Free amino acids in rat ocular tissues during postnatal development

Amino acid changes in the retina, vitreous, lens, iris-ciliary body and cornea of the rat eye were determined during postnatal growth. The amino acid concentrations of the ocular tissues showed varying profiles at various developmental stages. These results suggest a different timetable for development of each ocular tissue or indicate a synthesis of specific proteins in the postnatal period. Adult amino acid levels appeared to be fully reached on the 30th day after birth at the latest. Quantitatively the greatest changes were observed in taurine concentrations, which increased in all five ocular tissues during maturation. GABA changes paralleled those of taurine in the retina, whereas in the other ocular tissues GABA changes were very low. The greatest decrease in glutamic acid and aspartic acid concentration during postnatal development was in the lens, where these amino acids probably are needed for the synthesis of the lenticular proteins, the alpha-, beta-, and gamma-crystallines.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

L-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled L-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [3H]L-serine and 10 nM of [3H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 degrees C, for different periods up to 30 min. The uptake of [3H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na+, K+ or choline ions. At all postnatal ages studied, [3H]-GABA uptake showed a high activity in the presence of Na+ ions and at 30 degrees C. The values of Km were 90-489 microM in L-serine uptake. However, in the uptake of GABA the values of Km were 80-150 microM. The highest values of Vmax were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Cardiac phosphocreatine deficiency induced by GPA during postnatal development in rat

The effect of chronic administration of -guanidinopropionic acid (GPA) on the protein profiling, energy metabolism and right ventricular (RV) function was studied in the rat heart during the weaning and adolescence period. GPA was given in tap water (1–1.5%) using pair drink controls. The feeding of animals with GPA solution for a six week period resulted in elevation of heart to body weight ratio due to body growth retardation. GPA accumulated in the myocardium up to 67.37 ± 5.3 moles.g dry weight and the tissue content of total creatine, phosphocreatine and ATP was significantly decreased to 15%, 9% and 65% of control values respectively. Total activity of creatine kinase (CK) was not changed, but the proportion of mitochondrial (Mi) CK isoenzyme was decreased; the percentage of MB isoenzyme of CK was significantly higher. GPA treatment resulted in an elevation of the content of cardiac collagenous proteins and decrease of non-collagenous proteins in the heart; in parallel, a decrease of the collagen I to collagen III ratio was detected. The function of the RV was assessed using an isolated perfused heart with RV performing pressure-volume work. As compared to pair-drink controls, RV function was significantly impaired the GPA group: at any given right atrial filling pressure, the RV systolic pressure and the rate of pressure development were decreased by almost a factor of two. Elevation of the RV diastolic pressure with increasing pulmonary artery diastolic pressure was also significantly steeper in the GPA group which also showed decrease of cardiac output, especially at high outflow resistance. It may be assumed that chronic administration of GPA deeply influenced metabolic parameters, protein profiles and contractile function of the developing heart. On the other hand, concentrations of glucose, total lipids and triglycerides in blood plasma were not affected. All these data confirm the concept that the CK system is of central importance both for heart function and for the regulation of normal growth of cardiac myocytes.     

Polysomes during early postnatal development of brain in the rat

We have attempted to show eventual modifications in the brain protein synthesis apparatus of rat during the first three weeks after birth. Through this time we noted a steady decrease (about 60%) in the free polysomes, when expressed relative to tissue weight. This decrease does not correlate with changes in the polysome profile, indicating that no loss in the efficiency of protein synthesis was involved. Translation in a reticulocyte lysate also failed to reveal differences.     

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.     

Insulin-regulated protein kinases during postnatal development of rat heart

The control of glucose uptake and glycogen metabolism by insulin in target organs is in part mediated through the regulation of protein-serine/ threonine kinases. In this study, the expression and phosphotransferase activity levels of some of these kinases in rat heart ventricle were measured to investigate whether they might mediate the shift in the energy dependency of the developing heart from glycogen to fatty acids. Following tail-vein injection of overnight fasted adult rats with 2 U of insulin per kg body weight, protein kinase B (PKB), the 70-kDa ribosomal S6 kinase (S6K), and casein kinase 2 (CK2) were activated (30–600%), whereas the MAP/ extracellular regulated kinases (ERK)1 and ERK2 were not stimulated under these conditions. When the expression levels of the insulin-activated kinases were probed with specific antibodies in ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats, phosphatidylinositol 3-kinase (PI3K), PKB, S6K, and CK2 were downregulated (40–60%) with age. By contrast, ventricular glycogen synthase kinase-3β (GSK3β) protein levels were maintained during postnatal development. Similar findings were obtained when the expression of these kinases was investigated in freshly isolated ventricular myocytes, where they were detected predominantly in the cytosolic fraction of the myocytes. Compared to other adult rat tissues such as brain and liver, the levels of PI3K, PKB, S6K, and GSK3β were relatively low in the heart. Even though CK2 protein and activity levels were reduced by ∼60% in 365 day as compared to 1-day-old rats, expression of CK2 in the adult heart was as high as detected in any of the other rat tissues. The high basal activities of CK2 in early neonatal heart may be associated with the proliferating state of myocytes. J. Cell. Biochem. 71:328–339, 1998. © 1998 Wiley-Liss, Inc.     

