Synthesis and evaluation of new salicylaldehyde-2-picolinylhydrazone Schiff base compounds of Ru(II), Rh(III) and Ir(III) as in vitro antitumor, antibacterial and fluorescence imaging agents.

Reaction of salicylaldehyde-2-picolinylhydrazone (HL) Schiff base ligand with precursor compounds [{(p-cymene)RuCl2}2] 1, [{(C6H6)RuCl2}2] 2, [{Cp*RhCl2}2] 3 and [{Cp*IrCl2}2] 4 yielded the corresponding neutral mononuclear compounds 5-8, respectively. The in vitro antitumor evaluation of the compounds 1-8 against Dalton's ascites lymphoma (DL) cells by fluorescence-based apoptosis study and by their half-maximal inhibitory concentration (IC50) values revealed the high antitumor activity of compounds 3, 4, 5 and 6. Compounds 1-8 render comparatively lower apoptotic effect than that of cisplatin on model non-tumor cells, i.e., peripheral blood mononuclear cells (PBMC). The antibacterial evaluation of compounds 5-8 by agar well-diffusion method revealed that compound 6 is significantly effective against all the eight bacterial species considered with zone of inhibition up to 35 mm. Fluorescence imaging study of compounds 5-8 with plasmid circular DNA (pcDNA) and HeLa RNA demonstrated their fluorescence imaging property upon binding with nucleic acids. The docking study with some key enzymes associated with the propagation of cancer such as ribonucleotide reductase, thymidylate synthase, thymidylate phosphorylase and topoisomerase II revealed strong interactions between proteins and compounds 5-8. Conformational analysis by density functional theory (DFT) study has corroborated our experimental observation of the N, N binding mode of ligand. Compounds 5-8 exhibited a HOMO (highest occupied molecular orbital)-LUMO (lowest unoccupied molecular orbital) energy gap 2.99-3.04 eV. Half-sandwich ruthenium, rhodium and iridium compounds were obtained by treatment of metal precursors with salicylaldehyde-2-picolinylhydrazone (HL) by in situ metal-mediated deprotonation of the ligand. Compounds under investigation have shown potential antitumor, antibacterial and fluorescence imaging properties. Arene ruthenium compounds exhibited higher activity compared to that of Cp*Rh/Cp*Ir in inhibiting the cancer cells growth and pathogenic bacteria. At a concentration 100 µg/mL, the apoptosis activity of arene ruthenium compounds, 5 and 6 (~30 %) is double to that of Cp*Rh/Cp*Ir compounds, 7 and 8 (~12 %). Among the four new compounds 5-8, the benzene ruthenium compound, i.e., compound 6 is significantly effective against the pathogenic bacteria under investigation.

Characterization and chromosomal localization of the human homologue of a rat AMP-activated protein kinase-encoding gene: a major regulator of lipid metabolism in mammals.

AMP-activated protein kinase (AMPK) phosphorylates and inactivates acetyl-CoA carboxylase and beta-hydroxy beta-methylglutaryl-coenzyme A (HMG-CoA) reductase which are the major enzymes involved in fatty acid and lipid biosyntheses. The AMPK gene from rat (rAMPK) has recently been cloned [Carling et al., J. Biol. Chem. 269 (1994) 11442-11448]. In order to study the structure and function of the human AMPK gene (hAMPK), we have cloned the gene, and report in this communication its nucleotide (nt) sequence, tissue distribution and chromosomal location. Our results show that the ORF of hAMPK encodes 552 amino acids (aa) (62.250 kDa) and is highly conserved with rAMPK with identities of 97.3 and 90% at the aa and nt levels, respectively. The hAMPK gene bears homology to a yeast protein kinase-encoding gene (snf1) that regulates carbohydrate metabolism, and also with three other genes encoding SNF1-like kinases from different plant species, namely Arabidopsis thaliana, Hordeum vulgare and Secale cereale. As determined by fluorescent in situ hybridization of a human metaphase chromosome spread, hAMPK maps to chromosome 1p31. The size of the hAMPK transcript is 8.5 kb and the transcription start point (tsp) is located approx. 46 bp upstream from the ATG codon. While 10-15% of AMPK is alternatively spliced in most tissues of the rat, our RT-PCR analyses of the hAMPK mRNA did not reveal the presence of any alternatively spliced form of the gene in human tissues. An interesting aspect of AMPK is that its expression, unlike in rat liver, could not be detected in human liver, and thus the purported role of the gene in controlling fatty-acid synthesis in the human liver remains to be determined.

