Ovidio Bussolati

Employment of Confocal Microscopy for the Dynamic Visualization of Domes in Intact Epithelial Cell Cultures

Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 µM secretin or 10 µM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.

Endothelial cell injury induced by preservation solutions: a confocal microscopy study

BACKGROUND We evaluated the effects of standard preservation solutions on cultured human greater saphenous vein endothelial cells. METHODS Endothelial cells (eight strains) were preincubated for 6 or 24 hours at 4 degrees C in Celsior, Euro-Collins, St. Thomas Hospital II, and University of Wisconsin solutions, reincubated in warm oxygenated culture medium 199, and observed up to 48 hours. Culture viability was assessed through cell counting and confocal microscopy of calcein loaded cells. RESULTS Incubation in both Euro-Collins and St. Thomas, but not in Celsior or University of Wisconsin solutions, caused significant cells losses and diffuse morphological damages characterized by solution-specific distinctive alterations. Injury caused by 6-hour, but not by 24-hour treatment, was reversible. CONCLUSIONS The incubation with Celsior and University of Wisconsin solutions substantially preserved endothelial viability and proliferative capability. Conversely, a prolonged incubation in either Euro-Collins or St. Thomas solutions caused severe and potentially irreversible damage referable to the induction of, respectively, apoptotic or necrotic changes.

Thermodynamics and Transport

The total free energy change of a transport process or a series of processes may have numerous obvious components. Knowledge of these components frequently has practical implications for the interpretation of transport experiments. In addition, knowledge of the free energy changes associated with each component of transport is required in order to fully understand how transport contributes to the work performed by cells. It is proposed in this chapter that the free energy changes associated with the components of primary active transport processes may include much larger changes in the magnitude of enthalpy and entropy than are associated with ATP synthesis or hydrolysis. In addition to the free energy available from solute transport, the cell depends on transport occurring rapidly enough to do work that is useful to it. Transport proteins are the catalysts that permit solutes and the solvent to migrate across biomembranes much more rapidly than they can by permeating phospholipid bilayers. Although ordinary diffusion of solutes and the solvent may be relatively rapid over short distances in intracellular and extracellular aqueous solutions, such diffusion is virtually halted across thin biomembranes.

Effect of Nd:YAG laser light on post-extractive socket healing in rats treated with zoledronic acid and dexamethasone

Introduction The effect of low level laser therapy (LLLT) on the healing process could be useful for the prevention of post-extractive Bisphosphonate-related Osteonecrosis of the Jaws (BRONJ). The aim of the study was to investigate the effect of LLLT on the post-extractive socket healing in rats treated with zoledronic acid and dexamethasone. Material and Methods Thirty male Sprague-Dawley rats were divided in 4 groups: control group (C, n = 5), laser group (L, n = 5), treatment group (T, n = 10) and treatment plus laser group (T+L, n = 10). Rats of group T and T+L received zoledronate 0,1 mg/Kg and dexamethasone 1 mg/Kg every 2 days for 10 weeks. Rats of group C and L were infused with vehicle. After 9 weeks the first maxillary molars were extracted in all rats. Rats of groups L and T+L received laser therapy (Nd:YAG, 1064 nm, 1.25W, 15Hz, 5 min, 14.37 J/cm2) in the socket area at days 0, 2, 4 and 6 after surgery. At 8 days from extraction, the sockets were clinically assessed with a grading score and the wound area was measured with a dedicate software. Histomorphometric evaluation and western blot analysis of osteopontin and osteocalcin expression were performed. Results Group T+L showed a trend toward a better clinical grading score compared to group T (grade I 22% Vs 28 % - grade II 56% Vs 28% - grade III 22% Vs 44%, respectively). The average wound area was similar among the groups. Inhibition of osteoclastic alveolar bone resorption was found in groups T and T+L (P<0.001). Rats of groups L and T+L showed a significant higher expression of osteocalcin compared to rats of groups C and T (C=0.3993; L=1.394; T=0.2922; T+L=1.156; P=0.0001). The expression of osteopontin did not show significant differences in the groups treated with Nd:YAG compared to the ones that did not receive laser irradiation. Conclusion Our findings suggest that laser irradiation after tooth extraction can promote osteoblast differentiation, as demonstrated by the higher expression of osteocalcin. Thus, laser irradiation could be considered a way to improve socket healing in conditions at risk for MRONJ development.

Structure and Function of Transport Proteins That Form Solute Gradients

This chapter focuses on the transport ATPases in detail. Many of the known details of the structure and function of P- and F-type ATPases have been described. While the models developed for the thermodynamic coupling between ATP synthesis (or hydrolysis) and ion transport have conjectural components, they are in general founded on a large amount of experimental data and on the resultant ingenious hypotheses of several investigators. One needs to critically assess these impressive findings and insights in order to understand both what we do and what we do not know about how P- and F-type ATPases catalyze transport. While these general similarities in the structure and function of P- and F-type ATPases have undoubted importance, the differences in the details of their proposed structures and mechanisms of action are striking.

