Neonatal Steroid Treatment Reduces Catecholamine-Induced Increases in Pineal Serotonin N-Acetyltransferase Activity

Hydrocortisone acetate given to the neonatal rat diminishes subsequent elevations in pineal serotonin N-acetyltransferase (acetyl-coenzyme A:arylamine N-acetyltransferase; EC 2.3.1.5; NAT) activity produced by administration of catecholamines to the intact animal or to pineals in organ culture. The time required for development of this decrease in sensitivity varies inversely with age at treatment. A minimal dose of 200 micrograms of hydrocortisone acetate/rat is required to elicit this decreased response to agonist. Other glucocorticoids have qualitative effects similar to hydrocortisone acetate, but cholesterol and the gonadal steroids testosterone, estradiol, and progesterone are without effect. In addition to showing a smaller rise in NAT activity on stimulation, pineals from steroid-treated neonates also synthesize less N-acetylserotonin and melatonin from tryptophan. The decrease in NAT response to stimulation after steroid treatment appears due to actions beyond cyclic AMP generation and may involve inhibition of protein synthesis.     

Stimulation of Melatonin Synthesis in Ovine Pineals In Vitro

Static and superfused pineal slices (750 micron) have been used to study the control of melatonin synthesis by ovine pineals. Static incubates show a time-dependent accumulation of melatonin in the medium; this is significantly increased by stimulation with norepinephrine (NE) (10(-5) M), reaching 300% above control levels after 4 h. Perifused pineal slices show a rapid rise in melatonin release within 12-18 min in response to NE stimulation. This reaches a 3.5-4.5-fold increase in melatonin released within 30 min. Withdrawal of NE is associated with a rapid return to prestimulated levels within 12-18 min. These time-course characteristics compare favorably to those changes seen in vivo. The formation of [14C]melatonin from [14C]-tryptophan shows a linear increase with time. In the presence of NE (10(-5) M), the rate of synthesis is increased, albeit after an initial time lag of at least 30 min. The latter may reflect an N-acetyltransferase-independent mechanism of synthesis and release. In static incubations, propranolol (10(-5) M) inhibited NE-induced melatonin production by about 60%, but prazosin (10(-5) M) had no effect. As dibutyryl cyclic AMP (10(-3) M) stimulated melatonin production, it is concluded that beta-receptors are of primary importance to the control of melatonin production, as in the rat. The role of alpha 1-receptors is less clear, but the stimulatory action of phorbol 12-myristate 13-acetate on melatonin release implicates a receptor linked to phosphatidylinositol turnover.     

Rapid Nocturnal Increase in Ovine Pineal N-Acetyltransferase Activity and Melatonin Synthesis: Effects of Cycloheximide

Thirty minutes after the onset of darkness, ovine pineal arylalkylamine N-acetyltransferase, N-acetylserotonin, and melatonin increase 5- to 10-fold. No significant changes in hydroxyindole-O-methyltransferase, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxyindoleacetic acid, and 5-methoxytryptophol are detected at this time. Administration of cycloheximide inhibits the rise in N-acetyltransferase and N-acetylserotonin, but not melatonin. Unexpectedly, 5-methoxytryptophol increases after cycloheximide treatment. Taken together, these results, although consistent in part with a role for serotonin N-acetylation in the regulation of melatonin synthesis in sheep, indicate that an N-acetyltransferase-independent mechanism may also be involved.     

Effects of Adrenal Steroids on Basal Ganglia Neuropeptide mRNA and Tyrosine Hydroxylase Radioimmunoreactive Levels in the Adrenalectomized Rat

