Polyamine acetylations in normal and neoplastic growth processes

Summary The expression patterns of cytosolic and nuclear polyamine acetyltransferases were studied in normal and neoplastic growth processesin vivo andin vitro to evidentiate the roles played by these enzymes in cell proliferation. In regenerating liver, cytosolic spermidine/spermine N¹-acetyltransferase showed similar augments of mRNA level and enzymatic activity during the prereplicative period (4–8 h), whereas spermidine N⁸-acetyltransferase activity increased later (24 h) when DNA synthesis was maximally enhanced. In fibroblasts continuously dividing, the messenger for spermidine/spermine N¹-acetyltransferase rapidly accumulated after serum-stimulation. In cultured Morris hepatoma cells stimulated to logarithmic growth, spermidine N⁸-acetyltransferase activity remained at plateau for 1 day declining thereafter, while spermidine/spermine N¹-acetyltransferase activity immediately decreased. In Yoshida AH-130 hepatoma cells transplanted in rat peritoneum, spermidine N⁸-acetyltransferase and spermidine/spermine N¹-acetyltransferase activities rose, respectively, in concomitance with elevated proliferation-rate and quasi-stationary phase of growth. Since the expression of cytosolic and nuclear acetyltransferases underwent different temporal activation, an involvement of these enzymes in separate metabolic processes controlling normal and neoplastic growth may be suggested.     

The regulation of pineal serotonin N-acetyltransferase activity in newborn rats.

Rat pineal serotonin N-acetyltransferase activity increases 2–3 hr after birth and then decreases again. The activity at night is higher than that during the day in rats as young as 1 to 2 days old. Administration of the beta adrenergic blocker trimepranol to newborn or 2-day old rats at night lowers the elevated serotonin N-acetyltransferase activity. Hence, the activity is under adrenergic control even in the not yet innervated pineal gland of the newborn rats.     

Differential Regulation of Arylalkylamine N-Acetyltransferase Activity in Chicken Retinal Ganglion Cells by Light and Circadian Clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30–60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar)     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine decarboxylase activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N¹-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N¹-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N¹-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N¹-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

PINEAL SEROTONIN N-ACETYLTRANSFERASE ACTIVITY: PROTECTION OF STIMULATED ACTIVITY BY ACETYL-CoA AND RELATED COMPOUNDS

—Rat pineal serotonin N—acetyltransferase activity increases 30–70-fold at night in the dark and then decreases rapidly when animals are exposed to light. Activity of the enzyme is also stimulated by l -norepinephrine in organ culture. When homogenates of glands stimulated by dark in vivo or NE in vitro are incubated at 37°C, enzyme activity will also rapidly decrease. This decrease can be prevented by one of the cosubstrates of the enzyme, acetyl–CoA. Protection can also be conferred by cysteamine (β-mercaptoethylamine, HS–CH₂–CH₂–NH₂) which is the terminal portion of the CoA molecule. This protection mechanism could be involved in the physiological control of enzyme activity.     

A COMPARATIVE STUDY OF PINEAL ACETYL-CoA HYDROLASE AND N-ACETYLTRANSFERASE ACTIVITIES

pineal acetyl-CoA hydrolase is measurable at 4 days before birth. It increases rapidly to a maximum of 0.37 nmol/min/0.1 mg protein during the first week after birth, thereafter gradually decreasing and stabilizing at adult levels (0.27 nmol/min/0.1 mg protein) 3-4 weeks after birth. Unlike A/-acetyltransferase, the activity of acetyl-CoA hydrolase does not increase following treatment with isoproterenol, does not exhibit a circadian rhythm and is not inactivated on exposure of the animals to light at night. In addition, denervation of the pineal gland does not alter acetyl-CoA hydrolase activity.     