Importance of Biomembrane Transport

The study of biomembrane transport requires the consideration of its biophysics and physical chemistry, as well as its biology. Moreover, biomembrane transport is central to the functioning of all multicellular organisms regardless of whether it is considered at the subcellular, tissue, or systemic levels of their organization. Similarly, the study of biomembrane transport is as legitimate a component of investigations into mechanisms of development and differentiation as it is into the functioning of fully formed tissues and organs. Hence, there is scarcely a biological journal or a subsection within such journals from the biophysical to the evolutionary levels of investigation that does not contain articles on the subject of biomembrane transport. By analogy, numerous proteins have evolved over billions of years to catalyze transport of solutes and the solvent across the barriers formed by biomembranes. As the demand for different types of transport increased owing to evolution of numerous as well as more complex species, the needed transport processes also evolved. Similarly, as investigations have led to a fuller understanding of biology, the disciplinary boundaries that once helped to define ourselves now help more easily to recognize the various aspects of biology to which unfolding work may apply. Moreover, the contributions of these biomembrane transport processes to the physiological functioning of cells and organisms is rooted in the physical and chemical nature of biomembrane barriers and how the barriers may change in various physiological and pathophysiological conditions.

Regulation of Plasma Membrane Transport

Starting from the earliest studies on mammalian plasma membrane transport, it has become apparent that transport activities, such as enzyme activities, are subject to both short- and long-term regulation. In the past few years, the cloning and sequencing of cDNA for various transport proteins has allowed the exploration of the mechanisms of transport regulation in greater molecular detail. This chapter covers the study of the regulation of transport from the original kinetic experiments to the most recent work using transgenic animals. It also presents a description of the major factors through which the activity of transporters can be modulated. General principles are described in terms of regulation of activity due to changes in transport-driving force, modifications of endogenous transporters, or increase in the number of transporter molecules in the membrane. For each category, selected examples have been considered in detail. As far as possible, the physiological significance of these changes is also discussed. Finally, the chapter reviews the role of disorders of transport regulation in two pathological conditions of wide interest.

Biomembrane Composition, Structure, and Turnover

Much is being learned about how plasma membrane composition and structure are regulated. The anatomy of vesicular trafficking has been described reasonably well, and the biochemical processes that achieve proper targeting of lipids and proteins are beginning to be elucidated. Information within the structures of proteins is being discovered that helps to target them to, or retain them in the endoplasmic reticulum, various portions of the Golgi, lysosomes, endosomes, or the plasma membrane. Moreover, investigators in this field are beginning to study how the cell deciphers this information. The mechanisms by which the cytoskeleton and its associated motor proteins help to move vesicles to the correct destinations are also being described, as are the coat, docking, and fusion proteins that result in formation, recognition, and fusion of vesicles with the correct target compartments. One of the most challenging problems in the field appears to be to describe the processes by which lipids and proteins are sorted to apical membranes of polarized cells, since sorting to this compartment appears to differ mechanistically from much of what is known about sorting and vesicular trafficking. In contrast to the emerging nature of knowledge concerning modulation of the function of membrane proteins by membrane lipids and the cytoskeleton, much is already known about the biochemical actions of the proteins. The action of biomembrane transport proteins depends on the formation of a barrier to the free mixing of intracellular and extracellular constituents by the membrane lipid bilayer. Moreover, transport across the membrane barrier frequently results in or requires work.

Channel Proteins Usually Dissipate Solute Gradients

In contrast to transport proteins that form (Chapter 5) or propagate (Chapter 6) solute gradients by coupling transport to conspicuous sources of free energy, channel proteins appear to form pores through which ions pass by diffusion. The unusual characteristics of the proposed diffusion have been attributed to the narrowness of the channels and the effects on diffusion that such physical constraints produce. As the reader is by now aware, however, we view the transport catalyzed by channels as involving intimate contact between the transport protein and its substrate, as is the case for proteins that produce and propagate solute gradients. Moreover, it is likely to us that during their transport cycles, all of these proteins undergo at least small changes in their conformations that resemble such changes in enzymes and other transport proteins (e.g., Marban and Tomaselli, 1997 ). 1 This view also is supported by computer simulation of water migration across membranes via voltage-gated Na + channels. In this simulation, water and presumably Na + migration can occur when the channel protein is allowed to have the motion that it would have at 300°K but not when its motion is virtually frozen (Jakobsson, 1997). While the magnitudes of such protein motions that are needed for transport may vary widely among the proteins, we propose that virtually all transport proteins need to move to some extent in order to catalyze solute or solvent migration across biomembranes (see also Section III below).

Transport Proteins That Propagate Solute Gradients

If transport proteins migrated across membranes as carriers, solutes could conceivably drive them to one face of membranes with their total chemical potential gradients. In the case of, say, an antiporter, the protein could then follow its gradient back to the other face of the membrane carrying a different solute against its gradient. Transport proteins do not, however, migrate across membranes to catalyze transport. Rather, they undergo conformational changes while embedded in them. In the case of transport ATPases, such conformational changes are believed to be a part of the route of transfer of the free energy in phosphoric acid anhydride bonds to that of solute gradients. In contrast to some assessments, it is found in this chapter that the kinetics of antiport and symport may be at least as complex as the kinetics of primary active transport by P- and F-/V-type ATPases. Neither a ping-pong nor a two-site simultaneous model accounts well for the antiport catalyzed by anion exchange (AE) proteins. Moreover, this antiport may exhibit apparently hyperbolic kinetics as well as positive and negative cooperativity depending upon the conditions under which transport is measured. The function of AE proteins is further complicated in vivo owing to their also serving in some cases as channels for regulation of cellular volume. These transport functions may be regulated through interdependent associations of the proteins with other membrane proteins, the cytoskeleton, and cytosolic enzymes involved in intermediary metabolism.