Serotonin 5-HT1A receptor activation prevents phosphorylation of NMDA receptor NR1 subunit in cerebral ischemia

The mechanisms involved in the neuroprotective effect of serotonin 5-HT1Areceptor agonists on brain damage induced by ischemia remain to be fully elucidated.Given that serotonergic drugs may regulate N-methyl-D-aspartate (NMDA)receptor function, which is implicated in events leading to ischemia-induced neuronalcell death, this study sought to determine the effects of the selective 5-HT1Areceptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on thelevels of NMDA receptor NR1 subunit in gerbil hippocampus after transient globalcerebral ischemia. Pretreatment with 8-OH-DPAT (1 mg/kg) prevented the neuronalloss in CA1 subfield 72 h after ischemia. NMDA receptor NR1 levels in wholehippocampus were not affected 24 h after ischemia, but the levels of the subunitphosphorylated at the protein kinase A (PKA) site, pNR1(Ser897), were significantlyincreased, and this increase was prevented by the same 8-OH-DPAT dose, a probableconsequence of the increased phosphatase 1 (PP1) enzyme activity found inischemic gerbils pretreated with the 5-HT1A receptor agonist. The results suggestthat NR1 subunit phosphorylation plays a role in the neuroprotective effect of 8-OH-DPAT on cell damage induced by global cerebral ischemia in the gerbil hippocampusand support the potential interest of 5-HT1A receptor activation in the search for neuroprotective strategies (AU)

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Serotonin 5-HT1A receptor activation preventsphosphorylation of NMDA receptor NR1subunit in cerebral ischemia

The mechanisms involved in the neuroprotective effect of serotonin 5-HT1Areceptor agonists on brain damage induced by ischemia remain to be fully elucidated.Given that serotonergic drugs may regulate N-methyl-D-aspartate (NMDA)receptor function, which is implicated in events leading to ischemia-induced neuronalcell death, this study sought to determine the effects of the selective 5-HT1Areceptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on thelevels of NMDA receptor NR1 subunit in gerbil hippocampus after transient globalcerebral ischemia. Pretreatment with 8-OH-DPAT (1 mg/kg) prevented the neuronalloss in CA1 subfield 72 h after ischemia. NMDA receptor NR1 levels in wholehippocampus were not affected 24 h after ischemia, but the levels of the subunitphosphorylated at the protein kinase A (PKA) site, pNR1(Ser897), were significantlyincreased, and this increase was prevented by the same 8-OH-DPAT dose, a probableconsequence of the increased phosphatase 1 (PP1) enzyme activity found inischemic gerbils pretreated with the 5-HT1A receptor agonist. The results suggestthat NR1 subunit phosphorylation plays a role in the neuroprotective effect of 8-OH-DPAT on cell damage induced by global cerebral ischemia in the gerbil hippocampusand support the potential interest of 5-HT1A receptor activation in thesearch for neuroprotective strategies (AU)

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Serotonin 5-hT1A receptor activation prevents phosphorylation of NMDA receptor NR1 subunit in cerebral ischemia.

The mechanisms involved in the neuroprotective effect of serotonin 5-HT1A receptor agonists on brain damage induced by ischemia remain to be fully elucidated. Given that serotonergic drugs may regulate N-methyl-D-aspartate (NMDA) receptor function, which is implicated in events leading to ischemia-induced neuronal cell death, this study sought to determine the effects of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on the levels of NMDA receptor NR1 subunit in gerbil hippocampus after transient global cerebral ischemia. Pretreatment with 8-OH-DPAT (1 mg/kg) prevented the neuronal loss in CA1 subfield 72 h after ischemia. NMDA receptor NR1 levels in whole hippocampus were not affected 24 h after ischemia, but the levels of the subunit phosphorylated at the protein kinase A (PKA) site, pNR1(Ser897), were significantly increased, and this increase was prevented by the same 8-OH-DPAT dose, a probable consequence of the increased phosphatase 1 (PP1) enzyme activity found in ischemic gerbils pretreated with the 5-HT1A receptor agonist. The results suggest that NR1 subunit phosphorylation plays a role in the neuroprotective effect of 8-OH-DPAT on cell damage induced by global cerebral ischemia in the gerbil hippocampus and support the potential interest of 5-HT1A receptor activation in the search for neuroprotective strategies.