Abstract: To investigate the effects of type I (mineralocorticoid) and type II (glucocorticoid) receptor activation on striatal neuropeptide [preproenkephalin (PPE), preprotachykinin (PPT), and preprodynorphin (DYN)] mRNA and midbrain cholecystokinin (CCK) mRNA as well as striatal tyrosine hydroxylase radioimmunoreactivity (TH-RIC) levels, we administered either replacement levels of corticosterone (CORT; 0.5 mg/kg/day, s.c.) or pharmacological levels of deoxycorticosterone acetate (DOCA; a mineralocorticoid steroid with ability to bind to type I and type II receptors; 5 mg/kg, s.c.) to adrenalectomized adult male rats. After 1 week of recovery from adrenalectomy surgery, animals were injected daily with sesame oil or CORT for 1, 3, or 7 days or DOCA for 3 or 7 days and killed 16 h after the last injection. Adrenalectomy resulted in a decrease in all three striatal neuropeptide mRNA levels, compared with sham-operated rats. CORT replacement resulted in recovered PPE and PPT mRNA levels after 1 day and elevated PPE mRNA levels over those in sham-operated controls after 3 days. In contrast, DYN mRNA levels showed recovery after 7 days of CORT replacement. Results after DOCA treatment largely paralleled those after CORT replacement. There were no significant treatment effects on indirect markers of midbrain dopaminergic activity, i.e., CCK mRNA and TH-RIC. From these results we conclude that compared with striatal tachykinin and dynorphinergic neurons, enkephalinergic cells show greater sensitivity, whereas the dopaminergic system, including mesencephalic CCK, demonstrates an insensitivity to physiological CORT and to pharmacological DOCA treatment.     

Adrenocortical Steroids Modify Neurotransmitter-Stimulated Cyclic AMP Accumulation in the Hippocampus and Limbic Brain of the Rat

Glucocorticoid hormones are known to affect limbic system structures that have high levels of specific receptors for glucocorticoids, especially the hippocampus (HIPP). To understand how glucocorticoids may affect synaptic transmission, we have tested the effects of adrenal removal and glucocorticoid replacement on neurotransmitter-stimulated cyclic AMP accumulation in brain slices from the rat limbic system. Adrenalectomy (ADX) caused an enhancement of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in HIPP, amygdala (AMYG), and septum (SEP). In HIPP, ADX increased the cyclic AMP response to isoproterenol (ISOP) and decreased the response to histamine (HIST). In the AMYG and SEP, ADX did not affect significantly the action of ISOP, but ADX did decrease the response to HIST in AMYG. Administration of dexamethasone or corticosterone reversed the effects of ADX on cyclic AMP accumulation in the HIPP. The dexamethasone action on VIP-stimulated cyclic AMP accumulation takes place within 48 h and is most apparent in the mid-range of the VIP dose-response curve. These results demonstrate that glucocorticoids regulate neurotransmitter-stimulated cyclic AMP generation in a fashion that is specific, both for the neurotransmitter involved and for the brain regions affected.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Light Pulses (II)

Abstract: N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought responsible for melatonin circadian rhythms. The enzyme has properties of a circadian biological clock—its rhythm persists in constant conditions and it is precisely controlled by light and dark. Experiments are reported in which light pulses of 1 to 10 h duration were imposed on chicks during their dark-time. The effect of these pulses upon the NAT was measured and the effect of the pulses on subsequent NAT was also determined. The experiments support the conclusion that the amount and/or duration of dark-time NAT is limited. This finding is interpreted as supporting the idea that a fixed amount of some substance, an initiator, is synthesized during the subjective day.     

Organ culture of the goldfish pineal body

Summary The ultrastructure and biochemistry of the goldfish pineal organ were examined in expiants cultured for 1, 3, and 6 days. All four cell types (photoreceptor, supportive, ganglion, phagocytic) were identified; they exhibited many of the characteristics of these cells in vivo. Exceptions included a gradual disorganization of the outer segments and reduction of synaptic ribbons in photoreceptors with time in culture. In addition, there was a marked proliferation of endoplasmic reticulum in both photoreceptor and supportive cells. The indoles 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, 5-methoxytryptophol, and melatonin were separated in expiants by high performance liquid chromatography using electrochemical detection. Serotonin levels could be depleted by p-chlorophenylalanine and elevated by nialamide or by adding 5-hydroxytryptophan to the culture medium. These findings suggest that organ culture may be a useful model for study of regulatory processes related to the photoneuroendocrine functions of the teleost pineal organ.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Dark Pulses (III)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought to be responsible for melatonin circadian rhythms. The enzyme has circadian properties--its rhythm persists in constant conditions, and it is precisely controlled by light and dark. Experiments are reported in which 4-h light or dark pulses were imposed on chicks (Gallus domesticus) over a 24-h period. Pineal NAT profiles were measured during and subsequent to the pulses. The phase of the NAT cycle following pulses was plotted to obtain phase-response curves. Light pulses produced a maximum phase shift (advance of 5 h) 8 h after the expected time of lights-out; dark pulses produced a maximum phase shift (advance of 4 h) 3 h after the expected time of lights-out. Maximum phase delays (-2 h) occurred 1-2 h after the expected lights-out for light pulses and 8 h after expected lights-on for dark pulses.     

