Ontogeny of mitochondrial calcium transport in spontaneously hypertensive (SHR) and WKY rats

The current studies were designed to investigate calcium uptake by intestinal jejunal sacs as well as in intestinal mitochondria of spontaneously hypertensive rats and their genetically matched WKY control rats. Kinetics of jejunal calcium uptake by jejunal sacs of adult SHR and WKY rats showed a significant decrease in Vmax of calcium uptake in SHR (227 +/- 24 versus 423 +/- 22 nmol.g tissue-1.3 min-1) compared to WKY rats P less than 0.001. To explore the intracellular handling of calcium by the intestinal mitochondria, calcium uptake was characterized by intestinal mitochondria before (suckling and weanling periods) and after (adult period) development of hypertension. Calcium uptake by intestinal mitochondria was driven by ATP in the presence of succinate as a respiratory substrate. Calcium uptake was stimulated several fold by the presence of ATP compared to no ATP conditions. Maximal calcium uptake occurred between 15-30 min and was significantly greater in adult SHR and WKY rats compared to corresponding values in weanling and suckling rats. Maximal ATP dependent calcium uptake in adult, weanling and suckling WKY rats was significantly greater compared to corresponding mean values in each age group in SHR (P less than 0.001). Oligomycin (10 micrograms/mg protein) inhibited calcium uptake partially. Ruthenium red (0.25 microM), 1 mM sodium azide and 0.5 mM dinitrophenol inhibited calcium uptake by more than 80% in both SHR and WKY rats. Kinetic parameters for ATP stimulated calcium uptake at 10 s revealed a Vmax of 0.56 +/- 0.6, 3.46 +/- 0.23 and 3.95 +/- 0.52 nmol/mg protein/10 s in suckling, weanling and adult WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo study of the appearance and fluctuations of insulin binding sites in different tissues during rat development

The appearance and fluctuations of specific insulin binding sites in several tissues in vivo during rat development, have been determined. After intravenous administration of 125I-insulin to fetal, suckling and adult rats, changes on specific hormone uptake were observed depending on the tissues tested and on the age of animals. Thus, in liver, specific insulin uptake was much greater in 19 day-old fetuses and 10 day-old suckling animals than in adult rats. By contrast, brown fat and spleen insulin uptake was undetected in fetal animals but present in suckling rats, while lung insulin uptake was absent in the adults but present in fetal and suckling animals. Of interest were the specific insulin uptakes by three different muscle tissues. In fact, heart insulin uptake was much higher in younger animals than in adult rats, while in the diaphragm it was significantly smaller in all groups and in skeletal muscles hormone uptake was much smaller than in the other two muscle tissues and was even absent in the fetuses. In those tissues that had previously been shown to exhibit a specific insulin uptake, the iodinated hormone uptake decreased proportionally with simultaneous injection of increasing amounts of unlabelled insulin. These results indicate that insulin binding sites appear at different times and fluctuate in a different manner according to the tissues tested during rat development; this might be important in the stimulation of the functional activities of those tissues during perinatal age.     

Intestinal neuraminidase activity of suckling rats and other mammals. Relationship to the sialic acid content of milk.

1. The neuraminidase activity of homogenates of the mucosa of the middle and distal thirds of the small intestine of rats increased about 5-fold between birth and 4 to 8 days of age, and then gradually declined to the much lower adult activity by 24 days. No comparable changes occurred in the proximal third. 2. In 8-day-old rats, the neuraminidase activity of the middle and distal thirds of the small intestine was about 10 times greater than that of the proximal third, 20 times greater than that of the colon and at least 100 times greater than that of the liver, brain, gastric mucosa or pancreas. 3. In all other species investigated (mice, rabbits, cats and guinea pigs), the neuraminidase activity of the middle and distal thirds of the small intestine was greater in suckling animals than in adults. 4. The sialic acid content of rat milk increased about 2-fold between birth and 8 days post partum and then declined. 5. There was a highly significant positive correlation between the intestinal neuraminidase activity of suckling animals of various species and ages and the sialic acid content of milk obtained from the corresponding species and stage of lactation. 6. It is suggested that the intestinal neuraminidase of suckling mammals functions primarily to remove sialic acid from various components of milk, thus providing sialic acid for the synthesis of sialoglycoproteins and gangliosides by the young.     

Changes in adenosine receptors during differentiation of 3T3-F442A cells to adipocytes.