Opposing effects of AMPA and 5-HT1A receptor blockade on passive avoidance and object recognition performance: correlation with AMPA receptor subunit expression in rat hippocampus.

It has been suggested that antagonists at serotonin 5-HT1A receptors may exert a procognitive effect by facilitating glutamatergic neurotransmission. Here we further explored this issue by looking for the ability of a 5-HT1A antagonist to prevent the learning deficit induced by AMPA receptor blockade in two behavioural procedures in rats, and for concomitant molecular changes presumably involved in memory formation in the hippocampus. Pretraining administration of the competitive AMPA receptor antagonist, NBQX, produced a dose-related retention impairment in a passive avoidance task 24h later, and also impaired retention in a novel object recognition test when an intertrial interval of 3h was selected. Pretreatment with the selective 5-HT1A receptor antagonist, WAY-100635, prevented the learning deficit induced by NBQX in the two behavioural procedures. In biochemical studies performed on rat hippocampus after the retention tests, we found that learning increased the membrane levels of AMPA receptor GluR1 and GluR2/3 subunits, as well as the phosphorylated forms of GluR1, effects that were abolished by NBQX administration before the training session. Pretreatment with WAY-100635 counteracted the NBQX effects and restored the initial learning-specific increase in Ca2+/calmodulin-dependent protein kinase II (CaMKII) function and the later increase in GluR2/3 and phosphorylated GluR1 surface expression. Moreover, administration of WAY-100635 before object recognition training improved recognition memory 24h later and potentiated the learning-associated increase in AMPA receptor subunits. The results support the proposed utility of 5-HT1A antagonists in the treatment of cognitive disorders.

Acute and chronic effects of MDMA on molecular mechanisms implicated in memory formation in rat hippocampus: surface expression of CaMKII and NMDA receptor subunits.

Acute 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") treatment induces learning deficits in different animal models. In a passive avoidance learning task in rats, previous studies suggested a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII) and N-methyl-D-aspartate (NMDA) receptors in the acute learning impairment. As cognitive deficits by "ecstasy" in humans have been only reported in frequent recreational users, we examined whether a repeated MDMA treatment could induce in rats lasting molecular changes related to memory consolidation of passive avoidance. In rats with a pronounced 5-HT depletion by MDMA, the effect of another drug challenge was also examined. The surface expression in the hippocampus of NMDA receptor subunits, the scaffolding postsynaptic density protein PSD-95, phosphorylated CaMKII and protein phosphatase 1 (PP1) was measured. In rats repeatedly treated with MDMA (10 mg/kg) twice daily for 4 consecutive days, hippocampal 5-HT levels were markedly reduced 1 week later. At this time, neither learning performance was affected nor changes in membrane levels of NMDA receptor subunits, PSD-95, CaMKII and PP1 were found. In these rats, however, another drug challenge produced a rapid reduction in PSD-95 immunoreactivity and prevented the learning-specific increase in the NMDA receptor NR1 subunit and phosphorylated CaMKII. The results show no lasting change in learning-associated molecular events after a neurotoxic MDMA treatment. This drug only produces transient effects on early molecular events involved in memory consolidation, which do not appear to depend on endogenous 5-HT levels.

NMDA receptor subunit and CaMKII changes in rat hippocampus induced by acute MDMA treatment: a mechanism for learning impairment.

