Hsp27 protects the cytoskeleton and nucleus from the effects of 42 degrees C at pH 6.7 in CHO cells adapted to growth at pH 6.7.

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42 degrees C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42 degrees C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42 degrees C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42 degrees C-induced perinuclear collapse and resistance to 42 degrees C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.

Cellular localization of the bcl-2 protein and response to glucocorticoid stress.

We performed immunoelectronmicroscopy, immunofluorescence and subcellular fractionation studies of insect cells (Spodopetra frugiperda or SF9) infected with recombinant baculovirus containing bcl-2 cDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in pre-B cells carrying a bcl-2 gene activated by t(14;18) chromosomal translocation. By immunogold electron microscopy, bcl-2 was localized at several intracellular sites including the nuclear membrane, endoplasmic reticulum, mitochondria and plasma membrane. Immunofluorescence studies revealed the presence of the bcl-2 product throughout the cytoplasm, whereas biochemical fractionation studies indicated a similar pattern to that observed on electron microscopy. Our investigation clearly indicates that the bcl-2 product is expressed at several intracellular sites. Studies were also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in the distribution of bcl-2 protein were observed, more aggregated patches of gold labelled bcl-2 particles were found under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization necessary to prevent apoptosis.

Lack of endothelium-dependent relaxation in coronary resistance arteries of cholesterol-fed rabbits.

The endothelium-dependent contractile responses of subepicardial coronary resistance arteries (286 +/- 18 microns ID, n = 22) from rabbits fed either a 0.5 or 2.0% cholesterol-enriched diet or a control diet for 10-12 wk were determined under isometric conditions at the optimum length for active force production (Lo). After the development of tone with 29 mM K+-Krebs, arteries from control rabbits treated with acetylcholine (0.1-10 microM) showed a concentration-dependent relaxation, with a maximum decrease in tone of 63%. In contrast, coronary arteries from animals fed 0.5 and 2.0% cholesterol contracted to acetylcholine (approximately 210% increase in tone). A similar phenomenon was seen with arteries precontracted with 10 nM 9,11-methanoepoxy-prostaglandin H2 (U 46,619), a thromboxane A2 mimetic. The contractile responses to acetylcholine occurred in arteries in which the endothelium was structurally intact and which were devoid of plaque. Arteries from cholesterol-fed animals were poorly responsive to ADP (0.01-10.0 microM), whereas arteries from normal animals relaxed. All arteries relaxed to an equal degree when exposed to acidified nitrite, which produces nitric oxide (NO). The data suggest that as a result of hypercholesterolemia, there may be a dysfunction in the synthesis or release of endothelium-derived relaxing factor (EDRF) by the endothelial cells of coronary resistance arteries, rather than an abnormality of the smooth muscle cells per se.

Ultrastructure of human platelets following indium-111 labeling in plasma.

Platelets labeled with 111In have become increasingly useful in monitoring their role in hemostasis and response to vascular injury by external detection. Platelets (10(9) ml-1) labeled with 600 microCi 111In in saline have been shown previously to have an 'overdeveloped' canalicular system and to have aggregated dense bodies. We have labeled 1.35 X 10(9) human platelets with 367 to 1170 microCi 111In in plasma and studied their in vitro aggregability and ultrastructure by transmission electron microscopy for up to five days post labeling. Studies indicated that platelets labeled with 111In in plasma did not suffer from functional or ultrastructural changes keeping mitochondria, canalicular system, alpha granules and microtubules as structurally similar to those in unlabeled control platelets.

Model system to study interaction of platelets with damaged arterial wall. I. Inhibition of platelet adhesion to subendothelium by aspirin and dipyridamole.

An experimental model system has been developed to study the interaction of platelets with a damaged vessel wall, in vivo, without deep surgical intervention. Endothelium of the central ear artery of an anesthetized rabbit is damaged by placing artery forceps on the ear directly over the vessel. Forceps are removed 30 min later and blood flow resumes. After 30 min, blood is washed out with Tyrode's solution and the vessel is perfused with 1% glutaraldehyde solution. Tissue containing the vessel is then removed and further prepared for scanning and transmission electron microscopy. Damage to the endothelium observed by scanning electron microscopy included separation of adjacent endothelial cells (EC); partial destruction and lifting up of some EC, exposing subendothelium; and denudation of larger areas of endothelium. Disc-shaped platelets were seen clinging to some damaged endothelial cells. Platelets adhered, formed pseudopods, and spread over the surface of some areas of exposed subendothelium. The extent of platelet adhesion to exposed subendothelium was estimated by eight "blind" evaluators and the average taken. Aspirin (8 mg/kg, ip) significantly inhibited adhesion (P less than 0.05) when compared to controls. No other agent tested gave significant inhibition after a single treatment. Dipyridamole (1.5 mg/kg, ip) given five times on 3 successive days, inhibited adhesion significantly (P less than 0.001). Heparin (800 U/kg, iv) or dipyridamole (0.7 mg/kg, ip) enhanced the inhibitory effect of aspirin (8 mg/kg, ip), with either combined treatment giving P less than 0.001.

Early morphological changes in the endothelium of a peripheral artery of rabbits fed an atherogenic diet.

