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

Summary 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 counter-parts. Supported by NSF Grant GB-43215 to S.B. and NSF Grant GB-20919 to S.R.H.     

Phenotypic expression of photoreceptor and endocrine cell properties by cultured pineal cells of the newborn rat

Pineal glands of newborn rats were dissociated and maintained under cell culture conditions. The phenotypic expression of both photoreceptor and endocrine cell properties was investigated using immunohistochemical techniques (specific antibodies against opsin or serotonin). After one week in culture, a number of small round cells appeared on top of a sheet of flat epithelium. Among those cells, opsin-like immunoreactive cells were observed. These cells showed a neuron-like morphology with neuritic processes and often formed rosettes. Immunoreactivity was found on the plasma membrane of both the soma and cell processes. Serotonin-like immunoreactive cells were also differentiated in culture with two different morphological types of cells being found. One type resembled cultured serotonin-containing amacrine cells of the retina, and the other type had a flat, polygonal shape similar to that of pinealocytes. Both types of immunoreactive cells possessed fine neuritic processes. These results indicated that cell culture of rat pineal gland cells allowed expression of some properties, such as opsin synthesis and neuron-like morphology with long neuritic processes, that were not expressed in the intact rat 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.     

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.     

Morphometric analysis of photoreceptive,neuronal and endocrinal cell differentiation of avian pineal cells: an in vitro immunohistochemical study on the developmental transition from neuronal to photo-endocrinal property

Little is known about the developmental origin, determination and differentiation of different pineal immunoreactive cells in the avian group, and an experimental establishment is then required to explain the differentiation of cell types (i.e. photosensory, neural and secretory types). The present in vitro study suggests that the avian pineal organ is made up of multiple types of cells with different immunoreactivity at the ontogenic state (from embryonic day 9 to day 14), before it acquires the final photoendocrinal nature of the mature state. The morphometric analysis suggests that the developmental changes in the morphology of the quail pinealocytes appear to represent a condensed expression of the phylogenic development in the ontogeny. Several types of immunoreactive cells from a neuronal line were suppressed with maturation of developing pineal glands, while other cell types such as photoreceptive and endocrinal lines became more prominent. The melatonin level in the culture medium presented a high value up to 72 hr of culture, followed by a decrease as well as dampening of the level at the end of the culture possibly because the cultures were maintained in dark. The results of the present study, a combined analysis of morphometry and RIA, open a new line for research into the pineal development and cell differentiation.     

Meningeal calcification of the rat pineal organ

Summary The surface of the pineal organ of the rat is covered by a leptomeningeal tissue, the continuation of the corresponding meningeal layers of the diencephalon. The pineal leptomeninx consists of stratified arachnoid and of pia mater cells which follow the vessels into the pineal nervous tissue. The pineal arachnoid contains electron-lucent and electron dense cells differing from each other in their cytoplasmic components. Corpora arenacea of various size and density occur among these arachnoid cells and can grow into the pineal organ alongside pia mater tissue. Acervuli often form groups in circumscribed meningeal calcification foci. Concrements are absent or rare in the 1- and 2-month-old animal, while they are usually present in the 4- and 6-month-old rats.The electronmicroscopic localization of Ca-ions was studied in 2- and 4-month-old rats by potassium pyroantimonate cytochemistry. In the 4-month-old animals, arachnoid cells containing a varying amount of Ca-pyroantimonate deposits were found first of all around corpora arenacea, but there were also cells free of deposits in the close vicinity of the acervuli. Deposits were preferentially localized to the cytoplasm of electron dense arachnoid cells and to the cell membrane of electron-lucent cells. Most of the precipitates occurred in locally enlarged intercellular spaces. Here, microacervuli were found in 4-month-old animals suggesting that a calcium-rich environment was responsible for the appearance of the concrements. Intermediate stages between the small acervuli and large concentric corpora arenacea may indicate an appositional growth of the acervuli in the calcification foci. Occasionally, acervuli were also located inside meningeal cells.There was no sign of the formation of acervuli in the pinealocytes or elsewhere in the pineal nervous tissue proper, in the age interval (1- to 6-month-old animals) studied. These findings confirm the view that corpora arenacea can be produced in the rat by the pineal leptomeninx. The laboratory rat seems to be usefull in studying pineal calcification of the meningeal type.Supported by the Hungarian OTKA grant Nr. 1619 to B.V.     