RATIONALE: Cognitive deficits have been reported in recreational 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") users. In rats and other animal species, acute MDMA administration produces an impairment in passive avoidance and other learning tasks. Different studies have shown that this learning deficit is not strictly related to the pronounced serotonin (5-HT) depletion induced by the drug. OBJECTIVES: This study was aimed at determining if acute MDMA administration induces in the rat hippocampus early molecular changes related to memory impairment in a passive avoidance task. The membrane expression of key molecules in memory consolidation, such as the NR1 and NR2B subunits of the N-methyl-D-aspartate (NMDA) receptor, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1) was measured. Some of these studies were also performed after 5-HT depletion induced by the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA). METHODS: Neurochemical studies were performed in rats treated with MDMA and killed 90 min later and also in rats subjected to passive avoidance 30 min after MDMA treatment. Western blotting was used for measuring the levels of NMDA receptor subunits, CAMKII and PP1. Enzyme activity assays were also performed. RESULTS: In hippocampal membrane extracts, passive avoidance training increased NMDA receptor NR1 subunit expression as well as CaMKII levels and phosphorylated CaMKII. In untrained rats, MDMA reduced NR1 and NR2B protein levels, membrane CaMKII levels and enzyme activity, and enhanced PP1 levels and activity. In trained rats, MDMA prevented the learning-specific increase in NR1 subunit expression and membrane CaMKII/pCaMKII levels. After pronounced 5-HT depletion by PCPA, MDMA impaired passive avoidance retention to a similar extent and also prevented the training-associated changes in NR1 levels and CaMKII activity. CONCLUSIONS: Diminished function of hippocampal CaMKII and reduced levels of synaptic NMDA receptor subunits appear to be involved in the impairment of passive avoidance learning induced in rats by acute MDMA treatment.

Conformationally restricted PACAP27 analogues incorporating type II/II' IBTM beta-turn mimetics. Synthesis, NMR structure determination, and binding affinity.

To probe the importance of a proposed beta-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II' beta-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to 1H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM(10,11)]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an alpha-helical conformation within segment T7-L27. For residues S9-R12, our data seem more compatible with a segment of the alpha-helix than with the beta-turn previously proposed for this fragment. In compound 1 the alpha-helix, also spanning T7-L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.

BDNF mediates the neuroprotective effect of PACAP-38 on rat cortical neurons.

In primary cultures from rat cerebral cortex, pituitary adenylate cyclase-activating polypeptide (PACAP-38) exerted a protective effect on cell death induced by the excitotoxin NMDA in neuron-enriched cultures and also on apoptotic cell death induced by serum deprivation in mixed neuronal-glial cultures. The neuroprotective effect was already observed at subnanomolar concentrations of PACAP and was slightly more pronounced against excitotoxic cell death. BDNF protein expression was reduced by NMDA and much more markedly by serum deprivation (approximately 28 and 93% reduction respectively). In both cellular injury conditions, the diminished BDNF expression was significantly prevented by PACAP. When purified neuronal cultures were preincubated with an antiserum anti-BDNF, at a concentration without any intrinsic effect on cell viability, the neuprotective effect of PACAP was no longer observed. The results suggest that the neuroprotective effect of PACAP-38 is mediated, at least in part, by preventing the suppressed expression of a neurotrophin essential for cortical neuron survival.

Serotonin 5-HT(1A) receptor expression is selectively enhanced in the striosomal compartment of chronic parkinsonian monkeys.

Cynomolgus monkeys (Macaca fascicularis) were chronically treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) until stable parkinsonism was reached. Two months later, monkeys were sacrificed and monoamine content was measured in different brain regions of the lesioned monkeys and of age-matched controls. 5-HT(1A) serotonin receptor density was measured in coronal sections labeled with [(3)H]8-OH-DPAT. As expected, dopamine was virtually nonexistent in the caudate nucleus and putamen of MPTP-treated monkeys. Serotonin levels were significantly reduced in different brain regions, particularly in the raphe nuclei. 5-HT(1A) receptor density of control animals was high in the hippocampus, notably in the CA1 field and also in the raphe nuclei, and much lower in the striatum, where 5-HT(1A) receptors showed a patchy distribution which corresponded to striosomes with poor calbindin immunostaining. 5-HT(1A) receptor density was reduced in hippocampal fields and in the raphe nuclei of parkinsonian monkeys. Conversely, in the severely lesioned striatal nuclei 5-HT(1A) receptor density was increased at caudal levels of the striatum, particularly in the putamen. The results tend to support the possibility of an increased synthesis of 5-HT(1A) receptors in brain regions with higher neuronal cell death. Upregulation of this 5-HT receptor subtype in the limbic compartment of the striatum may represent a compensatory event for the serotonergic dysfunction and associated mental disorders in neurodegenerative diseases such as Parkinson disease.

