Further evidence for an involvement of nociceptin/orphanin FQ in the pathophysiology of Parkinson's disease: a behavioral and neurochemical study in reserpinized mice.

The contribution of nociceptin/orphanin FQ (N/OFQ) to reserpine-induced Parkinsonism was evaluated in mice. A battery of motor tests revealed that reserpine caused dose-dependent and long-lasting motor impairment. Endogenous N/OFQ sustained this response because N/OFQ peptide (NOP) receptor knockout (NOP(-/-) ) mice were less susceptible to the hypokinetic action of reserpine than wild-type (NOP(+/+) ) animals. Microdialysis revealed that reserpine elevated glutamate and reduced GABA levels in substantia nigra reticulata, and that resistance to reserpine in NOP(-/-) mice was accompanied by a milder increase in glutamate and lack of inhibition of GABA levels. To substantiate this genetic evidence, the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one (J-113397) simultaneously reduced akinesia and nigral glutamate levels in reserpinized NOP(+/+) mice, being ineffective in NOP(-/-) mice. Moreover, repeated J-113397 administration in reserpinized mice resulted in faster recovery of baseline motor performance which was, however, accompanied by a loss of acute antiakinetic response. The short-term beneficial effect of J-113397 was paralleled by normalization of nigral glutamate levels, whereas loss of acute response was paralleled by loss of the ability of J-113397 to inhibit glutamate levels. We conclude that endogenous N/OFQ contributes to reserpine-induced Parkinsonism, and that sustained NOP receptor blockade produces short-term motor improvement accompanied by normalization of nigral glutamate release.

[Analysis of central mechanisms involved in gastric mucosal integrity]. / A gyomornyálkahartya integritásban szerepet játszó centrális mechanizmusok analízise.

Beta-endorphin, deltorphin II, [D-Ala2, Phe4, Gly5-ol-enkephalin (DAGO) as well as endomorphin-1 and endomorphin-2 injected intracerebroventricularly (i.c.v.) induced gastroprotective action. It has been raised that endogenous opioids may have a central role in maintaining gastric mucosal integrity. Therefore we aimed to study the role of endogenous opioid system in the gastroprotective action induced by activation of alpha 2-adrenoceptors, nociceptin- and cannabinoid-receptors. Our results suggest that the non-selective opioid receptor antagonist naloxone (27 nmol i.c.v.) and the delta-opioid receptor antagonist naltrindole (5 nmol i.c.v.) abolished the mucosal protective effect of alpha 2-adrenoceptor agonists clonidine (470 pmol i.c.v.) and rilmenidine (45 pmol i.c.v.), nociceptin (1 nmol i.c.v.) and the cannabinoid receptor agonist anandamide (110 nmol i.c.v.). Based on our findings it can be raised that opioid system besides its well known regulatory functions might be involved in maintenance of gastric mucosal integrity.

Microinjections of nociceptin into the nucleus ambiguus elicit tachycardia in the rat.

Cardiovascular effects of activation of opioid receptor like receptors (ORL1 receptors) in the nucleus ambiguus were studied in urethane-anesthetized, adult male Wistar rats. Microinjections of nociceptin (0.31, 0.62, 1.25 and 2.25 mmol/L) into the nucleus ambiguus elicited increases in heart rate (17.5 +/- 4, 33.3 +/- 2.9, 16.5 +/- 1.5 and 13.9 +/- 2.7 beats/min, respectively) which were blocked by an ORL1 receptor antagonist. These results indicate that activation of ORL1 receptors in the nucleus ambiguus elicits tachycardia.

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor.

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe(4)-->(pF)Phe(4); (b) Ala(7), Ala(11)-->Aib(7), Aib(11); (c) Leu(14), Ala(15)-->Arg(14), Lys(15). Compared with the first series, N-terminus of the second series was changed from Phe(1) to Nphe(1). All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK(i)=9.33; pEC(50)=7.50). In particular, [(pF)Phe(4), Aib(7), Aib(11), Arg(14), Lys(15)] N/OFQ-NH(2) was found to be a highly potent agonist with pK(i)=10.78 in binding studies and pEC(50)=9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe(1), (pF)Phe(4), Aib(7), Aib(11), Arg(14), Lys(15)] N/OFQ-NH(2) was the best antagonist with pA(2)=8.39 and showed high binding affinity with pK(i)=9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.