Fetal Rat Pancreas in Organ Culture: Effects of Serum on the Development of the Endocrine Cells

Eighteen-day-old rat fetal pancreas was grown in organ culture for four days on medium consisting of tissue culture Medium 199 and varying concentrations of chicken serum. The glucagon and somatostatin concentration of the explants was decreased when the serum concentration of the medium was reduced from 50 to 10%. There was a further reduction in these hormones when the explants were cultured on Medium 199 alone. Explant insulin content was reduced only when serum was omitted from the medium. A serum factor tripeptide was not able to substitute for this serum requirement. Heat-inactivation of the serum resulted in a significant increase in medium insulin content and an increase in both the insulin and glucagon contents of the explants. This increase in hormone content was directly correlated with increases in the beta and alpha cell volumes of the explants. There was no change in the somatostatin content or delta cell volume of the explants grown on heat-inactivated medium. It is suggested that the serum is an important component of the culture media and is apparently required in high concentration for the development of the islet cells in vitro. The islet cell types differ in their requirement for serum. The alpha and delta require a higher concentration than do the beta cells.     

Glucocorticoids Increase Tyrosine Hydroxylase Activity in Cultured Murine Neuroblastoma

Cyclic AMP and glucocorticoids appear to have a role in regulating the activity of tyrosine hydroxylase (TH), as well as the expression of "morphological differentiation" in murine neuroblastoma. Monolayer cultures of C-1300 murine neuroblastoma (clone NBP2) were treated with the following compounds in ethanol: dexamethasone, triamcinolone acetonide, hydrocortisone, cortexolone, androstenedione, testosterone, estradiol-17 beta; or with the phosphodiesterase inhibitor Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone]. Treatment with either 200 micrograms/ml Ro20-1724 or 50 micrograms/ml dexamethasone produced significant increases in TH activity compared to alcohol controls (1.44 vs. 0.82 nmol 14CO2/mg protein/hr compared to 0.095). Triamcinolone acetonide or hydrocortisone also produced smaller, but significant, increases in TH activity compared to dexamethasone. When steroid activities were compared at 25 microM concentration and after 60 min of incubation (to maximize TH activity), triamcinolone acetonide was not as effective (62%) as dexamethasone. The relatively inactive glucocorticoid cortexolone produced a slight but significant increase, while the androgens androstenedione and testosterone and the estrogen estradiol-17 beta were without effect.     

Organ Culture of Foetal Rat Pancreas

The differentiation and growth of the foetal rat pancreas (20 days postcoitum) was studied in parabiotic organ culture with foetal adrenal tissue. In such co-cultures, characteristic pancreatic morphology was preserved and further acinar cell differentiation was fostered. Acinar cells continued to represent about 65% of the total explant volume following short-term incubation. The selective islet cell proliferation, previously observed in control pancreatic explants cultured alone, did not occur when adrenals were co-cultured. In addition, the amylase content of the incubation media and of the explanted pancreatic tissue remained high with adrenal co-culture, while the insulin content of the media and of the explanted tissue was markedly suppressed when compared to control pancreatic explants cultured alone. The effects of the adrenal in maintaining the differentiated acinar component of the pancreas and suppressing media insulin concentration diminished over extended incubation. The addition of adrenals to culture of foetal pancreatic explants after 6 days of control culture (at a time when differentiated acinar cells were not identifiable in the explant) did not result in redifferentiation of the acinar component, but did markedly depress media insulin content. Removal of adrenals after 4 days of co-culture resulted in an immediate rise in media insulin concentration and a rapid decline in pancreatic acinar mass. An adrenal-exocrine pancreatic axis is proposed and it is suggested that foetal adrenal secretions may play an important role in the development of the exocrine pancreas in vivo as well as in vitro.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.     