Effect of p-chloroamphetamine on 5-HT1A and 5-HT7 serotonin receptor expression in rat brain.

The aim of this study was to investigate if p-chloroamphetamine (PCA), which is neurotoxic to serotonin (5-HT) nerve terminals, was able to induce, like 3,4-methylenedioxymethamphetamine, a region-specific regulation of 5-HT1A receptor mRNA expression. The effect of PCA on the expression of 5-HT7 receptors, which share some pharmacological properties with 5-HT1A receptors, was comparatively studied. PCA (2 x 5 mg/kg) produced a lasting depletion of 5-HT content in the rat frontal cortex and hippocampus. In the hippocampus, the maximal 5-HT depletion was found on day 21 (-70%), whereas in the cortex, the highest 5-HT depletion was found on day 14 (-73%), with a partial but significant recovery on day 21. At the latter time point, 5-HT1A receptor mRNA expression was increased by 80% in the cortex and decreased by 50% in the hippocampus. The 5-HT1A receptor mRNA expression was also enhanced after exposure to PCA of rat cortical but not of hippocampal primary cultures. In regard to 5-HT7 receptor mRNA expression, the most remarkable change after PCA was the great increase (+200%) in the brain-stem. Binding studies to 5-HT1A receptors matched the changes in receptor mRNA expression. Gel shift assays revealed enhanced nuclear protein binding to the KB sequence with use of cortical but not hippocampal extracts of PCA-treated rats. Overall, the data show region-specific changes in 5-HT receptor-type expression that may not be entirely dependent on the neurotoxic effect of PCA on 5-HT terminals.

Implanted BDNF-producing fibroblasts prevent neurotoxin-induced serotonergic denervation in the rat striatum.

Degeneration of serotonergic fibers in the rat striatum was produced by local administration of the serotonergic neurotoxin 5, 7-dihydroxytryptamine (5,7-DHT) or the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), which is also toxic to serotonergic neurons. One week before neurotoxin administration, fibroblasts engineered to express the human BDNF gene were grafted into the mesencephalon, dorsal to the substantia nigra. Rats implanted with fibroblasts expressing the LacZ gene were used as controls, as well as sham-operated animals (not injected with any neurotoxin). After a survival period of 1 week, the serotonergic innervation of the striatum was assessed by measuring serotonin (5-HT) content and by immunohistochemical detection of 5-HT positive fibers. BDNF-producing cells prevented the striatal 5-HT loss induced by local administration of either 5,7-DHT or MPP(+), as well as the striatal dopamine (DA) loss induced by the latter neurotoxin. Grafting of fibroblasts carrying the BDNF or the Lac-Z gene did not modify striatal 5-HT or DA content in sham-operated animals. In 5, 7-DHT-lesioned rats, implanted or not with control Lac-Z fibroblasts, a striking reduction in the density of 5-HT immunoreactive fibers was observed. By contrast, the density of 5-HT fibers was similar in rats implanted with BDNF-producing fibroblasts as compared to sham-operated controls. The protective effect of BDNF on the damage to serotonergic terminals induced by the two neurotoxins suggests the interest of this neurotrophin in the treatment of behavioral disorders associated to neurodegenerative diseases.

Facilitation by 8-OH-DPAT of passive avoidance performance in rats after inactivation of 5-HT(1A) receptors.