Bone formation by rat calvarial cells grown at high density in organoid culture

Calvarial cells from day 21 rat fetuses were isolated by enzymatic digestion and grown at high density in an organoid culture system at the medium/air interface. In this type of culture, mineralization occurred as early as 7 days in vitro, as revealed by light and electron microscopic means. After about 18 days in vitro, most of the culture consisted of mineralized tissue. Mineralization was also achieved without β-glycerophosphate, but it was delayed by 2 to 3 days. Maximal alkaline phosphatase activity occurred at days 8 to 12 in vitro and then declined continuously during further cultivation. Two types of mineralization could be observed: (1) mineralization of a collagen-rich osteoid by typical apatite crystals; (2) mineralization of a nearly collagen-free matrix by amorphous material which was possibly secreted by the cells. The importance of higher cell densities for cell differentiation and formation of histotypic tissue in vitro is apparent, and it is indicated that cell-cell contacts and cell-matrix interactions may be prerequisites for the development of histotypic conditions similar to the in vivo situation.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis

Two S-antigen-specific rat T cell lines expressing the T helper cell surface phenotype (W 3/25+, OX 8-) have been isolated from the spleen and lymph node cells of retinal S-antigen-immunized Lewis rats, one of which displayed neither clinical nor histopathologic signs of experimental autoimmune uveoretinitis. The other rat had recovered from severe experimental autoimmune uveoretinitis for 2 mo before isolation of the cell line. Both lines are specific for S-antigen presented by histocompatible antigen-presenting cells, and also respond in vitro to several of the peptides produced by cyanogen bromide cleavage of bovine retinal S-antigen. The lesions induced by the i.v. transfer of from 1 to 10 X 10(6) viable line cells involve the retina and pineal gland, as is found when Lewis rats are immunized with immunopathogenic doses of S-antigen. Histologic examination of the eyes and pineal glands revealed pathologic lesions typical of experimental autoimmune uveoretinitis, and consisted of marked infiltration of the retina and surrounding tissues and the pineal gland by lymphocytes and inflammatory cells. T cells capable of mediating autoimmune disease are clearly present and readily isolated from both asymptomatic and convalescent animals. No significant differences in specificity for the cyanogen bromide peptides of S-antigen or cell surface phenotype were found in the T cell lines isolated from these two rats, nor was any difference found in the specificity or titer of serum antibodies taken from the original rats for the cyanogen bromide peptides of S-antigen.     

RADIOMETRIC ASSAY OF TOTAL TRYPTOPHAN HYDROXYLATION BY INTACT CULTURED PINEAL GLANDS

Abstract— A new method is described for the analysis of total tryptophan hydroxylation by intact tissue in culture. This method differs from classical measurements of tryptophan hydroxylase because it allows the detection of changes in tryptophan hydroxylation due to alterations in tryptophan transport, cofactor regeneration and protein synthesis. The rat pineal gland was studied. It was found to hydroxylate tryptophan linearly for a 48-h period. A direct proportional relationship between the concentration of tryptophan in the culture medium and the amount of tryptophan hydroxylated persists between 0.05 and 0.5 m m . As the medium concn of tryptophan is increased above 0.5 m m the amount of tryptophan hydroxylated decreases. Tryptophan hydroxylation is substantially inhibited by 1 m m p-chlorophenylalanine and 0.03 m m cycloheximide. No difference in the total amount of tryptophan hydroxylated was detected between male and female rat pineal glands, between chronically denervated and normal rat pineal glands, or between control glands and glands treated with 0.1-0.001 m m norepinephrine, 0.01 m m isoproterenol, or 0.01 m m mescaline.     

Muscle Differentiation in Cultured Pineal Body Cells of Neonatal Rats

Dissociated cells of pineal bodies of new-born rats were cultured to see what cell types would be differentiated during culture in vitro for about 4 weeks. In early stages of culture, about 10 days after inoculation, flattened cells with piliform processes, small round cells and small bubbling cells were distinguishable in the cultures. After about 2 weeks, neuronal cells with axon-like processes and multinuclear muscle-like cells were differentiated. On further culture, the latter cells differentiated into mature striated myotubes. The developmental origin of myotube formed in cell cultures of pineal body is discussed.     

Primary cultures of dispersed cells of rat pineal gland

Summary In vitro indole metabolism and ultrastructural morphology of the pineal gland of male rats were examined. A comparison of the effect of norepinephrine stimulation on indole synthesis in whole cultured glands and preparations of dispersed pineal cells is discussed. Our studies on the performance of dispersed cells during the first 24 h after preparation indicate a strong dependence of pineal cells upon physical attachment to the culture dish and probably also on cell-to-cell contact.     

