Pineal Glands In Organ Culture Research Articles

Cholera toxin-induced G sα down-regulation in neural tissue: studies on the pineal gland

Cholera toxin (CT) treatment (50 μg/ ml) was used to down regulate the α subunit of the stimulatory guanine nucleotide binding protein (Gsα) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces G sα by ≈ 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of Gβ, G iα or G oα. This effect on G sα was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of G sα. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP ( N 6,2′-O-dibutyryl adenosine 3′,5′monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced α- or β-adrenergic stimulation. α 1-Adrenergic activation of the pineal gland elevates [Ca 2+] i, which potentiates effects of cyclic AMP; in these studies the response to α-adrenergic activation was markedly increased in 48-h CT-treated glands, reflecting Ca 2+ potentiation of the effects of elevated levels of cyclic AMP. In contrast, the effects of the selective β-adrenergic agonist isoproterenol was reduced by ≈ 75%. These studies not only establish CT-induced G sα down-regulation as a new tool for the study of adrenergic signal transduction in the pineal gland, but indicate that this paradigm is probably useful in all neural tissue.

Photoneural control of the synthesis and phosphorylation of pineal MEKA (phosducin).

MEKA is an acidic 33-kilodalton phosphoprotein found in the retina and pineal gland. It is of interest because it forms a cytoplasmic heterotrimer with the beta gamma-complex of GTP-binding regulatory proteins (G proteins). Accordingly, MEKA may play a role in signal transduction. MEKA is phosphorylated on Ser73 by cAMP-dependent protein kinase. In the present report, MEKA was studied using an antiserum (Anti-32) against MEKA65-96, which can be used to estimate total MEKA and the phosphorylation state of MEKA. It was confirmed that MEKA is rapidly phosphorylated by adrenergic stimulation of pineal glands in organ culture. In addition, total (dephosphorylated) MEKA was observed to increase after a 6-h treatment with norepinephrine or (Bu)2 cAMP, an effect which was dependent upon new protein synthesis. In in vivo studies, it was found that the total amount of MEKA and MEKA phosphorylation were increased at night in the dark, a time when the pineal gland is adrenergically stimulated. The high level of phosphorylation was rapidly reduced when animals were exposed to light, which blocks neural stimulation of the gland. This report provides the first in vivo evidence that MEKA phosphorylation is under physiological control, and that MEKA synthesis is controlled by an adrenergic----cAMP mechanism which requires protein synthesis.

Inhibition of the Chick Pineal Rhythm in Rate of Thymidine Incorporation by Cyclic AMP

Chick pineal glands in organ culture showed a circadian rhythm in the rate of thymidine incorporation. Thymidine incorporation was very markedly inhibited when 3-isobutyl-l-methylxanthine (IBMX) was continuously present. When IBMX was added to cultures in control medium during the photoperiod of the second day in culture, the extent of inhibition of incorporation during that photoperiod increased with the increase in length of the photoperiod remaining. Incorporation did not resume at the start of a second photoperiod if IBMX was added within the first 10 h of the first photoperiod. Corresponding results were obtained with glands continuously cultured in constant darkness. Similar results were also obtained using glands treated with 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), 7 beta-acetoxy-8,13-epoxy-1 alpha, 6 beta, 9 alpha-trihydroxy-1abd-14-ene-11-one (forskolin), or 8-bromo-cyclic AMP, but not with 8-bromo-cyclic GMP. When glands cultured with IBMX were transferred to control medium, incorporation remained inhibited until the start of the next photoperiod. We conclude that the increase in the rate of thymidine incorporation at the start of each new photoperiod is dependent on a "switch" process that is inhibited by elevated concentrations of cyclic AMP.

