Effects of caffeine on heart mitochondria in newborn rats.

Caffeine consumption has been implicated in the development of cardiovascular disease. Therefore, in the present study, litters of rats were combined upon birth, and 8 pups were randomly assigned to each dam. Dams with pups were divided into 2 groups: group 1 received a 20% protein diet as a control, and group 2 received the 20% protein diet supplemented with caffeine (4 mg/100 g body weight). Pups from both groups were killed on days 11 and 15. Transmission electron microscopy revealed swollen, disrupted, degenerating mitochondria and intracellular edema in the hearts of rats in the caffeine groups when compared with those of the controls. Plasma Cu concentration was significantly decreased. These results indicate that early exposure to caffeine through maternal milk adversely affects cardiac mitochondria of rat pups and may be associated with decreased plasma Cu levels. It is unclear whether these results apply to the human infant. Interspecies extrapolation from rat to human must be made with caution.

Postnatal caffeine effects on copper, zinc, and iron concentrations in mammary gland, milk, and plasma of lactating dams and their offspring.

Beginning on the day of delivery and until the 15th or 22nd day of lactation, one group of dams received a 20% protein diet as a basal diet and one group received the basal diet supplemented with caffeine (4 mg/100 g body weight). A correlation between caffeine concentrations in the dams' plasma and milk was observed. In the caffeine group, levels of Fe and Cu in the dams' mammary glands at day 22 were decreased. Copper levels in the milk at day 15 and Fe and Zn levels in the milk at day 22 showed a significant decrease. Copper concentration in the plasma of 15-day-old pups showed a significant decrease also, but Zn and Fe concentration showed no difference between caffeine and noncaffeine control groups. The present study shows that the dams' consumption of a caffeine-containing diet influences trace elements of mammary glands, milk, and pups' plasma.

Effects of caffeine on bone cells and bone development in fast-growing rats.

BACKGROUND AND METHODS: Caffeine exerts harmful effects on fetal and suckling rats. To elucidate the effects of caffeine on bone in growing rats further, dams were fed normal laboratory chow until delivery. At birth, the litters were combined and eight pups were randomly assigned to each dam. Dams with the combined litters were divided into two groups. Dams of group 1 received a 20% protein diet as a control, and dams of group 2 received the 20% protein diet supplemented with caffeine (4 mg/100 g b.w.). Pups from both groups were killed on days 11, 15, 22, and 50. Parameters studied were ultrastructure of femoral osteoblasts, osteocytes, and osteoclasts; the number of osteocytes per area of bone in femoral histological cross sections; structural remodeling and shape of the lateral tibial metaphysis as revealed by scanning electron microscopy; and plasma Cu and Zn concentrations. RESULTS: Rats in the caffeine groups had significantly fewer osteocytes per area of femoral cross section, retarded structural remodeling of the lateral tibial metaphysis, osteoblasts and osteocytes with disrupted swollen mitochondria, and decreased plasma Cu and Zn levels when compared with controls. CONCLUSIONS: These results indicate that if young, rapidly growing rats are exposed to caffeine, disruption of osteoblasts and retarded bone development occur, which could be related to the decreased plasma Cu level in the young animals.

Combined effects of caffeine and alcohol during pregnancy on bones in newborn rats.

The combined effects of caffeine and alcohol on mineral contents of fetal mandibles and femurs were studied. Pregnant rats were divided into four groups: group 1, control; group 2, caffeine; group 3, alcohol; and group 4, caffeine-plus-alcohol. Alcohol (1.0 g ethanol/kg body weight) was intubated twice daily, beginning at day 9 of gestation. Caffeine (2 mg/100 g body weight) was given as a dietary supplement. Groups 1 and 2 were intubated with isocaloric sucrose solution. At birth, randomly selected pups were killed and the mandible and femur were removed and dried. Ca, P, Mg, Zn and hydroxyproline in these bones were measured. Notwithstanding the dams' intake of caffeine and alcohol administered separately, the present results suggest that the combination of caffeine and alcohol exhibited the most detrimental effects.

Effect of dietary caffeine and zinc on the activity of antioxidant enzymes, zinc, and copper concentration of the heart and liver in fast-growing rats.

