Arylamine N-acetyltransferase 1: a novel drug target in cancer development.

The human arylamine N-acetyltransferases first attracted attention because of their role in drug metabolism. However, much of the current literature has focused on their role in the activation and detoxification of environmental carcinogens and how genetic polymorphisms in the genes create predispositions to increased or decreased cancer risk. There are two closely related genes on chromosome 8 that encode the two human arylamine N-acetyltransferases--NAT1 and NAT2. Although NAT2 has restricted tissue expression, NAT1 is found in almost all tissues of the body. There are several single-nucleotide polymorphisms in the protein coding and 3'-untranslated regions of the gene that affect enzyme activity. However, NAT1 is also regulated by post-translational and environmental factors, which may be of greater importance than genotype in determining tissue NAT1 activities. Recent studies have suggested a novel role for this enzyme in cancer cell growth. NAT1 is up-regulated in several cancer types, and overexpression can lead to increased survival and resistance to chemotherapy. Although a link to folate homeostasis has been suggested, many of the effects attributed to NAT1 and cancer cell growth remain to be explained. Nevertheless, the enzyme has emerged as a viable candidate for drug development, which should lead to small molecule inhibitors for preclinical and clinical evaluation.

A novel human NatA Nalpha-terminal acetyltransferase complex: hNaa16p-hNaa10p (hNat2-hArd1).

BACKGROUND: Protein acetylation is among the most common protein modifications. The two major types are post-translational Nepsilon-lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational Nalpha-terminal acetylation catalyzed by NATs (N-terminal acetyltransferases). The major NAT complex in yeast, NatA, is composed of the catalytic subunit Naa10p (N alpha acetyltransferase 10 protein) (Ard1p) and the auxiliary subunit Naa15p (Nat1p). The NatA complex potentially acetylates Ser-, Ala-, Thr-, Gly-, Val- and Cys- N-termini after Met-cleavage. In humans, the homologues hNaa15p (hNat1) and hNaa10p (hArd1) were demonstrated to form a stable ribosome associated NAT complex acetylating NatA type N-termini in vitro and in vivo. RESULTS: We here describe a novel human protein, hNaa16p (hNat2), with 70% sequence identity to hNaa15p (hNat1). The gene encoding hNaa16p originates from an early vertebrate duplication event from the common ancestor of hNAA15 and hNAA16. Immunoprecipitation coupled to mass spectrometry identified both endogenous hNaa15p and hNaa16p as distinct interaction partners of hNaa10p in HEK293 cells, thus demonstrating the presence of both hNaa15p-hNaa10p and hNaa16p-hNaa10p complexes. The hNaa16p-hNaa10p complex acetylates NatA type N-termini in vitro. hNaa16p is ribosome associated, supporting its potential role in cotranslational Nalpha-terminal acetylation. hNAA16 is expressed in a variety of human cell lines, but is generally less abundant as compared to hNAA15. Specific knockdown of hNAA16 induces cell death, suggesting an essential role for hNaa16p in human cells. CONCLUSION: At least two distinct NatA protein Nalpha-terminal acetyltransferases coexist in human cells potentially creating a more complex and flexible system for Nalpha-terminal acetylation as compared to lower eukaryotes.

Population genetic diversity of the NAT2 gene supports a role of acetylation in human adaptation to farming in Central Asia.

The arylamine N-acetyltransferase 2 (NAT2) enzyme detoxifies a wide spectrum of naturally occurring xenobiotics including carcinogens and drugs. Variation at the NAT2 gene has been linked to the human acetylation capacity, either 'slow' or 'fast', which modifies susceptibility to cancer and adverse drug reactions. We investigated the possible influence of natural selection in shaping the acetylation phenotype and the NAT2 gene variability in six Central Asian populations, who are either long-term sedentary agriculturalists (two Tajik populations), recent sedentary agriculturalists (Kazakhs, Uzbeks) or nomad pastoralists (two Kirghiz populations). To this end, we sequenced the entire NAT2 coding exon, as well as genotyping nine intergenic SNPs covering a 200-kb region. Our results revealed that the two Tajik populations exhibited significantly higher proportions of slow acetylators than the nomadic populations. In addition, sequence-based neutrality tests yielded significantly positive values in Central Asian populations following an agriculturalist lifestyle, due to an excess of haplotypes at intermediate frequencies. Taken together, our data suggest that balancing selection, and/or directional selection on standing low-frequency alleles, have shaped NAT2 genetic diversity and the human acetylation phenotype in Central Asian agriculturalists. These results further support the hypothesis that a major transition in human lifestyle, such as the emergence of farming has dramatically changed human chemical environments and the selective pressures they imposed.

