Stimulated N-acetyltransferase Activity Research Articles

Dopamine receptor activation in bovine pinealocyte via a cAMP‐dependent transcription pathway

D1- and D2-dopamine receptors in the bovine pineal gland were previously identified and characterized. The data indicate that the density of D1-dopamine receptors far exceeded that of D2-dopamine receptors. In our previous study, the mRNA for both the D1- and D2-dopamine receptors which elucidated the status of dopamine and its possible involvement in the pineal function was identified. A selective D1-agonist enhanced N-acetyltransferase (NAT) activity and increased the melatonin level, whereas, a selective D2-agonist inhibited NAT activity and decreased the melatonin level. An attempt has been made in the present study to clarify the mechanism of dopamine in controlling melatonin production in bovine pineal. The level of intracellular cyclic 3',5'-adenosine monophosphate (cAMP) was determined after a 2-hr incubation of bovine pinealocytes with selected combinations of drugs. SKF 38393, a selective D1-agonist, enhanced intracellular level of cAMP, and its effect was blocked by SCH 23390, a D1-selective antagonist. In contrast quinpirole, a selective D2-agonist, inhibited forskolin-stimulated intracellular level of cAMP while its effect was blocked by a D2-selective antagonist, spiperone. In addition, the dopamine-dependent phosphorylation of the transcription factors, cAMP responsive element-binding protein (CREB) was investigated. Immunoblots showed that SKF 38393 enhanced CREB phosphorylation and the stimulatory effect was abolished by SCH 23390 whereas quinpirole inhibited forskolin-stimulated phosphorylated CREB production and the inhibitory effect was prevented by spiperone. Taken together with our previous data, the results indicate that activation of D1-dopamine receptor in bovine pinealocyte stimulates NAT activity and enhances melatonin level whereas activation of D2-dopamine receptor leads to an inhibitory effect and these stimulatory and inhibitory effects act, in part, via a cAMP-dependent transcription mechanism.

The stimulatory effect of mu- and delta-opioid receptors on bovine pinealocyte melatonin synthesis.

Mammalian pinealocytes synthesize and secrete melatonin. The synthesis of melatonin is regulated by several biogenic amine, amino acid and peptide transmitters. In our previous study, the delta- and mu-opioid receptors have been identified and characterized in bovine pinealocytes. In order to elaborate the function of different types of opioid receptors in regulating melatonin synthesis, we used a selective mu-opioid receptor agonist, Tyr-[D-Ala(2), N-methyl-phe(4), glycol(5)] (DAMGO), a selective delta-opioid receptor agonist, Enkephalin [D-Pen(2), D-Pen(5)], (DPDPE) and a selective kappa-opioid receptor agonist, ((+)-(5alpha, 7alpha, 8beta)-N-methyl-N-[7- (1-pyrrolidinyl)-1-oxaspiro [4,5] dec-8-yl]-benzene acetamide) (U69593) to investigate the activity of N-acetyltransferase (NAT) activity and melatonin secretion. The results of the present study show that both DAMGO and DPDPE stimulated NAT activity and increased the level of melatonin in cultured bovine pinealocytes. These stimulatory effects were blocked by naloxone, an opioid receptor antagonist. However, the kappa-opioid receptor agonist U69593 was unable to alter either the activity of NAT or the level of melatonin. In order to clarify the mechanism of how the activation of mu- and delta-opioid receptors in bovine pinealocytes leads to an increase in NAT activity, cyclic AMP levels were measured after bovine pinealocytes were treated with morphine, DAMGO and DPDPE. The results indicated that these stimulatory effects acted via induction of cAMP production. This study reveals that the stimulatory effect of opioid receptor on melatonin synthesis is mediated via the activation of adenylate cyclase system.

Effects of interferon-gamma and streptolysin O on hepatic procainamide N-acetyltransferase and various microsomal cytochrome P450-dependent monooxygenases in rats.

