Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours

Carbonic anhydrases (CAs) catalyse the hydration of C02 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. This review is focused on the distribution and role of CA isoenzymes in both normal and pathological gastrointestinal (GI) tract tissues. It has been known for many years that CAs are widely present in the GI tract and play important roles in several physiological functions such as production of saliva, gastric acid, bile, and pancreatic juice as well as in absorption of salt and water in intestine. New information suggests that these enzymes participate in several processes that were not envisioned earlier. Especially, the recent reports on plasma membrane-bound isoenzymes IX and XII have raised considerable interest since they were reported to participate in cancer invasion and spread. They are induced by tumour hypoxia and may also play a role in von Hippel-Lindau (VHL)-mediated carcinogenesis.

Expression of von Hippel-Lindau tumor suppressor and tumor-associated carbonic anhydrases IX and XII in normal and neoplastic colorectal mucosa

AIM:To analyze possible relationships between CA IX/ CA XII and pVHL expression in normal and neoplastic colorectal mucosa. METHODS: Immunohistochemical staining of 42 tissue specimens obtained from 17 cancer patients was performed to evaluate the distribution and semi-quantitatively assess the levels of CA IX, CA XII and pVHL. VHL mRNAs from 14 fresh-frozen tumors was amplified by RT-PCR and subjected to sequencing. CA9 and G412mRNA levels were analyzed by semi-quantitative RT-PCR in comparison with VEGFas an indicator of hypoxia that uncouples the pVHL control. RESULTS: Tumor tissues were associated with a borderline increase of CA IX staining signal and slight but significant decrease of CA XII immunoreactivity, whereas no association was found for pVHL. Sequence analysis of RT-PCR-amplified VHL mRNAs revealed no deletions/ mutations, suggesting that they were VHL-competent. We did not observe any correlation between pVHL and CA IX/CA XII proteins as well as between MEGFand CA9 mRNAs, but the tumor-associated changes in mRNA levels of VEGFand CA12 showed a significant inverse relationship. CONCLUSION: Our results indicate that CA9and CA12 are regulated by different intratumoral factors and that lack of apparent relationship between the levels of CA IX/CA XII and pVHL cannot be fully assigned to uncoupling of negative regulatory function of pVHL by tumor hypoxia signified by induced VEGF transcription. The interplay between the functional pVHL and CA IX/CA XII in colorectal tumors seems rather complex and is not evident merely at the expression levels.

Hydrochemical characteristics and the ecological effect of algal carbonic anhydrase in carbon cycle in the Taiyuan section of Fenhe River

Water scarcity and pollution pose a threat to the sustainable development of cities and society.Therefore,it is crucial to analyze the hydrochemical characteristics and carbon dynamics of waterdeficient areas.Taking the Taiyuan section of Fenhe River as the research object,we systematically explored the hydrochemical characteristics of surface water and its evolutionary processes,as well as the ecological effect of algal carbonic anhydrase in carbon cycle using the hydrochemical evolution method and correlation analysis.The ternary diagram demonstrates that the main water chemical type in Fenhe River was SO^(2-)_(4)·Cl^(-)-Na^(+).The Gibbs and end-member diagrams of each ion display that the chemical composition of surface water was mainly controlled by silicate decomposition.The chemical ions originated mainly from dissolution of some minerals,such as plagioclase,halite,dolomite,calcite,and gypsum.The diatoms had a lower CO_(2)requirement because they exhibited a higher abundance at a lower partial pressure of CO_(2)(p CO_(2)).However,high CO_(2)concentration had a positive effect on cyanobacteria,which reduced the active transport of HCO_(3)^(-),saved the energy needed for this part of active transport,and indirectly improved the overall photosynthetic efficiency of algae.Carbonic anhydrase(CA)activity was significantly negatively correlated with p CO_(2)and positively correlated with HCO_(3)^(-)concentration,indicating that CA in water promoted the conversion of CO_(2)to HCO_(3)^(-).The HCO_(3)^(-)generated from this process continued to participate in the erosion of silicate rocks,sequestering CO_(2)in the form of Ca CO_(3),which has a non-negligible impact on the carbon sink in the Fenhe River.These consequences may have important implications for the biogeochemical cycling occurring in urban water.

