Tullio Pozzan

The intracellular distribution of calcium

Abstract For a long time, the study of calcium distribution in neurons was plagued by artifacts and technological problems. Major progress in the field has now been made using a variety of techniques, such as electronprobe X-ray microanalysis of unfixed, rapidly frozen sections, subcellular fractionation and cell permeabilization, and by recently introduced indirect approaches ([Ca 2+ ] i topology revealed by fluorescent dyes and photoproteins; immunocytochemistry of Ca 2+ -binding proteins). The present interest is focused primarily on rapidly exchanging Ca 2+ pools that are responsible for the generation of [Ca 2+ ] i transients. These pools might reside in organelles (calciosomes and possibly others) indistinguishable by conventional EM, but molecularly and functionally distinct from the elements of the smooth endoplasmic reticulum (SER).

High tide of calcium in mitochondria

Mitochondria in intact cells can transiently accumulate calcium during cell stimulation. The heterogeneity of the response, the extremely high calcium concentrations reached in the mitochondrial matrix, and the ensuing modulation of secretion add further complexity to the spatiotemporal aspects of signalling through calcium ions.

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.

Chapter 8 Cytoplasmic Calcium in Phagocyte Activation

Publisher Summary The calcium ion has an unusual importance in biological phenomena. Changes in the cytosolic free calcium concentration [Ca 2+ ] i have evolved as a key intracellular messenger system to couple external stimuli to a variety of complex cellular responses. In phagocytic cells, (i.e., neutrophils and macrophages) changes in [Ca 2+ ] i follow stimulation of several receptors and are thought to be involved in the regulation of adherence, chemotaxis, phagocytosis, degranulation, and production of toxic oxygen metabolites. The challenge for future research in phagocyte physiology will be the discrimination of those responses that are [Ca 2+ ] i -activated and [Ca 2+ ] i -dependent from those simply associated with a change in [Ca 2+ ] i , the latter being due to the ligation of multifunctional receptors generating multiple signals and reactions. With the help of the new Ca 2+ chelators, which allow the manipulation of [Ca 2+ ] i in intact cells, answers to these questions are already becoming available. Yet nothing is known of the “missing messengers” that mediate phagocyte responses in those instances in which no generation of known intracellular signals has been documented.

Monoclonal antibodies against HLA class I antigens inhibit human lymphocyte proliferation but do not affect mitogen dependent second messenger generation.

Abstract The mechanism through which anti HLA class I monoclonal antibodies inhibit human lymphocyte proliferation induced by the policlonal mitogen phytohemoagglutinin was investigated. Anti HLA class I monoclonal antibodies inhibited mitogen stimulated DNA synthesis even when added several hours after phytohemoagglutinin. The extent of inhibition depended on the duration of the exposure of lymphocytes to the monoclonal antibodies. Anti HLA class I monoclonal Antibodies neither affected the rise in cytosolic fee Ca 2+ concentration nor the production of inositol phosphates induced by phytohemoagglutinin. These results demonstrate that anti HLA class I monoclonal antibodies inhibit lymphocyte proliferation at a step downhill of second messenger generation.

Frog brain expresses a 60 KDa Ca2+ binding protein similar to mammalian calreticulin

The present report was undertaken in an effort to characterize the nature of Ca2+ binding protein(s) in the central nervous system of less evolved vertebrates. In particular we investigated whether the brain microsomal fraction of Rana esculenta expresses calsequestrin, calreticulin and/or other related Ca2+ binding protein(s). We found that a 60 KDa protein having an NH2-terminal amino acid sequence similar to mammalian calreticulin is the major microsomal Ca2(+)-binding protein.

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.

Immunocytological identification of the microsomal calcium store of nonmuscle cells

The biochemical and functional similarities between skeletal muscle sarcoplasmic reticulum and the microsomal Ca2+ store of nonmuscle cells are discussed. It is shown that antibodies raised against two characteristic proteins of sarcoplasmic reticulum, Ca2+ ATPase and calsequestrin, recognize similar proteins in nonmuscle cells. The subcellular distribution of these two antigens was studied at the subcellular levels in ultrathin cryosections. In a variety of cell types these two proteins were found to be localized in small membrane enclosed vesicles, apparently distinct from other known organelles. We propose that these newly recognized structures (calciosomes) represent the functional equivalent of sarcoplasmic reticulum in nonmuscle cells.

Intracellular Source(s) of [Ca2+]i Transients in Nonmuscle Cells

A host of cellular functions are known to be regulated by the elevation of the cytosolic Ca2+ concentration [Caz+Ii, from the resting level around lo-’ M to higher values, usually in the micromolar range.’,’ For many years, the Ca2+ responsible for these changes has been known to originate from at least two different sources: the extracellular medium, where [Ca”] is millimolar; and intracellular storage organelle(s), whose calcium content can approach lo-’ M. Traditionally, some specific functions (for example, the release of neurotransmitters at nerve terminals) have been attributed primarily to the stimulation of Ca2+ influx via the activation of voltagegated Ca2+ channels; others (for example, striated muscle contraction) have been ascribed to release from intracellular store(s). In virtually all nonmuscle cells this latter phenomen has been recognized to be triggered by inositol1,4,5-trisphosphate (Ins-P,), a second messgener generated after the activation of a variety of surface receptors via the hydrolysis of a plasma membrane phospholipid, phosphatidylinositol4,5-biosphosphate.’ Activation of these same receptors causes increased Ca2+ influx through channels different from voltage-operated Ca2’ channels (VOCS), which have not yet been characterized in Other problems that at the moment appear still open are whether Ins-P, is the only intracellular messenger capable of inducing the release of Ca2+ from intracellular stores in nonmuscle cells; whether the intracellularevoked release of Ca2+ originates from one or multiple types of storage organelles; the cytological identification of these organelles; the effects the two processes (CaZ+ influx and release from intracellular stores) exert on the subcellular distribution of free CaZ+ in the various regions of the cytosol ( [Ca2+I1 topology). In this short review we will summarize the recent findings from our laboratories on these various problems.