Immunostimulants known to initiate cytokine production were found to inhibit processes of microsomal drug oxidation but to activate arylamine N-acetylation. The present study investigated the effects of immunstimulating doses of rat interferon-gamma (IFN gamma, 670,000 units ip) and streptolysin O (SLO, 100 HU/kg iv for 5 days) on hepatic cytosolic N-acetyltransferase (NAT) and microsomal cytochrome P450 (CYP)-dependent monooxygenases in male Wistar rats. Both IFN gamma and SLO activated NAT to 120% (P < 0.05) and 135% (P < 0.05), respectively. As expected, monooxygenases were depressed by IFN gamma (P < 0.05) and SLO, the ethylresorufin O-deethylase being the most susceptible enzyme. The results suggested that not only the toxin of gram-positive streptococcal bacteria SLO, but also the cytokine IFN gamma can stimulate NAT activity in rat hepatic cytosol. While the enhancing SLO effect on NAT could not be neutralized by the inhibitor of transcription actinomycin D, NAT stimulation by IFN gamma was abolished by actinomycin D and by the inhibitor of translation, cycloheximide. Obviously, SLO activated NAT independent of protein synthesis and different from IFN gamma-mediated pathways. Posttranslational processes might be involved in NAT stimulation in the rats.

Calcium potentiates cyclic AMP stimulation of pineal arylalkylamine N-acetyltransferase.

Pineal arylalkylamine N-acetyltransferase (N-acetyltransferase) controls large daily changes in melatonin production. It is generally thought that the activity of this enzyme is controlled by norepinephrine acting exclusively via elevation of cyclic AMP. However, norepinephrine also elevates pineal intracellular Ca2+ concentration ([Ca2+]i), and it is not known whether Ca2+ is involved in regulating N-acetyltransferase activity other than through its established role in cyclic AMP production. In this study, the issue of whether Ca2+ enhances the effects of cyclic AMP on N-acetyltransferase activity was investigated. The effects of cyclic AMP protagonists (isobutylmethylxanthine, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphothioate, Sp-diastereomer) were examined in combination with [Ca2+]i protagonists (A23187, ionomycin, and phenylephrine). All [Ca2+]i protagonists potentiated the effects of cyclic AMP protagonists. For example, ionomycin potentiated the effects of low concentrations of 8-bromoadenosine 3',5'-cyclic monophosphate, and A23187 potentiated the effects of isobutylmethylxanthine without altering cyclic AMP accumulation. These findings indicate that Ca2+ and cyclic AMP probably act physiologically in a coordinated manner to stimulate N-acetyltransferase activity; these second messengers could act directly at one or more sites or through indirect actions mediated by kinases.

Vasoactive intestinal peptide stimulates type II thyroxine 5′-deiodinase and N-acetyltransferase activities in dispersed pineal cells of euthyroid and hypothyroid rats

The effect of vasoactive intestinal peptide (VIP) on thyroxine type II 5′-deiodinase (5′-D) and N-acetyltransferase (NAT) activities were studied using pineal cells of euthyroid and hypothyroid rats. VIP activated 5′-D activity in a dose-dependent manner in both euthyroid and hypothyroid animals. However, basal and VIP stimulated activity was higher in pinealocytes from hypothyroid than in cells from euthyroid rats. VIP was also able to stimulate NAT activity but hypothyroidism did not induce modifications in its activity. Both 5′-D and NAT activities were stimulated not only by VIP, but also by isoproterenol, a β-adrenergic receptor agonist, and forskolin, a potent activator of adenylate cyclase activity. The results suggest that VIP may be involved in the physiological regulation of pineal 5′-D activity.

Involvement of alpha- and beta-adrenergic receptors in the regulation of rat pineal N-acetyltransferase activity during development.