Plant Carbonic Anhydrases： Structures, Locations, Evolution, and Physiological Roles

Carbonic anhydrases （CAs） are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3- and are ubiquitous in nature. Higher plants contain three evolutionarily distinct CA families, αCAs, 13CAs, and γCAs, where each family is represented by multiple isoforms in all species. Alternative splicing of CA transcripts appears common; consequently, the number of functional CA isoforms in a species may exceed the number of genes. CAs are expressed in numerous plant tissues and in different cellular locations. The most prevalent CAs are those in the chloroplast, cytosol, and mitochondria. This diversity in location is paralleled in the many physiological and biochemical roles that CAs play in plants. In this review, the number and types of CAs in C3, C4, and crassulacean acid metabolism （CAM） plants are considered, and the roles of the α and yCAs are briefly discussed. The remainder of the review focuses on plant βCAs and includes the identification of homologs between species using phylogenetic approaches, a consideration of the inter- and intracellular localization of the proteins, along with the evidence for alternative splice forms. Current understanding of βCA tissue-specific expression patterns and what controls them are reviewed, and the physiological roles for which βCAs have been implicated are presented.

Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2 to 7.4% in at least 12 chicken strains worldwide.To reveal the intrinsic factors causing crossed beaks,genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present studies.Crossed beak was induced by short length of unilateral mandibular ramus,and a total of 110differentially expressed genes were up-or down-regulated in the affected(short)mandibular condyle side as compared to the normal side.Carbonic anhydrase 2(CA2)and Carbonic anhydrase 13(CA13)were enriched in the carbonate dehydratase activity,and high-expressed in mandibular condyle and osteoblasts(P<0.05).However,both were low-expressed in short mandibular condyle side of affected birds(P<0.05).The carbonate dehydratase inhibitor experiments confirmed that there is positive association between the calcification and carbonic anhydrase isoenzymes.Quantitative analysis with cetylpyridinium chloride showed a decrease in calcification when the cells were transfected with an anti-CA13 shRNA.Our research suggested that CA2 and CA13 are down-calcified in shortside mandibular condyle,and caused mandibular ramus to grow slowly.CA2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.

Bioleaching of vanadium from stone coal vanadium ore by Bacillus mucilaginosus:Influencing factors and mechanism

Vanadium and its derivatives are used in various industries,including steel,metallurgy,pharmaceuticals,and aerospace engineering.Although China has massive reserves of stone coal resources,these resources have low grades.Therefore,the effective extraction and recovery of metallic vanadium from stone coal is an important way to realize the efficient resource utilization of stone coal vanadium ore.Herein,Bacillus mucilaginosus was selected as the leaching strain.The vanadium leaching rate reached 35.5%after 20 d of bioleaching under optimal operating conditions.The cumulative vanadium leaching rate in the contact group reached 35.5%,which was higher than that in the noncontact group(9.3%).The metabolites of B.mucilaginosus,such as oxalic,tartaric,citric,and malic acids,dominated in bioleaching,accounting for 73.8%of the vanadium leaching rate.Interestingly,during leaching,the presence of stone coal stimulated the expression of carbonic anhydrase in bacterial cells,and enzyme activity increased by 1.335-1.905 U.Enzyme activity positively promoted the production of metabolite organic acids,and total organic acid content increased by 39.31 mg·L^(-1),resulting in a reduction of 2.51 in the pH of the leaching system with stone coal.This effect favored the leaching of vanadium from stone coal.Atomic force microscopy illustrated that bacterial leaching exacerbated corrosion on the surface of stone coal beyond 10 nm.Our study provides a clear and promising strategy for exploring the bioleaching mechanism from the perspective of microbial enzyme activity and metabolites.