Molecular cloning, expression and chromosomal localisation of human AMP-activated protein kinase.

A cDNA encoding rat liver AMP-activated protein kinase (AMPK) was used to isolate human skeletal muscle AMPK cDNA clones. Human AMPK cDNA is more than 90% homologous to the rat sequence and predicts a protein of molecular mass 62.3 kDa, which closely agrees with the mass observed in Western blots of human tissues. AMPK antibodies were also shown to immunoprecipitate AMPK from human liver extracts. A cDNA probe was used to identify a 9.5kb transcript in several human tissues and to isolate human genomic clones. PCR mapping of rodent/human hybrid cell lines localised the human AMPK gene to chromosome 1, and fluorescent in situ hybridisation with a human genomic clone was used to sub-localise the human AMPK gene to 1p31.

Effect of chronic hypoxemia on the regulation of nitric-oxide synthase in the fetal sheep brain.

We tested the hypothesis that chronic hypoxemia modulates NO production of the fetal brain by altering its gene and protein expression. Chronically instrumented preterm fetal sheep were made hypoxemic by placental embolization for 21 days. Fetal oxygen content was measured to determine the level of hypoxemia. The expression of both eNOS and nNOS proteins and mRNA and enzyme activities of fetal sheep cerebrum were measured and compared between normoxic and hypoxemic animals. Our results show that in utero hypoxemia downregulates both Ca2+ dependent NOS activity and expression of eNOS protein and mRNA in the fetal sheep brain. In contrast, hypoxemia increased nNOS protein and mRNA levels in the cerebrum. This suggests that chronic hypoxemia has an opposing effect on eNOS and nNOS gene regulation. We propose that increased nNOS activity during chronic hypoxemia may excessively stimulate the neurons and contribute to fetal brain injury. On the other hand, downregulation of eNOS activity and expression may compromise the neuroprotective effect of eNOS and, therefore, further exacerbate brain injury.

Low-temperature-dependent expression of a rice gene encoding a protein with a leucine-zipper motif.

We have isolated from rice suspension cells three non-sequence-related cDNAs the expression of which is markedly induced by low, non-freezing temperature. Here we further characterize one of the cDNA clones, lip19. Expression of lip19 is positively regulated by low temperature, but not affected by high (40 degrees C) temperature. Sequencing and primer extension analyses showed that lip19 has a long (552 bp) 5' non-coding sequence followed by a single open reading frame specifying a protein of 148 amino acids. The deduced amino acid sequence of the protein, Lip19, shows at its amino-terminus a conserved basic region followed by a "leucine-zipper" domain. The reported sequence most similar to Lip19 is maize OCSBF-1, which is a bZip-type DNA binding protein. The possibility is suggested that Lip19 is a transcriptional factor that is positively controlled by low temperature.

Chorion releases a factor that inhibits oxytocin-stimulated myometrial contractility in the pregnant guinea pig.

