Ryszard Grygorczyk

Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations

Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom‐designed flow‐through chambers, we investigated the kinetics of cell swelling‐induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o− cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o− cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three cell types, the peak rates differed significantly (294 ± 67, 70 ± 22 and 17 ± 2.8 pmol min−1 (106 cells)−1, for A549, 16HBE14o− and NIH/3T3, respectively). The concomitant volume changes of substrate‐attached cells were analysed by a 3‐dimensional cell shape reconstruction method based on images acquired from two perpendicular directions. The three cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either cell volume expansion and the expected activation of stretch‐sensitive channels, or with the activation of volume‐sensitive, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid‐inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three cell types, with cytosolic calcium elevations. Furthermore, loading A549 cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of cell swelling. Lowering the temperature to 10°C almost completely abolished A549 cell swelling‐induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium‐dependent exocytosis plays a major role in mechanosensitive ATP release.

Membrane Reserves and Hypotonic Cell Swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o−, CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.

Ca2+‐dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca2+‐dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca2+ for the elevation of intracellular Ca2+ concentration ([Ca2+]i) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca2+‐dependent ATP release. We found that ATP release does not directly involve Ca2+ influx from extracellular spaces, but depends entirely on Ca2+ mobilization from intracellular stores. The [Ca2+]i response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)‐insensitive stores and a superimposed rapid spike due to Ca2+ release from TG‐sensitive endoplasmic reticulum (ER) Ca2+ stores. The latter could be abolished by hydrolysis of extracellular triphospho‐ and diphosphonucleotides with apyrase; blocking P2Y2/P2Y6 receptors of A549 cells with suramin; blocking UDP receptors (P2Y6) with pyridoxal phosphate 6‐azophenyl‐2′,4′‐disulfonic acid (PPADS); emptying TG‐sensitive stores downstream with TG or caffeine in Ca2+‐free extracellular solution; or blocking the Ca2+‐release inositol 1,4,5‐triphosphate receptor channel of the ER with 2‐aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca2+]i spike results from the autocrine stimulation of IP3/Ca2+‐coupled P2Y, predominantly P2Y6, receptors, accounting for ∼70% of total Ca2+‐dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress‐induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Nitric Oxide Signaling in Oxytocin‐Mediated Cardiomyogenesis

Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain‐2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G‐nitro‐l‐arginine‐methyl‐ester (l‐NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO‐sensitive guanylyl cyclases. The NO generator S‐nitroso‐N‐acetylpenicillamine (SNAP) reversed the l‐NAME inhibition of cell beating and GFP expression. In OT‐induced cells, l‐NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, α‐myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT‐induced P19Cl6‐GFP cells revealed ventricular CM coexpressing sarcomeric α‐actinin and GFP, with some cells exclusively expressing α‐actinin, most likely of the SM phenotype. The OT‐mediated production of CM, but not SM, was diminished by l‐NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of l‐NAME. Surprisingly, l‐NAME alone decreased the expression of anti‐stage specific embryonic antigen‐1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6‐GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.

Downregulation of Epithelial Sodium Channel (ENaC) by CFTR Co-expressed in Xenopus Oocytes is Independent of Cl− Conductance

Abstract. Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl− into the cell and is negligible when Cl− flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (Iamil) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced Iamil by up to 80%, demonstrating that ENaC is inhibited when Cl− is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of Iamil downregulation and the CFTR-mediated plasma membrane Cl− conductance, suggesting a direct correlation. However, Iamil downregulation was not affected when Cl− flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl− reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that Iamil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in Iamil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit Iamil. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl− transport across the plasma membrane.

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP‐activated, nucleotide‐dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR‐dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)‐affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR–ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR‐dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.

c-Fos expression in ouabain-treated vascular smooth muscle cells from rat aorta: evidence for an intracellular-sodium- mediated, calcium-independent mechanism

