The changes of IGF binding proteins after rhGH administration to patients totally dependent on parenteral nutrition

The objective was to study the effect of recombinant human growth hormone (rhGH) administration to patients with chronic malnutrition maintained on total parenteral nutrition (TPN) on the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) during a double-blind trial. After 1 week of TPN the patients were randomized into group I (placebo) or group II (rhGH). Samples were collected on the first day (start of the TPN) to measure basal values, the seventh day to study the effect of TPN and the 10th, 14th and 21st days to evaluate the rhGH effect. Basal laboratory evaluation, nutritional status and glucose tolerance were assessed using standard laboratory techniques. Radioimmunoassays were used to analyse IGF-I, free IGF-I (fIGF-I) and IGFBP1-3. Electrophoresis with Western ligand blotting and Western immunoblotting was applied to find the pattern of IGFBPs. TPN had no effect on the circulating IGF-I concentration and the pattern of IGFBPs present in the studied groups of patients. The rhGH administration led to significant increases of IGF-I, total IGFBP-3, glycosylated IGFBP-3 (39, 42 kDa) and the 29 kDa fragment of IGFBP-3 and the decrease of IGFBP-2 during the trial (P<0.05). The mean levels of IGFBP-1, fIGF-I and the parameters of nutritional status in group II during the trial were not significantly influenced by rhGH. However, it has been found that IGFBP-1 and fIGF-I levels were correlated with the levels of the weekly nitrogen balance of each patient in group II at the end of the trial. In spite of the significant changes of IGF-I, IGFBP-2, total IGFBP-3 and IGFBP-3 (29 kDa proteolytic fragment) after rhGH administration to patients with malnutrition, maintained on parenteral nutrition, the increase of nitrogen balance was seen only in patients who decreased their IGFBP-1 and increased bioavailable IGF-I as reflected by measurement of fIGF-I. The levels of IGFBP-1 may provide a useful marker of IGF-I bioavailability in monitoring the efficiency of the rhGH therapy in malnourished patients.     

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.     

Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating the inactive and the active groups, we found that positive and negative predictive values (PV(pos), PV(neg)) for clinical disease activity of total and free insulin-like growth factor-I (IGF-I) were 0.59, 0.90 and 1.00, 0.82 respectively. Acid-labile subunit (ALS) showed diagnostic merit similar to insulin-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity. Total IGF-I, IGFBP-3 and ALS possess a higher PV(neg) for the clinical disease activity. None of the parameters can at present be claimed to be superior to the others and thus all the measured parameters are recommended to be part of the evaluation of acromegalic patients.     

Gene regulation by NMDA receptor activation in the SDN-POA neurons of male rats during sexual development

The present study was designed to identify possible signaling pathways, which may play a role in prevention of neuronal apoptosis in the sexually dimorphic nucleus of the preoptic area (SDN-POA) after physiological activation of the N-methyl-D-aspartate (NMDA) receptor. Gene response to the blockage of the NMDA receptor by an antagonist (dizocilpine hydrogen maleate; MK-801) was screened after suppression subtractive hybridization (SSH). The results showed that differential screening after SSH detected the presence of some neurotrophic genes (RNA binding motif protein 3 (RBM3), alpha-tubulin) as well as apoptosis-related genes (Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase subunit III) in the SDN-POA of male rats, which were down-regulated by blocking the NMDA receptor. The RT-PCR products of the aforementioned genes in MK-801-treated males were significantly less than that in untreated males. In particular, the expression of Bcl-2 mRNA, including Bcl-2 protein, in male rats were significantly suppressed by MK-801 treatment. Moreover, the binding activity of nuclear factor kappaB (NFkappaB) was significantly higher in male rats than in females, but significantly diminished by blocking the NMDA receptor with MK-801 in male rats. No significant difference in cAMP response element-binding protein (CREB) binding activity was observed among untreated male, MK-801-treated male, untreated female and MK-801-treated female groups. These results suggest that genes regulated by NMDA receptor activation might participate in neuronal growth and/or anti-apoptosis, and support an important signaling pathway of NFkappaB activation and its target gene, Bcl-2, in preventing neuronal apoptosis in the SDN-POA of male rats during sexual development.     