Carbon Capture and Storage in the Biofouling Tubeworm Ficopomatus Enigmaticus

The study investigates the potential of the serpulid tubeworm Ficopomatus sp.,sourced from Jesolo Lido(Venice,Italy),for CCS(Carbon Capture and Storage).Specimens were processed to extract bioactive compounds using a 50%polypropylene glycol-water solvent system.Extracts were analysed and purified using column gel filtration chromatography,with fractions identified by TLC(Thin Layer Chromatography)and further characterized for antioxidant and antibacterial activities.Antioxidant activity was detected via DPPH(2,2-Diphenyl-1-Picrylhydrazyl)spraying on TLC plates,while antibacterial activity was evaluated using the antibiogram paper disk diffusion method.Enzyme activity in the fractions was previously confirmed through a bromothymol blue test followed by spectrophotometric analysis.The primary goal was to explore the CCS potential,using an experimental module involving Arduino Uno embedded microprocessor for the CO_(2) measurement and to confirm the conversion into insoluble carbonates(storage).The most active fraction,identified as S1,showed significant CCS action,confirmed by microscopic observation of calcareous deposits on treated sponges.These findings suggest that Ficopomatus sp.can be used in CCS research highlighting its potential for biotechnological applications in mitigating climate change.The paper underscores the importance of marine organisms in CCS and offers insights into innovative strategies for environmental conservation and carbon management.

Role of Carbonic Anhydrase as an Activator in Carbonate Rock Dissolution and Its Implication for Atmospheric CO_(2)Sink

The conversion of CO2 into H+ and is a relatively slow reaction. Hence, its kinetics may be rate determining in carbonate rock dissolution. Carbonic anhydrase (CA), which is widespread in nature, was used to catalyze the CO2 conversion process in dissolution experiments of limestone and dolomite. It was found that the rate of dissolution increases by a factor of about 10 after the addition of CA at a high CO2 partial pressure (Pco2) for limestone and about 3 at low Pco2 for dolomite. This shows that reappraisal is necessary for the importance of chemical weathering (including carbonate rock dissolution and silicate weathering) in the atmospheric CO2 sink and the mysterious missing sink in carbon cycling. It is doubtless that previous studies of weathering underestimated weathering rates due to the ignorance of CA as an activator in weathering, thus the contribution of weathering to the atmospheric CO2 sink is also underestimated. This finding also shows the need to examine the situ distribution and activity of CA in different waters and to investigate the role of CA in weathering.

Immobilization of carbonic anhydrase for facilitated CO2 capture and separation

Carbonic anhydrase(CA)as a typical metalloenzyme in biological system can accelerate the hydration/dehydration of carbon dioxide(CO2,the major components of greenhouse gases),which performer with high selectivity,environmental friendliness and superior efficiency.However,the free form of CA is quite expensive(~RMB 3000/100 mg),unstable,and non-reusable as the free form of CA is not easy for recovery from the reaction environment,which severely limits its large-scale industrial applications.The immobilization may solve these problems at the same time.In this context,many efforts have been devoted to improving the chemical and thermal stabilities of CA through immobilization strategy.Very recently,a wide range of available inorganic,organic and hybrid compounds have been explored as carrier materials for CA immobilization,which could not only improve the tolerance of CA in hazardous environments,but also improve the efficiency and recovery to reduce the cost of large-scale application of CA.Several excellent reviews about immobilization methods and application potential of CA have been published.By contrast,in our review,we stressed on the way to better retain the biocatalytic activity of immobilized CA system based on different carrier materials and to solve the problems facing in practical operations well.The concluding remarks are presented with a perspective on constructing efficient CO2 conversion systems through rational combining CA and advanced carrier materials.

