects were competitively inhibited by the 5-HT autoreceptor antagonists, metitepin and metergoline. (+/-)-Cyanopindolol also reduced the evoked release of 5-HT from slices of cortex from the rat. The inhibitor of the uptake of 5-HT, 6-nitroquipazine diminished the autoreceptor-mediated depression of release of 5-HT. In cortex tissue from the rat, 6-nitroquipazine reversed the decreased release of 5-HT, due to (+/-)-cyanopindolol, to a facilitation. The disinhibition of the release of 5-HT by autoreceptor antagonists was further enhanced by 6-nitroquipazine. Non-linear regression analysis of concentration-response curves for 5-COHT yielded the following pKd of endogenous 5-HT at the autoreceptor: 7.753 +/- 0.116. This value corresponds to the pKd of 5-HT at the 5-HT1B binding site. The 5-HT biophase concentration at the autoreceptor of 10(-8.220 +/- 0.132)M was markedly enhanced by 6-nitroquipazine (10(-6)M) to 10(-7.476 +/- 0.132)M. It is concluded that the 5-HT autoreceptor belongs to the 5-HT1B subtype of receptor; the corresponding 5-HT biophase concentration can be estimated quantitatively; 8-OH-DPAT decreased the evoked release of 5-HT through both 5-HT autoreceptors and alpha 2-heteroreceptors and (+/-)-cyanopindolol acts as partial agonist at the 5-HT autoreceptor.

Are presynaptic dopamine autoreceptors and postsynaptic dopamine receptors in the rabbit caudate nucleus pharmacologically different?

Slices of the rabbit caudate nucleus were preincubated with [3H]dopamine or [3H]choline and then superfused and stimulated electrically. Apomorphine reduced the stimulation-evoked overflow of tritium over the same concentration range, independently of whether slices had been pre-incubated with [3H]dopamine or with [3H]choline. Each of three antagonists--molindone, sulpiride and metoclopramide--increased the evoked overflow of tritium over the same concentration range in experiments with [3H]dopamine and those with [3H]choline. For each antagonist, the pA2 values against apomorphine obtained in [3H]dopamine experiments and in [3H]choline experiments were very similar. This study is a functional in vitro approach to receptor characterization, as opposed to radioligand binding studies or in vivo investigations. The results show that the dopamine receptor agonist apomorphine and three antagonists are unable to distinguish between the presynaptic, release-inhibiting dopamine autoreceptors and those postsynaptic dopamine receptors which, when activated, depress the release of acetylcholine. Although there are certainly more dopamine receptors in the caudate nucleus, these two physiologically important groups seem to be closely related.

The effects of intrahippocampal raphe and/or septal grafts in rats with fimbria-fornix lesions depend on the origin of the grafted tissue and the behavioural task used.