Evidence that interferon-γ alters pineal metabolism both indirectly via sympathetic nerves and directly on the pinealocytes

1. 1. Interferon-γ (IFN-γ) has been shown to suppress N-acetyltransferase (NAT) activity in cultured rat pineal glands when stimulated with isoproterenol (ISO). 2. 2. Conversely, IFN-γ has also been shown to increase the melatonin content of the rat pineal gland in organ culture. 3. 3. Circumstantial evidence leads to a hypothesis that the NAT suppressive effect may be due to the action of IFN-γ on the sympathetic nerve terminals. 4. 4. To test this hypothesis, pineal glands from intact (INT) and superior cervical ganglionectomized (SCGX) rats, which had been operated 5 days earlier, were cultured with either ISO or ISO + IFN-γ. 5. 5. The concentration of ISO was 10 −8 M and that of IFN-γ was 300 antiviral units/ml. 6. 6. The pineals were incubated for a total period of 5.5 hr, after which the activities of NAT and hydroxyindole- O-methyltransferase (HIOMT) and the levels of melatonin and cAMP were estimated. 7. 7. Suppression of NAT by IFN-γ was observed in the pineals from INT rats, but not in those from SCGX animals. 8. 8. IFN-γ significantly enhanced melatonin levels over those in ISO-stimulated pineals and culture media from the SCGX animals, but not from the INT animals. 9. 9. IFN-γ treatment had no effect on either the HIOMT activity or cAMP levels. 10. 10. The results indicate that the IFN-γ-induced NAT suppression requires the integrity of the sympathetic nerve terminals and the IFN-γ-induced enhancement of melatonin production is accomplished through its direct action on pinealocytes.

Interferon-γ Modulates Melatonin Production in Rat Pineal Glands in Organ Culture

The effect of interferon-gamma (IFN-gamma) on melatonin production was determined in rat pineal glands in organ culture. IFN-gamma enhanced the production of melatonin in the glands incubated with either 10 nM or 1,000 nM isoproterenol (ISO, a beta-adrenergic receptor agonist) but decreased the 100 nM ISO response. The enhanced melatonin production was accompanied by either unchanged or a lower level of N-acetyltransferase (NAT) activity, whereas the suppression was accompanied by a reduced NAT activity. IFN-gamma did not change the activity of the enzyme hydroxyindole-O-methyltransferase (HIOMT) in the ISO-stimulated pineal glands. The results indicate that IFN-gamma has a dual action on the pineal gland: One is to increase melatonin production through yet unknown mechanisms, and the other is to suppress NAT activity. These results also suggest that the pineal gland, under physiological stimulation, is modulated by IFN-gamma to increase melatonin production. A hypothetical model is proposed to explain the delicate regulation of pineal function by the immune system.

Treatment with forskolin for 8 hours during the day increases melatonin synthesis in the Syrian hamster pineal gland in organ culture: The long lag period is required for RNA synthesis

The exposure of organ-cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in melatonin levels when glands were collected in the second half of the dark period and incubated for 4 h. However, when glands were collected at the beginning of the dark period and incubated with the same drug, a significant increase in melatonin content was observed only after 6-8 h. Likewise, when glands were collected at the beginning of the light period, forskolin stimulated melatonin synthesis only after 6-8 h of incubation with the drug. These results support the existence of a relatively long lag period necessary for the induction of melatonin production in the Syrian hamster pineal gland. Experiments with actinomycin D indicate that RNA synthesis occurs during the lag period; thus, actinomycin D blocks the induction of melatonin synthesis by forskolin in glands collected at the beginning of the dark period and incubated for 8 h. In contrast, when pineal glands were collected from hamsters killed in the second half of the dark period, and incubated with forskolin for either 4 or 8 h, actinomycin D was unable to block the induction of melatonin production. These data suggest that RNA, presumably messenger RNA, which is necessary for increasing hamster pineal melatonin synthesis, is synthesized and accumulates during the first half of the night.

Effects of either forskolin, the 1,9-dideoxy derivative of forskolin, or 8-bromocyclic AMP on cyclic AMP and melatonin production in the Syrian hamster pineal gland in organ culture

The possible involvement of cyclic AMP in the regulation of melatonin production was investigated in cultured Syrian hamster pineal glands. Addition of forskolin, an adenylate cyclase activator, to the incubation medium caused marked increases in both cyclic AMP and melatonin levels in glands collected in the second half of the dark period 8-Bromocyclic AMP, an analogue of cyclic AMP, also increased melatonin production. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was ineffective in stimulating either cyclic AMP levels or melatonin production. These results support a primary role for cyclic AMP in the nocturnal increase of melatonin production in the Syrian hamster pineal gland.