Blockage of N-methyl-D-aspartate receptors decreases testosterone levels and enhances postnatal neuronal apoptosis in the preoptic area of male rats

Sexual dimorphism has been found in the preoptic area of the hypothalamus (POA), a major site of glutamate actions via N-methyl-D-aspartate (NMDA) receptors. The sexually dimorphic nucleus of the preoptic area (SDN-POA) of male rats exhibits about seven-fold greater nuclear volume than that of females. A naturally occurring neonatal neuronal apoptosis, that can be prevented by testosterone, may contribute to this sexual difference in SDN-POA nuclear volume. Since activation of NMDA receptors in the POA induces GnRH secretion, it may be involved in both elevation of serum testosterone and prevention of neuronal death in the SDN-POA. In the present study, protein expression of NMDA receptors in the POA of male and female fetuses was quantified on the day preceding the fetal testosterone peak (embryonic day 16; ED 16). Rats were then distributed in four groups: (1) untreated males, (2) untreated females, (3) males pretreated with MK-801 (a noncompetitive NMDA receptor antagonist), and (4) females pretreated with MK-801. Serum levels of testosterone were estimated on the afternoon of ED 18. Expression of Bcl-2 and Bax, as well as neuronal apoptosis in SDN-POA, were observed on postnatal day 8. The results showed that (1) expression of NMDA receptors in the POA of male fetuses was higher than that of females on ED 16; (2) levels of testosterone were lower in MK-801 pretreated male fetuses than in intact males on ED 18; (3) expression of Bcl-2 in the POA of MK-801 pretreated male rats was significantly less than that of control males; (4) the apoptotic incidence in the SDN-POA of MK-801 pretreated male rats was significantly greater than in control males, while there was no significant difference in apoptotic incidence in the SDN-POA between MK-801 pretreated and intact females. These results suggest that the NMDA receptor is highly expressed in prenatal male fetuses, and that it might play an important role in the elevation of testosterone levels. Moreover, activation of NMDA receptors may protect SDN-POA neurons from naturally occurring neuronal death, by modulating testosterone and/or Bcl-2 expression.     

Glutamate receptors in the cingulate cortex, hippocampus, and cerebellar vermis of alcoholics

This study tests the hypothesis that glutamate receptors are altered in the brains of alcoholics as a result of chronic alcohol neurotoxicity. Excessive release of the excitatory neurotransmitter glutamate may damage postsynaptic neurons by increasing calcium flux through N-methyl-D-aspartate (NMDA) receptor-gated ion channels. Alcohol has opposite effects on the NMDA receptor, depending on the duration of exposure. Acute exposure to alcohol inhibits ion flow through NMDA receptors, whereas chronic exposure upregulates the number of these receptors and thereby increases ion flow. Acute withdrawal from alcohol results in hyperexcitability and seizures in the presence of upregulated NMDA receptors, making postsynaptic neurons vulnerable to excitotoxic damage. For this study, 13 grossly and histologically normal brains from alcoholics and 13 brains from nonalcoholic controls were selected from our brain bank. The two groups were matched for age, postmortem interval, and storage time. Maximal binding and affinities of NMDA receptors were determined by quantitative autoradiography in the cingulate cortex, the cornu Ammonis of the hippocampus, and in the cerebellar vermis. Binding was determined with an agonist, L-[3H]glutamate, with a competitive antagonist, [3H]CGP-39653, and with an antagonist binding in the channel interior, [3H]MK-801. No significant differences were found in receptor densities or affinities between alcoholics and controls. Real differences were not likely to be obscured by nonalcohol-related variables because the groups were closely matched for age, autopsy delay, time in storage, and central nervous system medications. Various diseases causing acute and chronic hypoxia did not significantly affect receptor density or affinity. Liver diseases and thiamine deficiency were excluded. A long-lasting upregulation of the number or affinity of NMDA receptors is not a key feature of chronic alcoholics.     