It was postulated that chorion releases a substance necessary for the maintenance of uterine quiescence during pregnancy. A decrease in the release of this substance at the end of the pregnancy would be necessary for normal myometrial activation. This hypothesis was tested by demonstrating the ability of chorion to inhibit oxytocin-stimulated myometrial contractility in vitro. Tissues were obtained from timed pregnant Duncan-Hartley guinea pigs either at pre-term or near-term gestation. Myometrial strips were placed in organ baths for isometric tension measurement and contractions stimulated by oxytocin (10(-8) mol/l). Fetal membranes or conditioned medium from chorion were added directly to the organ bath. Near-term chorion and chorion conditioned-medium decreased oxytocin-stimulated contractile activity to 39% and 49% respectively. Neither pre-term nor near-term amnion reduced oxytocin-stimulated myometrial contractile activity. Relaxation induced by pre-term chorion was greater than near-term chorion (23% and 41% of the oxytocin-induced basal level respectively; P < 0.05). Further, chorion-induced relaxation was independent of the gestational age of the myometrium. Human chorion from a term, not-in-labour woman also inhibited oxytocin-stimulated guinea pig myometrial contractility. It was concluded that the chorion releases a substance or substances that reduce oxytocin-stimulated myometrial contractility and may be involved in the maintenance of uterine quiescence during pregnancy.

Natriuretic peptide-induced relaxation of myometrium from the pregnant guinea pig is not mediated by guanylate cyclase activation.

We tested both relaxation and cGMP generation by atrial (ANP), brain (BNP), and C-type natriuretic peptide (CNP) in oxytocin-stimulated myometrium from near-term pregnant guinea pigs to investigate the ability and mechanism of natriuretic peptides to inhibit myometrial contractility. Myometrial strips were contracted by 10(-8) M oxytocin, and relaxation to the cumulative addition (10(-9)-10(-6) M) of the natriuretic peptides measured. Maximal relaxation to BNP was significantly greater than to ANP (52 versus 32% respectively; p < 0.05), whereas CNP failed to produce relaxation. However, the increase in cGMP produced by BNP (10(-7) M) was significantly less than that produced by ANP (10(-7) M) (4.5 versus 7.0 times basal; p < 0.05); CNP did not increase myometrial cGMP. Anantin, a competitive blocker of the guanylate cyclase A receptor, significantly reduced the increase in cGMP produced by ANP and BNP, but had no effect on relaxation induced by either peptide. Rp-8-Br-cGMP, an inhibitor of the cGMP-dependent protein kinase, did not alter BNP-induced relaxation. The atrial natriuretic peptide-fragment 4-23 amide, a natriuretic peptide clearance receptor agonist, failed to inhibit oxytocin-stimulated myometrial contraction. We conclude that natriuretic peptide induced relaxation of oxytocin-stimulated myometrium from the pregnant guinea pig is not mediated by either guanylate cyclase A or B activation, is independent of the cGMP pathway, and does not involve clearance receptor activation. Our results suggest that natriuretic peptide-induced relaxation of pregnant myometrium is mediated via a novel mechanism.

Evidence that brain nitric oxide synthase is the major nitric oxide synthase isoform in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels.

