An alpha-actinin isoform which may cross-link intermediate filaments and microfilaments.

The G.3.5 antigen (named for the monoclonal antibody which recognizes it) has been characterized as an intermediate filament-associated protein found in a variety of tissue types, including human and rat astrocytes, rat skeletal and cardiac myocytes, fibroblasts, rat hepatocytes, and chicken and fish retinal tissues. Sequencing of proteolytic fragments indicated a high degree of similarity to alpha-actinin. Comparison of the G.3.5 antigen to alpha-actinin revealed that alpha-actinin and the G.3.5 antigen migrated similarly in reducing and non-reducing environments and had similar molecular masses (approximately 100,000). Overlay-immunoblotting assays indicated that the G.3.5 antigen and alpha-actinin could bind filamentous actin and desmin simultaneously. In contrast, immunocytochemistry indicated the G.3.5 antigen and alpha-actinin were immunologically distinct in tissue sections. The results of this study suggest that the G.3.5 antigen is an isoform of alpha-actinin which may serve to cross-link intermediate filaments to microfilaments, and that other isoforms of alpha-actinin may also share this property.

Rat glioma cell line as a model for astrogliosis.

In this investigation 9L rat glioma cells were grown on coverslips in culture, and when subjected to mechanical trauma, they were capable of mounting an astroglial response, without interaction with neurons, as evidenced by cell hypertrophy and increased immunostaining for glial fibrillary acidic protein and J1-31 antigen. It is of particular interest that this neoplastically transformed cell line is capable of mounting an astroglial response when subjected to mechanical trauma in vitro. The advantages of this model over in vivo models of central nervous system injury include the ease of reproducibility of results, and the ability to investigate molecular mechanisms under controlled conditions.

Multiple roles of intermediate filaments.

The widespread distribution, diversity of types, and highly conserved features of intermediate filaments (IFs) and the diversity of associated proteins indicate multiple roles for IFs in the nucleus and the cytoplasm of eukaryotic cells. Traditional functions include maintenance of cell shape, cell locomotion, movement and positioning of organelles and guiding cell development. Recent studies indicate some of these functions can now be expanded. Currently available information shows that IFs are important to nuclear function and cell division, to transport between the nucleus and cytoplasm, to the mechanical integrity of cells and tissues, to reorganization of the cytoplasm and chromatin in response to intercellular signals, and to embryonic and cellular differentiation.

Localization and characterization of an intermediate filament-associated protein.

A monoclonal antibody designated Mab G3.5 was used to test for the presence of reactive antigen in rat skeletal and cardiac muscle cells. Standard indirect labelling methods for immunofluorescence microscopy and immuno-gold localization for electron microscopy were applied to paraformaldehyde-fixed muscle tissues. In longitudinal sections, the reactive antigen was found to localize appropriately for Z-disks. In transverse sections of skeletal muscle, the antigen was apparent as a strongly fluorescent reticular network. Gold particles were observed by transmission electron microscopy in association with filamentous structures on either side of sarcomeres, and on either side of, but not adjacent to the Z-disk. This appearance is consistent with the exosarcomeric cytoskeleton and the known distribution of desmin. In competition binding experiments, neither antidesmin nor Mab G3.5 interfered with the binding of each other and co-localization was observed. Desmin (M(r) 52,000) differs in its relative molecular weight from the G3.5 antigen (M(r) 100,000), which was isolated and analysed by SDS-PAGE. The antigen was found to co-isolate with the cytoskeletal fraction from cell homogenates, and could be released from this fraction with the addition of reducing agents. Preliminary sequence analysis indicates that the G3.5 antigen may be an isoform of alpha-actinin which interconnects intermediate filaments and actin.

Exogenous superoxide dismutase and catalase promote recovery of function in isolated rat heart after regional ischemia and may be transported from capillaries into myocytes.

