An immunocytochemical study of thyrotropin releasing hormone in the porcine, ovine and rodent pineal gland

A biotin-avidin immunoperoxidase method was used to localize a pineal TRH-like compound in histological sections. TRH-like immunoreactivity was observed in porcine, ovine and rodent pineal glands. The immunoreaction was located on positively stained cell bodies and cellular processes throughout the glands. The exact location of the TRH (in pinealocytes, glial cells, or both) as well as the physiologic significance of the immunoreactive material remain to be elucidated.     

ROLE OF ADENOSINE 3'', 5''-MONOPHOSPHATE IN THE REGULATION OF CIRCADIAN OSCILLATION OF SEROTONIN N-ACETYLTRANSFERASE ACTIVITY IN CULTURED CHICKEN PINEAL GLAND

—When pineal glands of 10–12-day-old chicks were organ-cultured in darkness, serotonin N-acetyltransferase activity was low during the daytime, increased at midnight and then decreased to the daytime level the next morning. The pattern of increase and decrease of enzyme activity in cultured pineal glands was comparable to the circadian rhythm of N-acetyltransferase activity in vivo. When pineal glands were kept at a low temperature for 5 h prior to culture, the phase of autonomous rhythm of enzyme activity was delayed. When chicken pineal glands were cultured during the daytime for 6 h, derivatives of adenosine 3′, 5′-monophosphate (cyclic AMP), cholera toxin, a high concentration of KCl and phosphodiesterase inhibitors increased N-acetyltransferase activity 3–7-fold, indicating an involvement of cyclic AMP in the regulation of N-acetyltransferase activity in chicken pineal gland as has been shown in rat pineal gland. When pineal glands were cultured at night in darkness, cholera toxin or a high KCl did not enhance the night-time increase of the enzyme activity. Derivatives of cyclic AMP or phosphodiesterase inhibitors enhanced the autonomous night-time increase of N-acetyltransferase activity in an additive or more than additive manner in cultured pineal glands. These observations suggest that adenylate cyclase of pinealocytes is inactive during daytime, but is activated at night in darkness, which is transduced to the synthesis of N-acetyltransferase molecules. Catecholamines suppressed the basal level and the nocturnal increase of N-acetyltransferase activity via α-adrenergic receptor. The nocturnal increase of enzyme activity was prevented by cycloheximide or actinomycin D. Cocaine, which stabilizes cell membrane potential or light exposure, blocked the nighttime increase of N-acetyltransferase activity in cultured chicken pineal glands.     

Identification of the TRH-like peptides pGlu-Glu-Pro amide and pGlu-Phe-Pro amide in rat thyroid: regulation by thyroid status.

Rat thyroid contains thyrotropin-releasing hormone (TRH) and TRH-like peptides which react with TRH antisera. We have identified the TRH-like peptides in the thyroid and examined whether their levels are influenced by thyroid status. The peptides were extracted from the thyroid glands of five hyperthyroid rats and purified by ion-exchange chromatography on SP-Sephadex C25 and reversed-phase high performance liquid chromatography. The principal TRH-immunoreactive component exhibited the same retention on HPLC as synthetic pGlu-Glu-Pro amide and a secondary component corresponded to synthetic pGlu-Phe-Pro amide. In agreement with these assignments the main peptide was shown to be acidic when chromatographed on DEAE-Sephadex A25 and the second peptide neutral. The levels of TRH and TRH-like peptides in the thyroid were investigated in hyper-, hypo- and euthyroid rats. Hyperthyroidism was induced by chronic subcutaneous administration of triiodothyronine (T3) and hypothyroidism was produced by addition of propylthiouracil (PTU) to the drinking water. The amounts of the peptides were determined by radioimmunoassay with a TRH-antiserum, carried out after extraction from the tissues and purification by ion exchange chromatography. The mean concentration of TRH-like peptides in the thyroids of the hyperthyroid rats was 95.5+/-25.5 pmol/g, the mean concentration in the hypothyroid rats was 11.7+/-3.4 pmol/g, and in the euthyroid rats 17.6+/-3.2 pmol/g. The concentrations of TRH were less influenced by thyroid status: the values in hyper-, hypo- and euthyroid rats were 47.5+/-9.4, 42.1+/-6.3, and 17.2+/-1.6 pmol/g respectively. The results show that the levels of the TRH-like peptides in rat thyroid are highly sensitive to thyroid status, suggesting a possible involvement in thyroid regulation.     