In vitro functions of the rat diaphragm during postnatal development.

This study was designed to determine the developmental changes in the functional characteristics of the rat diaphragm. A total of 150 animals were studied at 1, 3, 5, 7 and 9 weeks of postnatal age. Body and diaphragm muscle weights were measured. Diaphragm strips were studied in an in vitro preparation to assess muscle contractile and endurance properties. Total diaphragm weight increased considerably, by a factor of 23 over the 9 week-period of study and was highly correlated with body weight (r = 0.93, P less than 0.01). However, the ratio of diaphragm-to-body weight decreased progressively with age. In comparison with those from older animals, diaphragms from 1 and 3 weeks old animals: (1) generated similar force normalized for muscle weight but a lower force normalized for fibre cross-sectional area (P less than 0.05), (2) had longer time-to-peak tension and one-half relaxation times (P less than 0.01) and (3) were more resistant to fatigue (P less than 0.01). The mechanisms underlying the diaphragm functional development were discussed.     

Morphometric study of the mandibular condyle of the rat during postnatal development

A morphometric technique for the use of the temporomandibular joint (TMJ) of the rat as an experimental model was developed to study TMJ growth from birth to 120 days of age. Experimental conditions for the reduction and embedding of the specimen, as well as for data collection were standardized. Morphometric data were obtained from projected drawings and the areas occupied by the various structures were determined by counting points with a specially constructed integration grid. The areas occupied by the layers making up the mandibular condyle remained relatively constant, forming an architectural pattern from the 30th postnatal day on. Between the 10th and 30th day they underwent modifications interpreted to be functional adaptations conditioned by changes in the animal's feeding habits.     

Distribution of estramustine-binding protein during postnatal development of rat brain

Estramustine-binding protein (EMBP) is expressed in several types of brain tumors, such as astrocytoma, ependymoma, and meningioma. It binds the cytotoxic drug estramustine with high affinity and is suggested to cause accumulation of the drug in EMBP-expressing tumor cells. In this study, the spatial distribution of EMBP in normal rat brain was studied with immunohistochemistry. Brains from male and female rats of different ages were used. EMBP was found in the cytoplasm of ependymal cells, in the leptomeninges, mainly the arachnoid, and in scattered neurons. Moreover, staining was seen in nuclei of choroid plexus cells, in the granular cell layer in the cerebellum, and in a few scattered endothelial cells. The nuclear staining was more frequent in younger animals. No obvious difference in EMBP expression between male and female rats was observed. The expression of EMBP in rat brain was confirmed with nested RT-PCR. Future studies are justified to elucidate the role of EMBP-like proteins in CNS and in brain tumors.     

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development

Changes in alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of alpha-amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.     

RNA synthesis in neuronal and glial cell nuclei from rat cerebral hemispheres during early postnatal development

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [³H]uridine) and in vitro (either by incubation of tissue slices with [³H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg²⁺-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn²⁺-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.     

Volume change of the ocular lens during accommodation

During accommodation, mammalian lenses change shape from a rounder configuration (near focusing) to a flatter one (distance focusing). Thus the lens must have the capacity to change its volume, capsular surface area, or both. Because lens topology is similar to a torus, we developed an approach that allows volume determination from the lens cross-sectional area (CSA). The CSA was obtained from photographs taken perpendicularly to the lenticular anterior-posterior (A-P) axis and computed with software. We calculated the volume of isolated bovine lenses in conditions simulating accommodation by forcing shape changes with a custom-built stretching device in which the ciliary body-zonulae-lens complex (CB-Z-L) was placed. Two measurements were taken (CSA and center of mass) to calculate volume. Mechanically stretching the CB-Z-L increased the equatorial length and decreased the A-P length, CSA, and lens volume. The control parameters were restored when the lenses were stretched and relaxed in an aqueous physiological solution, but not when submerged in oil, a condition with which fluid leaves the lens and does not reenter. This suggests that changes in lens CSA previously observed in humans could have resulted from fluid movement out of the lens. Thus accommodation may involve changes not only in capsular surface but also in volume. Furthermore, we calculated theoretical volume changes during accommodation in models of human lenses using published structural parameters. In conclusion, we suggest that impediments to fluid flow between the aquaporin-rich lens fibers and the lens surface could contribute to the aging-related loss of accommodative power. lens volume calculation; intralenticular fluid movement; presbyopia; mammalian lens