Long-Evans female rats sustained electrolytic lesions of the fimbria and the dorsal fornix and, two weeks later, received intrahippocampal suspension grafts of fetal tissue. The grafts were prepared from regions including either the medial septum and the diagonal band of Broca (septal grafts), or the mesencephalic raphe (raphe grafts), or from both these regions together (co-grafts). All rats were submitted to a series of behavioural tests (home cage and open-field locomotion, spontaneous alternation, radial-arm maze and Morris water maze performance) run over two periods after grafting (one to nine weeks and 20-35 weeks). Two weeks after completion of behavioural testing, histological (acetylcholinesterase and Cresyl Violet staining) and/or neurochemical (choline acetyltransferase activity, high-affinity synaptosomal uptake of choline and serotonin, noradrenaline, serotonin and 5-hydroxyindolacetic acid concentrations) verifications were performed on the hippocampus. Compared to sham-operated rats, lesion-only rats exhibited hyperactivity which was transient in a familiar environment (home cage) and lasting in an unfamiliar one (open field), decreased rates of spontaneous T-maze alternation, and impaired memory performance in both the radial-arm maze and the Morris water maze. These rats also showed decreased cholinergic and serotonergic markers with a maximal depletion in the septal two-thirds of the hippocampus. Noradrenaline concentration tended to be increased in the dorsal third of the hippocampus, but was not modified in the other two-thirds. While septal grafts specifically increased the cholinergic markers and raphe grafts the serotonergic ones, neither of these grafts produced a lasting effect on any behavioural variable. Conversely, the co-grafts, which increased both the cholinergic and serotonergic markers in the septal two-thirds of the hippocampus, completely normalized the Morris water maze probe trial performance, but failed to affect any of the other behavioural variables. Our present results confirm that grafts of fetal neurons injected into the denervated hippocampus may induce a neurochemical recovery that depends on the anatomical origin of the grafted cells, and that co-grafting two fetal brain regions allows the combination of their individual neurochemical properties. Furthermore, our results show that these neurochemical effects of the co-grafts may be involved in the recovery of behavioural function observed in the water maze. However, somewhat paradoxically, those effects appear inefficient for inducing any recovery in other behavioural tasks, even in the radial-arm maze; which is assumed to measure similar spatial functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Hippocampal amino acid concentrations after raphe and/or septal cell suspension grafts in rats with fimbria-fornix lesions.

Two weeks after infracallosal electrolytic fimbria-fornix lesions, Long-Evans female rats received intrahippocampal suspension grafts of either fetal septal or mesencephalic raphe tissue, or a mixture of both. Ten months after lesion surgery, the concentrations of alanine, aspartate, GABA, glutamate, glutamine, glycine, serine and taurine were determined in a dorsal, a "middle" and a ventral region of the hippocampus. We found neither the lesions nor the grafts to have significantly modified the concentration of these amino acids which, in all groups, presented a regional heterogeneity in their hippocampal distribution. GABA, glutamate and glutamine were highest in the ventral hippocampus, whereas the other amino acids were highest in the dorsal region. Our results (i) show that fimbria-fornix lesions do not result in lasting effects on hippocampal concentrations of the assessed amino acids, (ii) confirm the regional heterogeneity in the distribution of these amino acids in the hippocampus and (iii) demonstrate that cell suspension grafts of fetal septal or mesencephalic raphe tissue, as well as grafts of a mixture of both of these tissues, do not exert a non-specific effect on either of the amino acid concentrations measured. These data complete those of the preceeding paper [Kiss et al. (1990) Neuroscience 36, 61-72] concerning the effects of the same grafts on hippocampal cholinergic, serotonergic and noradrenergic markers, as well as on several behavioural variables.

The role of endothelial and non-endothelial prostaglandins in the relaxation of isolated blood vessels of the rabbit induced by acetylcholine and bradykinin.

Strips of rabbit extrapulmonary, coeliac and mesenteric arteries were mounted in organ baths for isotonic recording of changes in tissue length. The formation by the strips of the vasodilator prostaglandins PGI2 (measured as 6-keto-PGF1 alpha) and PGE2 was determined by specific radioimmunoassays. Removal of vascular endothelium initially increased and then permanently decreased the basal prostaglandin release of the tissues. Acetylcholine (ACh) relaxed strips of all three arteries if the endothelium was intact. ACh also stimulated the formation of PGI2 and PGE2 from all three tissues; about 60% of these prostaglandins originated from endothelial cells. Indomethacin caused complete inhibition of prostaglandins formation and a slight inhibition of the ACh-relaxation (not statistically significant). Complete inhibition of the ACh relaxation was achieved with nordihydroguaiaretic acid (NDGA). NDGA also partially inhibited prostaglandin formation. These data suggest that in blood vessels that are also prostaglandin-sensitive, the ACh relaxation is predominantly mediated by a non-prostaglandin endothelium-derived relaxing factor. Bradykinin was more potent that ACh in releasing prostaglandins from the same arteries. This release was activated in subendothelial components of the vascular wall. Neither this prostaglandin release nor the bradykinin-induced relaxations were significantly reduced in endothelium-denuded arteries. Indomethacin completely blocked the bradykinin-induced prostaglandin release and the bradykinin relaxation. NDGA caused a moderate inhibition of the bradykinin-induced prostaglandin release and slightly attenuated the bradykinin relaxation (neither effect of NDGA was statistically significant). Under all experimental conditions (control, indomethacin, NDGA) and with all three arteries there was a good correlation between the bradykinin-induced prostaglandin release and the respective mechanical response. No such correlation could be found for ACh. Prostaglandin-dependent relaxations of the coeliac and mesenteric artery are probably mediated by endogenous PGI2. The extrapulmonary artery is rather insensitive to PGI2 and is probably relaxed mainly by endogenous PGE2.