Recent studies suggest that nitric oxide (NO) may function as a neurotransmitter in the hypothalamus. In order to provide further evidence supporting this contention, we examined: (1) whether the hypothalamus displays significant NO synthase (NOS) activity and whether the activity is inhibited by an NOS inhibitor, (2) whether the different NOS isoforms [brain (b)-NOS, endothelial (e)-NOS and macrophage (m)-NOS] are expressed in the various nuclei of the hypothalamus of the random cycling adult female rat, (3) whether the NO donor molecule, sodium nitroprusside (SNP), regulates the heme-containing enzyme, guanylate cyclase in the preoptic area and medial basal hypothalamus of the random cycling adult female rat as well as the ovariectomized steroid (estradiol-17 beta)-treated rat. The results of the study showed that the preoptic area (POA) and medial basal hypothalamus (MBH) of the adult female rat displays significant NOS activity which can be dose-dependently inhibited by an NOS inhibitor. All three NOS isoform mRNA transcripts were present in the hypothalamus, with the order of expression being b-NOS > e-NOS > m-NOS. Immunohistochemical localization using monoclonal antibodies to the specific NOS isoform proteins revealed that b-NOS represented the major form of NOS in the hypothalamus based on density and distribution of immunostaining. b-NOS immunostaining was especially dense in the organum vasculosum laminae terminalis (OVLT), medial preoptic area (MPOA), supraoptic nucleus, and moderately dense in the arcuate nucleus/median eminence. The pattern and density of b-NOS staining closely mirrored our previously reported pattern of NADPH-diaphorase staining in the hypothalamus, and a polyclonal antibody to b-NOS yielded a similar staining pattern as that observed for the monoclonal antibody. In contrast to the dense staining observed for b-NOS in the hypothalamus, we observed no specific staining for m-NOS in the hypothalamus. e-NOS immunostaining, on the other hand, was present in the hypothalamus, but to a much lesser extent than b-NOS. Light e-NOS staining was observed in the OVLT, MPOA, supraoptic nucleus and arcuate nucleus/median eminence. That NO can regulate guanylate cyclase as a potential mediator of its effects was demonstrated using SNP which dose-dependently elevated cGMP levels in the POA and MBH of random cycling rats and estrogen-primed ovariectomized rats. The effect of SNP was due to its NO donor ability as it was blocked by the NO scavenger molecule, hemoglobin. Interestingly, hemoglobin alone caused a 50-60% reduction in basal cGMP levels, suggesting that endogenously produced NO regulates basal guanylate cyclase activity. Taken as a whole, the present study demonstrates that b-NOS is the major NOS isoform in the hypothalamus and it also provides evidence that cGMP may be a mediator of NO effects in the female hypothalamus as evidenced by the potent ability of SNP to elevate cGMP levels in the POA and MBH.

Evidence for a physiological role for nitric oxide in the regulation of the LH surge: effect of central administration of antisense oligonucleotides to nitric oxide synthase.

Antisense oligonucleotides of brain-nitric oxide synthase (b-NOS) and endothelial-NOS (e-NOS) were used in steroid-primed ovariectomized rats to examine the physiological role of nitric oxide in the regulation of the LH surge. Since macrophage-NOS (m-NOS) is not produced in the hypothalamus under normal conditions, the m-NOS antisense oligonucleotide was used as control for the possible toxicity of the phosphorothioated and propynylated antisense oligonucleotides used. Female rats were ovariectomized on day 70 of age and implanted with a third ventricle cannula on day 77 of age, injected with 5 micrograms of estradiol on days 84 and 85 of age at 17.00 h and with 1 mg progesterone or vehicle on day 86 at 09.00 h. Blood samples were collected between 13.00 and 19.00 h on day 86 of age via a jugular cannula inserted on day 85 of age. Antisense oligonucleotides (400 or 800 ng) or vehicle were injected in the third ventricle at 17.00 h on days 84 and 85 just before the estradiol injection and at 06.00 and 12.00 h on day 86. Neither the 400-ng nor the 800-ng dose of m-NOS AS had any effect on the steroid-induced LH surge. In contrast, central administration of the 400-ng dose of e-NOS AS and the 800-ng dose of b-NOS AS significantly attenuated the steroid-induced LH surge. The 40% reduction in LH by e-NOS AS and b-NOS AS was accompanied by a 33 and 28% reduction in their respective protein levels as shown by Western blots. The higher amount of b-NOS AS needed to reduce the LH surge is probably due to the high abundance of b-NOS in the hypothalamus as compared to e-NOS. As a whole, this study provides significant evidence for a physiological role of nitric oxide in mediating the steroid-induced LH surge.

Chronic hypoxia increases the NO contribution of acetylcholine vasodilation of the fetal guinea pig heart.