Melatonin secretion from goldfish pineal gland in organ culture

Pineal glands were removed from goldfish reared under 12L-12D at 25° for 2 weeks. These were incubated for 6 days under (1) normal 12L-12D (lights on 0600–1800 hr), (2) reversed 12L-12D (lights on 1800–0600 hr), (3) continuous dark, or (4) continuous light condition at 25°. The incubation medium was changed at 12-hr intervals (0600–1800 and 1800–0600 hr) and secreted melatonin (MLT) was measured by RIA. Under 12L-12D or reversed 12L-12D, MLT secretion was active in the dark phase and was suppressed in the light phase of a given photoperiod. Under a continuous dark condition, a large amount of MLT was secreted into the medium, although the amount gradually decreased. The MLT secretion was more active in the period corresponding to the dark phase of the acclimatory photoperiod than in the period corresponding to the light phase. This pattern in secretion remained for 4 days. Under a continuous light condition, MLT secretion was suppressed, but the secretion was rapidly increased after changing the photoperiod from the light to the dark condition. These findings clearly indicate that MLT secretion in the organ-cultured pineal gland is photosensitive. It is active under dark and inactive under light conditions. The existence of a circadian rhythm in MLT secretion is also suggested.

Role of Postsynaptic α‐Adrenergic Receptors in the β ‐Adrenergic Stimulation of Melatonin Production in the Syrian Hamster Pineal Gland in Organ Culture

The role played by postsynaptic alpha-adrenergic receptors in the stimulation of pineal melatonin production was investigated in the Syrian hamster. The studies were conducted using organ cultured pineal glands collected from both anatomically intact and superior cervical ganglionectomized hamsters. Results obtained indicate that phenylephrine, an alpha-adrenergic agonist, by itself has no effect in promoting melatonin production; however, it potentiates the stimulatory effects of isoproterenol, a beta-adrenergic agonist, on pineal melatonin production in nonoperated hamsters. Similar observations were obtained with pineal glands whose presynaptic terminals were removed by prior superior cervical ganglionectomy. However, a longer incubation time was required (4-6 hours vs. 2 hours) with pineal glands taken from ganglionectomized animals. Apparently, beta-adrenergic activation is an absolute requirement to stimulate pineal melatonin production, and an alpha-adrenergic receptor mechanism potentiates beta-adrenergic activation. In addition, the findings obtained with denervated pineal glands suggest that the regulation of pineal melatonin production by both alpha- and beta-adrenergic mechanisms is through receptors located on postsynaptic structures.

Stimulatory Effect of Isoproterenol but Not of Dibutyryl Cyclic AMP on N-Acetyltransferase Activity and Melatonin Content of Syrian Hamster Pineal Gland in Organ Culture

The purpose of this study was to compare the response of Syrian hamster pineal glands in organ culture either to isoproterenol, a beta-adrenergic agonist, or to dibutyryl cyclic AMP. When pineal glands were collected at night, hamsters were exposed to light for 30 min to depress pineal N-acetyltransferase (NAT) activity and melatonin values to low levels. Pineal glands were removed and placed in organ culture containing either isoproterenol or dibutyryl cyclic AMP and subsequent changes in NAT activity and melatonin levels were measured. At night, isoproterenol (10(-7) or 10(-6) M) induced an increase in the NAT activity and melatonin levels in both pineals and culture media. However, dibutyryl cyclic AMP was either ineffective or minimally effective in stimulating these parameters at either different incubation times (2, 4, and 6 h) or drug concentrations (0.1, 0.5, and 1.0 mM). Conversely, when rat pineal glands were incubated with either isoproterenol (10(-7)) or dibutyryl cyclic AMP (0.5 mM) dramatic rises in NAT activity and melatonin levels were observed. In another experiment, hamster pineal glands were collected from animals killed either late in the light period (19.00 h) or in the latter half of the dark period. Isoproterenol promoted NAT activity and melatonin production only in glands collected in the latter half of the dark phase.

Involvement of 5-lipoxygenase pathway in norepinephrine stimulation of rat pineal melatonin synthesis.

