In vivo electrochemical detection of 5-hydroxyindoles in the dorsal horn of the spinal cord: the contribution of uric acid to the voltammograms.

Treated carbon fiber electrodes were used with differential normal pulse voltammetry (DNPV) for in vivo determination of the relative participation of uric acid (UA) to peak 3 derived between 250-300 mV in the dorsal horn of the spinal cord of anesthetized rats. In vitro, treated carbon fiber electrodes respond linearly over a large range of concentrations of UA (oxidation potential around 250 mV) and 5-hydroxyindoleacetic acid (5-HIAA, oxidation potential around 280-290 mV), but are 3 to 4 times more sensitive to 5-HIAA than to UA. In vivo the question remains as to the exact nature of peak 3 because the difference between oxidation potentials of UA and 5-HIAA is not great enough to permit a separate monitoring of the two compounds. In normal rats, administration of the xanthine oxidase inhibitor allopurinol, produced a progressive decrease of the signal, which reached 64.3% of controls at 120 min (35.6% diminution) after injection, and then plateaued around this value for up to 2 h. The administration of the monoamine oxidase inhibitor (MAOI) clorgyline, produced a classical decay in the voltammograms due to a diminution of endogenous 5-HIAA; however, allopurinol injected 3 h after MAOI gave an additional decrease of peak 3 of about 28%. Finally, in rats pretreated with parachlorophenylalanine (pCPA), the residual peak (32.48% as compared to peak 3 of normal rats taken as 100%), the potential of which is shifted to near that of UA, could be decreased by allopurinol to a level of 9.6% of the peak in control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo electrochemical detection of 5-hydroxyindoles in rat somatosensory cortex: effect of the stimulation of the serotonergic pathways in normal and pCPA-pretreated animals.

Differential pulse voltammetry was used for the detection of 5-hydroxyindoles in the cerebral cortex of rats anaesthetized with urethane. The stimulation of the lateral hypothalamus or of the dorsal raphe nucleus induced a 10-40% increase in the amplitude of the signal. The signal recorded from p-chlorophenylalanine (pCPA)-pretreated animals was much smaller than in normal animals and could be increased by 5-HTP administration. The stimulation of the serotonergic pathways was ineffective in the pCPA-pretreated animals.

Differential pulse voltammetry in the dorsal horn of the spinal cord of the anesthetized rat: are the voltammograms related to 5-HT and/or to 5-HIAA?

Treated carbon fiber microelectrodes were used with the differential pulse voltammetry method for in vitro and in vivo determination of indoleamines. Under these conditions a peak of oxidation current which is characteristic of 5-hydroxyindoles is recorded at 280-300 mV. Treated carbon fiber microelectrodes respond in vitro linearly over a large range of concentrations of 5-hydroxytryptamine (5-HT) and of 5-hydroxyindoleacetic acid (5-HIAA), but are 5-8 times more sensitive to 5-HT than to 5-HIAA. In vivo, the question remains as to the exact nature of the peak because the oxidation potentials of 5-HT and 5-HIAA are close together and cannot be monitored separately. Pharmacological investigations were hence carried out in order to characterize the electrochemical signal detected at 300 mV in the dorsal horn of the lumbar spinal cord of chloral hydrate-anesthetized rats. Using 250 micron long carbon fiber microelectrodes, the electrochemical signal stabilizes at 30-90 min and the peak remains constant for up to 210 min. Administration of the monoamine oxidase inhibitor (MAOI) clorgyline produced a progressive decrease of the signal which reached a decrease of 33% of control at 180 min after injection. At this time biochemical measures demonstrated a 117% increase in 5-HT and a 32% decrease in 5-HIAA in the dorsal half of the spinal cord. Reserpine provoked an increase of 20% in the electrochemical peak and the 5-HIAA outflow blocker probenecid gave rise to a sustained plateau of about 60% above control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Laminar distribution of serotonergic innervation in rat somatosensory cortex, as determined by in vivo electrochemical detection.

In vivo differential pulse voltammetry was used for the detection of indoleamines during vertical electrode penetrations in rat first somatosensory cortex, for studying the laminar distribution of serotonin and/or its metabolites in that part of the cortex. The peak of current corresponding to 5-hydroxyindoles was maximum in the most superficial part of the cortex and diminished gradually in the deeper layers. These results suggest that the cortical serotonergic innervation is predominant in the superficial layers.

Selective immunocytochemical staining of mitral cells in rat olfactory bulb with affinity purified antibodies against N-acetyl-aspartyl-glutamate.

