Gillian Bullock

The oxygen paradox and the calcium paradox: two facets of the same problem?

An isolated rat heart preparation was used to study and compare the sudden tissue damage which may result from calcium repletion after a period of calcium-free perfusion with the effects of reoxygenation after a period of anoxic perfusion and with reperfusion after a period of ischaemia. Striking similarities in morphological and enzyme release characteristics exist for all three phenomena. Our results suggest that the primary lesion in the calcium paradox is the separation of the basement membrane from the sarcolemma and the energy-dependent loss of intracellular calcium to the extracellular space during the phase of calcium depletion. These changes, coupled with separation of opposing faces of the intercalated disc predispose the cell to the major damage which results from an energy independent, sudden, cellular influx of calcium during the phase of calcium repletion. In the case of the oxygen paradox the primary lesion would be the accumulation of cytoplasmic calcium and the loss of cellular and subcellular membrane control. While these anoxia or ischaemia-induced processes are related to the cellular availability of energy, the sudden damage induced by oxygen repletion would appear to be initiated by an energy-independent mitochondrial calcium uptake which is triggered by the reoxidation of the electron transport chain. Studies of such factors as the critical duration of calcium or oxygen depletion, the effects of stepwise calcium or oxygen repletion or the striking effects of hypothermia, reinforce the association between the calcium and oxygen paradoxes. While the basic principles underlying the cause of damage may be similar in both cases, fine differences exist in the progression, the nature and the extent of damage.

The preparation of mitochondria from muscle without the use of a homogeniser

Skeletal muscle from adult rats is difficult to homogenise and the excessive shearing forces needed raise the possibility that isolated subcellular organelles and particles are not truly representative of the in vivo state. In the case of ribosomes this is clear when sucrose density gradients show a preponderance of single ribosomes [l] although polyribosomes predominate in the intact tissue. It also seems likely to be the case with mitochondria since these organelles cover a wide range of sizes and preferential rupture of the larger particles during homogenisation could easily lead to enrichment with respect to the smaller members of the subcellular population. In the past, attempts have been made to overcome this difficulty by adding proteolytic enzymes to the tissue before homogenisation in order to make the cells less resistant to mechanical breakdown [2] . We have now shown that functional skeletal muscle mitochondria can be released in satisfactory yield by the use of proteolytic enzymes alone and that a mechanical homogeniser need not be used.

Rapid mitochondrial enlargement in muscle as a response to triamcinolone acetonide and its relationship to the ribosomal defect

Abstract A study of the morphology of the predominently white m. vastus lateralis of the male rat (250 g) showed that 2 hr after treating the animals with a high dose (20 mg/kg) of triamcinolone acetonide and 12 hr after a low dose (0·2 mg/kg) there was pronounced enlargement and proliferation of the mitochondria in a zone adjacent to the subsarcolemmal zone. There was, in addition, some destruction of mitochondria in deeper zones and some evidence of fragile membranes in the enlarged mitochondria. The mitochondrial enlargement has been quantified with the use of a modified Altmann stain and time and dose responses to triamcinolone acetonide constructed. Methandienone, (17α-methyl-Δ 1 -dehydrotestosterone) was shown to block the mitochondrial enlargement completely although ribosomes from pooled m. vastus lateralis, m. vastus medialis and m. glutens medius still partially retained a defect which significantly reduced their ability to incorporate amino acids into protein in vitro . Testosterone had virtually the opposite effect from methandienone at the same dose levels but its ability to allow ribosomes to function normally was to some extent dependent on the age of the animals. The above finding taken together with a disparity in the dose-response curves of mitochondrial enlargement and the ribosomal defect after triamcinolone acetonide treatment supports the view that these phenomena do not have a common cause and must be independent responses of the muscle cell to the action of the steroid.

A novel approach for enzyme histochemical and autoradiographic studies on single cells.

SynopsisA simple technique that does not involve the use of heat has been developed to fix cells or cell organelles. The cells or organelles are mixed with a bovine serum albumin solution, gelled by the addition of a suitable fixative, and then either embedded or frozen. The gelled mixture contains well preserved cells or organelles that are evenly dispersed, thus eliminating the problems of pellet packing. The technique was excellent for ultrastructural autoradiography where radioactive materials bound to plasma membranes or cytoplasmic nucleotides were being studied. Histochemical tests could be applied to the fixed embedded material. Light and electron microscopy could be done on the same well-mixed sample. Fixed frozen albumin samples cut with ease on a cryostat but there was ice crystal formation.