The effect of lipoxygenase inhibition, leukotriene agonists and antagonists, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was examined in the rat pineal gland in organ culture. To study melatonin secretion pineal explants were incubated for 6 h in tissue culture medium 199 with the different drugs. Melatonin concentration in the pineal gland and the medium was measured by RIA. Exposure of explants to norepinephrine (NE) brought about a 2- to 5-fold increase in both parameters, an effect that was reduced but not abolished, by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 10(-5) M). Lilly 171883 (10(-5) M) or FPL 55712 (10(-5) M; both antagonists of leukotrienes) reduced NE-induced melatonin production. Neither NDGA nor Lilly 171883 affected melatonin production in the absence of NE. Leukotrienes C4 and D4 increased melatonin release to the media at all concentrations tested (1-1,000 nM) with a maximum effect at 1 nM (leukotriene C4) and 10 nM (leukotriene D4). Significantly higher tissue melatonin concentrations as compared to controls were observed after exposure of pineal explants to 1 and 100 nM of leukotriene C4, or 100 nM of leukotriene D4. Another 5-lipoxygenase metabolite, 5-HETE, increased pineal melatonin content at concentrations of 1, 10 and 100 nM whereas only 1,000 nM stimulated melatonin release. These results suggest that the 5-lipoxygenase pathway plays a significant role in NE-stimulated melatonin production by the rat pineal gland.

Derivatives of [3H]serotonin in organ cultures of hamster pineal gland.

The purpose of this study was to determine the viability of the hamster pineal gland in organ culture and to test the effect of norepinephrine (NE) on [3H]serotonin derivatives. In this study, elevated levels of melatonin (7-fold, p less than .05), 5- hydroxytrytophol (5-fold, p less than .001), 5-methoxytryptophol (1.78-fold, p less than .05), and depressed levels of 5-hydroxyindoleacetic acid (3.8-fold, p less than .02) and methoxyindoleacetic acid (1.78-fold, p less than .05) were detected in the glands following the addition of NE to the medium. In a separate experiment, melatonin concentration in the media was also periodically measured by radioimmunoassay to determine the viability of the organ culture over a four-day period. The melatonin level on day 2 (2321 +/- 106 pg/gland) was significantly higher (p less than 0.01) than on day 3 (1542 +/- 86 pg/gland) or day 4 (805 +/- 39 pg/gland). The results of these experiments verify the viability of the hamster pineal organ culture and show that the gland responds to NE in vitro.

Light and agonists alter pineal N-acetyltransferase induction by vasoactive intestinal polypeptide.

Vasoactive intestinal polypeptide stimulated serotonin N-acetyltransferase activity in rat pineal glands in organ culture by a postsynaptic action that was independent of the beta-receptor. The magnitude of stimulation could be altered by environmental lighting conditions and by prior exposure to the agonist. Such up- and down-regulation, well known for catecholaminergic stimulation of this system, is compatible with a possible control of the pineal by vasoactive intestinal polypeptide as well as by catecholamines.

Biosynthesis of biopterin: adrenergic cyclic adenosine monophosphate-dependent inhibition in the pineal gland.

Pineal glands in organ culture synthesize and release biopterin and are able to maintain concentrations of biopterin occurring in vivo for up to 54 hours in vitro. The intracellular biopterin content is reduced 50 percent by treatment with l-norepinephrine or cyclic adenosine monophosphate derivatives, but not by d-norepinephrine. This is an indication that biopterin levels are regulated by an adrenergic cyclic adenosine monophosphate-dependent mechanism. The decline in tissue biopterin content, produced mainly by inhibited of biosynthesis, is maximal at 6 hours and is not associated with either an increase in biopterin release or a shift in the reduction state of the biopterin.

A biphasic effect of estradiol on serotonin metabolism in rat pineal organ cultures.

Physiological amounts of estradiol (1 nM) significantly increased serotonin, conversion to melatonin and 5-methoxytryptophol by rat pineal glands in organ culture whereas larger, pharmacological, doses (i.e. 1000 nM) impaired it significantly. The stimulatory but not the inhibitory effect of estradiol was blocked by the simultaneous addition of puromycin in the culture medium.

Hormone-induced increase of hydroxyindole.O.methyltransferase activity in the embryonic chick pineal gland in organ culture

The technique of organ culture was used to investigate the regulation of the increase of hydroxyindole.O.methyltransferase activity of the pineal gland during embryonic development. Glands of 15- and 17-day chick embryos developed small increases of enzyme activity when incubated in organ culture with a standard tissue culture medium. The increase of enzyme activity was markedly stimulated by a combination of hydrocortisone, somatotropin and thyroxine, and simulated the pre-hatch increase found in ovo . All three hormones appeared to be required for consistent development of maximal increases in enzyme activity but none was essential for some increase in activity.