To investigate the effect of chronic hypoxia (HPX) on vasodilation of the fetal heart, we exposed pregnant guinea pigs to room air or 12% O(2) for 4, 7, or 10 days. We excised hearts from anesthetized fetuses (60 +/- 3 days; 65-day gestation = term) and measured changes in both the coronary artery pressure of the isolated constant-flow preparation and endothelial nitric oxide synthase (eNOS) mRNA of fetal ventricles. Dilator responses to cumulative addition (10(-9)-10(-5) M) of acetylcholine and sodium nitroprusside in prostaglandin F(2alpha) (5 x 10(-6) M)-constricted hearts were similar among normoxia (NMX), 4-, 7-, and 10-day HPX (control). Nitro-L-arginine (L-NA, 10(-4)M), a NOS inhibitor, inhibited maximal acetylcholine dilation of hearts exposed to 10-day HPX greater than NMX, 4-, and 7-day HPX. Hypoxia (after 7 and 10 days) increased eNOS mRNA of fetal ventricles compared with NMX and 4-day HPX. 4-Aminopyridine (3 mM), a voltage-dependent K(+)-channel inhibitor, inhibited acetylcholine- but not sodium nitroprusside-induced dilation of NMX and 10-day HPX hearts to a similar magnitude. Glibenclamide (10(-5) M), an ATP-sensitive K(+)-channel inhibitor, had no effect on vasodilation. We conclude that chronic HPX increases the contribution of NO but does not alter K(+)-channel activation in response to acetylcholine-stimulated coronary dilation. Thus increases in NO production via upregulation of eNOS gene expression may be an adaptive response to chronic HPX in the fetal coronary circulation.

Application of a functional genomics approach to identify differentially expressed genes in human myometrium during pregnancy and labour.

The molecular mechanisms regulating uterine relaxation and contraction during pregnancy are poorly understood. In the present study, we used for the first time a functional genomics approach applying gene array technology to identify novel candidate genes involved in the regulation of uterine quiescence and contractility during pregnancy. The purpose of this approach was to obtain a molecular snapshot of the expression profile of gene transcripts as a function of the time dependent process regulating myometrial quiescence. Using this approach, we found several genes whose expression in human myometrium was altered with the onset of labour. For example, the expression of insulin-like growth factor (IGF)-II, calgranulin A and B, and G-protein coupled receptor were decreased while the expression of IGF-binding proteins, Ca(2+)/CaM binding protein kinase C substrate, and angiotensin converting enzyme were increased in the labouring, compared with non-labouring, pregnant myometrium. The differentially-expressed genes include several genes whose roles in myometrial quiescence are yet to be understood, although they have been reported to regulate vascular smooth muscle tone. Our findings illustrate the advantage of a functional genomics approach over a single gene analysis in identifying a large number of novel and potentially important genes mediating uterine smooth muscle contractile activity.

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.

Differential alterations in responsiveness in particulate and soluble guanylate cyclases in pregnant guinea pig myometrium.

OBJECTIVE: The mechanism underlying myometrial quiescence during pregnancy is unknown. Our group has previously shown that during pregnancy myometrial cyclic guanosine monophosphate content rises to several hundred times the nonpregnant levels, only to abruptly decline days before the onset of labor. Cyclic guanosine monophosphate plays an integral role in the relaxation of smooth muscle. The aim of this investigation was therefore to determine the effects of pregnancy on both soluble and particulate guanylate cyclase enzymatic activities and messenger ribonucleic acid expressions. STUDY DESIGN: Myometrium was obtained from randomly cycling adult nonpregnant guinea pigs and near-term (50-60 days' gestation) pregnant guinea pigs of similar chronologic age. Subcellular fractions were prepared by differential ultracentrifugation. Guanylate cyclase activity was determined by the conversion of guanosine triphosphate to cyclic guanosine monophosphate under basal or stimulated conditions in either the soluble guanylate cyclase or particulate guanylate cyclase fraction. A nitric oxide donor, S-nitroso- N-penacillamine, was used to activate soluble guanylate cyclase (n = 10 animals in each group). Several natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) and uroguanylin were used to stimulate the different particulate guanylate cyclase isoforms guanylate cyclase A, guanylate cyclase B, and guanylate cyclase C, respectively, in pregnant (n = 8) and nonpregnant (n = 6) animals. Cyclic guanosine monophosphate content was measured by radioimmunoassay, and enzymatic activity was expressed as picomoles of cyclic guanosine monophosphate per milligram of protein per minute. Total guanylate cyclase represented the sum of soluble guanylate cyclase and particulate guanylate cyclase activities for a tissue. To investigate whether the observed changes in guanylate cyclase activity were paralleled by changes in receptor expression, messenger ribonucleic acid levels of the genes for guanylate cyclase A and guanylate cyclase B isoforms were quantified by ribonuclease protection assay (n = 5 animals in each group). RESULTS: Under basal conditions particulate guanylate cyclase represented 78% (nonpregnant state) to 88% (during pregnancy) of the total guanylate cyclase activity in the guinea pig myometrium. Pregnancy further reduced myometrial soluble guanylate cyclase (both basal and stimulated by nitric oxide) relative to the nonpregnant state. Pregnancy selectively increased atrial natriuretic peptide-stimulated particulate guanylate cyclase activity (attributed to guanylate cyclase A), although it did not change basal myometrial particulate guanylate cyclase activity in general. Guanylate cyclase B (particulate guanylate cyclase stimulated by C-type natriuretic peptide) and guanylate cyclase C (particulate guanylate cyclase stimulated by uroguanylin) activities were unaltered by pregnancy. The selective increase in responsiveness of particulate guanylate cyclase to atrial natriuretic peptide during pregnancy was not paralleled by an increased in level of messenger ribonucleic acid for the gene for guanylate cyclase A. CONCLUSION: Pregnancy reduced the in vitro responsiveness of the myometrial soluble guanylate cyclase to nitric oxide while increasing the responsiveness of the particulate isoform to atrial natriuretic peptide and brain natriuretic peptide through a mechanism independent of any change in receptor expression.