Delayed epileptogenesis in nociceptin/orphanin FQ-deficient mice.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is implicated in many biological functions, including nociception, locomotor activity, stress and anxiety, drinking and food-intake. N/OFQ has also been reported to play a facilitatory role in acute kainate-induced seizures. The aim of the present study was to investigate its involvement in a chronic model of temporal lobe epilepsy, kindling epileptogenesis, using N/OFQ knock-out mice and their wild-type littermates as controls. Kindling development was retarded in N/OFQ-deficient mice, in that (compared with controls) they required a significantly greater number of stimulations and a significantly longer time in electrical seizures to reach kindling criteria. These data indicate that N/OFQ is involved in the development of kindling and that it may play a pro-epileptogenic role.

Urodynamic effects of intravesical nociceptin/orphanin FQ in neurogenic detrusor overactivity: a randomized, placebo-controlled, double-blind study.

OBJECTIVES: To evaluate the acute urodynamic effects of the neuropeptide nociceptin/orphanin FQ (N/OFQ) in a selected group of patients with neurogenic detrusor overactivity incontinence in a randomized, placebo-controlled, double-blind study. METHODS: The study involved 14 patients who presented with neurogenic detrusor overactivity due to spinal cord injury. They were randomized to receive intravesical infusion of 1 microM N/OFQ or the same dose of [desPhe(1)]N/OFQ (the placebo). The urodynamic parameters were the bladder capacity, volume threshold for the appearance of detrusor overactivity, and the maximal bladder pressure. The study was performed on a double-blind basis: neither the patients nor the doctors who performed the instillation could distinguish the solution containing N/OFQ from that containing [desPhe(1)]N/OFQ. Data are expressed as the mean +/- SD of seven determinations. Data were statistically analyzed using the Student t test for paired or unpaired data and P <0.05 was set as the criterion for a statistically significant difference. RESULTS: The two groups were well balanced with respect to mean age, male/female ratio, etiology of spinal cord disease, and years from the lesion. Also, the baseline mean values of bladder capacity, volume threshold for the appearance of detrusor overactivity, and maximal bladder pressure were similar. The intravesical infusion of the solution containing 1 microM N/OFQ produced the following changes: bladder capacity and volume threshold for the appearance of detrusor overactivity significantly increased from 139 +/- 48 mL to 240 +/- 61 mL, and from 84 +/- 32 mL to 201 +/- 68 mL, respectively. Maximal bladder pressure decreased from 81 +/- 25 cm H(2)O to 66 +/- 12 cm H(2)O; however, this difference was not statistically significant. The intravesical infusion of the solution containing 1 microM [desPhe(1)]N/OFQ did not produce any statistically significant modification of the urodynamic parameters. CONCLUSIONS: The results of this study confirm and extend previous results showing that N/OFQ, but not the placebo, elicits a robust acute inhibitory effect on the micturition reflex in patients with a neurogenic bladder. These findings apply nociceptin orphan peptide receptor agonists as potential novel drugs for the treatment of neurogenic urinary incontinence.

Colocalization of estrogen beta-receptor messenger RNA with orphanin FQ, vasopressin and oxytocin in the rat hypothalamic paraventricular and supraoptic nuclei.

The functional significance of the novel estrogen receptor beta in brain areas that exclusively contain the ERbeta receptor subtype such as the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus is not yet fully understood. The present study attempts to characterize the peptidergic nature of the ERbeta-containing neuronal population in the PVN and the SON using the double in situ histochemistry method in the female rat. Using this method, the ERbeta mRNA coexpressions with the novel opioid neuropeptide (orphanin FQ and its receptor ORL1) mRNA in addition to the previously reported neuropeptide (arginine vasopressin-AVP, oxytocin-OXY, corticotropin releasing hormone-CRH, enkephalin-ENK) mRNAs were assessed. In the PVN, roughly half of the ERbeta expression was colocalized with the prepro-orphanin FQ mRNA, which was comparable to the colocalization observed between the ERbeta and AVP mRNAs in the same region. In addition, there was 20% overlap between the ERbeta and ORL1 receptor mRNAs, and 10% overlap between the ERbeta and OXY mRNAs in the PVN. By contrast, the coexpression between the prepro-orphanin FQ and ERbeta mRNAs was less striking in the SON. Potential interactions between the ERbeta and the well-characterized AVP-OXY neurosecretory system as well as the novel OFQ-ORL1 opioid neuropeptide system may provide new leads for the functional significance of ERbeta, specifically in stress/autonomic responses.

Immunological challenge modulates brain orphanin FQ/nociceptin and nociceptive behavior.