Roles of insulin-like growth factor-I (IGF-I) and IGF-I binding protein-2 (IGFBP2) and -5 (IGFBP5) in developing chick limbs

Insulin-like growth factor-I (IGF-I) and the IGF-I binding proteins (IGFBPs) which modulate IGF-I action have been implicated in the development of the vertebrate limbs and skeleton. We have examined the distribution of IGF-I, IGFBP2 and IGFBP5 in developing chick limb buds and have investigated their functional roles and relationships during chick limb development. IGF-I and IGFBP2 are co-expressed throughout the lateral plate from which limbs form, although IGFBP2, unlike IGF-I, does not promote formation of rudimentary limb buds from non-limb-forming flank regions in vitro. During limb outgrowth, IGF-I is present in non-AER limb ectoderm, but little IGF-I is present in the AER itself, suggesting that restriction of endogenous IGF-I activity may be required for proper AER function. Consistent with this possibility, the ectoderm of mutant limbless and wingless wing buds, which fail to form an AER, continues to express IGF-I. We also found that the AER contains abundant IGFBP2 but that IGFBP2 is not present in limb subridge mesoderm. In contrast, IGFBP2 is present in the distal mesoderm of mutant limbless or wingless limb buds, which fail to grow out. This suggests that attenuation of IGFBP2 expression is controlled by the AER and that cessation of IGFBP2 expression may be necessary for the proliferation and suppression of differentiation of subridge mesoderm that is required for limb outgrowth to occur. Consistent with this possibility, we found that exogenous IGFBP2 inhibits the anti-differentiative activity of the AER in vitro. We also found that regions of cell death in the limb contain abundant IGF-I-immunoreactive cells, consistent with a role for IGF-I in apoptosis. During skeletogenesis, IGF-I and IGFBP2 are co-localized to the condensing central core of the limb, implicating these factors as potential regulators of the onset of chondrogenic differentiation. Intriguingly, we found that IGF-I and IGFBP2 have opposing effects on chondrogenesis, as IGF-I stimulates but IGFBP2 inhibits accumulation of cartilage matrix by micromass cultures in vitro. Long [R(3)] IGF-I, an analog of IGF-I that cannot bind IGFBPs, is more effective than IGF-I in stimulating matrix accumulation, consistent with a negative role for IGFBP2 in chondrogenesis. As the chondrocytes of the limb mature, IGF-I is present only in terminal hypertrophic chondrocytes, which undergo programmed cell death, while IGFBP2 becomes localized to prehypertrophic and hypertrophic chondrocytes, suggesting involvement in chondrocyte maturation. Consistent with this possibility, we found that exogenous IGFBP2 induces precocious expression of Indian hedgehog, a marker of prehypertrophy, in maturing chondrocytes in vitro. IGF-I and IGFBP2 are also present in the osteoblasts, clasts and nascent matrix of the long bones, consistent with roles in endochondral bone formation. Unlike in rodent limbs, IGFBP5 is not expressed by chick limb ectoderm or AER. IGFBP5 expression is highly localized to developing limb musculature and, later, to the developing skeletal elements where it is expressed by osteoblast precursers and osteoblasts. The results of this study suggest potential novel roles for IGF-I and IGFBP2 in several aspects of limb development including limb outgrowth and AER activity, programmed cell death, chondrogenesis and chondrocyte maturation.     

Regulation of the insulin-like growth factor axis by increasing cell number in intestinal epithelial (IEC-6) cells

Insulin-like growth factor binding protein-2 (IGFBP-2) production as a function of cell number by intestinal epithelial cells (IEC-6) was regulated such that the IGFBP-2 concentration in 24-h conditioned medium reached a maximum, which was maintained despite increasing cell number. Northern blot analysis revealed that this effect could largely be attributed to decreasing IGFBP-2 mRNA. In contrast to IGFBP-2, secretion of IGF-II and accumulation of IGF-II mRNA by IEC-6 cells correlated positively with cell number. The highest level of IGF-II protein detected by immunoblotting of conditioned medium occurred in post-confluent cells. IGF-I stimulated the cells to grow to a high cellular density and inhibited IGFBP-2 secretion in a concentration-dependent fashion. We conclude that expression of IGF-II and IGFBP-2 are regulated in IEC-6 cells by cellular density, and IGF-II may act as a survival factor at high cell density.     