We measured the amino acid concentrations in the afferent and efferent vessels of the liver in anaesthetized fed adult rats and in fed suckling rat pups. A much higher content of glutamine in the portal vein and the aorta than in hepatic veins suggests that this amino acid is actively taken up by the liver of fed suckling rat pups, conversely to what is found in adult rats. In an attempt to characterize further the mechanism(s) contributing to this enhanced glutamine uptake, we monitored the time course of 1 mM-glutamine transport into plasma-membrane vesicles purified from the livers of either adult or suckling rats. The concentrative Na+-dependent uptake of glutamine was lower in those vesicles obtained from pups than in those obtained from adult rats. Glutaminase and glutamine synthetase activities in livers from both experimental groups were also measured. Glutaminase and glutamine synthetase activities in suckling rats were about 3-fold higher and 2-fold lower respectively than those in adult rats. It is concluded that glutamine is a main nitrogen carrier to the liver in fed suckling rats. A high availability of this amino acid and an enzyme imbalance between glutamine-synthesizing and -degrading activities may account for the net uptake found in vivo.     

Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activities of enzymes of ketone-body utilization in brain and other tissues of suckling rats

1. The activities of 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase in rat brain at birth were found to be about two-thirds of those of adult rat brain, expressed per g wet wt. The activities rose throughout the suckling period and at the time of weaning reached values about three times higher than those for adult brain. Later they gradually declined. 2. At birth the activity of acetoacetyl-CoA thiolase in rat brain was about 60% higher than in the adult. During the suckling period there was no significant change in activity. 3. In rat kidney the activities of the three enzymes at birth were less than one-third of those at maturity. They gradually rose and after 5 weeks approached the adult value. Similar results were obtained with rat heart. 4. The activity of glutamate dehydrogenase (a mitochondrial enzyme like 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase) also rose in brain and kidney during the suckling period, but at no stage did it exceed the adult value. 5. Throughout the suckling period the total ketone-body concentration in the blood was about six times higher than in adult fed rats, and the concentration of free fatty acids in the blood was three to four times higher. 6. It is concluded that the rate of ketone-body utilization in brains of suckling rats is determined by both the greater amounts of the key enzymes in the tissue and the high concentrations of ketone bodies in the blood. In addition, the low activities of the relevant enzymes in kidney and heart of suckling rats may make available more ketone bodies for the brain.     

Pre- and postnatal development of differentiated functions in rat intestinal epithelial cells

A panel of monoclonal antibodies to intestinal cell surface components has been used to compare the expression of differentiation-specific antigens in the epithelial cells of fetal, suckling, and adult rat small intestine. Indirect immunofluorescence staining, and immunopurification of detergent-solubilized membrane proteins, followed by single- and two-dimensional slab gel electrophoretic analysis, have demonstrated that fetal intestinal cells (at day 21 of gestation) express most differentiation-specific markers typical of adult absorptive villus cells. A marked heterogeneity in antigen expression was observed among different villus cell populations in suckling rat intestine, and three cell surface components were identified which are exclusively present during this period of intestinal development. Striking changes in the patterns of antigen expression in crypt and villus cells, and variations in the apparent isoelectric points for most luminal membrane components, were associated with the maturation of the intestinal mucosa at weaning. These changes could not be prematurely induced by cortisone injection in newborn rats, suggesting that factors other than glucocorticoids are responsible for the postnatal development of the intestinal epithelium. These results suggest that basic differences in biological properties and regulatory mechanisms exist among intestinal epithelial cells at different stages of pre- and postnatal maturation.     

Age-related changes in calcium and phosphorus uptake by rat small intestine

The purpose of these experiments was to determine whether there are changes in intestinal Ca and P uptake with age and whether the regulation of Ca and P uptake changes with age. Experiments were performed in male Fischer 344 rats aged 2-3 months (young), 12-14 months (adult) and 22-24 months (old). Ca and P uptake were measured simultaneously by incubating everted intestinal sacs in a buffered salt solution containing radiolabeled 0.25 mM Ca and 1.0 mM P for 15 min. Ca uptake declined by over 50% with age in the duodenum, and P uptake showed a similar decline in both the duodenum and jejunum. The biggest decrease was seen between the young and adult age groups. These decreases in uptake were paralleled by decreases in serum 1,25-dihydroxyvitamin D with age. Administration of 1,25-dihydroxyvitamin D-3 increased Ca uptake by 50-65% in the duodenum and increased P uptake by 85-120% in the duodenum and jejunum of both young and adult rats. Although 1,25-dihydroxyvitamin D-3 increased uptake by about the same percentage in each age group, the maximal uptake was much greater in the young than in the adult. Feeding a low-Ca diet increased duodenal Ca uptake by 68% and increased serum 1,25-dihydroxyvitamin D over 2-fold in young rats. There was no significant increase in either parameter in adult rats fed a low-Ca diet. However, duodenal P uptake was stimulated by a low-Ca diet by 87% in young rats and by 51% in adult rats. These results demonstrate that there is an age-related decline in Ca and P uptake by the intestinal mucosa. In addition, there is decreased capacity of 1,25-dihydroxyvitamin D-3 and a low-Ca diet to stimulate intestinal uptake in the adult.     