Correlation of changes in pineal gland cAMP metabolism with light-mediated decrease in N-acetyltransferase activity

The present study investigated the correlation between the light-mediated decrease in rat pineal N-acetyltransferase (NAT) observed in vivo and changes in cAMP metabolism. While exposing dark-adapted rats to light for short time periods (0–10 min) resulted in a rapid decrease in pineal NAT activity, cAMP exhibited a biphasic response. Following light exposure for 30 s there was a 50% decrease in cAMP levels. However, after 6 min of light exposure the cyclic nucleotide levels had increased 2–3 times above control values. These responses were prevented by phenoxybenzamine pretreatment and the initial decrease was mimicked by i.v. propranolol administration. Examination of cAMP metabolic enzymes, adenylate cyclase and phosphodiesterase revealed an increase in adenylate cyclase activity following 6 min of exposure to light. We discussed how the results observed in vivo compare with those observed using cultured pinealocytes.     

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle

Summary The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied.In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed.Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only.Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.Abbreviations NAT N-acetyltransferase - LD cycle light-dark cycle - CT circadian time - LD xy light dark cycle comprising x h of light and y h of darkness     

Static and extremely low frequency electromagnetic field exposure: Reported effects on the circadian production of melatonin

The circadian rhythm of melatonin production (high melatonin levels at night and low during the day) in the mammalian pineal gland is modified by visible portions of the electromagnetic spectrum, i.e., light, and reportedly by extremely low frequency (ELF) electromagnetic fields as well as by static magnetic field exposure. Both light and non-visible electromagnetic field exposure at night depress the conversion of serotonin (5HT) to melatonin within the pineal gland. Several reports over the last decade showed that the chronic exposure of rats to a 60 Hz electric field, over a range of field strengths, severely attenuated the nighttime rise in pineal melatonin production; however, more recent studies have not confirmed this initial observation. Sinusoidal magnetic field exposure also has been shown to interfere with the nocturnal melatonin forming ability of the pineal gland although the number of studies using these field exposures is small. On the other hand, static magnetic fields have been repeatedly shown to perturb the circadian melatonin rhythm. The field strengths in these studies were almost always in the geomagnetic range (0.2 to 0.7 Gauss or 20 to 70 μtesla) and most often the experimental animals were subjected either to a partial rotation or to a total inversion of the horizontal component of the geomagnetic field. These experiments showed that several parameters in the indole cascade in the pineal gland are modified by these field exposures; thus, pineal cyclic AMP levels, N-acetyltransferase (NAT) activity (the rate limiting enzyme in pineal melatonin production), hydroxyindole-O-methyltransferase (HIOMT) activity (the melatonin forming enzyme), and pineal and blood melatonin concentrations were depressed in various studies. Likewise, increases in pineal levels of 5HT and 5-hydroxyindole acetic acid (5HIAA) were also seen in these glands; these increases are consistent with a depressed melatonin synthesis. The mechanisms whereby non-visible electromagnetic fields influence the melatonin forming ability of the pineal gland remain unknown; however, the retinas in particular have been theorized to serve as magnetoreceptors with the altered melatonin cycle being a consequence of a disturbance in the neural biological clock, i.e., the suprachiasmatic nuclei (SCN) of the hypothalamus, which generates the circadian melatonin rhythm. The disturbances in pineal melatonin production induced by either light exposure or non-visible electromagnetic field exposure at night appear to be the same but whether the underlying mechanisms are similar remains unknown.     

Characterization of arylalkylamine N-acetyltransferase (AANAT) activities and action spectrum for suppression in the band-legged cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae)