In this study, we examined the effect of Na+‐K+ pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain‐treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c‐Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c‐Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca2+ response element binding protein (CREB)‐1 and c‐Myc was not altered. Markedly augmented c‐Fos expression was also observed under Na+‐K+ pump inhibition in potassium‐depleted medium. Na+‐K+ pump inhibition triggered c‐Fos expression via elevation of the [Na+]i/[K+]i ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c‐Fos expression in high‐potassium‐low‐sodium medium and from the comparison of dose responses of Na+‐K+ pump activity, [Na+]i and [K+]i content and c‐Fos expression to ouabain. A fourfold increment of c‐Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na+]i but did not affect [K+]i. Augmented c‐Fos expression was also observed under VSMC depolarization in high‐potassium medium. Increments in both c‐Fos expression and 45Ca uptake in depolarized VSMC were abolished under inhibition of L‐type Ca2+ channels with 0.1 μM nicardipine. Ouabain did not affect the free [Ca2+]i or the content of exchangeable [Ca2+]i. Ouabain‐induced c‐Fos expression was also insensitive to the presence of nicardipine and [Ca2+]o, as well as chelators of [Ca2+]o (EGTA) and [Ca2+]i (BAPTA). The effect of ouabain and serum on c‐Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk‐1, serum response factor, CREB and activator protein‐1 transcription factors identified within the c‐Fos promoter. These results suggest that Na+‐K+ pump inhibition triggers c‐Fos expression via [Na+]i‐sensitive [Ca2+]i‐independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.

Arginine vasopressin-mediated cardiac differentiation: insights into the role of its receptors and nitric oxide signaling.

Despite the existence of a functional arginine vasopressin (AVP) system in the adult heart and evidence that AVP induces myogenesis, its significance in cardiomyogenesis is currently unknown. In the present study, we hypothesized a role for AVP in cardiac differentiation of D3 and lineage-specific embryonic stem (ES) cells expressing green fluorescent protein under the control of atrial natriuretic peptide (Anp) or myosin light chain-2V (Mlc-2V) promoters. Furthermore, we investigated the nitric oxide (NO) involvement in AVP-mediated pathways. AVP exposure increased the number of beating embryoid bodies, fluorescent cells, and expression of Gata-4 and other cardiac genes. V1a and V2 receptors (V1aR and V2R) differentially mediated these effects in transgenic ES cells, and exhibited a distinct developmentally regulated mRNA expression pattern. A NO synthase inhibitor, l-NAME, powerfully antagonized the AVP-induced effects on cardiogenic differentiation, implicating NO signaling in AVP-mediated pathways. Indeed, AVP elevated the mRNA and protein levels of endothelial NO synthase (eNOS) through V2R stimulation. Remarkably, increased beating activity was found in AVP-treated ES cells with down-regulated eNOS expression, indicating the significant involvement of additional pathways in cardiomyogenic effects of AVP. Finally, patch clamp recordings revealed specific AVP-induced changes of action potentials and increased l-type Ca2+ (ICa,L) current densities in differentiated ventricular phenotypes. Thus, AVP promotes cardiomyocyte differentiation of ES cells and involves Gata-4 and NO signaling. AVP-induced action potential prolongation appears likely to be linked to the increased ICa,L current in ventricular cells. In conclusion, this report provides new evidence for the essential role of the AVP system in ES cell-derived cardiomyogenesis.

Hemolysis is a primary ATP-release mechanism in human erythrocytes

The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

The Hydrogel Nature of Mammalian Cytoplasm Contributes to Osmosensing and Extracellular pH Sensing

Cytoplasm is thought to have many hydrogel-like characteristics, including the ability to absorb large amounts of water and change volume in response to alterations in external environment, as well as having limited leakage of ions and proteins. Some gel-like behaviors have not been rigorously confirmed in mammalian cells, and others should be examined under conditions where gel volume can be accurately monitored. Thus, possible contributions of cytoplasm hydrogel properties to cellular processes such as volume sensing and regulation remain unclear. We used three-dimensional imaging to measure volume of single substrate-attached cells after permeabilization of their plasma membrane. Permeabilized cells swelled or shrinked reversibly in response to variations of external osmolality. Volume changes were 3.7-fold greater than observed with intact cells, consistent with cytoplasms high water-absorbing capacity. Volume was maximal at neutral pH and shrunk at acidic or alkaline pH, consistent with pH-dependent changes of protein charge density and repulsive forces within cellular matrix. Volume shrunk with increased Mg(2+) concentration, as expected for increased charge screening and ionic crosslinking effects. Findings demonstrate that mammalian cytoplasm resembles hydrogel and functions as a highly sensitive osmosensor and extracellular pH sensor. Its high water-absorbing capacity may allow rapid modulation of local fluidity, macromolecular crowding, and activity of intracellular environment.