Characterization of the IGF axis in a rat liver-derived epithelial cell line

We have used the techniques of chemical cross-linking, Western blotting and immunoprecipitation-ligand blotting to demonstrate that insulin-like growth factor binding protein-2 (IGFBP-2) is associated with plasma membranes of an epithelial cell line derived from rat liver as well as being secreted into the medium by these cells. We demonstrate that these cells secrete IGF-I, but not IGF-II into serum free medium. Evidence from signalling, cell proliferation and cross-linking experiments indicate that these cells also express cell surface IGF-I receptors. Dose-response experiments indicate an enhanced biological activity of the IGF-I analogue des (1-3) IGF-I compared to wild-type IGF-I in both acute signalling experiments and longer-term (24 h) mitogenic assays. As this IGF-I analogue has lower affinity for IGFBPs, we believe that in this cell culture system, activity of IGF-I may be attenuated in the long and short term by the accumulation of IGFBP-2 in conditioned medium and by the presence of IGFBP-2 associated with the cell membrane and/or ECM.     

Maternal nutrition affects the ability of treatment with IGF-I and IGF-II to increase growth of the placenta and fetus, in guinea pigs

The aim of this study was to investigate how administration of IGF-I and IGF-II, during early to mid pregnancy, affects maternal growth and body composition as well as fetal and placental growth, in ad libitum fed, and in moderately, chronically food restricted guinea pigs. From day 20 of gestation, mothers (3-4 months old) were infused with IGF-I, IGF-II (565 microg/day) or vehicle for 17 days and then killed on day 40 of gestation. Maternal organ weights, fetal and placental weights were assessed. Treatment with IGFs did not alter body weight gain and had small effects on body composition in the mothers. Both IGF-I and IGF-II increased fetal and placental weights in ad libitum fed dams and IGF-I increased placental weight in food restricted dams. In conclusion, treatment with IGF-I during the first half of pregnancy stimulates placental growth in both ad libitum fed and food restricted guinea pigs without affecting maternal growth while fetal growth is stimulated by IGF treatment only in ad libitum fed animals.     

mRNA from NCB-20 cells encodes the N-methyl-D-aspartate/phencyclidine receptor: a Xenopus oocyte expression study.

The mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex.     

Insulin-like growth factor-I (IGF-I) colocalizes with IGF-binding proteins (IGFBPs) in mouse and rat ovary

The cellular localization of insulin-like growth factor (IGF)-I mRNA, IGF-I peptide, and IGF-binding proteins (IGFBPs) was examined in mouse and rat ovaries through use ofin situ hybridization and immunohistochemical methods. IGF-I mRNA was found to be most abundant in granulosa cells, although lower levels were also detected in cells of the theca interna, stroma, and corpus luteum. In contrast, IGF-I immunoreactivity was undetectable or low in granulosa cells, weak and variable in oocytes, high in theca interna and the corpus luteum, and highest in the stroma. Antibodies directed against IGFBP-2, 3, and 5 yielded similar patterns of immunoreactivity to that observed for IGF-I peptide. The results indicate that IGF-I is synthesized in ovarian follicles, and that IGF-I of ovarian or systemic origin becomes localized to sites containing IGFBPs in the ovary.     

Maturation of the hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty. I. Increased activation of N-methyl-D-aspartate receptors