1. Pretraining administration of 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT 0.1 mg kg(-1)), a 5-HT(1A) receptor agonist, or buspirone (1 mg kg(-1)), a 5-HT(1A) receptor partial agonist, markedly impaired passive avoidance retention in rats 24 h later. The effect of 8-OH-DPAT was prevented by the 5-HT(1A) receptor antagonists, NAN-190 and WAY-100635, at doses without any intrinsic effect. 2. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ 10 mg kg(-1)), an alkylating agent that inactivates different G-protein coupled receptors, impaired retention performance when given 48 h pretraining. The disruptive effect of EEDQ was reversed by 8-OH-DPAT or buspirone, given 30 min before training. 3. Non-specific actions did not account for 8-OH-DPAT-induced reversal of the EEDQ effect since no significant difference in locomotor activity or in pain threshold was found between rats receiving EEDQ or EEDQ+8-OH-DPAT. 4. When NAN-190 (1 mg kg(-1)) or WAY-100635 (0.5 mg kg(-1)) were given before 8-OH-DPAT to EEDQ-pretreated animals, the reversal by 8-OH-DPAT of EEDQ-induced retention impairment was still more pronounced. However, no EEDQ reversal by 8-OH-DPAT was found when 5-HT(1A) receptors were protected by WAY-100635 (10 mg kg(-1)) 30 min before EEDQ. 5. In the hippocampus of EEDQ-treated rats, 5-HT(7) receptors were less inactivated than 5-HT(1A) receptors and significant increases were found in 5-HT(1A) but not in 5-HT(7) receptor mRNA levels. Ritanserin and methiothepin (10 mg kg(-1) each), antagonists with higher affinity at 5-HT(7) than at 5-HT(1A) receptors, prevented the retention impairment induced by EEDQ but did not significantly protect against 5-HT(7) receptor inactivation. 6. The results indicate that the facilitatory effect of 8-OH-DPAT is not mediated through 5-HT(1A) receptors and suggest that other 8-OH-DPAT-sensitive receptors could be involved in the dual effect of 8-OH-DPAT on passive avoidance performance in rats.

Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors.

Among the recently cloned serotonin (5-hydroxytryptamine, 5-HT) receptors, the 5-HT6 subtype is of special interest for at least two reasons: 1) it is abundant in limbic areas which participate in the control of mood and emotion; and 2) some antidepressants and antipsychotics are potent 5-HT6 receptor antagonists. Studies using polyclonal anti-5-HT6 receptor antibodies and an antisense oligonucleotide were performed in order to investigate further the function(s) of 5-HT6 receptors in the rat brain. Immunocytochemistry at the light and electron microscope levels showed that 5-HT6 receptors are mainly confined to the dendritic compartment, suggesting that they could mediate 5-HT actions on neuronal firing. In some limbic areas, 5-HT6 receptor-like immunoreactivity is also associated with neuronal cilia with yet unknown functions. Continuous i.c.v. infusion with an antisense oligonucleotide for 3-4 days resulted in decreased 5-HT6 receptor-like immunostaining of the nucleus accumbens and anxiogenic behaviours in the social interaction and elevated plus maze tests. Selective 5-HT6 receptor ligands are eagerly expected to investigate further the potential implication of these receptors in limbic-dependent behaviours.

Anxiogenic-like effects and reduced stereological counting of immunolabelled 5-hydroxytryptamine6 receptors in rat nucleus accumbens by antisense oligonucleotides.

The physiological role of 5-hydroxytryptamine6 receptors in the central nervous system has not yet been elucidated. The high affinity of various psychotropic drugs for 5-hydroxytryptamine6 receptors has led to the suggestion that this receptor type may be a novel target in neuropsychiatry. We have found that continuous intracerebroventricular administration of a 5-hydroxytryptamine6 receptor antisense oligonucleotide, but not of a missense oligonucleotide, produced an anxiogenic-like response in rats using two different models of anxiety, the social interaction test and the elevated plus-maze. Neither oligonucleotide treatment modified locomotor activity, rectal temperature or food intake, suggesting a low or null neurotoxicity. The effectiveness of the treatment with the designed antisense oligonucleotide to block the synthesis of the protein encoded by the target mRNA was assessed by immunolabelling 5-hydroxytryptamine6 receptors in the nucleus accumbens, where this receptor is highly expressed, using previously characterized specific antibodies. The density of the immunostaining was quantified by means of an unbiased three-dimensional stereologic procedure, which revealed a significant reduction (-25%) in the number of immunolabelled neuronal elements. These results suggest that, in addition to other 5-hydroxytryptamine receptor subtypes, 5-hydroxytryptamine6 receptors in the nucleus accumbens may participate in anxiety-related neurobiological mechanisms.

