Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

OBJECTIVES: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

Gap26, a connexin mimetic peptide, inhibits currents carried by connexin43 hemichannels and gap junction channels.

Connexin mimetic peptides corresponding to short conserved extracellular loop sequences of connexins have been used widely as reversible inhibitors of gap junctional intercellular communication. These peptides also block movement of ATP and Ca(2+) across connexin hemichannels, i.e. hexameric channels yet to dock with partners in aligned cells and to generate the gap junction cell-cell conduit. By means of electrophysiology, we compared the effects of Gap26, a mimetic peptide corresponding to a short linear sequence in the first extracellular loop of connexin43, on connexin channel function in HeLa cells expressing connexin43. We demonstrate that Gap26 inhibited electrical coupling in cell pairs mediated by gap junctions after exposure for 30min. In contrast, Gap26 applied to single cells, inhibited hemichannel currents evoked in low Ca(2+) solution with a response time of less than 5min. The results further support the view that the likely primary and direct inhibitory effect of Gap26 is on connexin hemichannels, with gap junctions becoming inhibited later. The mechanism of action of Gap26 in blocking hemichannels and gap junction channels is discussed in the context of their different functions and locations.

Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous.

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.

Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

The effect of polystyrene beads on cyclic 3',5'-adenosine monophosphate concentration in leukocytes.

After incubation with polystyrene latex beads for 5 min. the cyclic 3',5'-adenosine monophosphate (cyclic AMP) content of human peripheral blood leukocyte suspensions was increased severalfold. Preparations enriched in mononuclear cells and containing only 0-20% polymorphonuclear leukocytes (PMN) and no visible platelets exhibited a quantitatively similar response. Purified fractions of cells containing 85-90% PMN responded to polystyrene beads with a much smaller increase in cyclic AMP content. Phagocytosis of paraffin oil emulsion in the unfractionated mixed human leukocyte preparation was associated with little or no change in cyclic AMP levels. There was no change in cyclic AMP content of rabbit alveolar macrophages or guinea pig PMN during phagocytosis of polystyrene beads. All of these observations are consistent with the view that particle uptake per se does not increase cyclic AMP levels in phagocytic cells. It seems probable that the increase in cyclic AMP concentration that results when unfractionated human blood leukocytes are incubated with polystyrene beads occurs in cells other than PMN.

Purification and properties of cytidine deaminase from normal and leukemic granulocytes.

Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70 degrees C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10(-5) M) than for ara-C (8.8 x 10(-5) M) or 5-azaC (4.3 x 10(-4) M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2'-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10(-8) M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52+/-1.86 x 10(3)/mg protein) than chronic myelocytic leukemia (CML) cells (1.40+/-0.70 x 10(3) U/mg protein) or acute myelocytic leukemia (AML) cells (0.19+/-0.17 x 10(3) U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation rather than a specific enzymatic defect in leukemic cells.

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

A deletion in chromosome 1 in cells of a transplantable granulocytic leukemia (GL-13) in guinea pigs.

Chromosomes were examined in leukocytes from a unique transplantable granulocyte leukemia (GL-13) of strain 13 guinea pigs. The GL-13 leukemia has many similarities to human chronic myelogenous leukemia (CML). It has remained predominantly diploid after 90 transplant generations and is characterized cytogenetically by a terminal deletion of the long arm of one of the #1 pair of chromosomes. No other chromosomal abnormality was observed. These findings lend further support to the conclusion that the GL-13 leukemia may be a useful model for the study of human CML.

Progressive loss of phenotypic proteins in mature granulocytes before the onset of blast crisis in human chronic myelogenous leukemia.

A combined high-performance liquid chromatography-sodium dodecyl sulfate-gel electrophoresis method for the study of the phenotypic protein patterns of mature blood granulocytes was previously described. With the use of this method in the present study, the progression of human chronic myelogenous leukemia (CML) from the stable to the blast crisis stage was shown to be accompanied by a progressive decrease in the amounts of cell membrane and granule phenotypic proteins in mature granulocytes. Survival time from the initial diagnosis was significantly shorter for CML patients whose levels of granulocyte phenotypic proteins were below the normal range compared with survival time for those patients whose levels were normal or higher than normal. The data suggest that these changes in mature granulocytes serve as useful diagnostic indicators of an impending blast crisis in CML patients.

Direct analysis of differentiation proteins in normal and leukemic human granulocytes by high-performance liquid chromatography.