Studies on bile secretion with the aid of the isolated perfused rat liver. II. The effect of two further pentacyclic triterpenes, asiatic acid and 22β-angeloyloxyoleanolic acid

Abstract The effect of 2 triterpene acids, 22β-angelolyoxyoleanolic acid and asiatic acid on the isolated perfused rat liver has been studied. From their structures it was predicted that 22β-angeloyloxyoleanolic acid would possess cholestatic properties while asiatic acid would be inactive. It was shown that 2mg of 22β-angeloyloxyoleanolic acid dissolved in ethanol produced cholestasis within 45 min while 10 mg of asiatic acid dissolved in ethanol had little effect on the bile flow rate. Changes in the rate of bile salt excretion closely followed those of the bile flow rate. At the end of the perfusion or when bile flow had ceased the livers were fixed for electron microscopic examination. The cholestasis produced was characterised by alterations of the canalicular membrane while cytoplasmic organisation remained unaffected. In the presence of asiatic acid the canalicular region appeared relatively normal and only minor changes to the cytoplasmic organelles were observed. The presence of 22β-angeloyloxyoleanolic acid had little effect in the changes in lactate and pyruvate levels caused by the metabolism of ethanol. Asiatic acid depressed the pyruvate level which led to an elevated lactate to pyruvate ratio but this had returned to control levels after 2 hours. These results suggest that the cholestatic triterpene acids exert their effect at the canalicular membrane, but the exact mechanism remains unclear.

The analysis of rat skeletal muscle mitochondria isolated by the method of tryptic lysis

1. Rat skeletal muscle mitochondria were isolated by the method of tryptic lysis developed by Bullock, Carter and White which does not utilise mechanical homogenisation. 2. These mitochondria were subjected to centrifugation on a discontinuous sucrose density gradient at 105000 × g for 45 min. Broken organelles (Band 1) did not penetrate into the gradient. The remaining mitochondria collected into three bands at the interfaces of the following sucrose molarities: 1.07 M/1.12 M, Band bands at the interfaces of the following sucrose molarities: 1.07 M/1.12 M, Band 2, 1.12 M/1.17 M, Band 3, and 1.17 M/1.22 M, Band 4. 3. Band 2 mitochondria had a respiratory control ratio of 16 and were smaller than those in Band 3 and Band 4. They also contained the lowest proportion of broken and swollen organelles. 4. Some mitochondria in all bands showed a shift from an aggregated to an open-mesh form during transition from State 4 to State 3. Band 2 contained the highest proportion of mitochondria capable of changing in this way.

Influence of methylxanthines and local anaesthetics on the metabolism of muscle and associated changes in mitochondrial morphology

Abstract The influence of methylxanthines and of a number of local anaesthetics and adrenaline-blocking agents on the metabolism of the isolated rat diaphragm has been investigated. Both caffeine and theophylline inhibited protein synthesis in the tissue and enhanced its endogenous respiration. The latter effect was counteracted by several local anaesthetics (butacaine, cinchocaine, amethocaine and marcaine) and by certain β-blocking agents (propranolol, oxprenolol and alprenolol), but these compounds by themselves enhanced respiration and inhibited protein synthesis by the tissue. By contrast with other agents toxic to muscle, neither the methylxanthines nor local anaesthetics produced much stimulation of lactate production. The change in mitochondrial morphology after caffeine treatment differed from that produced by the other agents although all three types of compound similarly enhanced the rate of respiration. Under the conditions studied many deleterious effects on the tissue were seen, observable particularly where caffeine was used in conjunction with some β-receptor blockers. Some of the changes, which were often similar to those reported as due to different respiratory states, could be reproduced by different concentrations of the ionophorous agent, valinomycin. The mechanism of the effects is discussed in the context of the influence of both groups of drugs on uptake of calcium and other ions by sarcoplasmic reticulum and mitochondria.

Morphology of immune lymphocytes in homograft lymph

Uropod-bearing lymphocytes (UBLs) resembling cytotoxic cells found in vitro were identified in lymph collected from rabbit hind-limbs bearing homografts. UBLs were found in lymph leaving the regional lymph node (efferent), both before and after rejection, but not in that draining the homograft (afferent) even though they were present in the graft tissue itself. UBLs may represent the sub-class of immune lymphocytes which induce necrosis of the grafted cells. Mitotic lymphocytes and cells resembling lymphocytes transformed by phytohaemagglutinin were also found in efferent homograft lymph.

Quantification and identification of particle movement in epidermis after thermal injury.

SynopsisSkin samples from rabbit hind limb were taken from controls and at 5 min, 2 and 6 hr after a mild thermal injury (60°C for 1 min). Large aggregates of intercellular particles, usually ribosomal in appearance, were seen in 6 hr samples and were accompanied by some peripheral aggregates of particles and by granule-coated vesicles. These structures were present in earlier samples to a lesser extent and were absent from control material. Quantitative assessment showed that intercellular particles apparently increased up to 6 hr whereas peripheral aggregation was maximum at 2 hr. Histochemical analysis confirmed that the particles contained ribonucleoprotein. Other larger particles were seen occasionally and contained carbohydrate. Lymph draining the site showed cellular changes, little change in enzyme activities, and no aggregates of particles.