Ornithine dec arboxylase—apparent lack of involvement in the induction of n-acetyltransferase in rat pineal gland

The potential role ornithine decarboxylase in the induction of serotonin- N-acetyltransferase in the rat pineal gland has been studied. The injection of isoproterenol (5 mg/kg, s.c.) resulted in an apparent induction of serotonin- N-acetyltransferase within 1 hr with a maximal response at 4 hr. No change in ornithine decarboxylase was observed in the first hour following isoproterenol administration. Rat pineal glands in organ culture responded to addition of 2 μM isoproterenol with a similar induction of N-acetyltransferase. There was a small but significant decrease in ornithine decarboxylase in the cultured rat pineal glands in response to isoproterenol. Additional experiments indicated that isoproterenol also did not elevate ornithine decarboxylase in the superior cervical ganglia under conditions known to induce tyrosine hydroxylase in this organ. The results of this study are consistent with our previous finding that ribosomal RNA synthesis is unaffected during the induction process in the pineal gland.

INFLUENCE OF PSORALEN ON MELATONIN FORMATION IN RAT PINEAL GLAND IN ORGAN CULTURE

8-Methoxypsoralen (10(-4) M) added to the medium of rat pineal glands in organ culture induces a five-fold increase of 14C-labelled N-acetylserotonin and melatonin formation when serotonin is used as 14C-labelled precursor. Addition of d-amphetamine (10(-5) M) to pineal glands also results in an increase of N-acetylserotonin and melatonin formation. This increase is enhanced by the addition of 8-methoxypsoralen (10(-5) M). In pineal glands pre-incubated for 24 h 8-methoxypsoralen (10(-4) M) caused an increased production of N-acetylserotonin and melatonin. This indicates that the effect of 8-methoxypsoralen is mediated through a postsynaptic event and not by release of noradrenaline or blocked re-uptake of amines to nerve terminals in the pineal gland. Further studies of the clinical significance of the effect of psoralen on pineal gland metabolism seems warranted.

Selective β-adrenoceptor antagonism of induced formation of 14C-N-acetylserotonin in rat pineal glands in organ culture

Stimulation of β-adrenoceptors in cultured rat pineal glands increased the formation of ( 14C)-N-acetylserotonin (NAcS) from ( 14C)-serotonin. The non-selective β-adrenoceptor antagonist dl-propranolol and the selective β 1-adrenoceptor antagonist practolol both decreased the response to the directly acting β-agonist terbutaline 2×10 −4 M, when measured as the amount of ( 14C)-NAcS released into culture medium during a 24 h period. A 1,000 times higher concentration was required in the medium using practolol than propranolol to decrease ( 14C)-NAcS formation 50% as compared to the level reached using terbutaline 2×10 −4 M by itself. Two different selective β 2-adrenoceptor antagonists were tried: butoxamine (0.05 mM–0.10 mM) and H 35/25 (0.01 mM–0.25 mM). Butoxamine was without effect in the concentrations tested and H 35/25 either did not affect or in the lowest concentration potentiated terbutaline induced ( 14C)-NAcS formation. The results indicate that the rat pineal gland β-adrenoceptors studied in organ culture respond similar to the β 1-adrenoceptor subgroup.

Metabolism of tryptophan and serotonin by the chick pineal gland in organ culture

Marked differences were seen between the metabolism of L-[3-14C] tryptophan and of [2-14C]serotonin by the intact chick pineal gland in organ culture. The major metabolite of tryptophan recovered by our procedures was melatonin, which accounted for about half the radioactivity recovered as metabolic products. In contrast, the principal product of serotonin metabolism recovered was hydroxyindoleacetic acid, and the yield of products derived through monoamine oxidase (EC 1.4.3.4) activity vastly exceeded that of melatonin. Metabolism of tryptophan yielded a much larger proportion of methlated metabolites among the products recovered than did metabolism of serotonin. However, the yield of methoxyindoleacetic acid from serotonin was greater than that from tryptophan. Serotonin formed endogenously and serotonin supplied exogenously appear to enter two or more largely distinct metabolic pools.