Differentiation of pinopsin-immunoreactive cells in the developing quail pineal organ: an in-vivo and in-vitro immunohistochemical study

The avian pineal organ contains several types of photoreceptors with different photopigments: rhodopsin, iodopsin, and pinopsin. We have previously examined the differentiation of both rhodopsin-like and iodopsin-like immunoreactive cells during pineal development in quail embryos to determine the onset of synthesis of specific proteins and their cellular localization. In the present study, we have performed pinopsin immunohistochemistry on in-vivo developing and in-vitro cultured pineal organs of quail embryos. The results were compared with those obtained with rhodopsin and iodopsin immunohistochemistry. In the developing pineal organs, pinopsin immunoreactivity was detected at embryonic day 8, i.e. five days earlier than rhodopsin-like and iodopsin-like immunoreactivities. It was localized exclusively in the protrusions extending into the lumen throughout development, whereas rhodopsin-like and iodopsin-like immunoreactivities were usually found both in cell bodies and processes. These differences were also observed under two different types of culture conditions (dissociated cell culture and organ culture) indicating that, in the avian pineal organ, the expression pattern of the pinopsin gene is basically different from those of the other two pineal photopigments. The present study suggests that pineal cells have a mechanism for the polarized transport of pinopsin molecules.     

Growth of neonatal rat uterine luminal epithelium on extracellular matrix

Summary We have developed a tissue culture system using an extract of basement membrane (extracellular matrix) which promotes the in vitro growth and development of uterine luminal epithelium from the 5-day-old rat. Uterine luminal epithelium, free of stroma, was obtained as short tubes by trypsinization of uterine segments followed by mechanical separation. Epithelial segments were grown in a serum-free medium on culture dishes coated with an extracellular matrix. After 2 days, rapid cell growth resulted in monolayer cultures, which subsequently formed organoid structures similar to differentiated uterine glands present in uterine tissue taken from older rats. Electron microscopy of cultures revealed columnar cells with basally located nuclei, apical microvilli, lateral membranes with interdigitations, desmosomes, and secretory Golgi complexes, all features found in functioning uterine epithelium in vivo. This model will allow the in vitro investigation of the development of uterine epithelium-specific functions free of the influence of stromal cell factors.     

Enzymatic and cellular study of a serotonin N-acetyltransferase phosphopantetheine-based prodrug

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) regulates the daily rhythm in the production of melatonin and is therefore an attractive target for pharmacologic modulation of the synthesis of this hormone. Previously prepared bisubstrate analogs show potent inhibition of AANAT but have unfavorable pharmacokinetic properties due to the presence of phosphate groups which prevents transfer across the plasma membrane. Here, we examine a bis-pivaloyloxymethylene (POM)-tryptamine-phosphopantetheine prodrug (2) and its biotransformations in vitro by homogenates and pineal cells. Compound 2 is an efficient porcine liver esterase substrate for POM cleavage in vitro although cyclization of the phosphate moiety is a potential side product. Tryptamine phosphopantetheine (3) is converted to tryptamine-coenzyme A (CoA) bisubstrate analog (1) by human phosphoribosyl pyrophosphate amidotransferase (PPAT) and dephosphocoenzyme A kinase (DPCK) in vitro. Compound 2 was found to inhibit melatonin production in rat pineal cell culture. It was also found that the POM groups are readily removed to generate 3; however, further processing to tryptamine-CoA (1) is much slower in pineal extracts or cell culture. Implications for CoA prodrug development based on the strategy used here are discussed.     

Tissue distribution and developmental expression of protein kinase C isozymes

Protein kinase C is a ubiquitous enzyme found in a variety of mammalian tissues and is especially highly enriched in brain and lymphoid organs. Based on biochemical and immunological analyses, we have identified three types of protein kinase C isozyme (designated types I-III) from rat brain. Monospecific antibodies against each of the protein kinase C isozymes were prepared for the determination of tissue distribution, subcellular localization, and developmental changes of these enzymes. The various protein kinase C isozymes were found to be distinctively distributed in different tissues: the type I enzyme in brain; the type II enzyme in brain, pituitary and pineal glands, spleen, thymus, retina, lung, and intestine; and the type III enzyme in brain, pineal gland, retina, and spleen. The rat brain enzymes were differentially distributed in different subcellular fractions. The type I enzyme appeared to be most lipophilic and was recovered mostly in the particulate fractions (80-90%) regardless of the EGTA- or Ca2+-containing buffer used in the homogenization. Significant amounts (30-40%) of the type II and III enzymes were recovered in the cytosolic fraction with EGTA-containing buffer. The expressions of different protein kinase C isozymes appear to be differently controlled during development. In rat brain, both type II and III enzymes were found to increase progressively from 3 days before birth up to 2-3 weeks of age and remained constant thereafter. However, the expression of the type I enzyme displayed a different developmental pattern; it was very low within 1 week, and an abrupt increase was observed between 2 and 3 weeks of age. In thymus, the type II enzyme was found to be maximal shortly after birth; whereas the same kinase in spleen was very low within 2 weeks of age, and a significant increase was observed between 2 and 3 weeks. These results demonstrate that protein kinase C isozymes are distinctively distributed in different tissues and subcellular locales and that their expressions are controlled differently during development.     