Contribution of the N-acetyltransferase 2 polymorphism NAT2*6A to age-related hearing impairment.

BACKGROUND: Age-related hearing impairment (ARHI) is the most common sensory impairment in older people, affecting 50% of those aged 80 years. The proportion of older people is increasing in the general population, and as a consequence, the number of people affected with ARHI is growing. ARHI is a complex disorder, with both environmental and genetic factors contributing to the disease. The first studies to elucidate these genetic factors were recently performed, resulting in the identification of the first two susceptibility genes for ARHI, NAT2 and KCNQ4. METHODS: In the present study, the association between ARHI and polymorphisms in genes that contribute to the defence against reactive oxygen species, including GSTT1, GSTM1 and NAT2, was tested. Samples originated from seven different countries and were combined into two test population samples, the general European population and the Finnish population. Two distinct phenotypes for ARHI were studied, Z(low) and Z(high), representing hearing in the low and high frequencies, respectively. Statistical analysis was performed for single polymorphisms (GSTM1, GSTT1, NAT2*5A, NAT2*6A, and NAT2*7A), haplotypes, and gene-environment and gene-gene interactions. RESULTS: We found an association between ARHI and GSTT1 and GSTM1 in the Finnish population sample, and with NAT2*6A in the general European population sample. The latter finding replicates previously published data. CONCLUSION: As replication is considered the ultimate proof of true associations in the study of complex disorders, this study provides further support for the involvement of NAT2*6A in ARHI.

The urinary metabolite profile of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is predictive of colon DNA adducts after a low-dose exposure in humans.

Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [(14)C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N(2)-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N(2)-glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N(2)-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.

Arylamine N-acetyltransferase aggregation and constitutive ubiquitylation.

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.

Biosynthetic pathways of the endocannabinoid anandamide.

Anandamide (=N-arachidonoylethanolamine) is the first discovered endocannabinoid, and belongs to the class of bioactive, long-chain N-acylethanolamines (NAEs). In animal tissues, anandamide is principally formed together with other NAEs from glycerophospholipid by two successive enzymatic reactions: 1) N-acylation of phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) by Ca2+-dependent N-acyltransferase; 2) release of NAE from NAPE by a phosphodiesterase of the phospholipase D type (NAPE-PLD). Although these anandamide-synthesizing enzymes were poorly understood until recently, our cDNA cloning of NAPE-PLD in 2004 enabled molecular-biological approaches to the enzymes. NAPE-PLD is a member of the metallo-beta-lactamase family, which specifically hydrolyzes NAPE among glycerophospholipids, and appears to be constitutively active. Mutagenesis studies suggested that the enzyme functions through a mechanism similar to those of other members of the family. NAPE-PLD is widely expressed in animal tissues, including various regions in rat brain. Its expression level in the brain is very low at birth, and remarkably increases with development. Analysis of NAPE-PLD-deficient mice and other recent studies revealed the presence of NAPE-PLD-independent pathways for the anandamide formation. Furthermore, calcium-independent N-acyltransferase was discovered and characterized. In this article, we will review recent progress in the studies on these enzymes responsible for the biosynthesis of anandamide and other NAEs.

Do cross-registry comparisons of Black and White Americans provide support for N-acetylation as an important determinant for urinary bladder and other tobacco-related cancers?

