Ultrastructural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion

Isolated rabbit hearts were perfused by the Langendorff technique, made ischemic and subsequently reperfused. It was found that ischemia results in: (i) aggregation of the intramembranous particles in the sarcolemma and (ii) extrusion of pure lipidic multilamellar structures (liposomes) from swollen mitochondria. Subsequent reperfusion resulted in further aggregation of the sarcolemmal intramembranous particles and disruption of the sarcolemma, which was attended by the formation of liposome-like structures. Intramembrane particle aggregation is explained in terms of lateral phase separation of the membrane lipids and a reduction of repulsive forces between the membrane proteins, both induced by a decrease in pH and an increase in Ca2+ concentration intracellularly. The formation and extrusion of the multilamellar structures are discussed in terms of destabilization of the bilayer which results in a structural blebbing-off of pure lipid.     

Ultrastructural demonstration of rabbit heart myocyte metabolism during fasting]

Thirty mature rabbits were completely deprived of food but water ad libitum for 10 days, then half of the animals were decapitated. The remaining animals were sacrificed 10 days after a sound diet. Histological, histochemical (estimation of glycogen, lipids, acid glycosamineglicans) and electron microscopic investigation of the myocardium was carried out. Degenerative and necrobiotic alterations were not revealed in muscular cells of the heart. At fasting accumulation of lipids and glycogen in the cardiac myocytes was evident. In fasting animals ultrastructure of the muscular cells of the heart demonstrated a certain decrease in synthetic processes. After a 10-day sound diet, the cardiac myocytes were actively involved into the process of intracellular regeneration. Increased lipid content in the rabbit myocardium, occurring at fasting, is connected with the necessity to provide the heart with an effective energy substrate required to preserve its contractile function and in this way adaptive-compensatory processes are demonstrated.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Control of glucose metabolism in the perinatal period

The central importance of glucose as a fuel for energy metabolism and growth of the fetus is clear as is the role of insulin in coordinating its utilisation by many fetal tissues. What is less clear is the qunatitative nature of the interaction between the fetus and placenta in organising glucose metabolism. Increasingly there is evidence that the fetus coordinates some of the supply of glucose to the placenta and that this is particularly important when uterine blood flow is reduced. It is unclear how this is regulated, but substrate cycles of glucose and lactate appear to make a significant contribution to carbohydrate metabolism in fetus and placenta. Another area as yet unresolved in the control of fetal glucose metabolism is the coordination of the changes that occur around the time of birth. Notable of these is the activation of glycogen mobilisation and of glucose synthesis and changes in the setting of glucose regulatory mechanism. These are briefly reviewed.     

Glucose metabolism in perinatal gonads of the rabbit

Metabolism of [U-14C]glucose was studied in the prenatal and neonatal rabbit ovary. Control tissues included the testis and female liver. No significant changes in glucose metabolism were observed in liver tissue. Mitosis and glucose oxidation were maximal in ovary and testis at 30 days post coitum and then declined dramatically by Day 8 after birth. Since mitosis is the primary physiological event in the gonad during the perinatal period these data suggest that glucose may be an important carbohydrate source for energy at this time.     

Ultrastructural changes in the mitochondria of Retzius' neuron exposed to hydrocortisone

The ultrastructure of the Retzius cell (gigantic neurone) mitochondria of the medical leech was investigated. After seven repeated injections of hydrocortisone into the coelomic cavity, the transformation of laminar cristae of mitochondria into tubule-vesiculated structures was discovered. A question of correlation between the structure and function independently of the systematical position of the organism is discussed.     

Catecholoestrogen synthesis and metabolism in the rabbit uterus during the periimplantation period

Microsomal oestradiol-2/4-hydroxylase (OE-2/4-H) and cytosolic catechol-O-methyltransferase (COMT) (EC 2.1.1.6) activity in the uteri of pregnant and pseudopregnant rabbits during the periimplantation period were studied. The apparent Km for the 4-hydroxylation of oestradiol (3.18 microM) was considerably less than for the 2-hydroxylation reaction (13.36 microM), whereas the Vmax were almost equal. This suggests that 4-hydroxyoestradiol (4-OH-OE2) is the predominant product of OE-2/4-H in the rabbit uterus. These reactions were inhibited by SKF-525A, indicating the involvement of cytochrome P450 dependent monooxygenases. Uterine cytosolic COMT utilized 2-hydroxyestradiol (2-OH-OE2) as the preferred substrate as compared to 4-hydroxyoestradiol (4-OH-OE2). Since the rabbit uterus has a considerable capacity to synthesize 4-OH-OE2 and a lower capacity to metabolize it, it could be suggested that more 4-OH-OE2 than 2-OH-OE2 could be available to the uterus for its physiological activities. Furthermore, an increase in OE-2/4-H in Day 6 pseudopregnant and pregnant uteri with a concomitant decrease in COMT suggests the involvement of catecholoestrogens in the implantation process in the rabbit.     

Rat liver glycogen metabolism in the perinatal period.

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen synthase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximum on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose or AMP on the respective phosphatases was considered. It was found that the Km of phosphorylase phosphatase for glucose in the prenatal period was 5--7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6--15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time. Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver. It is suggested that the rapid changes observed immediately after birth are due to an effect of gypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Ultrastructural features of heart insufficiency and its correction during the post-asphyxia period]

The ultrastructure of myocardium was studied in the experiments on rats during 24 hours after 6-min mechanical asphyxia with following clinical death. It has been established that the contractile cardiac apparatus of myocytes lesions on the intracellular edema background is the main while energy production process impairs insignificantly. The effectiveness of anti-hypoxia, membrane stability and protease inhibiting preparations for prevention of postresuscitation myocardium lesion was examined. The contrykal protective action, suppressing the proteolytic ferment hyperactivity was determined.     

Electromyographic correlates of the transition from aerobic to anaerobic metabolism in treadmill running

This study analysed the changes in electromyographic (EMG) activity of the vastus lateralis, biceps femoris and gastrocnemius muscles during incremental treadmill running. The changes in EMG were related to the lactate and ventilatory thresholds. Ten trained subjects participated in the study. Minute ventilation, oxygen consumption, carbon dioxide expired and the fraction of oxygen in the expired gas were recorded continuously. Venous blood samples were collected at each exercise intensity and analysed for lactate concentration. The EMG were recorded at the end of each exercise intensity using surface electrodes. The EMG were quantified through integration (iEMG) and by calculating the mean power frequency (MPF). The iEMG measurements were characterized by a breakpoint in the vastus lateralis and/or gastrocnemius muscles in eight of the subjects tested. However, the results indicated that blood lactate concentrations had already begun to increase in a nonlinear fashion before the iEMG breakpoint had been surpassed. Consequently, the occurence of the lactate threshold cannot be attributed solely to the change in motor unit recruitment or rate coding patterns demonstrated by the iEMG breakpoint. The ventilatory threshold was shown to be a far more reliable and convenient noninvasive predictor of the lactate threshold in comparison with EMG techniques. In conclusion, the EMG measurements used in this study (i.e. iEMG and MPF) were not considered to be viable noninvasive determinants of the aerobic-anaerobic transition phase in treadmill running.