Effect of converting enzyme blockade on isoprenaline- and angiotensin I-induced drinking.

1 Intravenous infusion of 0.072 mumol kg(-1) h(-1) (1Asp, 5-Ile) angiotensin I, 0.116 mumol kg(-1) h(-1) of (1-Asp beta-amid, 5-Val) angiotensin II or of (1-Asp, 5-Ile) angiotensin II, caused food-deprived and water-satiated rats to drink about 3.0 ml of water per animal. This indicates that angiotensin I has a 1.6 times stronger dipsogenic effect than the angiotensin II preparations when infused intravenously.2 The converting-enzyme-inhibitor SQ 20881 (Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) (1.0 mg/kg i.v.) had no intrinsic dipsogenic effect.3 SQ 20881 given in combination with angiotensin I or angiotensin II potentiated the dipsogenic effect of angiotensin I, but not that of the two angiotensin II preparations.4 The drinking induced by isoprenaline (100 mug/kg, i.m.) was potentiated by SQ 20881 to the same extent as drinking produced by angiotensin I infusion.5 Angiotensin I plasma levels were determined after angiotensin I infusion and isoprenaline application. Both were significantly raised by SQ 20881.6 It is concluded that one of the mechanisms which mediate the dipsogenic effect of isoprenaline is stimulation of the renin-angiotensin system and that increased plasma levels of angiotensin I may play a substantial role in this type of drinking.

The importance of endogenous prostaglandins other than prostacyclin, for the modulation of contractility of some rabbit blood vessels.

Helically cut strips of rabbit aorta, extrapulmonary artery, coeliac artery, and femoral artery were set up in organ baths. Contractions of the strips by noradrenaline and angiotensin II were recorded isotonically. The release of prostaglandins 6-keto-PGF1 alpha, E2, F2 alpha, D2 and thromboxane B2 from the strips was measured by means of sensitive and specific radioimmunoassays. All blood vessels released a characteristic pattern of cyclo-oxygenase products. Prostacyclin (PGI2, measured as 6-keto-PGF1 alpha) was the major compound formed, followed by smaller amounts of PGE2 and traces of PGF2 alpha, PGD2 and thromboxane A2 (measured as thromboxane B2). The pulmonary and the femoral artery had comparatively high abilities to synthesize PGE2. Contractions induced by noradrenaline increased prostaglandin release from the pulmonary artery but not from the other blood vessels. Angiotensin II-induced contractions were accompanied by a marked prostaglandin release from the coeliac artery. After angiotensin II, prostaglandin release was also enhanced in the pulmonary artery, but remained essentially unchanged in the aorta and femoral artery. Arachidonic acid markedly increased the levels of all prostaglandin formed. Indomethacin inhibited the formation of all prostaglandins below the detection limits of the respective radioimmunoassays. Indomethacin treatment induced a qualitatively similar shifting of the concentration-response curves of noradrenaline and angiotensin II in some vessels: the concentration-response curves remained unchanged for the aorta, were slightly shifted to the left of the pulmonary artery, were markedly shifted to the left for the coeliac artery, and were shifted to the right for the femoral artery. 7 Exogenous PGI2 strongly and concentration-dependently inhibited contractions induced by the approximate EC50 of noradrenaline in the coeliac artery, but was without effect on the other three preparations. PGE2 had no effect on noradrenaline-induced contractions of the aorta, inhibited those of the pulmonary and the coeliac artery, but markedly potentiated those of the femoral artery. PGF2 alpha significantly enhanced contractions of the femoral artery, but increased contractions of the other preparations were not significant. PGD2 was without effect on any preparation. 8 In conclusion, the contractility of the aorta does not seem to be modulated substantially by prostaglandins. The major prostanoid regulating the tone of the coeliac artery was found to be PGI2. The contractility of the pulmonary and especially the femoral artery is probably not modulated by PGI2 but rather by PGE2. 9 These observations suggest that in certain blood vessels, prostaglandins other than PGI2 are important endogenous modulators of contractility.