Delayed appearance of liver growth hormone binding sites and of growth hormone-induced somatomedin production during rat development

The present study was designed to determine whether the apparent paradox of high circulating growth hormone levels in the fetus and the minimal effect of this hormone on growth might reflect a diminished responsiveness of fetal target organs to GH. Specific uptake by rat liver of [125I] bGH was very low in fetuses as compared to suckling and adult rats. Also, liver uptake of the iodinated hormone decreased proportionally with the simultaneous injection of increasing amounts of growth hormone, but was not modified by the simultaneous injection of unlabelled chemically-related hormones. Since the water content is significantly greater in fetal than adult tissues, results were expressed by liver dry weight and again, [125I] bGH liver uptake continued to increase with age. After bovine growth hormone administration to adult rats, plasma somatomedin C concentrations increased significantly, while they had no effect in fetuses. These results suggest that reduced liver somatogenic binding sites in the fetus prevents growth hormone from inducing growth-promoting effects during intrauterine life.     

Transport of glycyl-L-proline in intestinal brush-border membrane vesicles of the suckling rat: characteristics and maturation

Transport of the dipeptide glycine-L-proline (Gly-L-Pro) in the developing intestine of suckling rats and its subsequent maturation in adult rats was examined using the brush-border membrane vesicles (BBMV) technique. Uptake of Gly-L-Pro by BBMV was mainly the result of transport into the intravesicular space with little binding to membrane surfaces. Transport of Gly-L-Pro in BBMV of suckling rats was: (1) Na+ independent; (2) pH dependent with maximum uptake at an incubation buffer pH of 5.0; (3) saturable as a function of concentration (apparent Km = 21.5 +/- 7.9 mM, Vmax = 8.6 +/- 1.5 nmol/mg protein per 10 s); (4) inhibited by other di- and tripeptides; and (5) stimulated and inhibited by inducing a negative and positive intravesicular membrane electrical potential, respectively. Similarly, transport of Gly-L-Pro in intestinal BBMV of adult rats was saturable as a function of concentration (apparent Km = 17.4 +/- 8.6 mM, Vmax = 9.1 +/- 2.1 nmol/mg protein per 10 s) and was stimulated and inhibited by inducing a relatively negative and positive intravesicular membrane potential, respectively. No difference in the transport kinetic parameters of Gly-L-Pro was observed in suckling and adult rats, indicating a similar activity (and/or number) and affinity of the transport carrier in the two age groups. These results demonstrate that the transport of Gly-L-Pro is by a carrier-mediated process which is fully developed at the suckling period. Furthermore, the process is H+-dependent but not Na+-dependent, electrogenic and most probably occurs by a Gly-L-Pro/H+ cotransport mechanism.     

Influence of intestinal microflora on the tryptic activity during lactation in rats

On comparing germ-free and conventional rats, inactivation of the tryptic activity was found to take place in the caecum of conventional adult rats only. A microbial intestinal inactivation of the tryptic activity was established in suckling conventional rats within 10 days after birth. At 3 weeks of age, suckling germ-free rats were found to have less faecal tryptic activity than their early-weaned littermates.     

Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.     