Tobacco smoking is an unequivocal risk factor for cancers of the larynx, lung, pancreas and urinary bladder. Whereas African-Americans demonstrate higher laryngeal, lung and pancreatic cancer rates than their Caucasian-American counterparts, they paradoxically have only approximately half of the urinary bladder incidences. One possible explanation is their N-acetyltransferase (NAT) status, since this enzyme is responsible for metabolism of arylamines in smoke and blacks are reported to have a higher rate for rapid acetylation than whites. However, other tobacco-related cancers are also linked to slow acetylation so that African-Americans might therefore also be expected to have lower incidences of other tobacco-related cancers. The present investigation was conducted with data from Cancer Incidence in Five Continents Vol VIII to assess whether there might be correlations between incidence rates for four major cancers across registries in the United States. Cluster analysis demonstrated clear separation of the white and black populations for all states, and significant correlations were observed between bladder and laryngeal cancers, and also for lung and laryngeal cancers, for both Blacks and Whites. Striking similarities in the plots for urinary bladder incidence against all three of the other cancers suggests the existence of a factor specific to the bladder. A review of black-white ratios for cancer incidences in all major body sites in both sexes and the published literature for NAT polymorphisms provided evidence that this might indeed be arylamine exposure, although other factors could also be involved.

Role of aromatic amine acetyltransferases, NAT1 and NAT2, in carcinogen-DNA adduct formation in the human urinary bladder.

The metabolic activation and detoxification pathways associated with the carcinogenic aromatic amines provide an extraordinary model of polymorphisms that can modulate human urinary bladder carcinogenesis. In this study, the metabolic N-acetylation of p-aminobenzoic acid (PABA) to N-acetyl-PABA (NAT1 activity) and of sulfamethazine (SMZ) to N-acetyl-SMZ (NAT2 activity), as well as the O-acetylation of N-hydroxy-4-aminobiphenyl (OAT activity; catalyzed by NAT1 and NAT2), were measured in tissue cytosols prepared from 26 different human bladder samples; then DNA was isolated for determination of NAT1 and NAT2 genotype and for analyses of carcinogen-DNA adducts. Both PABA and OAT activities were detected, with mean activities +/- SD of 2.9 +/- 2.3 nmol/min/mg protein and 1.4 +/- 0.7 pmol bound/mg DNA/min/mg protein, respectively. However, SMZ activities were below the assay limits of detection (< 10 pmol/min/mg protein). The levels of putative carcinogen-DNA adducts were quantified by 32P-postlabeling and averaged 2.34 +/- 2.09 adducts/10(8) deoxyribonucleotide phosphate (dNp). Moreover, the DNA adduct levels in these tissues correlated with their NAT1-dependent PABA activities (r = 0.52; P < 0.01) but not with their OAT activities. Statistical and probit analyses indicated that this NAT1 activity was not normally distributed and appeared bimodal. Applying the NAT1:OAT activity ratios (N:O ratio) allowed arbitrary designation of rapid and slow NAT1 phenotypes, with a cutpoint near the median value. Within each of these subgroups, NAT1 correlated with OAT (P < 0.05); DNA adduct levels were elevated 2-fold in individuals with the rapid NAT1 or NAT1/OAT phenotype. Examination of DNA sequence polymorphisms in the NAT1 gene by PCR have demonstrated that an NAT1 polyadenylation polymorphism is associated with differences in tissue NAT1 enzyme activity; accordingly, NAT1 activity in the bladder of individuals with the heterozygous NAT1*10 allele was 2-fold higher than in subjects homozygous for the putative wild-type NAT1*4 allele. Likewise, DNA adduct levels in the mucosa of the urinary bladder were found to be 2-fold (P < 0.05) higher in individuals with the heterozygous NAT1*10 allele (3.5 +/- 2.1 adducts/10(8) dNp) as compared to NAT1*4 homozygous (1.8 +/- 1.9 adducts/10(8) dNp). Thus, these data provide strong support for the hypothesis that NAT1 activity in the urinary bladder mucosa represents a major bioactivation step that converts urinary N-hydroxy arylamines to reactive N-acetoxy esters that form covalent DNA adducts.(ABSTRACT TRUNCATED AT 400 WORDS)

The putative tumour suppressor Fus-2 is an N-acetyltransferase.

