Thomas Haller

The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release

To test for a possible role of lysosomes in intracellular Ca2+ homeostasis, the effects of glycyl-L-phenylalanine-beta-naphthylamide (GPN), known to permeabilize these organelles by osmotic swelling, were studied in single MDCK cells. Fluorescence of acridine orange, rhodol green dextran, lysotracker green and FITC-dextran indicated that GPN (0.2 mmol/l) elicited a reversible permeabilization of lysosomes. Cytosolic Ca2+ ([Ca2+]i) as determined by Fura-2 fluorescence increased from 60 +/- 11 to 534 +/- 66 nmol/l (n = 41) in the presence of GPN. Whereas only a single intracellular Ca2+ release could be induced by GPN in a Ca(2+)-free perfusate, repetitive release could be evoked in Ca2+ containing solutions suggesting reuptake of Ca2+ into lysosomal stores. GPN-induced Ca2+ release was blunted after pretreatment with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, or repeated applications of ATP inducing Ca2+ release from inositol trisphosphate (InsP3) sensitive Ca2+ stores. The effect of ATP on Ca2+ release was, however, not abolished by preceding GPN treatment. GPN-induced Ca2+ release from lysosomes was independent of InsP3 formation or Ca(2+)-induced Ca2+ release, since it was unaffected by the phospholipase C inhibitor U-73, 122 or by caffeine and ruthenium red. These results suggest that Ca2+ largely accumulates in lysosomal vesicles. Moreover, these organelles seem to be part or functionally coupled with InsP3-sensitive Ca2+ stores.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Ficks law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca2+ concentration ([Ca2+])c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a “fusion-activated“ Ca2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200–500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X4. Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca2+ influx via vesicular Ca2+ channels regulates fusion pore expansion and vesicle content release.

Threshold calcium levels for lamellar body exocytosis in type II pneumocytes

Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca2+concentration ([Ca2+]i). In fura 2-loaded cells, [Ca2+]iwas selectively elevated by flash photolysis of a cell-permeant caged Ca2+ compound ( o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca2+influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca2+]ielevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca2+]iand thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca2+. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca2+ signal below ∼300 nmol/l. Our results suggest a highly Ca2+-sensitive step in LB exocytosis.

A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

1. The whole‐cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura‐2 and fluo‐3 were used to measure the whole‐cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin‐Darby canine kidney (MDCK) cells. 2. In a Ca(2+)‐free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)‐free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5‐trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)‐dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of ‐4.2 +/‐ 0.7 pA pF‐1. This Iin was unaffected by inositol 1,3,4,5‐tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N‐methyl‐D‐glucamine+ (NMG+), aminomethylidin‐trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl‐ with gluconate‐ or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG‐induced elevation of [Ca2+]i. SK&F 96365 (1‐[3‐(4‐methoxyphenyl)‐propoxyl]‐1‐(4‐methoxy‐phenyl)‐ethyl‐1H‐im idazole) HCl did not inhibit the TG‐induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365‐induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent methods, stimulates La(3+)‐inhibitable Ca2+ entry in MDCK cells. Ca2+ entry is at least, in part, mediated by a cation current, which is highly, but not exclusively, selective for Ca2+ over Na+ and insensitive to SK&F 96365.

Recent advances in alveolar biology: some new looks at the alveolar interface.

This article examines the manner in which some new methodologies and novel concepts have contributed to our understanding of how pulmonary surfactant reduces alveolar surface tension. Investigations utilizing small angle X-ray diffraction, inverted interface fluorescence microscopy, time of flight-secondary ion mass spectroscopy, atomic force microscopy, two-photon fluorescence microscopy and electrospray mass spectroscopy are highlighted and a new model of ventilation-induced acute lung injury described. This contribution attempts to emphasize how these new approaches have resulted in a fuller appreciation of events presumably occurring at the alveolar interface.

Pulmonary consequences of a deep breath revisited.

About two decades ago, a model was proposed for surfactant release by lung distension. This model implies rapid fusion of lamellar bodies (LBs) with the plasma membrane followed by quick release of surfactant into the alveolus, as reflected by immediate facilitation of lung inflation after a single deep breath. Recent experimental evidence indicates that this two-pool model (intracellular versus alveolar surfactant pool) has to be refined by introducing a third pool, which resides in fused but non-released LBs. Here we discuss the implication of this additional pool for strain-induced surfactant secretion and propose a revised model for the sequence of events following a single deep breath.

Spatio-temporal aspects, pathways and actions of Ca(2+) in surfactant secreting pulmonary alveolar type II pneumocytes.

The type II cell of the pulmonary alveolus is a polarized epithelial cell that secretes surfactant into the alveolar space by regulated exocytosis of lamellar bodies (LBs). This process consists of multiple sequential steps and is correlated to elevations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) required for extended periods of secretory activity. Both chemical (purinergic) and mechanical (cell stretch or exposure to an air-liquid interface) stimuli give rise to complex Ca(2+) signals (such as Ca(2+) peaks, spikes and plateaus) that differ in shape, origin and spatio-temporal behavior. This review summarizes current knowledge about Ca(2+) channels, including vesicular P2X4 purinoceptors, in type II cells and associated signaling cascades within the alveolar microenvironment, and relates stimulus-dependent activation of these pathways with distinct stages of surfactant secretion, including pre- and postfusion stages of LB exocytosis.

Long-term exposure to LPS enhances the rate of stimulated exocytosis and surfactant secretion in alveolar type II cells and upregulates P2Y2 receptor expression

Bacterial LPS is a potent proinflammatory molecule. In the lungs, LPS induces alterations in surfactant pool sizes and phospholipid (PL) contents, although direct actions of LPS on the alveolar type II cells (AT II) are not yet clear. For this reason, we studied short- and long-term effects of LPS on basal and agonist-stimulated secretory responses of rat AT II by using Ca(2+) microfluorimetry, a microtiter plate-based exocytosis assay, by quantitating PL and (3)H-labeled choline released into cell supernatants and by using quantitative PCR and Western blot analysis. Long term, but not short term, exposures to LPS led to prolonged ATP-induced Ca(2+) signals and an increased rate in vesicle fusions with an augmented release of surfactant PL. Most notably, the stimulatory effect of LPS was ATP-dependent and may be mediated by the upregulation of the purinergic receptor subtype P2Y(2). Western blot analysis confirmed higher levels of P2Y(2), and suramin, a P2Y receptor antagonist, was more effective in LPS-treated cells. From these observations, we conclude that LPS, probably via Toll-like receptor-4, induces a time-dependent increase in P2Y(2) receptors, which, by yet unknown mechanisms, leads to prolonged agonist-induced Ca(2+) responses that trigger a higher activity in vesicle fusion and secretion. We further conclude that chronic exposure to endotoxin sensitizes AT II to increase the extracellular surfactant pool, which aids in the pulmonary host defense mechanisms.

A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.

Background. Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells. Methods. Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (&Dgr;&PSgr;m) was measured by 5,5′,6,6′-tetrachloro- 1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide and 3,3′-dihexyloxacarbocyanine iodide fluorescence. Results. Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. &Dgr;&PSgr;m decreased significantly during cold ischemia and was sustained during reperfusion. The loss of &Dgr;&PSgr;m can be related to a reduced portion of mitochondria exhibiting high &Dgr;&PSgr;m. Conclusions. Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.