Nickel deficiency alters nickel flux in rat everted intestinal sacs

This study was conducted to determine nickel absorption in nickel-deficient rats. Jejunal segments obtained from dietary nickeldepleted (13 μg nickel/kg diet) and nickel-control (1 mg nickel/kg diet) adult rats from the first generation, and suckling pups from the second offspring were used. The nickel transfer across the intestinal epithelium and nickel uptake into the intestine were measured by use of everted jejunal sacs using a wide range of nickel concentrations administered on the luminal side (1.1 x 10^-8 M til 1.0 x 10^-4 M). Both the intestinal nickel transfer and nickel uptake were influenced by the dietary nickel supply in rat offspring, but not in the adult rats from the first generation. However, in nickel-deficient offspring, the nickel transfer across the small intestine was higher than in nickelcontrol offspring. This difference was greater using low intraluminal nickel concentrations than high nickel concentrations, and was significant at 1.1 x 10^-8 M, 6.1 x 10^-8 M, 5.1 x 10^-7 M, 1.0 x 10^-6 M, and 5.0 x 10^-6 M. Also, nickel uptake into the intestine was somewhat greater in nickel-deficient rat pups than in nickel-control pups, and significant using 1.1 x 10^-7 M and 1.0 x 10^-6 M nickel. A definite saturation type kinetic for the intestinal nickel absorption in relation to the intraluminal nickel concentration could not be observed.     

Dexamethasone control of the development of tryptophan oxygenase in young rats

Tryptophan 2, 3-dioxygenase activity follows a characteristic pattern of change during development of suckling rats; namely, it is appears 2 weeks after birth and then increases to the adult level by day 22. Glucocorticoids, potent inducers of the enzyme in adult rats, can induce its precocious appearance on day 8 or 10 after birth (1) or even on day 4 (2). The induced level is not more than the adult basal level. We investigated the conditions controlling the response to glucocorticoid before day 14 in suckling rats without using tryptophan. Pretreatment with dexamethasone for 2 days increased the induction by glucocorticoid, resulting in 2 to 3 times higher activity than the adult basal level. Actinomycin D and cycloheximide inhibited this enzyme induction.     

Transferrin degradation by gastrointestinal fluids of suckling and weanling rats

Dietary transferrins are postulated to play a number of biological roles in the developing gastrointestinal tract. A prerequisite for such roles is survival in the gastrointestinal lumen. To evaluate luminal transferrin digestion during development, 125I-transferrin was incubated in vitro with luminal fluid from the stomach and small intestine of 12-day old suckling and 31-day old weanling rats, followed by analysis of degradation products. At both ages, the rate of degradation to trichloroacetic acid soluble material was maximum in the mid-jejunum and lowest in the stomach. Transferrin hydrolysis by weanling fluid was 2-10 times greater than suckling depending upon the particular segment. Chromatography of small intestinal reaction mixtures on Sephacryl S-200 revealed label eluting between intact transferrin and free iodine: two such peaks were generated with suckling fluid and one with weanling. Electrophoresis on SDS-polyacrylamide gels showed two major bands of Mr 69K and 20K; the former was the predominant reaction product with suckling intestinal fluid and the latter with weanling. Both methods showed small amounts of apparently intact transferrin. Results indicate substantial yet incomplete luminal degradation of transferrin which is more pronounced in the weanling than in the suckling. This survival is compatible with potential biological functioning of dietary transferrin or one of its breakdown products within the gastrointestinal tract.     

Dopamine beta-hydroxylase in rat serum and lymph: changes with age and effect of cold exposure.

The pH optimum for rat serum dopamine beta-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating)(EC 1.14.17.1) was 4.0 in acetate buffer; other requirements were as reported by others. DBH activity in serum of 20-day-old fetuses is slightly higher than in that of their mothers. Levels of the enzyme in blood a few hours after birth are almost five times greater than in the adult, remain high during the suckling period, then drop rapidly during the 4th week after birth to about three times the adult level, which is then slowly reached over the next few weeks. These fluctuations in serum DBH activity coincide with the period of intense development and maturation of the sympathetic nervous system. There was not significant effect of cold exposure on blood DBH activity when newborn, suckling, weanling or adult warm- and cold-acclimated rats were exposed to cold. Similarly, exposure to cold that elicited two- to three-fold increases in O2 consumption failed to increase DBH activity in thoracic duct lymph. Therefore serum and lymph DBH activities are not sensitive indices of sympathetic secretory activity in the intact rat.     

Changes in sialic acid and fucose contents of enterocytes across the crypt-villus axis in developing rat intestine

Alterations in sialic acid and fucose contents of different populations of epithelial cells have been studied in suckling and adult rat intestine. The progression of cells from crypt base to villus tip is associated with an increase in sialic acid and a decrease in fucose levels of the cells in adult rats. In suckling pups, sialic acid is uniformly distributed along the length of villi, and fucose is richly (P less than 0.01) present in cryptic cells compared to that at the villus tip. Adult-type changes in sialylation and fucosylation of enterocytes across the crypt-villus axis were precociously produced by cortisone administration to suckling pups. Thyroxine treatment was less effective in influencing the glycosylation process in rat intestine.