Acetyltransferases are essential enzymes for a wide variety of cellular processes and mutations in acetyltransferase genes have been associated with the development of certain cancers. For this reason, we conducted a computerized sequence homology search for novel acetyltransferases. Here, we show that the putative tumour suppressor protein Fus-2 has homology to the catalytic domain of acetyltransferases. We demonstrate that Fus-2 can acetylate the N-terminus of proteins using a ping-pong mechanism and that it has a specificity for substrates. Consistent with other N-acetyltransferases, Fus-2 localizes to the cytoplasm, as shown by GFP-tag experiments. Since the Fus-2 gene maps to the chromosomal region 3p21.3, which contains at least one tumour suppressor gene, the N-acetyltransferase functions of Fus-2 may be relevant to its potential role in cancer.

Arylamine N-acetyltransferases - of mice, men and microorganisms.

Arylamine N-acetyltransferases (NATs) catalyse the transfer of an acetyl group from acetyl CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. These enzymes are polymorphic and have an important place in the history of pharmacogenetics, being first identified as responsible for the polymorphic inactivation of the anti-tubercular drug isoniazid. NAT has recently been identified within Mycobacterium tuberculosis itself and is an important candidate for modulating the response of mycobacteria to isoniazid. The first three-dimensional structure of the unique NAT family shows the active-site cysteine to be aligned with conserved histidine and aspartate residues to form a catalytic triad, thus providing an activation mechanism for transfer of the acetyl group from acetyl CoA to cysteine. The unique fold could allow different members of the NAT family to play a variety of roles in endogenous and xenobiotic metabolism.

SLI1 (YGR212W) is a major gene conferring resistance to the sphingolipid biosynthesis inhibitor ISP-1, and encodes an ISP-1 N-acetyltransferase in yeast.

ISP-1 (myriocin) is a potent inhibitor of serine palmitoyltransferase, the primary enzyme of sphingolipid biosynthesis, and is a useful tool for studying the biological functions of sphingolipids in both mammals and yeast (Saccharomyces cerevisiae). In a previous study, we cloned yeast multicopy suppressor genes for ISP-1, and one of these, YPK1/SLI2, was shown to encode a serine/threonine kinase which is a yeast homologue of mammalian SGK1 (serum/glucocorticoid-regulated kinase 1). In the present study, another gene, termed SLI1 (YGR212W; GenBank accession number CAA97239.1), was characterized. Sli1p has weak similarity to Atf1p and Atf2p, which are alcohol acetyltransferases. Although a sli1-null strain grew normally, the IC50 of ISP-1 for the growth of this strain was markedly decreased compared with that for the parental strain, indicating that Sli1p is a major contributor to ISP-1 resistance in yeast. On a sli1-null background, the increase in resistance to ISP-1 induced by YPK1 gene transfection was almost abolished. These data indicate that Sli1p co-operates with Ypk1p in mediating resistance to ISP-1 in yeast. Sli1p was found to convert ISP-1 into N-acetyl-ISP-1 in vitro. Furthermore, N-acetyl-ISP-1 did not share the ability of ISP-1 to inhibit the growth of yeast cells, and the serine palmitoyltransferase inhibitory activity of N-acetyl-ISP-1 was much lower than that of ISP-1. These data suggest that Sli1p inactivates ISP-1 due to its N-acetyltransferase activity towards ISP-1.

Effects of human arylamine N-acetyltransferase I knockdown in triple-negative breast cancer cell lines.

Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and ß-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers.

Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms.

N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues. Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood. To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T). Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A. Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A. No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles. These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity.

GSTT1 null genotype increases risk of premenopausal breast cancer.