The purpose of this study was to determine the relationship between concentrations of Zn and Cu and the activities of superoxide dismutase and glutathione peroxidase in the heart and liver of young rat pups whose dams were fed a diet supplemented with caffeine and/or Zn. Four groups of dams with their newborn pups were fed one of the following diets for 22 d: 20% protein basal diet; the basal diet supplemented with caffeine (2 mg/100 body wt); the basal diet supplemented with Zn (300 mg/kg diet); or the basal diet supplemented with caffeine plus Zn. The Cu levels in the livers of the pups were decreased by maternal intake of the caffeine and Zn diet. The maternal intake of the caffeine diet increased Mn-superoxide dismutase (MnSOD) activity and Cu, Zn-superoxide dismutase (Cu,ZnSOD) in the heart of the pups. On the other hand, the activity of Cu,ZnSOD was significantly reduced in the liver of pups whose dams consumed a caffeine, Zn, or caffeine plus Zn diet. Cu,ZnSOD activity in the liver of the pups seems to be correlated with Cu levels in the tissue. Selenium-dependent glutathione peroxidase (GSH-Px) activities in the heart and liver showed no difference among the groups. The effect of dietary caffeine and/or Zn on the activity of antioxidant enzymes in the heart and liver were different in young rats. The activities of these enzymes in the heart were lower than in the liver of 22-d-old rats. Our experiments indicate that the heart has limited defenses against the toxic effects of peroxides when compared to the liver.

Alteration of femoral structure in later life by chronically feeding caffeine during rapid growing period in newborn female rats.

The effects of caffeine intake in early life on bone structure later in life were studied in rats. At day 9 of gestation, dams were divided into 2 groups. Group 1 (control) received a 20% protein diet; group 2 received the 20% protein diet supplemented with caffeine (2 mg/100 g body weight). After birth pups were continuously fed their respective diets until day 93, when the diet of group 2 was replaced with a noncaffeine 20% protein diet. On day 388 animals from both groups were weighed, killed, and femora and mandibles were removed. Calcium, phosphorus, magnesium, zinc, hydroxyproline, and hexosamine concentrations were measured. Radiographs of some femora were taken and paraffin cross sections were made at the midshaft of others. Femora in the caffeine group were wider, periosteal bone area/total bone area was greater, the cross sectional area of femoral bone was smaller, and there were fewer osteocytes/bone area than in controls. Calcium, phosphorus, zinc, and hydroxyproline concentrations in the caffeine group were less in both bones of the caffeine group. These results indicate that if animals are exposed to caffeine during the rapidly growing period, changes occur in femoral bone which are similar to those that occur with aging.

Effects of chronic caffeine feeding on the activities of oxygen free radical defense enzymes in the growing rat heart and liver.

The purpose of the present study was to determine the relationship between concentration of Zn, Cu and Fe, and the catalase, glutathione peroxidase and superoxide dismutase activities in the heart and liver of newborn rats whose dams were fed a diet supplemented with caffeine. Heart Zn levels of the 22- and 30-day-old rats of the caffeine group showed a decrease, whereas liver Zn levels showed an increase compared to the control. Cu levels in the liver at day 22 in the caffeine group were less than in the control. Cu- and Zn-containing superoxide dismutase activities showed an increase in the hearts of the caffeine group compared to the control. The activity of catalase and glutathione peroxidase showed no difference in the heart and liver between the groups. The present study suggests the possible involvement of superoxide dismutase enzyme in the impairment of heart formation as a result of chronic caffeine intake in the early growing period.

Interaction between zinc and calcium in skeletal muscle in young growing rats.

The purpose of the present study was to determine whether zinc and calcium could interact at the tissue level. In the first part of the study, adult rats were injected with ZnCl2 dissolved in a physiological saline solution to determine the effects of Zn on Ca levels in various tissues. In the second part of the study, weaned rats (at day 22 postnatally) were fed a diet supplemented with Zn until day 50 and were then sacrificed. In both instances, blood, brain, heart, liver, and skeletal muscle were taken and analyzed. In the Zn-injected group, the brain, heart, and liver showed no interaction between Zn and Ca. The skeletal muscle, in contrast, showed a decrease in Ca in the homogenate, whereas Zn contents showed a significant increase at the sarcoplasmic reticulum (SR). Likewise, in the Zn-supplemented group, the Zn content of the SR vesicle of the skeletal muscle showed an increase, whereas Ca content of the pellet (14,000 g), which contains cell debris, nucleus, mitochondria, and SR vesicles of this group, showed a decrease. Current findings suggest antagonistic effects between Zn and Ca on this tissue. Zn may play a critical role in cellular function through the alteration of intracellular distribution of Ca in skeletal muscle.

Caffeine decreases zinc and metallothionein levels in heart of newborn and adult rats.

The purpose of the present study is to show that dietary caffeine, heart tissue Zn levels, and metallothionein (MT) concentration are all interrelated in newborn rats. Dams with eight pups in the control group were fed a 20% protein diet upon delivery, whereas dams in the experimental group were fed a 20% protein diet supplemented with caffeine (2 mg/100 g body weight). Offspring were killed at d 22 postbirth and the hearts and livers were removed to determine the Zn and MT concentrations. Hearts of the newborn rats in the caffeine group showed decreased Zn levels as well as decreased MT concentration. To explain the observed effects in newborn rats, the relationship between Zn and MT levels was studied in adult female rats. They were injected intraperitoneally with either ZnCl2 (20 mg/kg body weight) dissolved in saline solution or ZnCl2 and caffeine (4 mg/kg body weight) over a period of 2 d. Injection of ZnCl2 into adult female rats resulted in an increase in heart MT levels, whereas injection of caffeine caused decreased Zn levels and MT concentration. Current findings indicate that dietary caffeine intake during the lactational period by newborn pups causes a decrease of the heart Zn and MT levels.