Release of beta-lipotropin- and beta-endorphin-like material induced by angiotensin in the conscious rat.

1 The influence of the renin-angiotensin system on plasma beta-endorphin-like immunoreactivity (beta-EI) was investigated in the conscious rat by use of a radioimmunoassay for beta-endorphin without prior extraction.2 Intravenous infusion of angiotensin I, II or (des-1-Asp)angiotensin II (angiotensin III) caused a dose-dependent increase in plasma beta-EI, angiotensin III infusion being less effective than angiotensin I or II. The plasma adrenocorticotrophin (ACTH) levels too were elevated by angiotensin II. The receptor antagonist, saralasin, prevented the angiotensin II-induced beta-EI release as did dexamethasone pretreatment.3 Both the release of beta-EI and the pressor response to angiotensin I were abolished by the converting enzyme inhibitor, captopril (SQ 14225). In contrast, captopril did not affect the action of angiotensin II.4 In view of the appreciable cross-reactivity of beta-lipotropin (beta-LPH) in our assay, plasma beta-EI was analysed by Sephadex G-50 chromatography. In plasma extracts of angiotensin II-infused rats, immunoreactivity corresponding to human beta-endorphin comprised about 49% of the total immunoreactivity, whereas 51% co-migrated with human beta-LPH.5 The increase in plasma levels of beta-EI elicited by angiotensin II was diminished by about 35% in rats with a hereditary absolute lack of vasopressin (Brattleboro rats), when compared to normal rats.6 These results suggest that the renin-angiotensin system can stimulate the secretion of beta-LPH and beta-endorphin with ACTH from rat anterior pituitary. One link in mediating the response appears to be vasopressin. The physiological function remains to be defined.

Uptake kinetics and metabolism of 7-3H-dopamine in the isolated perfused rat heart.

1. The isolated, perfused rat heart accumulates dopamine by two distinct uptake mechanisms characterized by different kinetic constrants and different patterns of metabolite production (Uptake 1: Km 0.69 x 10(-6)M and Vmax (1.45 x 10(-9) mol/g)/min; Uptake 2: Km 5.9 x 10(-4)M and Vmax (0.14 x 10(-6) mol/g)/min).2. The metabolic fate of dopamine taken up by the isolated, perfused rat heart depends on the concentration of dopamine in the perfusion medium. At a very low perfusion concentration (0.047 x 10(-10) mol/ml) most of the radioactivity is stored as unchanged dopamine and the main metabolite is noradrenaline. With increasing perfusion concentrations deamination becomes the main metabolic pathway, deaminated metabolites accounting for more than 50% of the total radioactivity after perfusion with 2,614.4 x 10(-10) mol/ml for 16 minutes. The O-methylated, and the O-methylated deaminated metabolites are of minor importance at all perfusion concentrations.3. The resistance to wash out of the dopamine taken up by the isolated, perfused rat heart is dependent on the perfusion concentration used. At a concentration of 66.9 x 10(-10) mol/ml, 50% of the total activity is washed out during an 8 min wash period. Within the same time interval there is no wash out when a perfusion concentration of 0.042 x 10(-10) mol/ml is used.4. It is concluded that the metabolic fate of dopamine taken up at various perfusion concentrations reflects the distribution of dopamine within intra- and extraneuronal compartments in the hearts.