Biosynthesis of isorenin in cell cultures from pineal glands of adult and newborn rats

Renin-like activity (RA) was detected by bioassay and radioimmunoassay in culture media of pineal glands of male rats. The monolayer tissue culture of pineal cells from adult and newborn male white rats produced in 30 days, an amount of RA that was 20-fold greater than the RA in nonincubated pineal glands from rats of the same age. The cultured intact pineal glands from Long Evans and Brattleboro adult male rats produced in 7 days an amount of RA (radioimmunoassay) that was about 3 times greater than that found in nonincubated pineal glands from the same rat. These results suggest that cultured cells of rat pineal glands synthesize renin-like enzymes.     

S-antigen and glial fibrillary acidic protein immunoreactivity in the in situ pineal gland of hamster and gerbil and in pineal grafts: developmental expression of pinealocyte and glial markers.

Postnatal development of S-Ag and GFAP immunoreactivity in the in situ pineal glands of golden hamsters and gerbils was examined using the avidin-biotin-peroxidase immunohistochemical technique. S-Ag was present in the gerbil pineal gland on the first postnatal day (P1), whereas it did not appear in the hamster pineal until P6. GFAP-immunoreactive astrocytes were first observed in the hamster pineal gland on P7 and in the gerbil pineal gland on P10. The number of S-Ag-immunoreactive pinealocytes and GFAP-immunoreactive astrocytes in the pineal glands of hamsters and gerbils increased with increasing age from P7 to 3 weeks. By 4 weeks, strong S-Ag and GFAP immunoreactivity was observed in both hamster and gerbil pineal glands. GFAP-immunoreactive stellate astrocytes were distributed evenly throughout the gerbil superficial pineal gland, but they were more often located in the peripheral region of the hamster superficial pineal. For the pineal grafts, pineal glands from neonatal (3-5 day old) hamsters were transplanted into the third cerebral ventricle (infundibular recess or posterior third ventricle) or beneath the renal capsule of adult male hamsters. S-Ag immunoreactivity appeared in the pineal grafts within 1 week following transplantation. By 4 weeks the pineal grafts showed strong S-Ag immunoreactivity which was maintained until at least 12 weeks after transplantation. The time course of glial cell maturation in the cerebroventricular pineal grafts is generally parallel to the hamster pineal gland in situ before 4 weeks. By 12 weeks, however, more astrocytes differentiated and developed GFAP-immunoreactivity in the pineal grafts than in the in situ pineals. These studies have described the postnatal development of S-Ag and GFAP immunoreactivity in in situ pineal glands and in neonatal pineal grafts.     

TRH-like peptides

TRH-like peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) with neutral or acidic amino acid, like glutamic acid, phenylalanine, glutamine, tyrosine, leucin, valin, aspartic acid and asparagine. The presence of extrahypothalamic TRH-like peptides was reported in peripheral tissues including gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. Work deals with the biological function of TRH-like peptides in different parts of organisms where various mechanisms may serve for realisation of biological function of TRH-like peptides as negative feedback to the pituitary exerted by the TRH-like peptides, the role of pEEPam such as fertilization-promoting peptide, the mechanism influencing the proliferative ability of prostatic tissues, the neuroprotective and antidepressant function of TRH-like peptides in brain and the regulation of thyroid status by TRH-like peptides.     

Tyrosine hydroxylase and neuropeptide-Y immunoreactivity in pineal glands developing in situ and in pineal grafts

