In a previous study [Watanabe, H., W. Kuhne, R. Spahr, P. Schwartz, and H. M. Piper. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1344-H1352, 1991] metabolic inhibition (5 mM KCN + 5 mM 2-deoxy-D-glucose, for 2 h) was found to cause disintegration of F-actin filaments, cell retraction, and augmented paracellular macromolecule permeability in monolayer cultures of porcine aortic endothelial cells after a rapid depletion of ATP stores (90% in 5 min). These changes were reversible. In the present study, the nature of this cytoskeletal disintegration was investigated. 1) Disintegration of F-actin filaments within 2-h incubation under metabolic inhibition was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. 2) Cytosolic Ca2+ levels rapidly rose in metabolically inhibited cells; after 2 h a 10-fold increase was observed. 3) Presence of the Ca2+ ionophore A23187 (10 microM) mimicked the reversible effect of metabolic inhibition on F-actin filaments and monolayer permeability but not the extensive depletion of ATP stores. 4) Existence of the Ca(2+)-activatable actin-severing protein gelsolin in endothelial cells was demonstrated. The results show that during the reversible phase of endothelial energy depletion disintegration of F-actin filaments is only partial, since it is based on their fragmentation and not depolymerization. Increase in cytosolic Ca2+ levels seems to be the primary cause for the fragmentation, possibly through the activation of gelsolin.

Two stage specific cell-wall lytic enzymes (autolysins) from different strains of the unicellular, biflagellated green alga Chlamydomonas reinhardtii were isolated and purified to homogeneity. Quantitative and specific photometric assays for biological activity were worked out to follow fractionation and to establish lytic specificity and kinetics. The autolysins were studied for enzymatic properties and screened for biological activity towards several wall components obtained by salt extractions of sporangia and zoospores from C. reinhardtii. The autolysins are proteolytic enzymes, fragmenting proline-or hydroxyproline-containing polypeptides in structures like connective tissue. They attack predominantly selected domains within the walls of zoosporangia or gametes. The sporangial autolysins are not only site-and strain-specific but also stage-specific, whereas the gamete autolysins lyse cell walls of gametes as well as those of sporangia and zoospores.Lysis of cell walls is an essential event in the course of life of higher and lower plants [l]. It is the precondition for germination, growth, differentiation, fertilization and conjugation. In the vegetative and sexual cycles of the unicellular flagellated chlorophyte Chlamydomonas reinhardtii there are three stages in which cell wall lysis by the action of lytic enzymes (autolysins) occurs: (a) a sporangial autolysin releases the zoospores from the maternal sporangium; (b) a gamete autolysin dissolves the cell wall of the gamete prior to fusion; (c) an as-yet-unidentified autolysin opens the zygospores when the products of meiosis hatch. The autolysins involved in the first two processes seem to be extraordinarily specific. According to Schlosser [2], they are (a) strain-specific, i.e. they discriminate between cells of different strains of the genus Chlamydomonas; (b) stage-specific, i.e. they recognize the developmental stage of the algae. The sporangial autolysin dissolves only the wall of sporangia; the gamete autolysin acts on the walls of gametes, zoospores and sporangia as well.The gamete autolysin is secreted during agglutination of mating-type plus (mt') and minus (mt-) gametes. It is particularly interesting, because it produces protoplasts from zoospores [3]. It son of the two autolytic systems at the enzyme and the substrate level should provide a better insight into the mechanism of wall lysis, may explain the strain and stage specificity and should help to unravel the complicated composition of algal cell walls.We describe here the isolation and characterization of the sporangial autolysins from two cross-reacting strains of C. reinhardtii and the gamete autolysin of the same splitting group [2]. To follow purification and for enzyme kinetic studies, we have developed quantitative methods for the sporangial and gamete autolysin based on the liberation of wall fragments and protoplasts, respectively. Crude extracts and the purified autolysins were assayed for their hydrolytic activity towards synthetic substrates in order to characterize enzymat...

No abstract

The effect of the A2-adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) on macromolecule permeability (PM; indicator fluorescein isothiocyanate-labeled albumin) of endothelial cells was investigated using confluent monolayers of rat coronary microvascular endothelial cells (CEC) and porcine aortic macrovascular endothelial cells (AEC). In CEC, NECA (10(-7) M) increased PM by 39%. Similar results were obtained by isoproterenol (10(-6) M) and forskolin (10(-5) M). The effect of NECA could be antagonized by 8-phenyltheophylline (8-PT; 10(-5) M). In AEC, NECA (10(-7) M) caused an opposite effect in that it decreased PM by 26% as did isoproterenol (10(-6) M) and forskolin (10(-5) M). The response to NECA was abolished in the presence of 8-PT (10(-5) M). In AEC but not CEC, NECA could reduce the rise in PM caused by endothelial energy depletion (in the presence of 5 mM KCN and 5 mM 2-deoxy-D-glucose). It was common to AEC and CEC that NECA (10(-7) M), isoproterenol (10(-6) M), and forskolin (10(-5) M) stimulated production of adenosine 3',5'-cyclic monophosphate (cAMP). The stimulatory effect of NECA on production of cAMP could be antagonized by 8-PT (10(-5) M). In summary, the results indicate that in AEC and CEC PM is modulated by an A2-adenosine receptor-mediated stimulation of adenylate cyclase. The secondary effects of stimulation of adenylate cyclase are different in CEC and AEC, however, since it caused a reduction of PM in AEC, but an increase in CEC.

The dependence of macromolecule permeability (MP; indicator fluorescein isothiocyanate-labeled albumin) of endothelial cells on their energetic state was investigated using confluent monolayers of rat coronary microvascular endothelial cells and porcine aortic macrovascular endothelial cells. When oxidative and glycolytic energy productions were inhibited (5 mM KCN plus 5 mM 2-deoxy-D-glucose) 90% of the endothelial ATP contents were lost within 15 min, followed by a progressive increase of MP, disintegration of the actin cytoskeleton, and the opening of intercellular gaps. Elution of the blocker and a subsequent 3-h incubation in complete culture medium reversed the effects of 2-h metabolic blockade, completely for MP and partially for ATP levels. Coronary microvascular and aortic macrovascular endothelial cells responded similarly to energy depletion and repletion, the microvascular cells being more sensitive. The results demonstrate that 1) energetic inhibition augments endothelial macromolecule permeability when both oxidative and glycolytic energy production are inhibited, 2) increased macromolecule permeability in energy-depleted endothelial monolayers is caused by the opening of intercellular gaps, and 3) endothelial cells reversibly tolerate up to 2 h of almost complete ATP depletion.

No abstract

No abstract

No abstract