Membrane structure of caveolae and isolated caveolin-rich vesicles

 Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80–120 nm with an open porus of 30–50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30–50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles. Accepted: 16 September 1998     

Calcium metabolism in intact isolated thymocytes.

Isolated rat thymocytes incubated under proper metabolic conditions extrude Ca2+ previously taken up under metabolically unfavourable conditions. The extrusion can be supported by both respiratory and glycolytic energy but glycolysis seems to be more efficient for this purpose. La3+ (50--200 micron) and the ionophore A 23187 inhibit cell Ca2+ extrusion. Ruthenium Red (1--100 micron) does not influence cell Ca2+ extrusion while it inhibits the in situ mitochondrial cation uptake. All the results are consistent with a cell regulation model of Ca2+ content in which both plasma membrane and mitochondria co-operate, acting in opposite directions, in order to decrease cytosolic Ca2+ concentration. The possibility of Na+-Ca2+ hetero-exchange participation to cell Ca2+ homeostasis regulation is also discussed.     

Glycerolipid labelling kinetics in isolated intact chloroplasts.

Glycerolipid synthesis was studied in intact chloroplasts isolated from three different plant species. The sequential acylation of sn-glycerol 3-phosphate and lysophosphatidate (1-acyl-sn-glycerol 3-phosphate) was confirmed by monitoring the incorporation of oleate synthesized in situ into lysophosphatidate, phosphatidate and diacylglycerol. Lysophosphatidate was not only readily detected in these experiments, but was also present in the chloroplasts at the beginning of the time courses. The rate of glycerolipid synthesis depended primarily on sn-glycerol 3-phosphate supply, and given adequate sn-glycerol 3-phosphate, the proportion of newly synthesized fatty acids diverted into glycerolipids appeared to be determined by differing acyltransferase activities in the chloroplasts isolated from different plant species.     

AgNOR proteins from morphologically intact isolated nucleoli.

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.     

Lucigenin-derived chemiluminescence in intact isolated mitochondria

There are many data both in favor and against the use of lucigenin as a probe for superoxide anion (SA) in mitochondria, cells, and simple enzymatic systems. In the present work high concentrations (50-400 M) of lucigenin were used for continuous recording of rapid and reversible changes in the SA level in intact isolated mitochondria. The SA level in the presence of lucigenin rapidly and reversibly changed during the transition of the mitochondria from one functional state to another: under conditions of ATP synthesis from ADP and P_i, of Ca²⁺ accumulation, and of reverse electron transfer. Induction of a Ca²⁺,cyclosporin A-sensitive pore in mitochondria completely suppressed the lucigenin-derived chemiluminescence (LDC). The electron transfer in the Q-cycle of the respiratory chain complex III and high electric potential difference across the inner membrane of mitochondria were obligatory conditions for generation of a SA-dependent chemiluminescent signal. Based on our own and literature data, a scheme of LDC generation is suggested. The origin of superoxide anion detected in intact mitochondria with lucigenin is discussed.     

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.     

Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate.

1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogeneous precursors, 10 mM-propionate and 10mM-galactose, were linear for 1 h and were both stimulated by 1 muM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37 degrees, 22 degrees and * degrees C. Even under the best of the three conditions of storage that were tested (i.e. at 22 degrees C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceeded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 muM) and 2mM-butyrate accelerated the rate of glucose formation of liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 muM-glucagon. For lactate+pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1muM-glucagon and for both lactate+pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-cell gluconeogenesis by different mechanisms.     

Degradation of mucopolysaccharide in intact isolated lysosomes

The function of isolated lysosomes was studied by measuring mucopolysaccharide degradation. Cultured human diploid skin fibroblasts were grown in medium containing H235SO4 to label endogenous mucopolysaccharide. Lysosome containing preparations at various stages of purity were isolated from disrupted cells. These preparations degraded mucopolysaccharide as indicated by the release of radioactive sulfate. Degradation was temperature-dependent, required intact lysosomes, and was optimal when incubation was carried out at neutral pH in a buffer of low ionic strength. Lysosomes from Hurler fibroblasts were unable to carry out the degradative process. ATP at 0.5 mM was found to stimulate both the rate and the extent of mucopolysaccharide degradation; GTP, UTP, and CTP had similar effects, whereas the noncleavable ATP analog adenosine 5'-(beta gamma-imido)triphosphate gave no stimulation. The ATP stimulation was inhibited by nigericin. ATP also stimulated chloroquine accumulation in lysosomes, the magnitude of which was used to measure the change in intralysosomal pH. The presence of ATP was associated with acidification of lysosome pH by 0.23 units. Acetyl coenzyme A was also found to stimulate lysosome function. This reagent, however, had no effect on chloroquine accumulation and thus appears to stimulate mucopolysaccharide degradation by a mechanism different than that caused by ATP.     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

Membrane retrieval in epithelial cells of isolated thyroid follicles.

Follicles from rat and pig thyroid glands were isolated by digestion with collagenase. The epithelial cells of isolated follicles maintain their structural and functional polarity as shown by incorporation of 3H-leucine and autoradiography. To trace the fate of surface membrane, isolated follicles were opened, stimulated with thyrotropin and incubated for various time intervals with cationized ferritin (CF), uncharged dextran, native ferritin (NF), and latex spheres (0.5 mum in diameter) which were either pre-coated with CF or added together with CF. Uncharged dextran and native ferritin did not bind to the luminal cell membrane, were taken up in small amounts and accumulated in lysosomes; anionic NF was not found in Golgi cisternae in contrast to uncharged dextran which occassionally reached a few Golgi stacks. CF bound rapidly and in clusters to the luminal plasmalemma, preferentially to coated pits, was taken up by endocytosis, accumulated in lysosomes after 5 min and reached the Golgi cisternae after 30 min. Latex spheres were taken up by engulfment through fusion of microvilli and reached the lysosomes. CF particles coating the latex spheres may detach at this station and reach the Golgi cisternae. The findings show that the route of small tracers depends on the charge of the tracer, in agreement with results obtained by Farquhar [8]. Vesicles carrying NF can be traced to lysosomes only, whereas vesicles containing uncharged dextran or - more conspicuously -CF also fuse with Golgi membranes. Large tracers (latex beads) reach only the lysosomes, but CF taken up with them may move to Golgi cisternae.