Summary Postnatal development of the innervation of the pineal gland in situ as well as the reinnervation of pineal grafts by tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined using the avidin-biotin-peroxidase immunohistochemical technique. TH-immunoreactive nerve fibers appeared in the pineal gland on the second postnatal day (P2) in both hamsters and gerbils. NPY-immunoreactive nerve fibers first appeared in the pineal gland of gerbils on P2 and in the hamsters on P3. By the seventh postnatal day (P7), the pineal glands of both hamsters and gerbils were richly innervated by TH- and NPY-fibers that appeared as smooth fibers or fibers with sporadic varicosities. By the age of 4 weeks, the innervation of the pineal glands of hamsters and gerbils by TH-and NPY-fibers was fully developed. Abundant TH- and NPY-fibers formed a dense meshwork in the parenchyma of the superficial and deep pineals. The great majority of the fibers bore a large number of varicosities. More NPY-fibers were found in the pineal glands of gerbils than hamsters. NPY fibers were distributed evenly throughout the pineal glands of the gerbil, but they were more often located in the central region of the superficial pineal of the hamster. For the pineal grafts, superficial pineals from neonatal and 4-week-old hamsters were transplanted to different sites in the third cerebral ventricle (infundibular recess, posterior third ventricle) or beneath the renal capsule. The pineal grafts from 4-week-old donors appeared to undergo severe degeneration and eventually disappeared. The pineal grafts from neonatal hamsters, however, successfully survived and became well integrated into their new locations. Abundant TH-and NPY-fibers in the host brain were found surrounding the pineal grafts placed in the third cerebral ventricle, but were only rarely seen entering the parenchyma of the grafts. A few TH-fibers were demonstrated in the renal grafts 4 weeks after transplantation. These studies describe the postnatal development of the innervation of the pineal glands in situ by TH-and NPY-nerve fibers, and demonstrate a lack of reinnervation of cerebroventricular pineal grafts by TH and NPY fibers from adjacent host brain.Portions of the results of this paper were previously reported in abstract form at the 1990 Meeting of The American Association of Anatomists (Anat Rec 226:57A)     

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

Summary 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.     

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.     

Postnatal appearance of luteinizing hormone-releasing hormone (LHRH)-like material in the pineal gland of the rat

Summary Immunoreactive luteinizing hormone-releasing hormone (LHRH)-like material has been demonstrated in the pineal gland of the adult rat. The objective of the present study was to examine the ontogenetic development of this LHRH-like substance in the rat pineal with the peroxidase-antiperoxidase (PAP) method of Sternberger. LHRH-like immunoreactive material was not observed in pineal glands of newborn rats. The amount of material increased progressively from the 6th–12th day of postnatal development. On day 12, the amount of LHRH-like immunoreactivity was consistent and comparable in all pineal glands of male and female animals examined.Supported by NIH Grant 1 R01 HD-12956     

Determination of rat pineal gland melatonin content by a radioimmunoassay.

Melatonin content in individual rat pineal glands was measured by radioimmunoassay (RIA). The RIA used can very reliably detect as little as 50 pg of melatonin. The various precursors, analogues, and the metabolite of melatonin (6-hydroxymelatonin) which were tested for cross-reactivity were not recognized by the antibody. The effects on melatonin levels in rat pineal glands following the administration of L-tryptophan, 5-hydroxy-L-tryptophan, serotonin, N-acetylserotonin, melatonin and pargyline are also presented.     

Chronic exposure to 60-Hz electric fields: Effects on pineal function in the rat

As a component of studies to search for effects of 60-Hz electric field exposure on mammalian endocrine function, concentrations of melatonin, 5-methoxytryptophol, and serotonin-Nacetyl transferase activity were measured in the pineal glands of rats exposed or sham-exposed at 65 kV/m for 30 days. In two replicate experiments there were statistically significant differences between exposed and control rats in that the normal nocturnal increase in pineal melatonin content was depressed in the exposed animals. Concentrations of 5-methoxytryptophol were increased in the pineal glands of the exposed groups when compared to shamexposed controls. An alteration was also observed in serotonin-N-acetyl transferase activity, with lower levels measured in pineal glands from exposed animals.     

Pineal glands of immature rats: rise and fall in N-acetyltransferase activity in vitro

N-acetyltransferase activity in the pineal glands of immature rats (12--14 days old) shows a spontaneous rise and fall when the glands are placed in organ culture. The peak of the 28-fold change occurs 5--16 hr after the cultures are initiated. This observation can be interpreted in two ways: (1) the pup pineal gland is responding to norepinephrine released when nerve endings degenerate in culture, or (2) the pup pineal gland has an innate ability which is responsible. Whatever the mechanism, the phenomenon is associated with the development of pineal gland function since pineal glands of adult rats do not show a spontaneous rise and fall in identical experiments.     

Photoperiod affects amplitude but not duration of in vitro melatonin production in the ruin lizard (Podarcis sicula)

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.     