The effect of an atherogenic diet on the endothelium of the central artery of the rabbit ear was studied by scanning and transmission electron microscopy. Examination of the inner surface of the artery after only 5 weeks on the diet revealed morphological changes including irregularly shaped cells, breaks at intercellular junctions, swelling of the membrane over the central part of the cells, and occasional holes in the cells. By 9 weeks more severe damage was seen including lifting off of cells and some holes. In addition, arrays of dark spots were seen by scanning electron microscopy in some cells in arteries of rabbits fed the diet for 5 to 9 weeks. The dark spots may correspond to abnormally large vacuoles seen inside endothelial cells by transmission electron microscopy. The central ear artery and aorta of some rabbits were tested for their ability to convert arachidonic acid into prostacyclin and thromboxane A2 by radioimmunoassay of the stable metabolites 6-keto-prostaglandin F1 alpha and thromboxane B2. The vessels from rabbits fed the atherogenic diet did not differ from vessels of rabbits on the control diet in their production of either metabolite.

Backscattered electron imaging to visualize arterial endothelial detachment in the scanning electron microscope.

The endothelium of the central ear artery of an anesthetized rabbit was damaged by placing artery forceps on the ear over the vessel for 30 min. After removal of the forceps, blood was allowed to flow through the injured vessel for 30 min. Blood flow was then stopped, blood was washed out of the artery in situ and initial fixation with 1% glutaraldehyde was begun. Specimens of the vessel containing the damaged region were postfixed in 3% cacodylate buffered glutaraldehyde and prepared for SEM examination after methenamine silver staining or following post fixation with osmium. Backscattered electron imaging (BEI) of silver stained specimens with inverted signal polarity showed "black" endothelial nuclei similar to darkly stained nuclei of conventional light microscope slide preparations. Secondary electron imaging (SEI) of corresponding sites stained with silver also showed endothelial nuclei. An interruption of the normal pattern of nuclei indicated a detached area of endothelium where subendothelium was exposed. Platelets were observed adhering to the surface of the exposed subendothelium. Injury to the endothelium was seen more readily in silver stained preparations as voids in the nuclear pattern as compared with conventional SEI preparations, postfixed with osmium, which did not show well defined nuclei.

Prostacyclin production in rabbit arteries in situ: inhibition by arachidonic acid-induced endothelial cell damage or by low-dose aspirin.

The central artery of the rabbit ear was perfused in situ and effluent fractions from the artery were assayed for 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha) and thromboxane B2 (TxB2), the stable metabolites of prostacyclin (PGI2) and TxA2, using specific radioimmunoassays. These metabolites of arachidonic acid (AA) were not detected in the effluent during infusion of Tyrode's solution but both metabolites were detected when small amounts of AA were infused into the artery. Examination of the arteries by scanning electron microscopy revealed that high concentrations of AA which caused a short burst of 6-K-PGF1 alpha and TxB2 production damaged the endothelial cells while lower concentrations which stimulated continuous production did not cause damage. When a non-damaging concentration of AA was infused into an artery that had previously received a damaging concentration, PG production was greatly reduced. Pretreatment of the rabbits with 4 mg/kg acetyl-salicylic acid (ASA) inhibited 6-K-PGF1 alpha production by the rabbit ear artery in response to AA and 70% inhibition was still evident 18 hours after ASA.

A model system for studying initial events in atherosclerosis.

A model system has been developed which employs the central artery of the rabbit ear, and allows for the study of 1) ultra-structural changes in the arterial endothelium and 2) formation and release of prostacyclin and thromboxane from the arteries in situ. Use of the model should be helpful in evaluating the initial events in atherosclerosis. Both prostacyclin and thromboxane (detected by radioimmunoassay) were formed by arteries in situ in response to infusion of sodium arachidonate.

Model system for the study of inital damage to arterial endothelial cells in situ by scanning electron microscopy.

The central artery of the rabbit ear was injected with either 1 ml of control or test solutions in a model system which allowed for the study of effluent fractions coming from a segment of the artery and for the examination of the vessel by electron microscopy. The procedure allowed for either mixing of blood with the test solution ("flow by") or injection of the solutions in the absence of blood ("no flow by"). Effluent fractions were collected and assayed for 6-keto-PGF1 alpha, and indicator of PGI2 production. After the collection of effluent fractions, the artery was perfused with 1% glutaraldehyde to begin fixation. Ultrastructural damage to endothelial cells was seen, by SEM, in a graded response to sodium arachidonate solutions at concentrations between 6.6 and 3.3 mM ("flow by"); and between 3.3 and 0.83 mM ("no flow by"). Lower concentrations of arachidonate did not cause damage. Damage included enucleation, separation or denudation of endothelial cells. Sodium linoleate produced similar damage. A burst of PGI2 production appeared to be associated with arachidonate-damaged endothelium. Injection of sodium arachidonate at lower concentrations (0.33 mM and 0.165 mM) resulted in continuous production of PGI2 over a period of 1 to 5 minutes.

Fatty acids and the initial events of endothelial damage seen by scanning and transmission electron microscopy.

A method was developed for observing changes in the endothelial cells in rabbit ear veins in vivo by scanning electron microscopy. Injection of fatty acids into the ear vein caused damage to the endothelium. The first signs of damage seen were marked bulges in the nuclei and loss of the rhomboidal shape of the endothelial cells. More severe damage included loss of nuclei, leaving holes in the cytoplasm. Some parts of the damaged endothelium showed complete separation of cells from each other and exposure of sub-endothelial tissue to which platelets with pseudopodia were adhering. Damage to the endothelium was produced by arachidonic, linoleic, gamma-linolenic, 8,11,14-eicosatrienoic, 5,8,11,14,-eicosatetraenoic or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acids. The effect of arachidonic acid was not prevented by pre-treating the animals with aspirin. It appears that damage produced by the fatty acids is non-specific.