The NAT2, GSTM1 and GSTT1 genes are known candidate cancer susceptibility markers and have been investigated in breast cancer susceptibility with conflicting results. We conducted a case-control study to investigate the role of NAT2, GSTM1 and GSTT1 in premenopausal breast cancer. Women with the GSTT1 null genotype were found to have a significant 3.15-fold increased risk of breast cancer (95% CI = 1.7-5.8), while GSTM1 and NAT2 genotypes were not associated with breast cancer risk. Our results suggest that the GSTT1 null genotype may play a role in early onset breast cancer.

Properties of clock-controlled and constitutive N-acetyltransferases from chick pineal cells.

The pineal gland synthesizes its hormone melatonin (O-methyl-N-acetylserotonin) from serotonin. Acetyl-CoA: serotonin N-acetyltransferase (SNAT), the enzyme that catalyzes the committed step in this biosynthesis, is largely restricted to the pineal gland and is regulated by adrenergic and circadian mechanisms. Another enzyme, acetyl-CoA: arylamine N-acetyltransferase (ANAT), having an apparently similar activity, is also present in the pineal. This enzyme, however, is not rhythmically regulated. SNAT activity of cultured chick pineal cells was obtained without ANAT after ammonium sulfate precipitation. ANAT activity was retained without SNAT activity after pre-incubation at 37 degrees C. Thus, each enzyme could be examined independently. Overlap in substrate specificity between the two enzymes was minimal. Kinetic analysis of the separated enzyme activities revealed that while SNAT operates via a random or ordered bi bi mechanism, ANAT catalysis occurs through a ping pong bi bi mechanism with substrate inhibition by acetyl-CoA. By size-exclusion chromatography, ANAT was confirmed to be 30-35 kDa, and SNAT was estimated at 15-20 kDa. Taken together, these results indicate that the two enzymes differ in their structure, reactivity, stability, and mechanism of catalysis.

Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells.

Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3',5'-monophosphate (cAMP) in cultured retinal cells entrained to a daily light-dark (LD) cycle, as well as the role of Ca(2+) and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the nocturnal increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca(2+) influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca(2+) channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca(2+) influx, cAMP formation, and AANAT activity in retinal cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.

Ontogeny of a diurnal rhythm in arylalkylamine-N-acetyltransferase mRNA in rat pineal gland.

Melatonin synthesis in the pineal gland of adult rats is linked to cAMP-dependent transcriptional and post-transcriptional regulatory mechanisms affecting its rate-limiting enzyme, the arylalkylamine-N-acetyltransferase (AA-NAT). During development of the pineal gland, neuronal control gains access to the earlier matured cAMP-signaling pathway to shape the day-night rhythm in AA-NAT enzymatic activity. By semiquantitative in situ hybridization we analyzed if the developmental onset of a rhythmic AA-NAT activity is correlated to a temporally parallel onset in AA-NAT transcription. We found that AA-NAT mRNA levels in rat pineal gland become rhythmic at postnatal day 5. Thus, AA-NAT gene transcription in rat pineal gland starts to show day-night differences shortly prior to the appearance of a rhythmic AA-NAT activity.

Food access schedule and diet composition alter rhythmicity of serum melatonin and pineal NAT activity.

This study investigated the effect of dietary composition and food access schedule on the rhythmicity of serum melatonin and pineal N-acetyltransferase (NAT) activity. Wistar rats maintained on a 12:12 h light-dark cycle were assigned to two dietary groups: a group fed rat chow and a group fed a choice between a protein-rich and a carbohydrate-rich diet. Each dietary group was further divided based on feeding schedule, with food available between 0800 and 1600 h or ad lib access to food. Regardless of dietary condition, total food and carbohydrate intake of rats having free access to food was higher than under the restricted food access schedule. Protein intake of rats fed the dietary choice was lower with the restricted access than in the free access. In rats fed the dietary choice, melatonin levels and NAT activity were significantly decreased with restricted access compared to free access. Such results were not found in rats offered restricted chow. This study suggests that the rhythms of melatonin secretion and NAT activity can be altered by dietary composition.