Development of rhythmic melatonin secretion from the pineal gland of embryonic mummichog (Fundulus heteroclitus)

The pineal gland of vertebrates produces and secretes the hormone melatonin in response to changes in the light-dark cycle, with high production at night and low production during the day. Melatonin is thought to play an important role in synchronizing daily and/or seasonal physiological, behavioral, and developmental rhythms in vertebrates. In this study, the functional development of the pineal melatonin-generating system was examined in the mummichog, Fundulus heteroclitus, an euryhaline teleost. In this species, the pineal gland contains an endogenous oscillator, ultimately responsible for timing the melatonin rhythm. Oocytes from gravid females were collected and fertilized in vitro from sperm collected from mature males. Skull caps containing attached pineal glands were obtained from F. heteroclitus embryos at different embryonic stages and placed in static or perfusion culture under various photoperiodic regimes. Rhythmic melatonin secretion from pineal glands of embryonic F. heteroclitus embryos exposed to a 12L:12D cycle in static culture was observed at five days post-fertilization. The ontogeny of circadian-controlled melatonin production from F. heteroclitus pineal glands exposed to constant darkness for five days was also seen at day five post-fertilization. These data show that early development of the pineal melatonin-generating system in this teleost occurs prior to hatching. Pre-hatching development of the melatonin-generating system may confer some selective advantage in this species in its interactions with the environment.     

Cultivation of mammalian pineal cells: retention of organization and function in tissue culture

By means of a newly developed method of cultivating pineal tissue in vitro, the types of cells which comprise rat pineal glands have been identified. Previous in vitro studies have involved short-term culture more suitably called "organ culture" and provide no means of assessing the contribution of a putative "pineal" cell versus any other cell type found in the cultures. Short-term outgrowths of minced rat pineal glands provided a reproducible and easily dissociated source of pineal-derived cells. In monolayer culture these cells continued to have pineal enzyme activities which were sensitive to pineal-activating substances, and the cells aggregated to mimic the lobular organization of intact glands. Two types of aggregates were found, each composed of a single morphological cell type. In addition to the transient appearance of skeletal muscle straps, connective tissue and neural/glial tissue was consistently found. The cell types are discussed in relation to their in vivo counterparts.     

Distribution of growth associated protein (GAP-43) immunoreactivity in nerve fibers supplying the pig pineal gland

An immunohistochemical study of the pig pineal gland was carried out using monoclonal mouse antiserum against growth-associated protein GAP-43. The pineal glands were obtained from the 3, 5, 8 weeks old piglets. The immunopositive nerve fibers were observed in the pineal gland as well as in the habenular and the posterior comissural areas. They formed a dense network in the habenular area and the proximal part of the pineal gland. In the comissural area and in the apical part of the gland. single positive fibers were observed. The obtained results may suggest a difference in the plasticity of innervation between the particular regions of the pineal gland.     

Ultrastructural observations at pineal gland capillaries in four rodent species.

The fine structure of the capillaries of the pineal glands of the rat, mouse, chinchilla, and ground squirrel were investigated. The pineal endothelial cells in the rat, mouse and ground squirrel were often composed of attenuated cytoplasmic portions which contained numerous fenestrations, in contrast to pineal capillaries in the chinchilla which were lined by thick non-fenestrated endothelial cells. Marked morphological differences were also apparent in terms of the types of vesicles within the cytoplasm and abutting on the cell surface of pineal endothelial cells from the various species investigated. The interendothelial junctions exhibited remarkable species differences with the chinchilla pineal possessing typical tight endothelial junctions while those in the rat, mouse and ground squirrel lacked such endothelial cell associations. Generally, capillary lining cells in the chinchilla pineal resembled similar cells within the brain, while endothelial cells in pineal glands of rat, mouse and ground squirrel were more typical of those found in other endocrine organs. Species differences in the structure of the pineal capillaries may represent physiological differences as well.     

The in vitro activation of cyclic AMP production by either forskolin or isoproterenol in the Syrian hamster pineal during the day is not accompanied by an increase in melatonin production

The role of cyclic AMP in the regulation of melatonin production was investigated in cultured Syrian hamster pineal glands. Forskolin markedly increased cyclic AMP production in pineal glands collected either late in the light period or in the dark period. The effect of forskolin was synergistically enhanced by 3-isobutylmethylxanthine, a phosphodiesterase inhibitor; however, increase in cyclic AMP after isoproterenol was only apparent in the presence of 3-isobutylmethylxanthine. Since beta-adrenergic agonists are able to stimulate melatonin production late in the dark period only, these data suggest that, in the hamster pineal gland, there may be intracellular mechanisms in addition to a cyclic AMP increase required for induction of melatonin production by beta-adrenergic agonists.