Orphanin FQ/Nociceptin (OFQ/N), an endogenous peptide found throughout the central nervous system, has been attributed with a wide range of functions, including modulation of motivational and emotional behavior, but most prominently, facilitation of hyperalgesia. It has also been shown that brain OFQ/N is stimulated during locally-induced peripheral inflammation, a condition well known to increase pain sensitivity. However, few studies have addressed whether specific immunological challenge using T-cell dependent and independent stimuli alters OFQ/N gene activation in the brain. Consequently, male C57BL/6J mice were challenged with 5 microg of lipopolysaccharide (LPS) or a T-cell-activating bacterial superantigen, Staphyloccocal enterotoxin A (SEA), and levels of brain OFQ/N precursor, pNOC, mRNA were analyzed by semi-quantitative RT-PCR. In addition, nociceptive thresholds were examined in immunologically challenged mice using the hotplate test. Initial results on a combined region of the brain containing various limbic components, revealed increased levels of pNOC mRNA in response to SEA challenge, but not to LPS. Further analysis of more discrete brain regions revealed increased pNOC mRNA in the hypothalamus and amygdala in response to SEA. Interestingly, challenge with SEA, but not LPS, significantly reduced hindpaw-lick latency in the hot plate test, although this effect was observed only if the hotplate environment was unfamiliar, suggesting an interaction between immunological stimulation and novelty-induced stress. Since SEA induces various cytokines, including TNF-alpha, these results are consistent with a growing literature documenting the effects of cytokines on nociceptive functions, and a possible involvement of the OFQ/nociceptin system.

Pharmacological properties of nociceptin/orphanin FQ-induced stimulation and inhibition of cyclic AMP formation in distinct layers of rat olfactory bulb.

1. We recently reported that nociceptin/orphanin FQ (N/OFQ) inhibited forskolin-stimulated adenylyl cyclase activity and increased basal enzyme activity in membranes of the external plexiform layer (EPL) and granule cell layer (GRL), respectively, of the rat main olfactory bulb. In the present study we have characterized the pharmacological profile of the inhibitory and stimulatory responses by examining the effects of various N/OFQ receptor agonists and antagonists. 2. N/OFQ(1 - 13)NH(2) fully mimicked the inhibitory and stimulatory effects of N/OFQ with EC(50) values of 0.9 and 6.5 nM, respectively. N/OFQ(1 - 7) was inactive at concentrations up to 1 microM, whereas Ac-RYYRIK-NH(2) and [Phe(1)Psi(CH(2)NH)Gly(2)]N/OFQ(1 - 13)-NH(2) behaved as partial agonists in eliciting both responses. 3. The nonpeptidyl N/OFQ receptor antagonist J-113397 competitively counteracted the inhibitory and stimulatory effects of N/OFQ with pA(2) values of 8.63 and 8.70, respectively. Similarly, the peptidyl antagonist [Nphe(1)]N/OFQ(1 - 13)NH(2) potently antagonized the two effects with pA(2) values of 8.03 and 8.45, respectively. None of the antagonists per se affected adenylyl cyclase activity. 4. These data show that in distinct layers of rat olfactory bulb both the inhibitory and stimulatory effects of N/OFQ on cyclic AMP formation display pharmacological properties consistent with the involvement of N/OFQ receptors.

Inhibition by nociceptin on excitatory non-adrenergic non-cholinergic response in guinea pig airways.

AIM: To study the effect of nociceptin (NC), a newly discovered heptadecapeptide, and U-50488H, a kappa-opioid receptor agonist, on excitatory non-adrenergic non-cholinergic (eNANC) constriction responses in guinea pig isolated bronchus. METHODS: An eNANC response was induced by electric field stimulation (EFS) in the preparation via activation of the sensory nerve terminals. The effect of NC and U-50488H was analyzed on the response. RESULTS: Nociceptin 0.001 - 0.1 micromol/L inhibited the eNANC constriction which was induced by EFS but not by capsaicin in guinea pig bronchus. The constriction inhibited by NC 0.01 micromol/L was (43 +/- 31) % compared with the control. After pretreatment with naloxone 0.1 micromol/L, the constriction was inhibited by (46 +/- 28) %, without marked change compared with the above figure. IC50 (95 % of confidence limits) was 6.12 (3.8 - 9.9) nmol/L. U-50488H also inhibited the EFS-evoked eNANC constriction and the effect was abolished after pretreatment with naloxone. IC50 (95 % of confidence limits) was 1.08 (0.5 - 2.2) micromol/L. Capsaicin 0.01 - 1 micromol/L caused a cumulative constriction response in the preparation. Moreover, the effect of capsaicin was not affected by pretreatment with NC 0.01 micromol/L or U-50488H 0.1 micromol/L. The constriction induced by exogenous neurokinin A, were also unaffected by treatment with NC 0.01 micromol/L or U-50488H 0.1 micromol/L in isolated bronchus. CONCLUSION: Nociceptin inhibits EFS-induced eNANC constriction, which is not reversed by naloxone, while U-50488H inhibits EFS-induced eNANC response via activation of opioid receptor in guinea pig airways.