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaC1. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5＇ franking region of 1.4 kb from the carbonic anhydrase （CAH） gene ofD. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem （GT）n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site （TSS） of the CAH gene was determined by 5＇ RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar （bialaphos resistance） gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

Cloning and Bioinformation Analysis of Carbonic Anhydrase Gene FsCA1 of Tibet Wild Buckwheat

A carbonic anhydrase( CA) transcript was obtained from the Contig library according to the published sequencing information of the buckwheat transcripts. The full length of the CA gene was amplified by reverse transcription PCR( RT-PCR). The bioinformatics analysis showed that the full length of Fs CA1 gene was 1233 bp and open reading frame was 978 bp,and encoding 325 amino acids. The molecular weight was 35. 11 ku and the isoelectric point was 7. 59; there were 9 α helices,6 β folds,many randon coil and extension chain,containing one signal peptide and one transmembrane region,having a 2 amino acid conserved domains with typical beta-type carbonic anhydrase. Subcellular localization showed that the protein is most likely to appear in the chloroplast. The three-dimensional structure model of Fs CA1 was built by homologous modeling method,indicating that the homo-octamer of buckwheat CA and pea CA could match well,so it can be inferred that buckwheat CA is also homo-octamer. Real-time quantitative PCR was used to detect the expression of Fs CA1 in different organs of buckwheat.The results showed that Fs CA1 had the highest expression level in leaves,then in the stems,and the lowest in roots.

Effect of Modified HIF-1<i>α</i>Linked to Carbonic Anhydrase IX Inhibitor and Glycosylated Cisplatin on Solid Tumors: Short Review

Many cancer cells in solid tumors are hypoxic or pseudohypoxic and create acidic environment for malignancy progression. Under low oxygen conditions (hypoxia), hypoxia-inducible factors (HIFs) play pathological roles in cancer cell survival and spreading. HIF regulates several genes such as genes of glucose transporters that enhance anaerobic glycolysis, angiogenesis, erythropoiesis and carbonic anhydrase IX (CA-IX). CA-IX is a cell-surface glycoprotein that catalyzes the hydration of CO2 to protons and bicarbonate ions (respiratory acidification). This process is involved in adaptation to acidosis and implicated in cancer progression. Therefore, CA-IX inhibitors (such as sulfonamide-based compounds) showed hoping results in reduction malignancy progression. The article aims to reversal the malignant hypoxic environment in solid tumors to create a condition of weakness within the cancer for further focused cisplatin potency. This article suggests the use of modified synthesized HIF as a drug delivery molecule for both carbonic anhydrase IX inhibitor and glycosylated cisplatin that damages the DNA of malignant cell. HIF molecule has high affinity to bind with CA IX-expressing malignant cells, which is followed by cell entrance via endocytosis. Once the HIF-Cisplatin-CA-inhibitor complex enters the cell, the carbonic anhydrase inhibitor will improve the cellular pH that makes the environment unsuitable for HIF 1α function and it may be ubiquitinated. So, the raise in target genes transcription will be arrested. On the other hand, once the synthetized HIF is degraded, the cisplatin molecules will be released inside the malignant cell and start to damage its DNA. This approach may be a good solution for many solid tumors.

Overexpression of carbonic anhydrase 1 in pterygium

Carbonic anhydrase （CA） is a kind of zinc enzyme that reversibly catalyzes hydration of CO2. Sixteen CAisoenzymes have been described in mammals . These isozymes play physiological roles in erythrocytes, including CO2 transport, ion secretion, pH regulation and so forth. CA1 and 2 are both cytosolic enzymes that are found in large quantities in erythrocytes. Excluding hemoglobin, CA I is the most abundant protein in erythrocytes

Effect of Hyperglycemia on Erythrocyte Carbonic Anhydrase and Lactic Acid in Type II Diabetic Subjects

