Bm-iAANAT3: Expression and characterization of a novel arylalkylamine N-acyltransferase from Bombyx mori.

The arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a "versatile generalist", most likely, functioning in amine acetylation - a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests.

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine alpha-hydroxylating monooxygenase (PHM).

Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

Physician offered prayer and patient satisfaction.

OBJECTIVE: While there is ongoing debate about the role of physician-offered prayer during the physician-patient encounter, many physicians feel inclined to include prayer in their practices. This randomized-controlled trial evaluated patients' acceptance of physician-offered prayer in a family practice setting, and the impact of physician-offered prayer on patient satisfaction with the physician-patient encounter. METHOD: Subjects were 137 patients in an urban, largely African American, Southeastern family medicine practice who were randomized to receive usual care plus an offer of physician-led prayer or usual care alone. Satisfaction surveys were administered following the clinical encounter. The outcomes of interest were the rate of acceptance of physician-offered prayer and the impact of the prayer offer on patient satisfaction. Personal characteristics and satisfaction scores for patients accepting prayer were compared to those for patients declining prayer. RESULTS: Over 90% of patients accepted the offer of prayer. The offer of prayer had no significant impact on patient satisfaction scores. The number of patients declining prayer was too low to permit comparison of prayer decliners with acceptors. CONCLUSIONS: This small pilot trial demonstrated that patient responses to spiritual interventions by physicians can be evaluated using randomized study designs. A large majority of patients accepted an offer of physician-led prayer, but no significant short-term impact on patient satisfaction was detected. Future research with larger sample sizes and more diverse patient populations should evaluate the effects of physician-offered prayer on the physician-patient relationship. Difficulties in conducting such research are discussed.

Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine alpha-amidating monooxygenase.

Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding alpha-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)(amidation) that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.

Comparison of computer-based and paper-based imagery strategies in learning anatomy.

This study evaluated the use of computer-based interactive imagery on students' achievement scores when compared with paper-based static imagery. It also assessed students' perceptions about the two imagery strategies and their different components. Sixty-four freshmen veterinary students (50 females, 14 males), enrolled in a comparative anatomy course, volunteered to participate in the study. This study used a pretest/posttest comparison group design and data was examined by analysis of covariance (ANCOVA). A close-ended questionnaire was administered to collect students' perceptions about the two imagery strategies. The mean difference in students' perceptions between the two strategies was analyzed using a two-tailed paired t-test. No significant differences were observed between computer-based interactive imagery and paper-based static imagery in the immediate recall of anatomical information. There was a significant difference in students' opinions toward the two strategies: students perceived computer-based interactive imagery as a better strategy in the assimilation of anatomical information than paper-based static imagery.

Using computer-based interactive imagery strategies for designing instructional anatomy programs.

In an effort to design and implement effective anatomy educational programs, this study was conducted to evaluate students' perceptions toward using two computer-based self-directed instructional modules (e.g., digestive system and canine skull) that were designed utilizing interactive imagery strategy for teaching and learning veterinary anatomy. Sixty-eight freshmen veterinary students and one graduate student participated in this study. Open-ended and close-ended questionnaires were administered to evaluate the utilization of computer-based interactive imagery strategy in developing anatomy instructional programs, and to collect data about the students' perceptions toward the use of interactive images in teaching and learning of anatomy. Means and standard deviations were calculated and analyzed for close-ended items. The open-ended questionnaire items were analyzed to identify shared patterns or themes in the students' experience after using the two instructional anatomy modules. Students reported positive attitudes toward the interactive imagery strategy used in the development of computer-based anatomy modules. Based on our findings, this study outlines the characteristics of effective instructional images that will serve as guidelines for the preparation and selection of anatomical images, as well as, how to utilize these images to develop computer-based instructional anatomy programs. Students perceived interactive imagery as an effective design strategy that helped them learn anatomical concepts.