In the male rat the timing of puberty can be estimated by the rapid increase in testicular weight occurring between 25-50 days of age. We found that elongated spermatids, the most mature germ cells identified using flow cytometry, were first seen at 25 days (4% of the testicular cells), while an adult proportion (63%) was attained by 45 days of age. We have shown previously that hypothalamic explants could release GnRH in a pulsatile fashion at a frequency increasing around the age of 25 days, thus consistent with the time of onset of puberty. Since pulsatile GnRH secretion could be suppressed by MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor activation, we postulated that an increased activation of those receptors could be involved in the neuroendocrine mechanism that activates pulsatile GnRH secretion at the onset of puberty. Such a concept was supported by the NMDA-induced release of GnRH, which was observed using 1 mM NMDA at 25 days, while a dose of 20-50 mM was required at 15 or 50 days of age. MK-801 could provide an index of NMDA receptor activation, since the antagonistic effect of MK-801 is use dependent. This particular property was confirmed by the inability of MK-801 (5 pM) to block the depolarization (veratridine)-induced release of GnRH in the presence of 0.001 mM NMDA, while partial or complete suppression was obtained in the presence of 0.1 and 10 mM NMDA, respectively. Using explants obtained at 5, 10, 15, 20, 25, 30, 35, and 50 days of age, the lowest concentrations of MK-801 that blocked the veratridine-induced release of GnRH were, respectively, 10(7), 10(7), 10(7), 10(3), 10, 10(2), 10(4), and 10(8) pM. In contrast, there was no age-related difference in sensitivity to the inhibitory effect of Mg2+, a noncompetitive NMDA receptor antagonist which is not use dependent. The pulsatile secretion of GnRH occurred at a similar frequency at 25 and 50 days of age (4.7 and 5.4 pulses/3.5 h, respectively) but it was suppressed by a lower MK-801 concentration at 25 days (10(4) pM) than at 50 days (10(8) pM). These data indicate that the NMDA receptors involved in the control of pulsatile GnRH secretion are markedly and transiently activated around the time of onset of puberty in the male rat.     

Reduced serum insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 levels in adults with inflammatory bowel disease

In the present study, the changes in circulating IGF-1 and its binding protein IGFBP-3 were determined in adult patients with active inflammatory bowel disease (IBD) in order to assess the effect of this inflammatory condition on the IGF system. IGF-1 and IGFBP-3, as well as interleukin-6 (IL-6) were measured in serum obtained from 22 consecutive newly diagnosed patients (mean age 41.3 years) with active IBD, including 10 patients with Crohn's disease (CD), and 12 with ulcerative colitis (UC). For comparison the same parameters were determined in 30 healthy volunteers matched for age, sex and Body Mass Index (BMI). Serum IGF-1 and IGFBP-3 levels were similar in the two subgroups of patients and the values from all patients were combined for comparison with those from the control group. The mean (+/- SD) serum IGF-1 concentration (178 +/- 91 ng/ml) in the patients with IBD was lower compared with that in the controls (227 +/- 79 ng/ml, P<0.035). Similarly, the mean IGFBP-3 concentration in the patients was lower than in the controls (1.6 +/- 0.6 ng/ml vs 3.2 +/- 0.7 ng/ml respectively, P<0.001), Serum IL-6 levels were higher in the patients compared with the controls (5.5 +/- 4.2 vs 0.65 +/- 0.11 pg/ml, P<0.0001). The reduced IGF-1 and IGFBP-3 levels in patients with active IBD suggest that this systemic inflammatory condition is associated with a degree of acquired GH resistance, possibly induced by inflammatory cytokines.     

Protective Effect of Riluzole on MPTP-Induced Depletion of Dopamine and Its Metabolite Content in Mice

The neuroprotective effects of riluzole (2-amino-6-trifluoromethoxy benzothiazole), a Na⁺ channel blocker with antiglutamatergic activity, MK-801, a blocker of N-methyl-D-aspartate (NMDA) receptors and monoamine oxidase (MAO) inhibitor pargyline were compared in the model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced depletion of dopamine and its metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) levels in mice. The mice received four intraperitoneal injections of MPTP (10 mg/kg) at 1-hr intervals and then the brains were analyzed at 1, 3 and 7 days after the treatments. Dopamine and DOPAC levels were significantly decreased in the striatum from 1 day after MPTP treatments. A severe depletion in dopamine and DOPAC levels was found in the striatum 3 and 7 days after MPTP treatments. Riluzole dose-dependently antagonized the MPTP-induced decrease in dopamine and DOPAC levels in the striatum. Pargyline also protected against MPTP-induced decrease in dopamine levels in the striatum. However, this drug showed no significant change in the striatal DOPAC levels. On the other hand, MK-801 failed to protect against MPTP-induced decrease in dopamine levels in the striatum. However, MK-801 reversed the MPTP-induced decrease in DOPAC levels. These results suggest that riluzole can protect against MPTP-induced striatal dopamine and DOPAC depletion in mice. This protective effect may be caused by inactivation of voltage-dependent Na⁺ channels by riluzole. Furthermore, the present study suggests that the activation of NMDA receptors does not mainly contribute to MPTP-induced neurodegeneration, whereas MAO, especially MAO type B(MAO-B) plays a crucial role in MPTP-induced degeneration of the nigrostriatal dopaminergic neuronal pathway.     