Effect of pretreatment with 6-hydroxydopamine on the uptake and metabolism of catecholamines by the isolated perfused rat heart.

1. Isolated rat hearts from control and 6-hydroxydopamine pretreated animals were perfused with (3)H-noradrenaline or (3)H-dopamine, either at a low perfusion concentration (1.50 x 10(-10) mol/ml (3)H-dopamine; 1.18 x 10(-10) mol/ml (3)H-noradrenaline) or a high perfusion concentration (296.69 x 10(-10) mol/ml (3)H-noradrenaline, 327.45 x 10(-10) mol/ml (3)H-dopamine) for 8 minutes.2. At the low perfusion concentration, the total activity, the radioactivity in the alumina eluates (sum of (3)H-dopamine, (3)H-noradrenaline and deaminated catechol metabolites) and the concentration of (3)H-dopamine. (3)H-noradrenaline and the deaminated catechol metabolites were decreased in the hearts of the pretreated rats as compared with the controls. The O-methylated amine metabolites were increased. The deaminated O-methylated metabolites were increased in the experiments with (3)H-noradrenaline and decreased in the (3)H-dopamine experiments.3. Uptake of (3)H-dopamine and (3)H-noradrenaline by the hearts of 6-hydroxydopamine pretreated rats was decreased to a much smaller extent when perfused with the high concentration than with the low concentration.4. At the high perfusion concentration there was a significant difference between control and pretreated animals with regard to the total radioactivity and the radioactivity in the alumina eluates only. The absolute and relative amounts of metabolites were not significantly changed by pretreatment with the exception of the deaminated catechol metabolites in the (3)H-dopamine experiments.5. It is concluded that neuronal Uptake 1 is greatly impaired in the hearts from rats pretreated with 6-hydroxydopamine, but extraneuronal Uptake 2 remains intact.

The adenosine receptor-mediated inhibition of noradrenaline release possibly involves an N-protein and is increased by alpha 2-autoreceptor blockade.

The stimulation-evoked overflow of [3H]-noradrenaline from slices of the rabbit hippocampus is inhibited by alpha 2-autoreceptors as well as by adenosine (A1)-receptors. Slices of rabbit hippocampus were labelled with [3H]-noradrenaline, superfused continuously and stimulated twice electrically (rectangular pulses; 2 ms, 3 Hz, 24 mA, 5 V cm-1). Treatment of hippocampal slices with N-ethylmaleimide (NEM, 30 microM; 30 min), which functionally disturbs certain N-proteins, decreased the inhibitory action of adenosine receptor agonists like (-)-N6-(R-phenylisopropyl)-adenosine ((-)-PIA) and adenosine on noradrenaline release. Release inhibition caused by (-)-PIA (0.03-1 microM) was antagonized by NEM in a non-competitive manner in the absence and in the presence of the alpha 2-adrenoceptor antagonist yohimbine. The adenosine receptor antagonist 8-phenyltheophylline significantly increased the evoked noradrenaline release by about 15% in control slices by diminishing the inhibitory action of endogenous adenosine. In NEM-treated slices this effect of 8-phenyltheophylline was not seen. In the presence of (-)-PIA (0.1 microM), i.e. under conditions of an increased inhibitory tone, release facilitation by 8-phenyltheophylline was decreased by NEM compared to that in the respective controls. Occupation of the A1-receptor with (-)-PIA prior to and during the NEM treatment did not protect the A1-receptor-coupled signal transduction system from being affected by NEM. In the presence of the alpha 2-adrenoceptor antagonist yohimbine, the inhibitory action of (-)-PIA was strongly increased. The above results suggest the involvement of a regulatory N-protein in the A1-receptor-mediated inhibition of noradrenaline release and an interaction between the alpha 2-autoreceptor and the A1-receptor-coupled signal transduction system, possibly at the level of a N-protein.