Glutathione, S-substituted glutathiones, and leukotriene C4 as substrates for peptidylglycine alpha-amidating monooxygenase.

The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.

Studies on the mechanism of diarrhoea induced by Escherichia coli heat-stable enterotoxin (STa) in newborn calves.

Enterotoxigenic Escherichia coli (ETEC) produces a heat-stable enterotoxin (STa) that binds to and activates a putative intestinal receptor, guanylate cyclase, causing an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). Using flow cytometry and 125I-STa binding assays, we studied the distribution of STa-receptors on enterocytes isolated from different segments of the newborn calf's intestinal tract. We also investigated the effect of STa on the intracellular levels of cGMP and ion transport to the intestinal lumen. More STa-receptors were found on enterocytes prepared from the ileum than on enterocytes obtained from the other segments of the intestinal tract. Guanylate cyclase activity was higher in the ileum of STa-challenged calves than in the ileum of control calves. No changes were observed in the guanylate cyclase activity of the other intestinal segments of the STa-challenged and control calves. Na+ levels, as measured by atomic absorption spectroscopy, were significantly increased in the luminal contents of the ileum of STa-challenged calves, whereas serum Cl- levels were significantly lower in the STa-challenged calves than in control calves. This study supports previous observations on the role of guanylate cyclase in the initiation of STa-induced secretory diarrhoea and suggests that Na+/Cl- coupling may be the major mechanism for the loss of ions in the diarrhoeal response that is mostly induced in the ileum of newborn calves.

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Evidence that neuronal nitric oxide synthase but not heme oxygenase increases in the hypothalamus on proestrus afternoon.

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.

Characterization of the interaction of Escherichia coli heat-stable enterotoxixn (STa) with its intestinal putative receptor in various age groups of mice, using flow cytometry and binding assays.

BACKGROUND AND PURPOSE: Enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) is a major cause of diarrhea in young animals. Age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa was investigated. METHODS: Four age groups (2-day-, 1- and 2-week-, and 2-month-old) of Swiss Webster mice were studied (8 to 10 mice/group). Flow cytometry and radiolabeled STa (125I-STa) assays were used as reliable quantitative measures for characterization of STa-enterocyte receptor interaction. RESULTS AND CONCLUSIONS: Interaction of STa with its putative receptor was stronger for enterocytes of 2-day-old mice. Scatchard analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at higher numbers on enterocytes from 2-day-old (7.2 nmol/mg) than older (0.30, 0.36, and 0.40 nmol/mg for 1-week-, 2-week-, and 2-month-old mice, respectively). Additionally, receptors from 2-day-old mice had greater affinity for STa (Kd = 75 nM) than did receptors from older mice (Kd = 125, 1,430, and 1,111 nM for 1-week-, 2-week-, and 2-month-old mice, respectively). Density of STa receptors on enterocytes and their affinity to STa may determine extent of binding and severity of the secretory response, and may explain the high susceptibility of newborn animals and human infants to STa-mediated diarrhea.

Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Insulin modulates intestinal response of suckling mice to the Escherichia coli heat-stable enterotoxin.

Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used. Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants.

Age-dependent variation in the density and affinity of Escherichia coli heat-stable enterotoxin receptors in mice.

Enterotoxigenic strains of Escherichia coli that produce heat-stable enterotoxin (STa), are a major cause of diarrheal disease worldwide. Resistance to diarrheal disease in human infants and newborn animals has been attributed to a gradual turnover in the intestinal brush border membrane receptors to bacterial pili. In this study, we demonstrated age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa. Flow cytometry and radiolabeled-STa (125I-STa) assays were used as more reliable quantitative measures for the characterization of STa-enterocyte receptor interaction. These assays indicated a stronger interaction of STa with its putative receptor on the enterocytes of the 2-day-old suckling mice than with enterocytes from 1-week, 2-week and 2-month-old mice. Scatchard plot analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at a higher number on enterocytes from the 2-day-old mice than enterocytes of the older mice. Additionally, receptors from the 2-day-old mice had a greater affinity for STa ligand than receptors from the older mice. Density of STa receptors on enterocytes and their affinity to STa may determine the extent of binding and severity of secretory response. This may further explain the increased susceptibility of newborn animals and human infants to STa-mediated diarrheal disease.