Central mechanisms subserving the impaired growth hormone secretion induced by persistent blockade of NMDA receptors in immature male rats.

Recently, we have reported in immature female rats that short-term blockade of glutamate receptors of the N-methyl-D-aspartic acid (NMDA) subtype by the noncompetitive antagonist MK-801 induced a reduction of growth rate, basal and stimulated growth hormone (GH) release and plasma somatomedin C levels. In the present study, we investigated in immature male rats the mechanism(s) through which agonists and antagonists of glutamate receptors affect GH secretion. In 21-day-old male rats, administration of MK-801 (0.2 mg/kg i.p.b.i.d.) for 10 days induced a significant impairment of growth rate, which was unrelated to a significant reduction of food intake. GH secretion from anterior pituitary fragments of MK-801-treated rats was not significantly reduced under basal conditions but was significantly less under stimulation by 40 mM K+. Incubation of dispersed pituitary cells of 31-day-old rats with N-methyl-aspartic acid (1 and 100 microM), alone or associated with MK-801 (1 microM) did not change GH secretion. Semi quantitative densitometric analysis of hypothalami of MK-801-treated rats evidenced a clearcut decrease in the intensity of GHRH-like immuno-reactivity (LI) staining in the median eminence (ME), whereas no difference was observed in the ME-somatostatin (SS)-LI. Finally, GHRH mRNA but not SS-mRNA, evaluated by slot-blot hybridization, was reduced in the hypothalamus of MK-801-treated rats. These and our previous data would demonstrate that NMDA glutamate receptors play an important role in the neuroendocrine control of GH secretion in the rat, and suggest an action mediated by GHRH-secreting neurons.     

Stability of [3H]MK-801 Binding Sites Following Chronic Ethanol Consumption

Previous work has demonstrated that short periods (1–2 weeks) of exposure to ethanol produce an upregulation of the N-methyl-D-aspartate (NMDA) receptor complex in hippocampus; an alteration that appears to be associated with the development of physical dependence, because a return to control levels occurs over a 24- to 48-hr abstinence period. Prolonged periods of chronic ethanol treatment (CET; 4–8 months of treatment) have been shown to produce severe and permanent alterations in the morphological and functional characteristics of hippocampal pyramidal neurons. Several lines of research have demonstrated that the NMDA receptor complex is involved in excitotoxic cell loss during certain pathological states. On the basis of this evidence, we hypothesized that prolonged ethanol exposure would be accompanied by an enduring increase in NMDA receptors and that NMDA receptor binding in cells surviving CET would be altered. To test this hypothesis, we measured the binding characteristics of the NMDA receptor complex in a variety of brain structures following CET. Animals were fed a nutritionally complete, ethanol-containing diet for 28 weeks and then allowed a 48-hr abstinence period. A control group was fed the same diet, except sucrose was isocalorically substituted for ethanol. We first examined the effect of CET on the binding properties of a noncompetitive antagonist to the NMDA receptor channel, [³H]diclozipene ([³H]MK-801). Next, as an indirect examination of NMDA receptor function, we measured the ability of glutamate to stimulate channel opening and thus [³H]MK-801 binding. In all brain structures examined, neither the K_d nor the B_max of [³H]MK-801 binding to the NMDA receptor was altered following CET. In addition, no effect of treatment was seen on the ability of glutamate to stimulate [³H]MK-801 binding.     

