Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

Three, non-cytosolic Ca2+ pools were characterized in intact PC12 cells. The first pool, sensitive to both inositol 1,4,5-trisphosphate and caffeine (Zacchetti, D., Clementi, E., Fasolato, C., Zottini, M., Grohovaz, F., Fumagalli, G., Pozzan, T., and Meldolesi, J. (1991) J. Biol. Chem. 266, 20152-20158) accounts for approximately equal to 200 microM of Ca2+/liter of cell water (less than 30% of total exchangeable Ca2+) and takes up Ca2+ from the cytosol via a Ca(2+)-ATPase, blocked by thapsigargin. A second pool, approximately equal to 400 microM/liter, is insensitive to both inositol 1,4,5-trisphosphate, caffeine, and thapsigargin and is released by the Ca2+ ionophore ionomycin. This pool is probably heterogeneous and its intracellular localization and physiological roles remain undefined. The third pool, approximately equal to 170 mumoles of Ca2+/liter, was discharged by the combination of ionomycin together with a substance that collapsed intracellular pH gradients, such as monensin or NH4Cl. This indicates that the pool is acidic, at variance with the first two. When exocytosis was stimulated, the size of this pool declined, indicating its primary residence within secretory granules. In the conditions of our experiments no major transfer of Ca2+ among the pools seemed to occur. This is the first comprehensive description of non-cytosolic Ca2+ pools investigated in intact neurosecretory cells by non-invasive procedures.

Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.

Astrocytes play a crucial role in proper iron handling within the central nervous system. This competence can be fundamental, particularly during neuroinflammation, and neurodegenerative processes, where an increase in iron content can favor oxidative stress, thereby worsening disease progression. Under these pathological conditions, astrocytes undergo a process of activation that confers them either a beneficial or a detrimental role on neuronal survival. Our work investigates the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes. Our data confirm that the main source of iron is the non-transferrin-bound iron (NTBI) and show the involvement of two different routes for its entry: the resident transient receptor potential (TRP) channels in quiescent astrocytes and the de novo expressed divalent metal transporter 1 (DMT1) in activated astrocytes, which accounts for a potentiation of iron entry. Overall, our data suggest that at rest, but even more after activation, astrocytes have the potential to buffer the excess of iron, thereby protecting neurons from iron overload. These findings further extend our understanding of the protective role of astrocytes under the conditions of iron-mediated oxidative stress observed in several neurodegenerative conditions.