The feasibility of the use of reverse-phase high-performance liquid chromatography (RP-HPLC) for detecting differences in the protein phenotypes of highly purified fractions of normal and chronic myelogenous leukemic (CML) mature granulocytes isolated from peripheral blood was determined. At least 21 protein peaks were consistently and reproducibly detected in the RP-HPLC profiles of acetonitrile-trifluoroacetic acid extracts of normal granulocytes. This assay takes only 60 minutes to perform and can be done on 4 X 10(6) granulocytes (approximately the number of granulocytes in 1 ml normal blood). Furthermore, sodium dodecyl sulfate-gel electrophoresis of the RP-HPLC fractions provides a second dimension to the analysis of the polypeptide pattern of these cell lysates. Analyses of subcellular fractions by these methods revealed that most of the major peaks in the RP-HPLC profiles of intact granulocytes originate mainly from the granule and membrane fractions. Although the protein phenotypes of mature granulocytes were remarkably uniform among normal individuals, those of mature granulocytes obtained from the blood of CML patients were consistently abnormal and varied considerably among individual patients. The results indicate that the approach used here could have useful application in the study of abnormal granulocyte differentiation in leukemia.

Transplantable granulocytic leukemia in strain 13 guinea pigs.

The occurrence of a granulocytic leukemia in 1 of 40 female strain 13/N guinea pigs given N-nitroso-N-butylurea continuously in their drinking water for 21 weeks is reported here. This leukemia has been successfully transplanted in this guinea pig strain for 13 transplant generations by i.p. inoculation of leukemic blood or marrow cells. Macroscopically and microscopically, this leukemia resembles the chronic myelogenous form in humans. Histochemical studies showed, however, that unlike the human leukemic cells those in the leukemic guinea pigs are alkaline phosphatase positive. Electron microscopic studies of the guinea pig leukemic cells revealed the presence of numerous intracisternal A-type particles that are not found in corresponding normal leukocytes.

Isolation and partial characterization of glycolipids and a carbohydrate from the secondary granules and plasma membrane of polymorphonuclear leukocytes.

Chloroform/methanol extracts of the secondary granule and plasma membrane fractions of polymorphonuclear leukocytes have been shown to contain both non-polar and polar carbohydrate-containing materials. The ratio of the polar to the non-polar material was much higher in the plasma membrane than the secondary granule fraction. The non-polar material contains at least two ceramide-like glycolipids and accounts for most of the broad band of periodic acid/Schiff-positive material which migrates at the dye front in sodium dodecyl sulfate electrophoretic gels of granule and plasma membrane extracts. The polar material appears to be a single substance containing no fatty acids or sialic acid and is composed of glucose, hexosamine and a carboxylic acid derivative of pentose. Expressed on a per mg of protein basis, the amount of carbohydrate associated with the polar material in the plasma membrane fraction was about five times that of the secondary granule fraction.

Differences in protein and glycoprotein patterns between mature and immature neutrophil leukocytes.

The post-nuclear particulate fractions of highly enriched mature and immature neutrophil preparations were dissolved in buffer containing sodium dodecyl sulfate and analyzed for protein and glycoprotein components by polyacrylamide gel electrophoresis. The electrophoretic patterns of mature and immature neutrophils showed pronounced differences in both protein and glycoprotein bands. There were 12 prominent protein bands in the patterns of mature neutrophils which also stained for glycoproteins. With one exception, these bands were either greatly reduced in intensity or undetectable at corresponding positions in the patterns of immature cells. The patterns of immature neutrophils revealed three prominent glycoprotein bands, two of which were absent and the third was greatly reduced in intensity at corresponding position in the mature cell patterns. The results indicate that a number of new glycoproteins appear in the later stages of neutrophil maturation, whereas other glycoproteins, present in immature cells, are either lost or greatly reduced in amount. These changes are presumably related to the development of specific cell functions that also appear in later stages of cell maturation.

Changes in unsaturated fatty acids in serum lipids of hamsters infected with schistosomes (S. mansoni).

Measurements have been made of the fatty-acid composition of lipids in normal hamster sera compared with sera of hamsters infected with Schistosoma mansoni. The results show a consistent decrease of linoleic and eicosapentaenoic acids, and an increase in arachidonic acid in the phosphatidylcholine and lysophosphatidylcholine of the infected animals. Arachidonic acid was also increased in cholesterol ester, and docosahexaenoic acid was raised in free fatty acid and in phosphatidylcholine in sera from infected animals.

Partial purification of an intercalated disc-containing cardiac plasma membrane fraction.

The sarcolemma of cardiac muscle cells contains a specialised junctional region, the intercalated disc which includes three types of intercellular junction, the macula and fascia adherens and the nexus or gap junction. To facilitate the isolation of these junctions a procedure for the partial purification from mouse hearts of a subcellular fraction containing the intercalated disc region of the sarcolemma was developed. This involved investigating methods of tissue disruption that preserve the integrity of the intercalated disc and minimise myofibrillar entrapment of organelles. Examination of the distribution of marker enzymes showed that relative to the homogenate the intercalated disc fraction prepared by sucrose density centrifugation was only enriched 1.5- to 3-fold in 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas mitochondrial and sarcoplasmic reticulum marker enzymes were low. The properties of the intercalated disc-containing fraction were compared with the vesicular sarcolemmal fractions devoid of junctional complexes prepared by other methods.