Background: Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to and H+. CA plays a crucial role in CO2 transport, acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin and also facilitates lactate shuttling across the monocarboxylate transporters (MCT). The study aimed to investigate the influence of hyperglycemia on erythrocyte carbonic anhydrase activity and lactic acid in type II diabetic patients. Method: Red blood cell carbonic anhydrase activity was determined in washed lysed-hemolysate by the action of the enzyme on the substrate p-nitrophenyl acetate. The absorbance of released p-nitrophenol was recorded at 345 nm. Glycated Hemoglobin was determined by ion exchange method (Spectrum Diagnostic Kit). Blood glucose, lactate, cholesterol and triglyceride were determined using Accutrend GCT meter (Roche, Germany) with cobias® test strips. Results: The present study showed that hyperglycemia significantly (p < 0.05) increased both erythrocyte carbonic anhydrase activity and blood lactate level in type II diabetic patients. Conclusion: We may conclude that hyperglycemia may be responsible for the increased activity of carbonic anhydrase activity and blood lactate concentration.

Prognostic significance of carbonic anhydrase IX expression in clear cell renal cell carcinoma

Objective To evaluate the prognostic significance of carbonic anhydrase IX ( CA IX) expression in patients with clear cell renal cell carcinoma ( ccRCC) . Methods CA IX excression in a cohort of 120 patients with ccRCC was evaluated by P-V immunohistochemistry

Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality

Carbon neutrality is widely concerned and highly valued by many countries.Biosphere has always maintained the balance between oxidized organic substances and assimilated organic matter,resulting in netzero carbon dioxide(CO_(2)) emissions and maintaining its own carbon neutrality.Nature has set a good example for human beings to coordinate oxygen(O_(2)) balance and CO_(2)balance,and achieve carbon neutrality.How does photosynthetic oxygen evolution initiate carbon and water neutrality?My synthesis shows that photo system Ⅱ functions as carbonic anhydrase to catalyze the reaction of CO_(2)hydration under physiological conditions,and CO_(2)hydration coupled with chemical equilibrium,H^(+)+HCO_(3)^(-)→1/2O_(2)+2e^(-)+2H^(+)+CO_(2),occurs in a photosystem Ⅱ corecomplex.Meanwhile,I focused on the revisiting of four classical heavy oxygen(O^(18)) labeling experiments and found that bicarbonate can promote photo synthetic oxygen evolution,and that photo synthetic oxygen evolution can alternately come from bicarbonate and water,not only water.Bicarbonate photolysis and water photolysis account for half of the photo synthetic oxygen evolution respectively,which can well explain the bicarbonate effect,Dole effect and plants’ environmental adaptability.Photosynthetic oxygen evolution initiated the journey of water metabolism and carbon metabolism in nature,which led to the coupling as 1:1(mol/mol) stoichiometric relationship between the reduction of CO_(2)and oxidation of organic carbon,coordinated the evolution of the atmosphere,hydrosphere,lithosphere and biosphere,and realized "carbon neutrality" in the whole Earth system.

Inorganic Carbon Utilization in Some Marine Phytoplankton Species

In order to learn the ways and possible utilization mechanisms of dissolved inorganic carbon (DIC) in marine phytoplankton species under carbon-replete or -limited conditions, the activity of extracellular carbonic anhydrase (CA) was assayed in different pH, CO 2 and DIC concentrations. Extracellular CA in Amphidinium carterae and Prorocentrum minimum was detected under carbon-replete conditions, while in Melosira sp., Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira rotula, Emiliania huxleyi and Pleurochrysis carterae, CA activity was assayed under conditions of carbon limitation. No CA activity was found even under carbon-limited conditions in Chaetoceros compressus, Glenodinium foliaceum, Coccolithus pelagicus, Gephrocapsa oceanica and Heterosigma akashiwo. In species without extracellular CA activity, the direct HCO - 3 uptake was investigated using a pH drift technique and the anion exchange inhibitor 4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) in a closed system. The result showed that direct HCO - 3 transport might occur by an anion exchange mechanism in species Coc. pelagicus and G. oceanica. Of the 13 species investigated, only H. akashiwo did not have the potential for direct uptake or extracellular CA-catalyzed HCO - 3 utilization.