Effects of caffeine on the DNA and protein synthesis of the protein-energy malnourished neonatal cardiac muscle cells in culture.

1. The growth of cardiac cells derived from newborn rats whose dams were either malnourished or malnourished with caffeine during pregnancy was inhibited in culture over the period of 5 days as compared to that of the normally nourished cells. 2. Cells derived from malnourished rats with caffeine added to their diets showed a greater inhibition than those from the malnourished rats not given caffeine. 3. Both DNA and protein synthesis showed an inhibition due to caffeine in a dose-dependent manner using normally nourished cells. 4. In the presence of exogenous 2 mM caffeine, the degree of percent inhibition of DNA and protein synthesis of cells derived from rats malnourished with caffeine was less than that from the rats with malnutrition alone. 5. The present data indicated that malnutrition combined with caffeine during pregnancy exerted a greater negative effect on the nature of cell growth than malnutrition alone and these cells became less sensitive to exogenous caffeine.

Effect of caffeine and zinc on DNA and protein synthesis of neonatal rat cardiac muscle cell in culture.

The effect of caffeine and/or zinc on DNA and protein synthesis of purified neonatal-rat ventricular cardiac myocytes was studied. Caffeine (0.2-2 mM) inhibited both DNA and protein synthesis of the cells. Addition of EDTA in the growth medium inhibited both DNA and protein synthesis. Without caffeine and in the presence of lower concentrations of caffeine (0.2 mM) in the growth medium, 10 microM of zinc concentration reversed DNA synthesis, which was inhibited by the chelating agent (EDTA). Higher concentrations of caffeine (2 mM) in the growth medium completely abolished sensitivity of cardiac myocytes to zinc. Additional zinc supplementation to the growth medium of cardiac myocytes did not alter the rate of protein synthesis. The present results suggest that the effect of caffeine on cardiac myocytes may be associated with the zinc-dependent enzymes involved in DNA synthesis.

Effect of caffeine on zinc absorption and Zn concentration in rat tissue.

The purposes of the present study were to determine whether caffeine has an effect on zinc absorption and tissue levels of Zn. Under anaesthesia, one side of the intestine of female rats was connected to infusion pumps and an infused solution (first caffeine and later Zn solution) was collected from the other side of the intestine using either 300 mm or the whole small intestine to determine Zn absorption. In a further study, different doses of caffeine solution were injected directly into the femoral vein and a saline or Zn solution was infused into the intestine to measure Zn absorption and tissue Zn concentrations. The results consistently showed that the caffeine solution infused into the intestine did not affect intestinal Zn absorption nor was absorption affected by the direct injection of caffeine into the vein. In contrast, injection of different doses of the caffeine solution significantly decreased Zn tissue levels for the heart only. Calcium concentrations in the heart tissue were also decreased, but not magnesium concentrations. Tissue Zn levels recovered immediately on infusion of a Zn solution into the intestine.

Interaction between caffeine intake and heart zinc concentrations in the rat.

The purpose of the present study was to determine the levels of zinc in the hearts of growing post-weaning offspring, fetuses and their dams chronically fed caffeine. A further study was conducted to determine the distribution of Zn in subcellular heart fractions affected by acutely injecting caffeine into the veins of the adult rats. After delivery pups were raised on a 200 g protein/kg diet until day 22 of weaning. On day 22 randomly selected male offspring from each litter were divided into two groups. Group 1 was fed continuously on the same diet as a control, whereas in the experimental group offspring were fed on a 200 g protein/kg diet supplemented with caffeine (20 mg/kg). On day 49 the animals were killed and Zn, calcium and magnesium concentrations of the hearts were measured. In the second series of studies pregnant dams were randomly divided into two groups. Group 1 was fed on a 200 g protein/kg diet from day 3 of gestation, whereas in the experimental group dams were fed on the diet supplemented with caffeine. On day 22 of gestation the fetuses were surgically removed. The Zn, Ca and Mg concentrations of hearts of fetuses and dams were determined. In the third phase a caffeine solution was injected into the vein. After 45 min the hearts were removed and Zn levels in the subcellular fractions determined. The hearts of the growing offspring fed on a caffeine-supplemented diet consistently showed decreased Zn and Ca levels compared with the non-caffeine group.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex.

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.