Arylalkylamine N-acetyltransferase (AANAT), constituting a large family of enzymes, catalyzes the transacetylation from acetyl-CoA to monoamine substrates, although homology among species is not very high. AANAT in vertebrates is photosensitive and mediates circadian regulation. Here, we analyzed AANAT of the cricket, Dianemobius nigrofasciatus. The central nervous system contained AANAT activity. The optimum pHs were 6.0 (a minor peak) and 10.5 (a major peak) with crude enzyme solution. We analyzed the kinetics at pH 10.5 using the sample containing collective AANAT activities, which we term AANAT. Lineweaver-Burk plot and secondary plot yielded a K_m for tryptamine as substrate of 0.42 µM, and a V_max of 9.39 nmol/mg protein/min. The apparent K_m for acetyl-CoA was 59.9 µM and the V_max was 8.14 nmol/mg protein/min. AANAT of D. nigrofasciatus was light-sensitive. The activity was higher at night-time than at day-time as in vertebrates. To investigate most effective wavelengths on AANAT activity, a series of monochromatic lights was applied (350, 400, 450, 500, 550, 600 and 650 nm). AANAT showed the highest sensitivity to around 450 nm and 550 nm. 450 nm light was more effective than 550 nm light. Therefore, the most effective light affecting AANAT activity is blue light, which corresponds to the absorption spectrum of blue wave (BW)-opsin.     

Guanylyl cyclase activity during photoperiodic flower induction in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It is known that the level of cGMP is modulated in plant cells in response to a number of stimuli but intracellular events dependent on cGMP metabolism are not clear. Guanylyl cyclases (GCs) are enzymes which are responsible for synthesis of cGMP in eukaryotic and prokaryotic cells. To collect evidence for the participation of cGMP in light signal transduction we isolated enzyme with guanylyl cyclase activity from Pharbitis nil and analysed its level and activity during photoperiodic flower induction. Soluble proteins were isolated from seedlings of a model short-day plant P. nil, partly purified and identified by in vivo and in vitro enzyme assay. In green plants enzyme activity amounted to 484 nmol cGMP/min/mg protein, whereas in etiolated plants it was three times lower (158 nmol cGMP/min/mg protein). Analyse cyclase consists of a single polypeptide of Mr 40 kDa. In order to determine if changes in guanylyl cyclase activity occurred in response to a long, inductive night, we measured enzyme activity in 4-h intervals and observed its increase at 4, 8 and 16 h of darkness. This pattern also fits well with changes in the endogenous cGMP level during a 16 h long flower inductive night. Immunocytochemical analysis confirmed these observations and revealed that changes in the GC level during light/dark conditions appeared. During 16 h long inductive night the strongest signal was observed in cotyledons after 4 and 16 h of the darkness. A high level of fluorescence was generally distributed in mesophyll, however, it was also observed in guard cells. Staining was apparently absent in the veins and cotyledon body. Furthermore, the location inside the cell was analysed. The protein was immunolocalized preferentially in the cytosol, chloroplasts and peroxysomes. Taken together, these data demonstrate in Pharbitis nil the presence of an enzyme which is able to convert GTP to cGMP. Because its level and activity are affected by light we believe that GC/cGMP play a substantial role in light/dark dependent process in plants, such as photoperiodic flower induction.     

Photoperiod modifies daily maps of light and dark sensitivity for N-acetyltransferase activity in pineal glands of 3-week oldGallus domesticus

Summary N-acetyltransferase (NAT) activity in pineal glands exhibits a circadian rhythm with peak activity occurring in the dark-time. We previously showed that inGallus domesticus chicks pretreated with LD12:12, NAT activity was increased by dark exposure (peak dark sensitivity occurred during the expected dark-time) or decreased by light at night (peak light sensitivity occurred early in the night during the time of dark sensitivity). In this study we mapped dark sensitivity vs time (for NAT activity increase in response to 2 h dark pulses), and light sensitivity vs time (for NAT activity decrease in response to 10 min or 30 min light pulses) over a cycle for 3-week old chicks,Gallus domesticus, pretreated with long (LD16:8) or short photoperiod (LD8:16). Sensitivity to light was increased in the second 8 h after L/D by LD8:16. Sensitivity to dark was increased in the first 8 h after L/D by LD16:8.Abbreviations LD16:8 a light-dark cycle consisting of 16 h of light alternating with 8 h of dark - LD8:16 a light-dark cycle consisting of 8 h of light alternating with 16 h of dark - DD constant dark - LL constant light - L/D lights-off - D/L lights-on - NAT pineal serotonin N-acetyltransferase - NAT activity is given in nmoles/pineal gland/h - chick used here to denote a young bird of either sex of the speciesGallus domesticus from hatching to three weeks of age     

Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus

Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings.     