Effect of chronic antidepressant treatment on transcription factor binding activity in rat hippocampus and frontal cortex.

1. The effect of chronic antidepressant administration on CRE-, SP1- and GRE-binding activity was studied in rat hippocampus and frontal cortex. 2. Fluoxetine and desipramine (3 and 10 mg/kg/day respectively) were given to rats for 21 consecutive days. The animals were killed 3 hr after the last injection and nuclear extracts were prepared to perform the DNA-protein reaction with consensus CRE, SP1 and GRE oligonucleotides. 3. Gel-shift assays showed that CRE-binding activity was increased in both frontal cortex and hippocampus by chronic fluoxetine treatment. Desipramine, however, only enhanced this activity in the frontal cortex. 4. Chronic fluoxetine decreased SP1-binding activity in the two selected brain regions. Again, desipramine only produced a significant reduction in the frontal cortex. 5. GRE-binding in the hippocampus was only enhanced by desipramine. Since chronic desipramine, and not fluoxetine, is able to increase hippocampal glucocorticoid receptor (GR) expression, interactions of GR with CREB and SP1 may determine the lack of effect of desipramine on binding activity of the two latter transcription factors in this brain region. 6. Overall, the results show a differential and region-specific effect of chronic, and not acute, antidepressant treatment on the DNA-binding activities studied and are consonant with the possible role of changes in gene expression in the mechanism of antidepressant action.

Differential regulation by methylenedioxymethamphetamine of 5-hydroxytryptamine1A receptor density and mRNA expression in rat hippocampus, frontal cortex, and brainstem: the role of corticosteroids.

The present study examined the effects of repeated administration to rats of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") on 5-hydroxytryptamine1A (5-HT1A) receptor density and mRNA expression in the hippocampus, frontal cortex, and brainstem. As expected, 7 days after subacute MDMA administration (20 mg/kg i.p. twice daily for 4 consecutive days) 5-HT content was markedly reduced (-70%) in the hippocampus and the frontal cortex. 5-HT1A receptor density was increased in the frontal cortex by 23% and decreased in the hippocampus and the brainstem by 25%. These changes correlated with an enhanced or diminished 5-HT1A receptor mRNA expression in the three regions studied. To examine the influence of corticosteroids on these changes, adrenalectomized (ADX) rats received the same dosage regimen as above. Adrenalectomy by itself did not modify 5-HT content in the brain regions examined and increased 5-HT1A receptor density in the hippocampus (+20%) but produced no change in the frontal cortex and brainstem. Adrenalectomy also prevented MDMA-induced changes in receptor number in the hippocampus and brainstem but not in the frontal cortex. Dexamethasone (1 mg/kg/day i.p.) administered for 7 consecutive days reversed the effects of adrenalectomy in the hippocampus but not in the frontal cortex. In the brainstem, MDMA no longer reduced 5-HT1A receptor number in ADX rats, but a significant reduction was restored when ADX animals received the glucocorticoid treatment. The present data show that MDMA may affect 5-HT1A receptors in a regionally dependent manner, notably through a drug effect on corticosterone release, which attenuates 5-HT1A receptor gene transcription selectively in the hippocampus.

Inotropic and chronotropic effects of 4-(4'-n-butylaniline)-7,8- dimethoxy-5H-pyrimido[5,4-b]indole in guinea-pig atria.