The regulation by alpha- and beta-adrenergic receptor agonists of rat pineal N-acetyltransferase (NAT) activity during development was studied. We found that isoproterenol, a beta-adrenergic agonist, exhibited a weak stimulatory effect on pineal NAT activity during the first 3 weeks of age. Indeed, the drug did not mimic the effect of darkness on pineal NAT activity until the fourth week of age. The results might be explained by the existence of other mechanisms, in addition to those that involve beta-adrenergic receptors, which regulate pineal NAT activity during early postnatal development. In line with this, we determined that phenylephrine, an alpha 1-adrenergic agonist, stimulated NAT activity during both the day and at night in 2-week-old rats, while exhibiting no effect in adult rats. Methoxamine, an alpha 1-adrenergic agonist more selective than phenylephrine, also activated NAT activity in young, but not in adult, rats. Moreover, prazosin, a selective alpha 1-adrenergic receptor blocker, prevented pineal NAT activation by norepinephrine or darkness at night in 2-week-old rats, but not in adult animals. The results suggest that alpha 1-adrenergic receptors may be more important than beta-adrenergic receptors in regulating rat pineal NAT activity during development.

Involvement of alpha- and beta-adrenergic receptors in the regulation of rat pineal N-acetyltransferase activity during development

The regulation by alpha- and beta-adrenergic receptor agonists of rat pineal N-acetyltransferase (NAT) activity during development was studied. We found that isoproterenol, a beta-adrenergic agonist, exhibited a weak stimulatory effect on pineal NAT activity during the first 3 weeks of age. Indeed, the drug did not mimic the effect of darkness on pineal NAT activity until the fourth week of age. The results might be explained by the existence of other mechanisms, in addition to those that involve beta-adrenergic receptors, which regulate pineal NAT activity during early postnatal development. In line with this, we determined that phenylephrine, an alpha 1-adrenergic agonist, stimulated NAT activity during both the day and at night in 2-week-old rats, while exhibiting no effect in adult rats. Methoxamine, an alpha 1-adrenergic agonist more selective than phenylephrine, also activated NAT activity in young, but not in adult, rats. Moreover, prazosin, a selective alpha 1-adrenergic receptor blocker, prevented pineal NAT activation by norepinephrine or darkness at night in 2-week-old rats, but not in adult animals. The results suggest that alpha 1-adrenergic receptors may be more important than beta-adrenergic receptors in regulating rat pineal NAT activity during development.

Serotonin N‐acetyltransferase activity in chicken retina: In vivo effects of phosphodiesterase inhibitors, forskolin, and drugs affecting dopamine receptors

A role of D2-dopaminergic neurotransmission in the regulation of melatonin biosynthesis in retina was studied in vivo in chickens. The nighttime rise in serotonin N-acetyltransferase (NAT)--the penultimate and key regulatory melatonin-synthesizing enzyme--was potently inhibited by both acute light exposure and agonists of dopamine D2-receptor (quinpirole, bromocriptine, and apomorphine). Spiroperidol, a selective dopamine D2-receptor blocker, increased the enzyme activity in light-exposed chickens, but had no effect in animals kept in darkness. Inhibitors of cyclic nucleotide phosphodiesterase, aminophylline, and 3-isobutyl-1-methylxanthine given peripherally, along with a direct adenylate cyclase activator forskolin injected directly into the eye, mimicked the action of darkness, and markedly enhanced the retinal NAT activity when administered to animals maintained in an illuminated environment. Dopamine D2-receptor agonists had no effect on aminophylline-stimulated enzyme activity, whereas spiroperidol enhanced it. Forskolin-driven NAT activity was suppressed by quinpirole. Spiroperidol and aminophylline given alone at different times of day under light conditions stimulated NAT activity, and their effects were mainly additive when given in combination. SCH 23390, a selective D1-dopamine receptor antagonist, did not affect the rise in NAT activity of chicken retina produced by either darkness or by aminophylline. The results provide further evidence that dopamine, acting via D2-receptors, mediates the inhibitory effects of light on the cyclic AMP-dependent dark-evoked induction of NAT activity in chicken retina.