Polyclonal antibodies were raised in rabbits against N-acetyl-aspartyl-glutamate (NAAG) coupled to bovine serum albumin (BSA) with carbodiimide and were purified by affinity chromatography sequentially over BSA-agarose and NAAG-agarose resins. Solid-phase RIA revealed a distinct pattern of specificity of the antibodies for N-acetylated acidic peptides, with highest signal obtained for NAAG, and essentially no immunoreactivity demonstrable for aspartate or glutamate. Coronal sections through carbodiimide-fixed rat olfactory bulb were incubated with the purified antiserum and antigen localization visualized by the avidin-biotin peroxidase techniques. Immunoreactivity was restricted to the mitral cells, the major excitatory projection neurons of the lateral olfactory tract, a putative glutamatergic pathway. Immunoreactivity was selectively blocked by preincubation of the antibody with 1 microgram/ml of NAAG-BSA. These results demonstrate a specific neuronal localization of NAAG-like immunoreactivity and support the candidacy of NAAG as a neurotransmitter of the lateral olfactory tract.

Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues: findings with a novel liquid phase radioimmunoassay.

Antibodies were raised in rabbits against the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific alterations in the levels of N-acetyl-aspartyl-glutamate in the nervous system of the dystrophic mouse.

Determinations of N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG) levels were obtained by ion-exchange HPLC from 10 regions of the male dystrophic mouse brain as well as from those of non-dystrophic littermate controls. Similar to previous studies in the rat, NAA levels in control mice were distributed rather uniformly while NAAG levels exhibited a pronounced rostrocaudal gradient, with highest levels found in the lumbar spinal cord. Contrary to a recent report, we found no significant alterations in gross brain or spinal cord levels of NAA. In contrast, levels of NAAG were substantially and differentially reduced in several regions of the dystrophic mouse nervous system. These results demonstrate a pathological dissociation between NAA and NAAG, whose levels are known to display differential regional, ontogenetic and phylogenetic patterns. In addition, they may represent an ability of neural tissue to differentially regulate their steady-state levels, if indeed they can be shown to be biosynthetically related. The pronounced and non-uniform NAAG reductions observed in the dystrophic CNS underscores recent suggestions of a role for the neuropeptide in central systems involved in the control of motor function.

In vivo electrochemical detection of 5-hydroxyindoles in rat cerebral cortex and spinal cord: differential effects of p-chloroamphetamine, probenecid and clorgyline.

Differential pulse voltammetry associated with carbon fiber microelectrodes was used to detect the 300 mV signal which is known to reflect the concentration of 5-hydroxyindoles in the spinal cord and cerebral neocortex of rats anesthetized with urethane or chloral hydrate. The intraperitoneal injection of p-chloroamphetamine resulted in an increase in the amplitude of the signal in the neocortex but not in the spinal cord. Administration of clorgyline did not consistently modify the signal monitored in the neocortex whereas it decreased in the spinal cord. Probenecid induced a larger increase in 5-hydroxyindoles in the neocortex than in the spinal cord. These results demonstrate that different parts of the serotonergic system might be differentially sensitive to drugs affecting serotonin metabolism.

Pre or post-operative medical treatment with nafarelin in stage III-IV endometriosis: a French multicenter study.

OBJECTIVES: To determine the effectiveness of a 6-month course of nafarelin in the treatment of stage III-IV endometriosis and to determine if pre-operative use of nafarelin facilitates surgery. DESIGN: Prospective, multicenter, clinical trial. SETTING: Eight university hospitals and two private practice institutions in France. PATIENTS: Fifty-five patients with stage III and IV endometriosis. Two were excluded. INTERVENTIONS: The severity of endometriosis was assessed at the time of laparoscopy and patients were randomized to have either laparosopic surgery at that time following 6 months of nafarelin therapy (n=28), or laparoscopic surgery following 6 months of nafarelin therapy (n=25). All had 200 microg intranasal nafarelin twice a day for 6 months and a second look laparoscopy. MAIN OUTCOME MEASURE: Clinical efficacy, tolerance to the treatment. RESULTS: Efficacy and tolerance to the treatment were the same in both groups. AFS scores compared on both laparoscopies were significantly better if nafarelin was given prior to surgery (P=0.007). CONCLUSIONS: This preliminary study shows that in cases of combined medico-surgical treatment for stage III-IV endometriosis, preoperative medical treatment with GnRH-a gives a better AFS score improvement, but no conclusion was possible whether preoperative treatment facilitates surgery.