N-methyl-D-aspartate receptors mediate endogenous opioid release in enteric neurons after abdominal surgery

BACKGROUND & AIMS: We tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors mediate surgery-induced opioid release in enteric neurons. METHODS: We used mu opioid receptor (muOR) internalization as a measure of opioid release with immunohistochemistry and confocal microscopy. MuOR internalization was quantified in enteric neurons from nondenervated and denervated ileal segments of guinea pig after abdominal laparotomy with and without pretreatment with NMDA-receptor antagonists acting at different recognition sites (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,b] cyclohepten-5,10-imine (MK-801) or (D) 2-amino-5-phosphopenoic acid (AP-5) at .5, 1 mg/kg; 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazinol [4,5-]quinoline-5-oxide choline (MRZ 2/576) or 8-chloro-1,4-dioxo-1,2,3,4-tetrahydropyridazinol [4,5-]quinoline choline salt (MRZ 2/596) at .3, 1 mg/kg, or with an antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (1, 3 mg/kg). To determine whether NMDA stimulation induces opioid release, (1) ilea were exposed to NMDA (100 micromol/L) and D-serine (10 micromol/L) with or without the antagonist MK-801 or AP-5 (50 micromol/L); and (2) neuromuscular preparations of the ileum were stimulated electrically (20 Hz, 20 min) with or without MK-801 or AP-5 (50 micromol/L). RESULTS: MuOR endocytosis induced by abdominal laparotomy was inhibited significantly by NMDA-receptor antagonists in nondenervated and denervated ileal segments, but not by the AMPA-receptor antagonist. MuOR endocytosis in neurons exposed to NMDA or electrical stimulation was prevented by NMDA-R antagonists. CONCLUSIONS: Abdominal laparotomy evokes local release of glutamate that results in endogenous opioid release through the activation of peripheral NMDA receptors. This suggests an interaction between the glutamatergic and opioid systems in response to the noxious and perhaps mechanosensory stimulation of surgery.     

丹皮酚对缺糖缺氧再灌注损伤大鼠海马神经元EAA、NMDA受体表达的影响及机制探讨

目的观察丹皮酚对缺糖缺氧（OGD）再灌注损伤大鼠海马神经元兴奋性氨基酸（EAA）、N．甲基．D．天门冬氨酸（NMDA）受体表达的影响,探讨该药的神经保护作用机制。方法原代培养新生大鼠海马神经元8～12d后随机分为4组,其中对照组正常换液培养;模型组、丹皮酚组、地佐环平（MK-801）组均制备OGD再灌注损伤模型,在此基础上丹皮酚组于每个再灌注时间点前分别加入丹皮酚,MK-801组则加入MK-801溶液;倒置相差显微镜下观察神经元一般形态学变化,用MTr法测定神经元存活率,分别采用放射配基结合实验及RT-PCR法测定NMDA受体结合力、NR1亚基mRNA表达,以氨基酸分析仪检测EAA含量。结果模型组神经元肿胀或变形,突起缩短并逐渐消失,胞质内颗粒变性明显、甚至完全崩解;丹皮酚组及MK-801组神经元形态改变均较模型组减轻,胞体完整,突起较清楚,基本保持完整。与对照组比较,模型组神经元存活率明显降低,NMDA受体结合力、NR1亚基mRNA表达及E从含量明显升高,而丹皮酚组及MK_801组上述指标均较模型组明显改善（P均〈0．05）。结论丹皮酚对离体培养的大鼠海马神经元OGD再灌注损伤具有保护作用,其作用机制可能与抑制EAA及NMDA受体表达有关。     

A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty

The physiological role of N-methyl-D-aspartate (NMDA) receptors in controlling LH secretion and the initiation of puberty was investigated using two specific antagonists, MK-801 and DL-2-amino-5-phosphono valeric acid (AP-5). Single daily sc injections of MK-801 (0.1-0.2 mg/kg BW), a noncompetitive NMDA receptor antagonist, given to prepubertal rats significantly delayed but did not prevent the timing of puberty, as determined by the age at vaginal opening and first ovulation. Infusion of MK-801 (5 micrograms/h) via osmotic minipumps for 4 days inhibited the postovariectomy rise of LH secretion in prepubertal rats. Both MK-801 (0.2 mg/kg BW, sc) and AP-5 (4 x 30 mg, iv), a competitive NMDA receptor antagonist, blocked the estradiol-induced LH surge in prepubertal ovariectomized rats. These results demonstrate that blockade of NMDA receptors can prevent the development of enhanced LH secretion in female rats undergoing sexual maturation. Moreover, they support the view that activation of NMDA receptors significantly contributes to the physiological initiation of the pubertal process.