B. Neufang

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

1 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‐PGF1α) and PGE2 was determined by specific radioimmunoassays. 2 Removal of vascular endothelium initially increased and then permanently decreased the basal prostaglandin release of the tissues. 3 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 prostaglandin 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. 4 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). 5 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. 6 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.

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

1 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‐PGF1α, E2, F2α, D2 and thromboxane B2 from the strips was measured by means of sensitive and specific radioimmunoassays. 2 All blood vessels released a characteristic pattern of cyclo‐oxygenase products. Prostacyclin (PGI2, measured as 6‐keto‐PGF1α) was the major compound formed, followed by smaller amounts of PGE2 and traces of PGF2α, PGD2 and thromboxane A2 (measured as thromboxane B2). The pulmonary and the femoral artery had comparatively high abilities to synthesize PGE2. 3 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. 4 Arachidonic acid markedly increased the levels of all prostaglandin formed. 5 Indomethacin inhibited the formation of all prostaglandins below the detection limits of the respective radioimmunoassays. 6 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. PGF2α 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.

The endothelium-dependent vasodilator effect of acetylcholine: characterization of the endothelial relaxing factor with inhibitors of arachidonic acid metabolism.

Acetylcholine (ACh) caused concentration-dependent relaxation of strips of rabbit thoracic aorta precontracted with noradrenaline if the endothelium was intact. More than ten-fold higher concentrations of ACh also stimulated the release of prostacyclin (PGI2) and PGE2 from the strips. De-endothelialized strips released much smaller amounts of prostaglandins and contracted slightly to ACh. The endothelium-dependent vasodilation was resistant to cyclooxygenase inhibition by indomethacin, flurbiprofen and diclofenac. However, it could be reversed by six different inhibitors of lipoxygenase (nordihydroguaiaretic acid, phenidone, eicosatetraynoic acid, nafazatrom, compound BW 755C and caffeic acid). BW 755C and caffeic acid, a selective inhibitor of 5-lipoxygenase, had comparatively weak effects on the relaxation. Eicosatetraynoic acid, which probably does not inhibit C-5-lipoxygenase, completely reversed the effect of ACh. It is concluded that ACh relaxes strips of rabbit aorta by a mechanism in involving a non-cyclooxygenase metabolite of arachidonic acid of endothelial origin. This compound is probably not a product of C-5-lipoxygenase.

Effects of septal and/or raphe cell suspension grafts on hippocampal choline acetyltransferase activity, high affinity synaptosomal uptake of choline and serotonin, and behavior in rats with extensive septohippocampal lesions

At 31 days of age, Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 14 days later, received intrahippocampal suspension grafts prepared from the region including the medial septum and the diagonal band of Broca (Group S, n = 11), from the region including the mesencephalic raphe (Group R, n = 11) or from both regions together (Group S+R, n = 11). Sham-operated (Group Sham, n = 9) and lesion-only (Group Les, n = 11) rats served as non-grafted controls. Seven Sham, 7 Les and 8 rats from each transplant group were tested for home cage activity (6 months after grafting) and radial maze performance (between 7.5 and 8.5 months post-grafting). One month after completion of behavioral testing, the dorsal hippocampi of these rats were prepared for measuring choline acetyltransferase (ChAT) activity and high affinity synaptosomal uptake of both [3H]choline and [3H]serotonin. The remaining rats were used for histological verifications on brain sections stained for acetylcholinesterase (AChE). The lesions increased locomotor activity, impaired radial maze learning and, in the dorsal hippocampus, reduced AChE positive staining, decreased ChAT activity (-73%) as well as high affinity uptake of both choline (-81%) and serotonin (-82%). Neither type of transplant produced any significant behavioral recovery. However, septal transplants increased hippocampal AChE positivity, restored ChAT activity and enhanced choline uptake to 116% and 70% of the values found in sham-operated rats, respectively; they had no significant effect on uptake of serotonin. Transplants from the raphe region had weak effects on hippocampal AChE positivity, increased both the ChAT activity and the choline uptake to 70% ad 38% of the sham-operated rats, respectively, and produced an (over)compensation of the serotonin uptake which reached 324% of the values found in sham-operated rats. The co-transplantation of both regions resulted in restoration of ChAT activity (113% of sham-operated rats values), choline uptake (83% of sham-operated rats) and serotonin uptake (129% of sham-operated rats). Our neurochemical data show that after extensive denervation of the hippocampus, intrahippocampal grafts of fetal neurons may foster a neurotransmitter-specific recovery which depends upon the anatomical origin of the grafted cells: a graft rich in serotonergic neurons overcompensates the serotonergic deficit, a graft rich in cholinergic neurons attenuates the cholinergic deficit, whereas a mixture of both types of grafts produces recovery from both types of deficits. Thereby, both the feasibility and the interest of the co-grafting technique are confirmed.(ABSTRACT TRUNCATED AT 400 WORDS)

Regional distribution of arachidonic acid metabolites in rat brain following convulsive stimuli

Seizures were induced in female Wistar rats by electroconvulsive shock (ECS) or administration of pentetrazole (PTZ). Brain content of various prostanoids measured by radioimmunoassay showed time-dependent changes after the induction of convulsions; highest levels were found for PGD2 followed by PGF2 alpha, PGE2, TXB2 and 6-keto-PGF1 alpha. Analysis of the various arachidonic acid metabolites in seven parts of the rat brain dissected according to the method of Glowinski and Iversen revealed the largest increases in hippocampus and cerebral cortex and smaller ones also in hypothalamus and corpus striatum both after ECS and PTZ. The ratios of the different cyclo-oxygenase products remained virtually the same in whole brain as well as in those regions where the formation of prostaglandins was markedly elevated. 15-keto-13,14-dihydro-PGF2 alpha also increased simultaneously in parallel to its parent compound, PGF2 alpha and was detected in significant amounts only in hippocampus and cerebral cortex. However, concentrations of 15-keto-13,14-dihydro-PGF2 alpha in these brain regions as well as in whole brain represented only 3-10% of the amounts found for PGF2 alpha. Thus, the metabolizing enzymes 15-hydroxy-PG-dehydrogenase and delta 13-PG-reductase seem to be of minor importance for the inactivation of prostanoids in brain tissue.