The lipid composition of plasma membrane subfractions originating from the three major functional domains of the rat hepatocyte cell surface.

1. The neutral and phospholipid compositions of three rat liver plasma membrane subfractions originating predominantly from the three major functional domains of the hepatocyte viz the blood sinusoidal, contiguous and bile canalicular fractions, were determined. 2. The sinusoidal and canalicular plasma membrane subfractions, both of which were vesicular, contained a higher lipid to protein weight ratio than the contiguous plasma membrane subfraction that consisted of membrane strips, junctional complexes and some larger vesicles. The three plasma membrane subfractions contained a similar neutral lipid to phospholipid ratio. The highest unesterified cholesterol content was associated with the canalicular plasma membrane subfraction. 3. The phospholipid profiles of the three subfractions were generally similar. However, the canalicular plasma membrane subfraction contained a higher proportion of sphingomyelin than the other subfractions. 4. Correlations between the neutral and phospholipid composition of the subfractions and membrane integrity and function are discussed, especially with respect to a possible role of lipids in governing the resilience of the canalicular plasma membrane to the action of bile salts.

Immunological evidence that plasma-membrane 5'-nucleotidase is a transmembrane protein.

Antibodies inhibiting specifically plasma membrane-bound 5'nucleotidase were used to determine the disposition of the enzyme in various lymphoma cell lines. Fluorescent Fab fragments of the inhibiting antibodies bound to MF2s and MOPC 173 plasmacytoma cells, whereas no fluorescence was observed with P 1798 thymoma cells in which the enzyme was absent. The relative potency of the antiserum in inhibiting the enzyme in right-side-out and inside-out plasma membrane vesicles prepared from the above cell lines indicated that various group of antigenic determinants were present. One group of antigenic determinants was present at the external face of the cell, and a second was associated with the inner surface of the membrane. A third group of antigenic determinants was located in the vicinity of the active site of the enzyme and it is the group that varied in the various plasmacytoma cells studied. The results are interpreted as immunological evidence that 5'nucleotidase is a transmembrane glycoprotein.

Biogenesis of plasmalemmal glycoproteins. Intracellular site of synthesis of mouse liver plasmalemmal 5'-nucleotidase as determined by the sub-cellular location of messenger RNA coding for 5'-nucleotidase.

1. Free and membrane-bound mouse liver polyribosomes were separated by prolonged density-gradient centrifugation of the post-mitochondrial supernatant. RNA was extracted from free and membrane-bound polyribosomes and mRNA purified by oligo(dT)-cellulose column chromatography. 2. Antisera against purified mouse liver plasma membrane 5'-nucleotidase and moust albumin were prepared and characterized. 3. Microinjection of equivalent amounts of mRNA from free and membrane-bound liver polyribosomes into Xenopus laevis oocytes indicated by immuno precipitation and sodium dodecylsulphate gel electrophoresis a higher proportion of mRNA coding for 5'-nucleotidase and serum albumin in membrane-bound polyribosomes than free polyribosomes. 4. Although small, significant amounts of serum albumin and 5'-nucleotidase were also coded for by mRNA purified from free polyribosomes. The results suggest that in vivo, mRNA in mouse liver membrane-bound polyribosomes codes for the synthesis of 17 times more 5'-nucleotidase than does the mRNA in free polyribosomes.

Fractionation of granule proteins of granulocytes by copper chelate chromatography.

The tendency of lactoferrin to associate with other proteins at low salt concentrations defeated attempts to fractionate the acetate-extracted granule proteins of guinea pig granulocytes by ion-exchange chromatography. This problem was overcome by using copper-chelate chromatography at high salt concentrations to separate lactoferrin from the other granule proteins. Lysozyme and a 'cationic protein' were purified to apparent homogeneity in the same operation. The chromatographic profile of proteins extracted from purified secondary granules differed from that of proteins extracted from total granules chiefly in the absence of a substantial 'cationic protein' peak.

Development of surface antigen during maturation of bone marrow neutrophil granulocytes.

The presence of neutrophil surface antigens on maturing bone marrow granulocytes was examined by indirect immunofluorescence and cellular immunoadsorption using heterologous antibody to mature neutrophil granulocytes. The results show that bone marrow neutrophils possess surface antigens that appear during cell maturation from myeloblasts. Fluorescent antibody capping and patching, indicators of antigen mobility, were more pronounced in mature than in immature cells. Neutrophil surface antigen development could also be demonstrated during granulocyte maturation in vitro.