In vivo activation of pineal N-acetyltransferase but not type II thyroxine 5′-deiodinase by phenylephrine in young rats

The regulation by α- and β-adrenergic agonists of pineal N-acetyltransferase (NAT) and type II thyroxine 5′-deiodinase (5′-D) in rats at either 2 or 6 weeks of age was studied. The pattern of stimulation was different because NAT activity could be clearly activated by an α-adrenergic agonist, phenylephrine, only in 2-week-old rats. However, isoproterenol, a β-adrenergic agonist, was able to stimulate NAT activity in rats at both 2 and 6 weeks of life. On the other hand, phenylephrine was always ineffective in stimulating 5′-D activity, while isoproterenol clearly activated it at both ages. These results strongly suggest a role for α-adrenergic receptors, in addition to β-adrenergic receptors, in regulating rat pineal NAT activity during development.

Effect of 6‐Methoxy‐2‐Benzoxazolinone on the Activities of Rat Pineal N‐Acetyltransferase and Hydroxyindole‐O‐Methyltransferase and on Melatonin Production

A naturally occurring compound, 6-methoxybenzoxazolinone (6-MBOA), present in grasses, has been shown to induce sexual maturation in a number of rodent species. The structural similarity of 6-MBOA and melatonin has led researchers to suspect that 6-MBOA might induce its progonadal effects by directly altering pineal function. Previous studies have shown that 6-MBOA has the properties of a weak beta-adrenergic agonist capable of stimulating rat pineal N-acetyltransferase (NAT) activity at pharmacological concentrations of 10(-3) M. In the present study we have examined the effect of 6-MBOA on both the pineal melatonin synthesizing enzymes, namely, NAT and hydroxyindole-O-methyltransferase (HIOMT) as well as on melatonin production, in organ cultured rat pineal glands. In addition, we have also examined the ability of 6-MBOA to displace a ligand from rat brain alpha- and beta-adrenoceptors. Our results confirm that 6-MBOA stimulates NAT activity and melatonin production at the high concentration of 10(-3) M. It appears to have no effect on HIOMT activity. A competition study shows that 6-MBOA is able of displacing ligands at the alpha- and beta-adrenoceptors but only at concentrations greater than 10(-4) M. Whether such high concentrations of 6-MBOA reach the pineal of rodent in their natural habitat is unknown. However, if 6-MBOA does mediate progonadal effects by altering pineal function it would be expected that 6-MBOA would ultimately inhibit the effects of melatonin. The possibilities are that the high melatonin levels induced by 6-MBOA cause desensitization of melatonin receptors or that 6-MBOA is an antagonist at the level of the melatonin receptor, thus restricting the inhibitory effects of melatonin on the reproductive system.

Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87).

The activity of arylalkylamine N-acetyltransferase (EC 2.3.1.87), the rate-controlling enzyme in melatonin synthesis is stimulated approximately equal to 100-fold by an adrenergic cyclic AMP mechanism in both neonatal and adult rat pineal glands. This stimulation is blocked in the adult gland by the depolarizing agents ouabain (1 microM) and K+ (80 mM) (Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H. (1975) Mol. Pharmacol. 11, 241-255). In the present study pineal glands obtained from prenatal to adult rats were used; it was found that K+ (80 microM) inhibited the adrenergic stimulation of N-acetyltransferase activity at all ages but that ouabain (1 nM to 1 mM) treatment was not inhibitory early in development. In contrast, in the neonate, ouabain (1-100 nM) enhanced adrenergic induction of N-acetyltransferase activity, and ouabain treatment alone (1-1000 nM) stimulated N-acetyltransferase activity. A small stimulation was also seen at one concentration (1 nM) in the adult. Analysis of the development of high affinity ouabain binding sites and Na+,K+-ATPase activity in the intact pineal gland indicated that the developmental pattern of both resemble the development of ouabain inhibition of the adrenergic stimulation of N-acetyltransferase activity. All are low for the first few days of life, gradually increase during the next 3 weeks of life, and then approach adult levels. Similarly, ouabain (1 nM to 1 mM) had no effect on 86Rb uptake in the 2-day-old gland but blocked (IC50 congruent to 20 nM) 86Rb uptake in the adult gland. These findings indicate ouabain probably has little inhibitory effect on the norepinephrine stimulation of N-acetyltransferase activity in the neonatal because a high affinity ouabain binding form of Na+,K+-ATPase activity, similar to the alpha + form identified in rat brain, is at very low levels in the pinealocyte. Accordingly, it appears that an ouabain-insensitive mechanism in the neonatal gland maintains membrane potential and that this mechanism plays a less important role in the adult. The explanation of why ouabain alone stimulates N-acetyltransferase activity and why it enhances the effects of norepinephrine in the neonatal pineal gland might be that ouabain acts on surviving neural elements present in the gland to cause the net release of a transmitter, perhaps norepinephrine, which then stimulates N-acetyltransferase activity.