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [3H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [3H]choline accumulation and the electrically evoked [3H]acetylcholine ([3H]ACh) release were assessed in hippocampal slices. The release of [3H]ACh was measured in presence of atropine (muscarinic antagonist, 1 microM), physostigmine (acetylcholinesterase inhibitor, 0.1 microM), oxotremorine (muscarinic agonist, 0.01 microM-10 microM), mecamylamine (nicotinic antagonist, 10 microM), methiothepin (mixed 5-HT1/5-HT2 antagonist, 10 microM), 8-OH-DPAT (5-HT1A agonist, 1 microM), 2-methyl-serotonin (5-HT3 agonist, 1 microM) and CP 93129 (5-HT1B agonist, 0.1 microM-100 microM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [3H]choline was reduced to 46% of normal and the release of [3H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [3H]ACh release and was significantly more effective in grafted (-70%) than in sham-operated (-56%) or lesion-only (-54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [3H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the [3H]ACh release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [3H]ACh release. In contrast, CP 93129 dose-dependently decreased [3H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.

Graft-derived cholinergic reinnervation of the hippocampus prevents a lasting increase of hippocampal noradrenaline concentration induced by septohippocampal damage in rats

Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 2 weeks later, received into the dorsal hippocampus grafts prepared from the septal area (rich in cholinergic neurons; Group Sep) or from the mesencephalic raphe (poor in cholinergic neurons; Group Rap) of rat fetuses. Lesion-only (Group Les) and virtually intact (Group Sham) rats served as controls. Between 9.5 and 10.5 months after grafting surgery, we found the lesions to decrease choline acetyltransferase activity (ChAT), high affinity synaptosomal uptake of [3H]choline (HACU) and serotonin concentration ([5-HT]), as well as to increase the noradrenaline concentration ([NA]) in the dorsal hippocampus. Raphe grafts increased [5-HT] to 456% of normal, but had only weak or no effects on the other lesion-induced modifications in brain neurochemistry. Septal grafts dramatically increased ChAT activity and HACU, enhanced [5-HT], and reduced [NA] to near-normal levels. We also found a significant negative correlation between HACU and [NA] in rats with lesions, whether grafted or not. These data show that grafts providing the denervated hippocampus with a new cholinergic innervation might be able to exert inhibitory effects on the lesion-induced increase of [NA]. Since such an increase is indicative of sympathetic sprouting, the finding of reduced [NA] in rats with graft-derived cholinergic reinnervation of the hippocampus is in line with the hypothesis that hippocampal cholinergic denervation plays a crucial role in the induction of sympathetic sprouting. However, our data do not allow to distinguish whether grafts rich in cholinergic neurons inhibited the sympathetic sprouting itself, or rather reduced the NA content of sprouted fibers.

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

1 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 μM, 60 min incubation) and [3H]‐dopamine (0.1 μM, 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. 2 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, 10μM) did not affect the accumulation of [3H]‐5‐HT; 6‐nitroquipazine (1 μM) 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 μM) reduced the tritium accumulation to 65% whereas 6‐nitroquipazine (1 μM) was ineffective. The combination of both drugs (1 μM each) led to a further decrease to about 15%. 3 The uptake of [3H]‐dopamine into hippocampal slices was blocked by both nomifensine (1 μM) and 6‐nitroquipazine (1 μM) whereas in caudate nucleus slices only nomifensine (1, 10 μM) 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 1 μM or 10 μM 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 μM) of tritiated 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.

The endothelium‐dependent relaxation of rabbit aorta: effects of antioxidants and hydroxylated eicosatetraenoic acids

Acetylcholine (ACh) induced concentration‐dependent relaxations of rabbit aortic strips precontracted with noradrenaline. The relaxations were abolished if the endothelium of the strips was disrupted. Three different antioxidants (butylated hydroxytoluene, dithiothreitol and α‐tocopherol) reversed the endothelium‐dependent vasodilatation in a concentration‐dependent manner. However, the antioxidant ascorbic acid did not alter the vasodilatation. The hydroxylated eicosatetraenoic acids 5‐HETE, 12‐HETE, 15‐HETE and 5,12‐diHETE had no effect on aortic strips under basal or induced tension. These results suggest, that (non‐cyclo‐oxygenase) oxidation processes, insensitive to the action of ascorbic acid, represent a crucial step in the endothelium‐dependent dilatation of rabbit aorta by ACh. The hydroxylated fatty acids tested are unlikely to mediate this relaxation.

Sympathetic Sprouting: Time Course of Changes of Noradrenergic, Cholinergic, and Serotonergic Markers in the Denervated Rat Hippocampus

Abstract: As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria‐fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high‐affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5‐hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1–2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5‐hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near‐normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear‐cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high‐affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery. It is concluded (a) that hippocampal noradrenaline concentration is a more sensitive marker for sympathetic sprouting than high‐affinity noradrenaline uptake and (b) that functional in vitro studies on hippocampal sympathetic ingrowth appear to fit optimal conditions in the ventral hippocampus at a delay of at least 40 weeks after surgery.