An in vitro model of 1-methyl-4-phenyl-pyridinium (MPP+) toxicity: incubation of rabbit caudate nucleus slices with MPP+ followed by biochemical and functional analysis.

1. Slices of rabbit caudate nucleus were preincubated for up to 24 h in vitro in the presence of the neurotoxic compound 1-methyl-4-phenyl-pyridinium (MPP+). Subsequently the levels of endogenous monoamines in the slices were determined by h.p.l.c. with electrochemical detection. MPP+, in concentrations higher than 32 nM significantly diminished the dopamine levels within the slices in a concentration- and time-dependent manner; at 32 microM the depletion was more than 95%. The concentration of the major metabolite of dopamine, dihydroxyphenyl acetic acid (DOPAC) was decreased at concentrations of MPP+ that did not alter dopamine levels. Thus, MPP+ increased the dopamine/DOPAC ratio. 2. In contrast, both 5-hydroxytryptamine (5-HT) levels and 5-HT/5-hydroxyindolacetic acid (5-HIAA) ratios were increased at nanomolar concentrations of MPP+. 5-HT was significantly reduced only at 32 microM. 3. The dopamine uptake inhibitor nomifensine reduced the depletory effect of MPP+ on dopamine and DOPAC content. 4. Following 24 h pretreatment with MPP+, the uptake of [3H]-dopamine into rabbit caudate nucleus slices was either enhanced (at 0.32 microM, 1 microM and 3.2 microM MPP+) or reduced (at 32 microM MPP+). 5. Preincubation of slices with 10 microM MPP+ for only 1 h increased their 3H-labelling (in contrast to 24 h pretreatment) whereas after 9 h no net increase was detectable. After 1 and 9 h MPP+ pretreatment, much less deaminated metabolites of [3H]-dopamine were found in the incubation medium of MPP+ treated slices than in the medium of control slices. These findings suggest that MPP+ strongly inhibits the enzyme monoamine oxidase (MAO) within dopaminergic (and 5-hydroxytryptaminergic) terminals before destroying them. 6. To validate the proposed in vitro model functionally, the electrically evoked release of [3H]-acetylcholine ([3H]-ACh) was investigated in MPP+ treated slices and controls. MPP+ reduced both the facilitatory effect of the D2-receptor antagonist domperidone and the inhibitory effect of the catecholamine uptake inhibitor nomifensine on [3H]-ACh release; effects compatible with a diminished inhibitory dopaminergic input on cholinergic neurones. 7. These findings also show that the terminal region of dopaminergic neurones, the caudate nucleus, is a site for MPP+ toxicity. The present in vitro model may be useful for investigating the effects of MPP+ and its interaction with other drugs under defined conditions.

False labelling of dopaminergic terminals in the rabbit caudate nucleus: uptake and release of [3H]-5-hydroxytryptamine.