The effects of infusing superoxide dismutase (SOD) and catalase (CAT) into the coronary circulation were investigated in isolated, working rat hearts prior to and during a 15 minute episode of regional ischemia followed by 30 minutes reperfusion. Aortic output, left ventricular pressure and dP/dT were recorded. Compared to untreated hearts, SOD and CAT significantly improved function during reperfusion, but had no effect during the pre-ischemic or the ischemic period. To investigate possible transport of SOD and CAT into rat myocytes, cryotome sections of isolated, Langendorff perfused rat hearts were exposed to rabbit antibody prepared against the exogenous SOD and CAT. Bound antibody was detected by the indirect-fluorescent antibody test. The interior of myocytes from rat hearts exposed to SOD and CAT bound antibodies prepared against these enzymes, whereas myocytes from rat hearts not exposed to exogenous SOD and CAT only bound the CAT antibodies. This indicates the anti-SOD we prepared is specific for exogenous SOD, and also suggests exogenous SOD can gain access to the cytoplasm of myocytes from the coronary circulation.

Evidence for transcytosis of exogenous superoxide dismutase and catalase from coronary capillaries into dog myocytes.

Infusion of superoxide dismutase (SOD) and catalase (CAT) into the coronary circulation protects myocardial tissue from free radical injury and improves recovery of myocardial function after a short episode of ischaemia. To investigate the ultrastructure of myocardium treated with SOD and CAT, these enzymes were injected into the left atrium of dogs prior to and during 15 min of regional myocardial ischaemia, allowing 30 min of reperfusion, and then fixing the tissue for electron microscopy. The exogenous SOD + CAT was found to promote recovery of both function and structure in these hearts. In addition, electron dense material was unexpectedly found in vesicles of capillary endothelia, between capillaries and myocyte, and in vesicles within myocytes. This occurred only in hearts treated with SOD and/or CAT, suggesting SOD and CAT was concentrated and transported across the capillary endothelium and into myocytes. The rate of transcytosis, as measured by the number of intra-endothelial vesicles, was increased in tissue subjected to ischaemia and reperfusion in the presence of SOD and CAT. These observations suggest transcytosis of SOD and CAT is an important part of the process by which these enzymes provide protection to myocardium during reperfusion after ischaemia.

Inhibitors of adenosine catabolism improve recovery of dog myocardium after ischemia.

The effects of inhibitors of adenosine catabolism on contractile function and metabolites were assessed during 15 minutes of ischemia followed by 30 minutes of reperfusion in the open-chest dog heart. As compared to sham treatment, pretreatment with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and dipyridamole (DP) protected contractile function during ischemia, and improved recovery of high energy phosphate content and contractile function during reperfusion following ischemia. Testing EHNA and DP in a free-radical generating system indicated both compounds have some scavenging ability, suggesting the effect of EHNA + DP may not be on adenosine nucleotide metabolism alone. Comparison of end diastolic segment lengths to contractile function indicated the results were not affected by changes in preload resulting from peripheral vasodilation.

Ultrastructure, peroxisomes and lipid peroxidation in reperfused myocardium.

Reperfusion injury was studied in dog myocardium using a transmission electron microscope and 3,3'-diaminobenzidine (DAB) to locate areas of peroxidatic activity. Dark electron dense DAB reaction product was observed in peroxisomes and damaged mitochondria. These results suggest attack by reactive oxygen species on mitochondrial membranes, which might result in the formation of lipid peroxides and prostaglandin-like compounds. It is suggested that the release of lipid peroxide or prostaglandins from the injured cells may contribute to reactive hyperaemia, ventricular fibrillation and angina.

Visualization of endocytotic pits in capillary endothelia of dog heart by scanning electron microscopy.

Endocytotic transport may provide a mechanism for myocyte uptake of agents which ameliorate the effects of myocardial ischaemia. Examination of the inner capillary surface by transmission electron microscopy (TEM) is tedious and slow, while use of scanning electron microscopy permits the examination of greater areas in less time. Methods to obtain oblique exposures of capillaries and provide sufficient resolution to observe endocytotic pits were explored. Partial success was achieved using glutaraldehyde fixed tissue, treated with 30% glycerol, frozen-fractured under hexane at -85 degrees C, then dehydrated with acetone at -25 degrees C, critical-point dried and lightly coated with gold. Ultrastructural detail comparable with TEM images of heart tissue was obtained, but the resolution appeared limited by the coating process. It was not possible to verify stimulation of endocytosis by AMP in coronary capillary endothelia with scanning electron microscopy, due to this limitation.