Neuroendocrine integrative mechanisms in mammalian pineal gland: effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.     

In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects?

In vitro cell culture experiments have lead to the consensus in the literature that certain PUFAs have a selective cytotoxic or anti-proliferative effect on tumour cells and a minimal, or no effect on normal cells. Re-examination of key publications showed that when normal cells were used for comparison, they were generally not from the same cell, tissue, or species type as the tumour cells. Recently, investigations have included more appropriate normal control cells, and though tumour specific cytotoxic/anti-proliferative PUFA effects are found in some cell types, in others the normal cells are more sensitive. Cell type differences were found in the relative ability of individual PUFAs to act. However, within a cell type differences in susceptibility were influenced by grade and stage of tumour, immortalisation and tumourigenic status, cell culture media and cell plating density. Together these results suggest that the consensus is not valid, and that susceptibility to PUFA is cell type specific, and alters during neoplastic progression. Furthermore, the cytotoxic/anti-proliferative effect induced by both n-3 and n-6 PUFAs on a wide variety of cell types, associated with an increase in lipid peroxidation in vitro, cannot account for the in vivo data on the relationship between dietary fat and certain cancers. However, the effects of PUFAs and their metabolites on cell signalling pathways may explain the in vivo data.     

Perfusion culture of fetal human hepatocytes in microfluidic environments

Various types of bioreactors composed of microstructured PDMS (Polydimethylsiloxane) layers have recently been fabricated for perfusion culture of mammalian cells such as adult rat hepatocytes. As a new feature of those bioreactors, in this study, cultivation of fetal human hepatocytes (FHHs) was attempted, because they have high possibility to mature in vitro with preserving their normality, which is suitable for inplantation of liver tissue equivalents reconstituted in vitro. During the perfusion culture in the PDMS bioreactors for 1 week, cells showed good attachment, spreading and reached their confluence over the channels. In addition, their albumin production was significantly enhanced in the perfusion culture using the PDMS bioreactors up to about four times during the FHH perfusion culture when compared in dish-level static culture. Hep G2 cell cultures were also performed and have also shown under perfusion conditions an enhanced cell activity multiplied by 2 compared to static conditions. Although, the cellular activities of FHH cells are still low even compared to those of the Hep G2 cells, the conclusions of this work is encouraging toward future liver tissue engineering based on in vitro propagation and maturation of hepatocyte progenitors combined with microfabrication technologies.     

Meningeal calcification of the rat pineal organ. Finestructural localization of calcium-ions

The surface of the pineal organ of the rat is covered by a leptomeningeal tissue, the continuation of the corresponding meningeal layers of the diencephalon. The pineal leptomeninx consists of stratified arachnoid and of pia mater cells which follow the vessels into the pineal nervous tissue. The pineal arachnoid contains electron-lucent and electron dense cells differing from each other in their cytoplasmic components. Corpora arenacea of various size and density occur among these arachnoid cells and can grow into the pineal organ alongside pia mater tissue. Acervuli often form groups in circumscribed meningeal "calcification foci". Concrements are absent or rare in the 1- and 2-month-old animal, while they are usually present in the 4- and 6-month-old rats. The electronmicroscopic localization of Ca-ions was studied in 2- and 4-month-old rats by potassium pyroantimonate cytochemistry. In the 4-month-old animals, arachnoid cells containing a varying amount of Ca-pyroantimonate deposits were found first of all around corpora arenacea, but there were also cells free of deposits in the close vicinity of the acervuli. Deposits were preferentially localized to the cytoplasm of electron dense arachnoid cells and to the cell membrane of electron-lucent cells. Most of the precipitates occurred in locally enlarged intercellular spaces. Here, microacervuli were found in 4-month-old animals suggesting that a calcium-rich environment was responsible for the appearance of the concrements. Intermediate stages between the small acervuli and large concentric corpora arenacea may indicate an appositional growth of the acervuli in the calcification foci.(ABSTRACT TRUNCATED AT 250 WORDS)