[Sensitivity of gastric parietal cells to histamine after administration of enprostil and ranitidine during 4 weeks in healthy subjects]. / Sensibilité des cellules pariétales gastriques à l'histamine après administration pendant 4 semaines d'enprostil et de ranitidine chez le sujet sain.

Parietal cell sensitivity is increased in patients with active duodenal ulcer. It has been shown to increase in healthy volunteers after a 4-week treatment with H2-receptor blockers. Metaanalyses of therapeutic trials have indicated that time to first relapse was longer after ulcer healing with prostaglandins (PG) than after healing with H2-receptor blockers, which might be due to a different effect of the two treatments on parietal cell sensitivity. OBJECTIVES--To study, in healthy volunteers, basal gastric acid secretion, acid secretory responses and parietal cell sensitivity to histamine before and after a 4-week treatment with enprostil (E) and ranitidine (R). METHODS--This was a randomized double-blind double-dummy crossover study. Twelve male healthy volunteers (22-44 years) were randomly assigned to receive a 4-week treatment with either E (35 micrograms bid) or R (150 mg bid). After a 2-month washout period, they were crossed to the alternate treatment. Basal acid output (BAO), acid secretory responses to low-dose histamine infusion (histamine dihydrochloride 2.5 micrograms/kg/h) (LDAO), to a high-dose histamine infusion (25 micrograms/kg/h) (HDAO) and parietal cell sensitivity (PCS = LDAO/HDAO x 100) were measured 24 hours before the first administration and 72 hours after the last administration of each medication. RESULTS--All secretory parameters were similar before treatment with E and R. As compared to pretreatment values: a) HDAO tended to increase after both treatments (NS); b) LDAO (m +/- sem) slightly increased after R (18.4 +/- 2.6 vs 13.9 +/- 3.3 mEq) (NS) but not after E (12.5 +/- 1.8 vs 13.2 +/- 1.9 mEq); c) PCS (m +/- sem) was unchanged after R (36 +/- 4 vs 33 +/- 6) but decreased after R (30 +/- 6 vs 36 +/- 0.5; P < 0.02). When secretory parameters after treatment with R and treatment with E were compared, the difference was significant for LDAO (P < 0.02) and PCS (P < 0.05). CONCLUSIONS--If also found in patients with duodenal ulcer disease, these results might, at least partly, explain the lesser propensity of the ulcers to relapse early after healing with PG than after healing with H2-receptor blockers. However enprostil has been with-drawn from the market and it is now well established that one factor influencing the relapse rate is the efficiency of the treatment given to achieve ulcer healing in eradicating Helicobacter pylori. Consequently, the interest of our results is presently mostly physiological.

Neurochemical and immunocytochemical studies on the distribution of N-acetyl-aspartylglutamate and N-acetyl-aspartate in rat spinal cord and some peripheral nervous tissues.

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.

Synaptosomal transport of radiolabel from N-acetyl-aspartyl-[3H]glutamate suggests a mechanism of inactivation of an excitatory neuropeptide.

This study was undertaken to explore in synaptosomal preparations the disposition of N-acetyl-aspartyl-glutamate (NAAG), an endogenous acidic dipeptide neurotransmitter candidate. Radiolabel from N-acetyl-aspartyl[3H]glutamate was taken up rapidly into an osmotically sensitive compartment by rat brain synaptosomal preparations in a sodium-, temperature-, and time-dependent manner. HPLC analysis of the accumulated radiolabel indicated that the bulk of the tritium cochromatographed with glutamic acid and not with NAAG. In contrast, [14C]NAAG, labeled on the N-terminal acetate, was not taken up by the synaptosomal preparation. All effective inhibitors of synaptosomal, Na+-dependent [3H]glutamate uptake were found to exhibit similar potency in inhibiting uptake of tritium derived from [3H]NAAG. However, certain alpha-linked acidic dipeptides, structurally similar to NAAG, as well as the potent convulsant quisqualic acid inhibited synaptosomal transport of [3H]NAAG but were ineffective as inhibitors of [3H]glutamate transport. Together with a demonstration of disparities between the regional accumulation of radiolabel from [3H]NAAG and high-affinity [3H]glutamate uptake, these data suggest the presence in brain of a specific peptidase targeting carboxy-terminal glutamate-containing dipeptides that may be coupled to the Na+-dependent glutamate transporter. These findings provide a possible mechanism for NAAG inactivation subsequent to its release from nerve endings.