Adrenal-mediated depression of n-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h - lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h - 5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.

Histamine mediation of nicotine effects on postganglionic sympathetic neuroeffector junctions

The sympathomimetic action of nicotine was investigated using preparations of the rat pineal gland and the rabbit pulmonary artery. Nicotine (0.01–1 mM) did not stimulate N-acetyltransferase activity in pineal glands which contained functional sympathetic nerve endings. In the pulmonary artery preparation the previously described contractile effects of nicotine were antagonized by the antihistaminic compound chlorpheniramine. It is therefore postulated that histamine is involved in the sympathomimetic effects of nicotine on the pulmonary artery.

Beta adrenergic-blockers decrease adrenergically stimulated N-acetyltransferase activity in pineal glands in organ culture

Treatment with 10 μM l-propranolol caused a rapid decrease in the high level of acetyl coenzyme A: serotonin N-acetyltransferase (EC 2.3.1.5, N-acetyltransferase) activity in pineal glands cultured in the presence of 1 μM norepinephrine. d-Propranolol was ineffective except at high concentration (1 mM). The same stereospecificity was also observed in organ culture when d- or l-propranolol was tested as a blocker of the norepinephrine-induced increase in N-acetyltransferase activity. Practolol (10–300 μM), a relatively specific β 1,-blocker, also initiated a decrease and inhibited the norepinephrineinduced increase in N-acetyltransferase. Butoxamine (10–300 μM), a relatively specific β 2-blocker, had no significant effect in either experimental situation. The percentage decrease in N-acetyltransferase activity after 40 min of treatment with l-propranolol was strongly dependent on temperature. At 40° C enzyme activity decreased by 92% but at 34° C enzyme activity decreased by only 50%. l-Propranolol (20 μM) did not inhibit the increase or initiate a decrease in N-acetyltransferase activity in pineal glands treated with 1 mM N 6,O 2 -dibutyryl adenosine cyclic 3',5'-monophosphate. The fast response observed when l-propranolol was added to the culture medium of norepinephrinetreated glands was not blocked by cycloheximide treatment. Concentrations of ouabain or K +, which are known to completely inhibit the norepinephrine-stimulated increase in N-acetyltransferase activity, caused only a small decrease in induced N-acetyltransferase activity, similar to that due to cycloheximide treatment.

Input and output signals in a model neural system: the regulation of melatonin production in the pineal gland.

The production of melatonin has been studied using organ cultures of pineal glands incubated with methionine-methyl-3H. Melatonin-O-methyl-3H was extracted from cultured pineal glands and incubation media, and the activity of N-acetyltransferase was measured. This is the first of two enzymes necessary for the conversion of serotonin to melatonin in the pineal. The treatment of pineal glands with norepinephrine or dibutyryl cyclic AMP increased the release of melatonin-O-methyl-3H into the incubation media and the concentration of melatonin-O-methyl-3H in the glands. These treatments also resulted in the stimulation of N-acetyltransferase, as compared to untreated glands. The transduction of neural information to biochemical, signals which regulate the melatonin pathway appears to involve the release of norepinephrine, which stimulates N-acetyltransferase activity through an adenyl cyclase-cyclic AMP mechanism, as evidenced by these and other studies discussed. In the present study the effects of harmine were studied. This hallucinogen is known to inhibit monoamine oxidase and stimulate melatonin production. Harmine was observed to stimulate N-acetyltransferase. This observation raises the possibility that an important action of this psychotropic drug may be on mechanisms which convert neural activity into biochemical events.