Contractility, ATP, and creatine phosphate during myocardial ischaemia and reperfusion: the effects of adenosine and inhibition of adenosine catabolism in the dog heart.

Provision of AMP or adenosine to heart cells during recovery from episodes of myocardial ischaemia accelerates physiological, biochemical, and structural recovery. Inhibition of adenosine loss from the tissue during ischaemia should have a similar effect. This hypothesis was tested in dog heart by infusion of adenosine and inhibitors of adenosine catabolism prior to, during, and following ischaemia. Post-ischaemic recovery of ATP and contractile function was accelerated significantly by adenosine and by inhibitors of adenosine catabolism both singly and in combination. Contractility and ATP levels during ischaemia were also increased by these inhibitors.

Recovery of myocardial function after ischaemia: the effects of AMP and inhibition of endocytosis.

Recovery from ischaemia in heart tissue can be accelerated by addition of precursors of ATP such as AMP to the coronary circulation. Endocytosis in capillary endothelia is also stimulated by AMP; therefore endocytosis may be important in the transport of AMP from the circulation into myocytes. Alternatively, the increase in endocytotic transport itself could be responsible for accelerated recovery, irrespective of the stimulating agent. The effects of sham, AMP, cytochalasin-D (an inhibitor of endocytosis), and cytochalasin-D + AMP infusates given prior to, during, and following a 15 min ischaemic episode, were examined. AMP accelerated biochemical and functional recovery after episodes of ischaemia and stimulated endocytosis in coronary capillaries. Cytochalasin-D strongly inhibited contractility before, during, and after ischaemia, and similarly depressed ATP and creatine phosphate levels. Cytochalasin-D also strongly inhibited endocytosis and caused swelling of the capillary endothelium. When cytochalasin-D and AMP were provided together, the beneficial effects of AMP were only partially inhibited by cytochalasin-D. In fact, AMP was able to reverse most of the effects of cytochalasin-D including the inhibition of endocytosis. This suggests accelerated recovery of ischaemic myocytes requires precursors of ATP such as AMP, and the stimulation of endocytosis may abet transport of these precursors, or may be a spurious phenomenon.

Changes in metabolite levels and morphology of teleost ventricular myocytes due to hypoxia, ischaemia, and metabolic inhibitors.

Since fish hearts are resistant to the effects of hypoxia, comparison of the effects of hypoxia and ischaemia on fish and mammalian hearts may lead to better understanding of ischaemic injury in mammalian hearts. The ultrastructure and levels of ATP, creatine phosphate, and lactic acid were examined in hearts obtained from largemouth bass. Bass hearts were subjected to conditions of normoxia, ischaemia, hypoxia, and hypoxia in the presence of fluoride and cyanide. ATP levels remained stable during hypoxia and ischaemia, but fell during hypoxia in the presence of fluoride or fluoride plus cyanide. Changes in creatine phosphate and lactic acid indicated ATP was produced during hypoxia and ischaemia by glycolysis, by rephosphorylation from creatine phosphate, and by oxidative phosphorylation with oxygen obtained from myoglobin or the atmosphere. Ultrastructural changes were found similar to those reported in ischaemic mammalian heart, consisting of inter- and intracellular swelling, glycogen depletion, and mitochondrial alterations. Comparison of metabolic rates between fish and mammalian hearts suggests the lower rate in fish hearts may be the chief factor which permits stable ATP levels during hypoxia and ischaemia, and thus provides resistance to these conditions.

AMP stimulation of endocytotic transport from canine coronary circulation into myocytes.