Superoxide generation links nociceptin/orphanin FQ (NOC/oFQ) release to impaired N-methyl-D-aspartate cerebrovasodilation after brain injury.

BACKGROUND AND PURPOSE: Although activation of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to altered cerebrovascular regulation after traumatic brain injury, the effects of such injury on the vascular response to NMDA itself has been less well appreciated. The newly described opioid nociceptin/orphanin FQ (NOC/oFQ) elicits pial artery dilation, at least in part, in a prostaglandin-dependent manner and is released into cerebrospinal fluid after fluid percussion brain injury (FPI). Generation of superoxide anion (O(2)(-)) occurs after FPI, and a byproduct of cyclooxygenase metabolism is the generation of O(2)(-). This study was designed to determine whether NOC/oFQ generates O(2)(-), which in turn could link NOC/oFQ release to impaired NMDA-induced pial artery dilation after FPI. METHODS: Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O(2)(-) generation. RESULTS: Under non-brain injury conditions, topical NOC/oFQ (10(-)(10) mol/L, the concentration present in cerebrospinal fluid after FPI) increased superoxide dismutase-inhibitable NBT reduction from 1+/-1 to 20+/-3 pmol/mm(2) but had no effect itself on pial artery diameter. Indomethacin (5 mg/kg IV) blunted such NBT reduction (1+/-1 to 6+/-2 pmol/mm(2)), whereas the NOC/oFQ receptor antagonist [F/G] NOC/oFQ (1-13) NH(2) (10(-)(6) mol/L) blocked NBT reduction. [F/G] NOC/oFQ (1-13) NH(2) and indomethacin also blunted the NBT reduction observed after FPI (1+/-1 to 15+/-1 versus 1+/-1 to 4+/-1 versus 1+/-1 to 4+/-1 pmol/mm(2) for sham, NOC/oFQ antagonist, and indomethacin-treated animals, respectively). NMDA (10(-)(8) and 10(-)(6) mol/L)-induced pial artery dilation was reversed to vasoconstriction after FPI, and [F/G] NOC/oFQ (1-13) NH(2) attenuated such vasoconstriction (sham 9+/-1% and 16+/-1% versus FPI -7+/-1% and -12+/-1% versus FPI-[F/G] NOC/oFQ (1-13) NH(2)-pretreated animals -2+/-1% and -3+/-1%). Indomethacin and the free radical scavengers polyethylene glycol superoxide dismutase and catalase also partially restored NMDA-induced vasodilation. CONCLUSIONS: These data show that NOC/oFQ, in concentrations present in cerebrospinal fluid after FPI, increased O(2)(-) production in a cyclooxygenase-dependent manner and contributes to such production after FPI. These data show that NOC/oFQ contributes to impaired NMDA-induced pial artery dilation after FPI. Therefore, these data suggest that cyclooxygenase-dependent O(2)(-) generation links NOC/oFQ release to impaired NMDA-induced cerebrovasodilation after brain injury.

Nocistatin inhibits food intake in rats.

Nocistatin, a product of the same precursor as nociceptin/orphanin FQ (N/OFQ), has been shown to antagonize effects of N/OFQ. N/OFQ stimulates feeding, most probably by inhibiting activation of neurons containing oxytocin (OT) and vasopressin (VP), peptides considered as satiety factors, and implicated in the development of conditioned taste aversion (CTA). The present study was designed to investigate whether intracerebroventricularly (ICV) injected nocistatin (a) affects deprivation- and N/OFQ-induced feeding, (b) causes CTA, and (c) induces activation of hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, as well as OT and VP neurons present in these regions. C-Fos immunohistochemistry was used as a marker of cellular activation. Nocistatin (1-3 nmol) significantly reduced food intake in deprived rats during the first and second hour post-injection. Doses of 1-3 nmol suppressed N/OFQ-induced feeding. Nocistatin at the highest (3 nmol) dose did not cause CTA. It also did not affect activation of the PVN or SON. In nocistatin-treated animals, the percentage of Fos-positive OT and VP neurons was similar to controls. We conclude that nocistatin antagonizes the influence of N/OFQ on feeding and suppresses deprivation-induced food consumption through mechanisms other than aversion. Nocistatin does not, however, activate the PVN or SON. It does not exert its effects via VP or OT neurons.