Effect of dietary insulin on the response of suckling mice enterocytes to Escherichia coli heat-stable enterotoxin.

Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of 2-day-old Swiss Webster suckling mice were used. For this study, 5, 10, 25 and 50 micrograms of insulin was given orally to half the mice in each group for 7 days. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, a suckling mouse assay in which 1 microgram of STa was given to all mice in insulin-treated and control groups was performed. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin-treated groups compared to control mice (P < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an up-regulatory effect on the STa-receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction, which is a major cause of diarrhea in newborn animals and human infants.

Application and evaluation of the alamarBlue assay for cell growth and survival of fibroblasts.

Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentiation. In this paper, we describe the application of alamarBlue, a new and versatile metabolic dye, for the detection of Swiss 3T3 fibroblast proliferation and/or survival. As a redox indicator, alamarBlue is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number. Various assay parameters were optimized for a 96-well format to achieve a detectable range of fibroblast cell number from 100 to 20,000 cells/well, which is similar to that obtained with traditional (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and [3H]thymidine assay techniques. Standard (reference) curves generated with a known fibroblast stimulator were used to facilitate quantitation and comparison of unknown test substances. The alamarBlue assay offers the advantages of technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity. We anticipate that this simple and versatile alamarBlue assay, when used alone or in conjunction with other bioassays, will be a useful tool for investigating the complex mechanisms of cellular proliferation.

Gaseous transmitters and neuroendocrine regulation.

Recent work has demonstrated that the brain has the capacity to synthesize impressive amounts of the gases nitric oxide (NO) and carbon monoxide (CO). There is growing evidence that these gaseous molecules function as novel neural messengers in the brain. This article reviews the pertinent literature concerning the putative role of NO and CO as critical neurotransmitters and biological mediators of the neuroendocrine axis. Abundant evidence is presented which suggests that NO has an important role in the control of reproduction due to its ability to control GnRH secretion from the hypothalamus. NO potently stimulates GnRH secretion and also appears to mediate the action of one of the major transmitters controlling GnRH secretion, glutamate. Evidence is presented which suggests that NO stimulates GnRH release due to its ability to modulate the heme-containing enzyme, guanylate cyclase, which leads to enhanced production of the second messenger molecule, cGMP. A physiological role for NO in the preovulatory LH surge was also evidenced by findings that inhibitors and antisense oligonucleotides to nitric oxide synthase (NOS) attenuate the steroid-induced and preovulatory LH surge. CO may also play a role in stimulating GnRH secretion as heme molecules stimulate GnRH release in vitro, an effect which requires heme oxygenase activity and is blocked by the gaseous scavenger molecule, hemoglobin. Evidence is also reviewed which suggests that NO acts to restrain the hypothalamic-pituitary-adrenal (HPA) axis, as it inhibits HPA stimulation by various stimulants such as interleukin-1 beta, vasopressin, and inflammation. This effect fits a proinflammatory role of NO as it leads to suppression of the release of the anti-inflammatory corticosteroids from the adrenal. Although not as intensely studied as NO, CO has been shown to suppress stimulated CRH release and may also function to restrain the HPA axis. Evidence implicating NO in the control of prolactin and growth hormone secretion is also reviewed and discussed, as is the possible role of NO acting directly at the anterior pituitary. Taken as a whole, the current data suggest that the diffusible gases, NO and CO, act as novel transmitters in the neuroendocrine axis and mediate a variety of important neuroendocrine functions.

Regulation of gonadotrophin-releasing hormone (GnRH) secretion by heme molecules: a regulatory role for carbon monoxide?