The effect of the catecholamine uptake inhibitor nomifensine and of the 5-hydroxytryptamine (5-HT) uptake blocker 6-nitroquipazine on the accumulation of [3H]-5-HT (0.1 microM, 60 min incubation) and [3H]-dopamine (0.1 microM, 30 min incubation) into slices of hippocampus and caudate nucleus of the rabbit was investigated. In addition, the influence of nomifensine on the electrically evoked [3H]-5-HT release from caudate nucleus slices and of nomifensine and 6-nitroquipazine on [3H]-5-HT released from caudate nucleus slices was analysed. In hippocampal slices, which contain practically no dopaminergic but densely distributed 5-hydroxytryptaminergic and noradrenergic nerve terminals (ratio of dopamine:5-HT:noradrenaline about 1:30:25), nomifensine (1, 10 microM) did not affect the accumulation of [3H]-5-HT; 6-nitroquipazine (1 microM) reduced [3H]-5-HT uptake to about 35% of controls. In the caudate nucleus, however, where dopamine is the predominant monoamine (ratio of dopamine:5-HT:noradrenaline about 400:25:15) nomifensine (1, 10 microM) reduced the tritium accumulation to 65% whereas 6-nitroquipazine (1 microM) was ineffective. The combination of both drugs (1 microM each) led to a further decrease to about 15%. The uptake of [3H]-dopamine into hippocampal slices was blocked by both nomifensine (1 microM) and 6-nitroquipazine (1 microM) whereas in caudate nucleus slices only nomifensine (1, 10 microM) reduced the accumulation of [3H]-dopamine. The combination of both drugs was not more effective than nomifensine alone. The different effects of both uptake inhibitors in the hippocampus and caudate nucleus suggest a neurone specific rather than a substrate specific mode of action. 4 In caudate nucleus slices incubated with [3H]-5-HT and superfused continuously the electrically evoked 5-HT release was diminished by the D2-dopamine receptor agonist LY 171555 and enhanced by the D2-receptor antagonist domperidone. If, however, the labelling of caudate nucleus slices was performed in the presence of I microM or 1O microM nomifensine, the modulation of 5-HT release via D2- receptors was reduced or abolished, respectively. In the hippocampus both LY 171555 and domperidone were completely ineffective in modulating 5-HT release regardless of the absence or presence of nomifensine. 5 The present results indicate that an inverse cross labelling of [3H]-5-HT into dopaminergic and of [3H]-dopamine into 5-hydroxytryptaminergic terminals may occur despite the low concentration (0.1 microM) oftritiated transmitters used. Such cross labelling, as demonstrated with the incubation period of 60 min in the caudate nucleus, may falsely indicate the existence of D2-dopamine receptors modulating [3H]-5-HT release. If both 5-hydroxytryptaminergic and dopaminergic terminals are present within the brain region under investigation false labelling can be corrected using neuronally specific uptake inhibitors.

Protein kinase C activation and alpha 2-autoreceptor-modulated release of noradrenaline.

1 Effects of phorbol esters on the evoked noradrenaline release were studied in slices of the rabbit hippocampus, labelled with [3H]-noradrenaline, superfused continuously with a medium containing the reuptake inhibitor cocaine and stimulated electrically for 2 min (stimulation parameters: 2 ms, 24 mA, 5 V cm-1, 3 or 0.3 Hz). 2 The electrically-evoked overflow of [3H]-noradrenaline in the slices was increased in a concentration-dependent manner by the protein kinase C (PKC) activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB). Phorbol esters, which do not activate PKC, 4-O-methyl-TPA and 4 alpha-PDB, showed no effect on neurotransmitter release. The effect of 4 beta-PDB was abolished in the presence of tetrodotoxin and in the absence of calcium. The PKC inhibitor polymyxin B inhibited the evoked noradrenaline release. 3 In the presence of 4 beta-PDB the inhibitory effects of the alpha 2-adrenoceptor agonist clonidine or the facilitatory effects of the alpha 2-adrenoceptor antagonist yohimbine seemed to be modified only by changes in the concentration of noradrenaline in the synaptic region. At a stimulation frequency of 3 Hz the inhibitory action of clonidine was reduced whereas the facilitatory effect of the yohimbine was even slightly enhanced by the phorbol ester. At 0.3 Hz and in the presence of 4 beta-PDB the effect of clonidine remained and that of yohimbine was strongly enhanced. 4 Pretreatment of the slices with islet-activating protein or N-ethylmaleimide significantly reduced the enhancement of noradrenaline release caused by 4 beta-PDB. It is possible that a regulatory N-protein is involved in steps following PKC activation. 5 These results suggest that PKC participates in the mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals of the rabbit hippocampus and that effects on the autoinhibitory feedback system were not responsible for the 4 beta-PDB-induced increase of neurotransmitter release.