Circadian clock component,LHY, tells a plant when to respond photosynthetically to light in nature

The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY (irLHY) and NaTOC1 (irTOC1). We characterized growth and light‐ and dark‐adapted photosynthetic rates (A_c) throughout a 24 h day in empty vector‐transformed (EV), irLHY, and irTOC1 plants in the field, and in NaPhyA‐ and NaPhyB1‐silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced A_c earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field. irLHY, but not EV plants, responded to light in the night by rapidly increasing A_c. Under controlled conditions, EV plants rapidly increased A_c in the day compared to dark‐adapted plants at night; irLHY plants lost these time‐dependent responses. The role of NaLHY in gating photosynthesis is independent of the light‐dependent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light‐mediated in native tobacco.     

Effect of constant and fluctuating temperature on daily melatonin production by eyecups from Rana perezi

We analysed the effect of daily temperature cycles in relation to constant temperature on day/night melatonin synthesis in frog eyecups in culture. Eyecups were cultured for 24 h under 12L:12D photoperiod and two thermal regimes, constant temperature (25, 15 and 5 °C) and thermoperiod (WL/CD, thermophase coinciding with photophase and cryophase coinciding with scotophase; and CL/WD, cryophase coinciding with photophase and thermophase coinciding with scotophase). A negative correlation between ocular serotonin N-acetyltransferase activity and culture temperature for both diurnal and nocturnal activities has been observed. This effect of increased ocular activity at low temperature is more pronounced than the well-known stimulatory effect of darkness, and it does not depend on the photoperiod phase. The lack of interactions between the phase of photoperiod and culture temperature indicates that the effects of both factors are independent. Nighttime temperature is the key factor in determining the amplitude of the melatonin rhythm in the Rana perezi retina. However, daytime temperature can not counteract the inhibitory effect of light on ocular melatonin synthesis. Accepted: 22 June 1995     

The rise and fall of pineal N-acetyltransferase in vitro: Neural regulation in the developing rat

In rats, the pineal gland has a rhythm in the activity of the enzyme, N-acetyltransferase (NAT), which is thought responsible for daily cycles of melatonin synthesis. Neonatal rat pineal glands, but not those of adult rats, have a single cycle that is observed in vitro during the first day of organ culture. The neural regulation of the cycle was investigated using neonatal rats with adult rats used for comparison. Prior treatment of rat pups with constant light did not abolish the cycle in vitro though it did abolish the in vivo rhythm. Removal of the superior cervical ganglia did not abolish the in vivo rhythm that was measured the first day after surgery, but ablation of the ganglia did abolish the rhythm if several days or more elapsed after surgery. Extirpation of the superior cervical ganglia abolished the in vitro NAT cycle in pup pineal glands as did the pharmacological equivalent, injection of 6-hydroxydopamine. Propranolol, a beta blocking agent, prevented the occurrence of the cycle in vitro.     

Design,Synthesis and In Vitro Evaluation of Novel Benzo[b]thiophene Derivatives as Serotonin N-acetyltransferase (AANAT) Inhibitors

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is the penultimate enzyme in melatonin (5-methoxy-N-acetyltryptamine) biosynthesis. It is the key-enzyme responsible of the nocturnal rhythm of melatonin production in the pineal gland. Specific AANAT inhibitors could be useful for treatment of different physiopathological disorders encountered in diseases such as seasonal affective disorders or obesity. On the basis of previous works and 3D-QSAR studies carried out in our laboratory, we have synthesized and evaluated four novel benzo[b]thiophene derivatives designed as AANAT inhibitors. Compound 13 exhibited high inhibitory activity (IC₅₀=1.4?μM) and low affinities for both MT1 (1100?nM) and MT2 (1400?nM) receptors.