Peripheral modulation of rat knee joint afferent mechanosensitivity by nociceptin/orphanin FQ.

The peripheral effects of nociceptin were examined in normal and acutely inflamed rat knee joints by analyzing single unit recordings from articular primary afferents in response to normal and extreme rotation of the knee. Bolus close intraarterial injection of nociceptin (0.01, 1 and 100 microM) caused a sensitization of normal and inflamed knee joint afferents in response to movements in the normal working range of the joint. When the joint was hyper-rotated, nociceptin had no significant effect on afferent discharge rate in normal knees, however, in inflamed joints the top dose of the neuropeptide caused a decrease in articular mechanosensitivity. These findings suggest that nociceptin seems to be involved in the control of peripheral nociceptive mechanisms, although the behaviour of the peptide is dependent upon the inflammatory status of the tissue.

Suppression of acute and chronic opioid withdrawal by a selective soluble guanylyl cyclase inhibitor.

Previous studies have shown that activation of N-methyl-D-aspartate (NMDA) receptors and formation of nitric oxide (NO) contributes to the hyperactivity of locus coeruleus (LC) noradrenergic neurons and behavioural symptoms seen during opioid withdrawal. However, the role of soluble guanylyl cyclase (sGC), the 'physiological' target of NO, in this phenomenon is unclear. In this study, the effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a highly selective sGC inhibitor, on the naloxone-precipitated morphine withdrawal was examined using differential normal pulse voltammetry (DNPV) to measure LC activity, in vivo microdialysis to measure glutamate/aspartate release response, and behavioural assessment to evaluate withdrawal symptoms. In halothane-anaesthetized rats, acute intracerebroventricular (i.c.v.) morphine (10 microg) reduced the catecholamine oxidation current (CA.OC) (54.5+/-4.9% of baseline). Naloxone (2 mg/kg, i.v.) reversed this action of morphine and produced a rebound increase in CA.OC (136.1+/-6.0% of baseline), representing acute morphine withdrawal. Administration of ODQ (200 nmol, i.c.v.) blocked this response without affecting acute morphine action. In animals chronically treated with morphine (15 microg/microl/h, i.c.v., 5 days), naloxone significantly increased both the CA.OC signal (270.0+/-19.6% of baseline) and the release of L-glu (193+/-30.4%) and L-asp (221.5+/-28.4%) above baseline. These responses were attenuated in animals pretreated with ODQ. In unanaesthetized chronic morphine dependent rats, ODQ treatment suppressed the signs of withdrawal precipitated by naloxone (10 mg/kg). Taken together, the results of this study suggest that sGC plays an intermediary role in the genesis of LC neuronal hyperactivity and behavioural signs of morphine withdrawal.

Nociceptin stimulates thyrotropin secretion in rats.

Effects of nociceptin on thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) secretion in rats were studied. Nociceptin (150 microgram/kg) was injected intravenously and rats were serially decapitated after the injection. The effects of nociceptin on TRH release from the hypothalamus and TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormones were measured by individual radioimmunoassays. TSH was determined by enzyme immunoassay. TRH contents in the hypothalamus decreased significantly after nociceptin injection, whereas plasma TRH concentrations showed no changes. Plasma TSH concentrations increased significantly in a dose-related manner. The TRH release from the hypothalamus was enhanced significantly in a dose-related manner with the addition of nociceptin. The TSH release from the anterior pituitary in vitro was not affected by the addition of nociceptin. The plasma thyroxine and 3,3',5-triiodothyronine levels did not change significantly after nociceptin administration. The inactivation of TRH by plasma or hypothalamus in vitro after nociceptin injection did not differ from that of controls. The findings suggest that nociceptin acts on the hypothalamus to stimulate TRH and TSH secretion.

Role of zinc in blockade of excitotoxic action of quinolinic acid by picolinic acid.

This study examined whether picolinic acid (PIC) inhibits quinolinic acid (QUIN)-induced excitotoxicity through zinc chelation. Injection of QUIN into the nucleus basalis magnocellularis significantly depleted cortical choline acetyltransferase activity 7 days post injection and PIC inhibited this response. Zinc augmented the QUIN- but not NMDA-induced response. When PIC was co-administered with zinc, PIC failed to attenuate the QUIN-induced response. The inhibition of QUIN-induced cholinergic toxicity by PIC may involve chelation of zinc.