Recent studies suggest that carbon monoxide (CO), which is produced in significant quantities in many brain regions including the hypothalamus, may function as a neurotransmitter. The purpose of the present study therefore was to access whether CO is capable of regulating the secretion of the hypothalamic releasing factor, gonadotropin-releasing hormone (GnRH). To this end, medial basal hypothalami were obtained from estrogen-primed ovariectomized adult rats and incubated in vitro for a 1 hour preincubation period followed by a 30 minute incubation with either vehicle or test compounds. The media was then collected for GnRH determination by RIA. Hematin, a heme molecule cleaved by heme oxygenase (HO) to yield CO, dose-dependently stimulated GnRH release with 50 microM being the lowest effective dose. The effect of hematin was not due to a toxic effect as all groups responded to KCL stimulation at the end of the experiment. The effect of hematin required HO cleavage as evidenced by the fact that the HO inhibitor, zinc protoporphyrin IZ (ZnPP), blocked the effect of hematin on GnRH release. The effect of hematin appeared to be due to its conversion to CO, as the CO scavenger molecule, hemoglobin, completely reversed the effect of hematin. Finally, the effect of hematin did not appear to be due to the generation of the other HO-generated product (biliverdin) since biliverdin dose-dependently inhibited GnRH release. Taken as a whole, the present studies provide evidence that CO is capable of modulating hypothalamic GnRH release in the female rat, suggesting that CO may function as a neurotransmitter in the hypothalamus.

Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing hormone neurons and co-localization with N-methyl-D-aspartate receptors.

The neurotransmitter glutamate plays an important role in the control of gonadotropin-releasing hormone (GnRH) secretion. Recent evidence suggests that the novel transmitter nitric oxide may also play a role in controlling GnRH release and may be an important mediator of glutamate effects. To explore the role of nitric oxide in these events, the present study determined the distribution of the enzyme which catalyzes nitric oxide production, nitric oxide synthase (NOS) in the hypothalamus, its association with GnRH neurons, and whether NOS neurons contain NMDA receptors. NOS was localized by staining hypothalamic sections from female rats for NADPH-diaphorase activity. Specific antibodies for GnRH and the NMDAR1 receptor subunit were used for double-staining to determine NOS association with GnRH neurons and the presence of NMDA R1 receptor subunits in hypothalamic NOS neurons. The studies showed intense NOS cell body and fiber staining in the organum vasculosum of the lamina terminalis (OVLT) where numerous GnRH cell bodies are located. Other major GnRH cell body sites such as the median preoptic nucleus (MPN) and medial preoptic area (MPOA) displayed moderate staining of NOS cell bodies and fibers. Intense NOS staining was also observed in the median eminence, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus of the hypothalamus. While no GnRH neurons were found to double stain for NOS in the hypothalamus, GnRH neurons were frequently surrounded by NOS neurons in the OVLT, MPN and MPOA with potential contacts between NOS and GnRH neurons in these areas. In addition, there was significant overlap of GnRH and NOS fibers in the lateral portion of the internal zone of the median eminence where GnRH fibers and terminals converge. Double-staining studies for NADPH-diaphorase and NMDA R1 receptor subunit showed that many NOS neurons in the OVLT, MPOA, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus co-localize the NMDA R1 receptor subunit. Localization of NMDA R1 receptor subunit immunoreactivity in B-NOS neurons in the hypothalamus was further confirmed by using combined immunohistochemistry-in situ hybridization. Finally, the functional importance of this co-localization was shown by the finding that central administration of a nitric oxide synthase inhibitor blocked the ability of NMDA to induce LH secretion. Taken as a whole, these studies provide evidence which support a role for nitric oxide as an important regulator of GnRH neurons in the female. They also suggest that hypothalamic NOS neurons are targets for glutamate regulation as evidenced by co-localization of the NMDA R1 receptor subunit.