Nitrogen in Relation to Photosynthetic Capacity and Accumulation of Osmoprotectant and Nutrients in Brassica Genotypes Grown Under Salt Stress

Different strategies of the application of nutrients are required to overcome the adverse effects of mustard （Brassica juncea L.） in response to NaCl stress. The objective of the present study was to determine if different added levels of nitrogen （N） in growth medium could alleviate the adverse effects of salt stress on photosynthetic capacity and accumulation of osmoprotectants and nutrients. 14 days mustard seedlings of salt-sensitive （cv. Chuutki） and salt-tolerant （cv. Radha） genotypes were fed with： （i） 0 mmol L^-1 NaCl ＋ 0 mg N kg^-1 sand （control）, （ii） 90 mmol L^-1 NaCl ＋ 30 mg N kg^-1 sand, （iii） 90 mmol L^-1 NaCl ＋60 mg N kg^-1 sand, （iv） 90 mmol L^-1 NaCl ＋90 mg N kg^-1 sand and （v） 90 mmol L^-1NaCl＋ 120 mg N kg^-1 sand. Under the condition of salinity stress, N application caused a significant ameliorative effect on both genotypes with respect to growth attributes [fresh weight （FW） and dry weight （DW）] and physio-biochemical parameters [percent water content （WC）, net photosynthetic rate （PN）, stomatal conductance （gs）, total chlorophyll （Ch1）, carbonic anhydrase （CA） activity and malondialdehyde （MDA）, nitrogen （N）, potassium （K） and sodium （Na） contents, and K/Na ratio] and yield attributes （number of pods/plant, seeds/pod and seed yield/plant）. The salt-tolerant genotype exhibited maximum value for growth, physio-biochemical and yield attributes at 60 mg N kg 1 sand than that of salt-sensitive genotype. These results suggest that application of N may ameliorate most of the attributes and prove to be a physiological remedy to increase the tolerance against the ill effects of salt stress in Brassicas.

Effects of carbon anhydrase on utilization of bicarbonate in microalgae:a case study in Lake Hongfeng

A bidirectional labeling method was established to distinguish the proportions of HCO3- and CO2 utiliza- tion pathways of microalgae in Lake Hongfeng. The method was based on microalgae cultured in a medium by adding equal concentrations of NaH13CO3 with different 613C values simultaneously. The inorganic carbon sources were quantified according to the stable carbon isotope composition in the treated microalgae. The effects of extracellular carbonic anhydrase （CAex） on the HCO3 and CO2 utilization pathways were distinguished using acetazolamide, a potent membrane-impermeable carbonic anhydrase inhibitor. The results show utilization of the added HCO3- was only 8% of the total carbon sources in karst lake. The proportion of the HCO3- utilization path- way was 52% of total inorganic carbon assimilation. Therefore, in the natural water of the karst area, the microalgae used less bicarbonate that preexisted in the aqueous medium than CO2 derived from the atmosphere. CAex increased the utilization of inorganic carbon from the atmosphere. The microalgae with CAex had greater carbon sequestration capacity in this karst area.

Targeting neuronal nitric oxide synthase as a valuable strategy for the therapy of neurological disorders

The management of neurological disorders have huge and increasing human and economic costs. Despite this, there is a scarcity of effective therapeutics, and there is an extreme urgency for new and real treatments. In this short review we analyze some promising advancements in the search of new bioactive molecules targeting neuronal nitric oxide synthase （nNOS）, an enzyme deputed to the biosynthesis of nitric oxide （NO）. In different conditions of neuronal damages, this molecule is overproduced, contributing to the pathogenesis and progression of neuronal diseases. Two main approaches to modulate nNOS are discussed： a first one consisting in the direct inhibition of the enzyme by means of small organic molecules, which can be also active against other different targets involved in such diseases. A second section is dedicated to molecules able to prevent the formation of the ternary complex N-methyl-D-aspartate （NMDA）type glutamate receptors, postsynaptic density-95 （PSD95） protein-nNOS, which is necessary to activate the latter for the biosynthesis of NO.