Serum affects the characteristics of excitatory amino acid-binding sites on Müller cells.

The binding of [3H]L-aspartate to membranes obtained from primary cultures of chick retinal Müller cells (glia) was studied Cells seeded in low-serum-containing medium (1%) and maintained in this condition showed an increased number of binding site from 1 to 5 days in vitro (DIV), when compared with controls cultured in medium containing 10% serum; these changes were not reversed by the addition of 10% serum after 48 h in vitro. Increased binding at this age was due to the expression of a low affinity binding system, competitively inhibited by the glutamate uptake blocker L-aspartate-beta-hydroxamate, suggesting that high serum might inhibit the expression of uptake sites at precise maturation stages. Experiments showed the effect was due to a thermolabile serum component. The increase in binding sites is parallel in time to both an increase in aspartate uptake and the initiation of synaptogenesis in the whole retina. Our results suggest that the presence of serum at defined stages in retinal development, could result in the elevation of extracellular glutamate and the concomitant excitotoxic death of neuronal cells, due to a decreased glutamate uptake by glial cells.

Mechanisms of altered LH secretion in neonatally oestrogenized male rats.

It is well known that the control of LH secretion depends on the steroid milieu during the postnatal period. In this study LH secretion was analysed in adult male rats injected neonatally with 500 micrograms oestradiol benzoate (1) after orchidectomy, (2) after selective elimination of androgens by destruction of Leydig cells with ethylene dimethane sulphonate (EDS), and (3) after removal in orchidectomized animals of Silastic capsules containing testosterone. In addition, (4) in vivo and in vitro LH secretion in response to LHRH agonist and antagonists, (5) the hypothalamic LHRH content, (6) the basal and stimulated in vitro LHRH release, and (7) the LH responses after administration of naloxone (2 mg/kg), alpha-methyl-p-tyrosine (alpha-MPT; 250 mg/kg), N-methyl-D-aspartic acid (NMDA, 15 mg/kg) or kainic acid (KA; 15 mg/kg) were also examined. Our data indicated that (1) the LH response after orchidectomy, after EDS administration and after removal of Silastic capsules containing testosterone was diminished in oestrogenized male rats, (2) the pituitaries from oestrogenized males retained responsiveness to LHRH, (3) hypothalamic LHRH content was reduced in oestrogenized males, but the hypothalamus from oestrogenized males released more LHRH than those of control groups both under basal conditions or after depolarization, (4) alpha-MPT decreased LH secretion only in oestrogenized males, and (5) NMDA and KA stimulated LH only in oestrogenized males. We conclude that in oestrogenized male rates the loss of sensitivity to the negative feedback action of testosterone on LH secretion was not due to decreased pituitary responsiveness to LHRH stimulation or to the inherent damage of LHRH neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of excitatory amino acid pathways in the control of pituitary function in neonatally oestrogenized male rats.

It is well known that in the rat neonatal manipulation of the sex steroid environment results in altered hypothalamic-pituitary function in adulthood which implies an abnormal prolactin secretion and gonadotrophin response to orchidectomy. The present paper analyses the involvement of excitatory amino acid pathways in the disturbed gonadotrophin and prolactin secretion in male rats neonatally injected with oestradiol benzoate (500 micrograms on the day of birth). In the first experiment, 60-day-old control and oestrogenized male rats (intact or orchidectomized a week before) were killed 15 min after injection of vehicle, N-methyl-D-aspartic acid (NMDA; 15 mg/kg) or kainic acid (KA; 15 mg/kg). In the second experiment, prepubertal males were killed 15 min after injection of vehicle or NMDA. In the third experiment 30-, 45-, 60- and 90-day-old intact control and oestrogenized males were killed 15 min after injection of vehicle or KA. In the fourth experiment, control and oestrogenized males were sham-orchidectomized, orchidectomized or orchidectomized and implanted with Silastic capsules at 30 days of age and killed on day 90, 15 min after vehicle or KA injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed excitotoxic neurodegeneration induced by excitatory amino acid agonists in isolated retina.

Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in isolated chick embryo retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either the N-methyl-D-aspartate (NMDA) or non-NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non-NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the isolated chick embryo retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non-NMDA agonists kainate (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release.(ABSTRACT TRUNCATED AT 250 WORDS)