Provision of ATP precursors before, during, and after an episode of myocardial ischaemia accelerates repletion of ATP levels and functional recovery of the tissue. To investigate ultrastructural effects of such provision, canine hearts were perfused in situ with sham and AMP solutions. Perfusion was begun 15 min before induction of ischaemia and continued during the 15 min ischaemic and 15 min reperfusion period. Some hearts were then treated with LaOH to label endocytotic vesicles, and the hearts were fixed for electron microscopy. A dramatic and significant increase was found in the number and size of capillary endothelial and subsarcolemmal vesicles in hearts treated with AMP, but not in hearts treated with sham additives. Endocytosis and exocytosis across capillary endothelia, and endocytosis into myocytes is suggested as a mechanism for rapid transport of ATP precursors from the coronary circulation into myocytes.

Changes in morphology of endocytotic vesicles in cardiac capillary endothelial cells and myocytes of rats after treatment with adenine and AMP.

Isolated rat hearts were perfused with media containing no additions (sham), adenine, or AMP to determine if adenine or AMP would affect post-ischaemic myocyte ultrastructure. The most significant change induced by both adenine and AMP was a dramatic increase in the number and size of endocytotic vesicles apparent in capillary endothelia and myocytes. This observation suggests endocytosis as a possible transport mechanism for AMP or adenine, or other large polar molecules including purine nucleotide precursors, from the coronary circulation across capillary endothelia and into myocytes.

Accelerated recovery of ischemic canine myocardium induced by AMP. Preliminary report.

Experiments were conducted on 18 dogs using an in situ blood-perfused canine heart model. Intracoronary infusion of AMP resulted in increased ATP and total adenine nucleotide levels. On reperfusion following a 15-min period of ischemia, ATP and total adenine nucleotide levels were significantly higher than control. Most important, contractile function recovered more rapidly in the AMP-treated dogs. It is therefore concluded that the delayed functional recovery noted after periods of ischemia is likely to be a direct result of delayed ATP resynthesis.

Contractile activity and oxidative metabolism in single adult myocytes during normoxia and hypoxia.

Contractile activity and redox metabolism were measured by optical techniques in single myocytes isolated from adult rats. Observations were made during periods of perfusion with normoxic and hypoxic solutions. Contractile activity was determined by quantitation of light transmitted through myocytes, and the state of redox metabolism was determined by measurement of flavoprotein fluorescence. Contractile activity was found to decrease gradually and in proportion to the duration of hypoxia, and this decline followed a similar but more abrupt decline in flavoprotein fluorescence which indicated reduction of the electron transport system. This observation suggests the decline in contractile activity was due to a decrease in the oxidative formation of ATP, and also indicates the response of isolated myocytes to hypoxia is dissimilar to the response of in vivo myocytes to conditions of ischaemia.

Sensitivity of flavoprotein fluorescence to oxidative state in single isolated heart cells.

The contribution of flavoprotein fluorescence to redox sensitive cellular autofluorescence was studied in single isolated adult rat heart cells. Fluorescence was measured quantitatively under conditions stimulating flavoprotein fluorescence in cells subjected to no inhibitors (sham), to cyanide, to 2,4-dinitrophenol, and to hypoxia. It was found that fluorescence apparently due to flavoproteins could be measured in single cells, and the fluorescence was sensitive to the redox state of the cell. Comparison with NADH fluorescence from single cells indicated that, while flavoprotein fluorescence was less intense, excitation of flavoproteins caused less bleaching of fluorescence when compared to the effect of excitation on NADH fluorescence.

Release of lactate dehydrogenase during isolation of adult rat heart cells.

In order to determine the least injurious method of cell isolation, effluent perfusates from isolated rat hearts were examined for lactate dehydrogenase activity during cellular isolation procedures. Correlation of these results with the perfusates, with modifications in the perfusates, and with the yield of intact cells isolated, indicated that perfusion of isolated hearts with solutions containing bovine serum albumin and/or collagenase can result in severe cellular injury. These effects were significantly modified by the presence of calcium ion. These results indicated that the concentration of calcium ion during isolation is critical to successful isolation of calcium tolerant, functional adult heart cells.

Functional role of collateral flow in the ischaemic dog heart.

Studies were carried out in four different canine preparations to determine the effect of collateral flow on early changes induced by acute segmental ischaemia. The results of these experiments indicated that collateral flow provided a limited and variable amount of protection. When collateral flow was eliminated, myocardial cell injury was more severe and less variable.