Steroids as Potential Modulators of Thyroid Hormone Activity in Anuran Metamorphosis

SYNOPSIS. Anuran metamorphosis is controlled by the complexinteraction of several hormones. Although thyroid hormone isthe major stimulatory hormone in metamorphosis and likely regulatesall of the metamorphic genes directly, other hormones are involvedin regulating thyroid hormone secretion and activity. Corticoids(in particular corticosterone) and the sex steroids (especially17ß estradiol) all potentially regulate thyroid hormoneactivity both by affecting hypothalamic and pituitary controlof thyroid hormone secretion and by interacting with thyroidhormones peripherally. Although there is likely a role for endogenouscorticoids in anuran metamorphosis, the role for endogenousestrogens remains to be shown. The role of these "modulators"must be fully understood, if our understanding of hormonal controlof metamorphosis is to be complete.     

Effects of Methotrexate on Biopterin Levels and Synthesis in Rat Cultured Pineal Glands

Abstract: Culture of rat pineal glands in methotrexate (0.5, 5, or 10 μM) for 6 or 24 h did not alter pineal tetrahydrobiopterin (85–90% of total biopterin in cultured glands), except for a decrease of 30% after 24 h culture in 10 μM methotrexate. However, pineal dihydrobiopterin and/or biopterin (10–15% of total biopterin) was increased by methotrexate up to 2.5-fold. Biopterin detected in the culture medium following pineal culture was also increased to a similar extent after methotrexate treatment and appeared to represent leakage of pineal dihydrobiopterin and/or biopterin. Culture of glands in 5 μM methotrexate did not alter the conversion of [U-¹⁴C]-guanosine to [¹⁴C]biopterin, suggesting that pineal tetrahydrobiopterin synthesis was not altered by methotrexate. Complete inhibition of dihydrofolate reductase activity measured in pineal homogenates was obtained following culture of glands in all concentrations of methotrexate studied. Therefore, dihydrofolate reductase and dihydrobiopterin do not appear to be involved in a major biosynthetic pathway for pineal tetrahydrobiopterin from GTP, although they may have a minor role in tetrahydrobiopterin synthesis.     

Mouse Genital Ridges in Organ Culture: The Effects of Temperature on Maturation and Experimental Induction of Teratocarcinogenesis

Abstract. Testicular teratomas can be induced experimentally by grafting genital ridges from male mouse fetuses to the testes of adults. A high incidence of teratomas occurs in genital ridges grafted to scrotal testes, but not in genital ridges grafted to testes maintained at body temperature. Genital ridges were cultured at 32° C or 37° C prior to grafting to the testes to determine the effect of temperature on the incidence of teratomas. Genital ridges cultured at 32° C for two or three days produced a high incidence of teratomas when grafted to the testes, in contrast to genital ridges cultured at 37° C for two or three days which produced a low incidence. Histologically, genital ridges cultured at 32° C contained disorganized testicular tubules and were retarded in development. Genital ridges continued to develop in vitro at 37° C, but were histologically different from genital ridges maturing in the fetus. Genital ridges cultured at 32° C for 10 to 12 days did not develop teratomas in vitro or after grafting to the testes. Further characterization of temperature effects in vitro may lead to a better understanding of teratocarcinogenesis in vivo.     

Effects of blinding on the ultrastructure of mouse pinealocytes with particular emphasis on the dense-cored vesicles

Summary The mammalian pineal is thought to produce an antigonadotropic principle under conditions of reduced photoperiod, constant darkness or blinding by optic enucleation. A number of previous studies on mammalian pineals have suggested that the dense-cored vesicles present in pinealocytes may represent morphological evidence of secretory activity.In the present study the ultrastructure of pinealocytes was studied in adult Charles River CD-1 mice blinded by optical enucleation. By one month following optic enucleation the mean number of dense-cored vesicles in the cytoplasm of pinealocytes adjacent to pericapillary spaces had significantly decreased by 55% when compared with intact controls, and remained at this low level at two months and six months. A relative increase in the proportion of large agranular vesicles and an increased number of large, irregular vacuoles was observed also in the pinealocytic polar processes of blinded mice. When compared to control mice the pinealocytic Golgi regions appeared to be hypertrophied in blinded mice. The apparent stimulation of pinealocytic organelles coupled with the observed decrease in dense-cored vesicles suggest an increased synthesis and release of secretory product.Supported in part by NIH Grant No. HD 08759