Cardiotonic effect of 4-(4'-n-butylaniline)-7,8-dimethoxy- 5H-pyrimido[5,4-b]indole (B11) was investigated in isolated cardiac tissue preparations. The action of this agent on force of contraction, beating frequency and cyclic nucleotide phosphodiesterase (PDE) activity was studied. Amrinone was used for comparison. B11 produced concentration-dependent (5 x 10(6)-1 x 10(-4)M) positive inotropic and positive chronotropic responses in guinea-pig atrial tissues. The potency of B11 was greater than that of amrinone. The cardiotonic effects of B11 were not modified by beta-adrenoceptor blockade. Carbachol inhibited the positive inotropic effect of B11. The activity of B11 was increased in desensitized left atrial tissues. B11 inhibited the activities of PDE isoenzymes (type I, II, IV and V) from dog heart ventricle and PDE type IV from guinea-pig heart ventricle nonselectively. It is concluded that B11 possesses potent positive inotropic activity in guinea-pig atria, and the effect is probably mediated by a non-selective inhibition of PDE activity.

Inhibition of inflammation and gastric damage in rats by copper (II) complexes.

Anti-inflammatory activity and the effect on gastric damage of some copper(II) complexes has been investigated. In a preliminary screening the compounds were studied on the carrageenin paw edema model and two complexes showed inhibitory action on acute as well as subacute models of inflammation. This activity does not seem to be related to an inhibitory effect on prostaglandin biosynthesis or with leukocyte migration into inflamed tissue. These complexes showed an inhibitory effect on volume exudate in experimental pleurisy and on the production of superoxide anions. These inhibitory effects could explain, at least in part, their anti-inflammatory activity. Also, these complexes protected gastric mucosa against the damage induced by HCl and by oral administration of indometacin.

VIP suppression in the intestine and cerebral cortex following administration of VIP antiserum to newborn rats.

The administration of vasoactive intestinal peptide (VIP) antiserum to newborn rats significantly reduced the VIP content, both in the cerebral cortex and in intestinal epithelial cells. The decrease was observed at postnatal days 14 and 21 and also in 90 day-old animals. The neonatal treatment produced a significant increase in the density of high- and low-affinity binding sites for VIP in the cerebral cortex at post-natal days 14 and 21 whereas in the intestinal epithelial cells only the low-affinity binding sites were up-regulated at the same time points. VIP suppression induced by neonatal administration of the corresponding antiserum may represent a useful approach to further characterize the physiological role of this neuropeptide.

Differential effects of the novel analgesic, S 12813-4, on the spinal release of substance P- and calcitonin gene-related peptide-like materials in the rat.

The possible inhibitory control by the novel analgesic S 12813-4 (3-(2-(4-phenylpiperazine-1-yl)-ethyl)-2-oxo-2,3- dihydrooxazolo(b)pyridine) of spinal neurones containing substance P (SP) and/or calcitonin gene-related peptide (CGRP) was assessed in vitro and in vivo in the rat. S 12813-4 (10 nM-0.1 mM) did not affect the spinal release of CGRP-like material (CGRPLM) but inhibited in a concentration dependent manner the K(+)-evoked overflow of SP-like material (SPLM) from slices of the dorsal half of the rat lumbar enlargement. The inhibitory effect of 10 microM S 12813-4 on SPLM release was not additive with that of Na (0.1 mM), and could be prevented by the alpha 2-adrenoceptor antagonist idazoxan (10 microM). Similarly, idazoxan (10 microM) suppressed the inhibition by intrathecally administered S 12813-4 (10 microM) of the spinal outflow of SPLM in halothane anaesthetized rats whose intrathecal space was perfused with an artificial cerebrospinal fluid. These data suggest that the analgesic effect of S 12813-4 might involve some alpha 2-adrenoreceptor-mediated control of SPLM release within the spinal cord. Whether this control concerns SP-containing primary afferent fibres (presynaptic inhibition) or SP-containing interneurones and/or bulbo-spinal SP-ergic pathways (postsynaptic inhibition) deserves further investigations.