Mammalian AMP-activated protein kinase is homologous to yeast and plant protein kinases involved in the regulation of carbon metabolism.

In mammals, an AMP-activated protein kinase (AMPK) phosphorylates both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro and has been proposed to play a major role in the regulation of lipid metabolism in vivo. We report here the primary sequence of rat AMPK and show that antibodies raised against synthetic peptides based on the deduced sequence of AMPK immunoprecipitate AMPK activity from rat liver extracts. AMPK has a remarkable degree of sequence identity to the proteins encoded by the yeast SNF1 gene and the plant RKIN1 gene. SNF1 protein kinase activity is essential for release of genes from glucose repression in Saccharomyces cerevisiae. Expression of cRKIN1 in yeast snf1 mutants restores SNF1 function. These results indicate that AMPK, SNF1, and RKIN1 form part of a family of protein kinases that have been highly conserved throughout evolution. Our results suggest that AMPK may be involved in the regulation of a wide range of metabolic pathways.

Increased nitric oxide synthesis is not involved in delayed cerebral energy failure following focal hypoxic-ischemic injury to the developing brain.

This study addressed the hypothesis that the delayed impairment in cerebral energy metabolism that develops 10-24 h after transient hypoxia-ischemia in the developing brain is mediated by induction of increased nitric oxide synthesis. Four groups of 14-d-old Wistar rat pups were studied. Group 1 was subjected to unilateral carotid artery ligation and hypoxia followed immediately by treatment with the nitric oxide synthase (NOS) inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg). Group 2 underwent hypoxia-ischemia but received saline vehicle. Group 3 received L-NAME without hypoxia-ischemia, and group 4, saline vehicle alone. At defined times after insult, the expression of neuronal and inducible NOS were determined and calcium-dependent and -independent NOS activities measured. Cerebral energy metabolism was observed using 31P magnetic resonance spectroscopy. At 48 h after insult, the expression of inducible NOS increased, whereas neuronal NOS at 24 h decreased on the infarcted side. Calcium-dependent NOS activity was higher than calcium-independent NOS activity, but did not increase within 36 h after insult, and was significantly inhibited by the administration of L-NAME. However, L-NAME did not prevent delayed impairment of cerebral energy metabolism or ameliorate infarct size. These results suggest that the delayed decline in cerebral energy metabolism after hypoxia-ischemia in the 14-d-old rat brain is not mediated by increased nitric oxide synthesis.