Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3.

Intracellular signaling pathways that are involved in protection of vascular smooth muscle cells (VSMC) from apoptosis remain poorly understood. This study examines the effect of activators of cAMP/cGMP signaling on apoptosis in non-transfected VSMC and in VSMC transfected with c-myc (VSMC-MYC) or with its functional analogue, E1A-adenoviral protein (VSMC-E1A). Serum-deprived VSMC-E1A exhibited the highest apoptosis measured as the content of chromatin and low molecular weight DNA fragments, phosphatidylserine content in the outer surface of plasma membrane and caspase-3 activity (ten-, five-, four- and tenfold increase after 6 h of serum withdrawal, respectively). In VSMC-E1A, the addition of an activator of adenylate cyclase, forskolin, abolished chromatin cleavage, DNA laddering, caspase-3 activation and the appearance of morphologically-defined apoptotic cells triggered by 6 h of serum deprivation. In non-transfected VSMC and in VSMC-MYC, 6 h serum deprivation led to approximately six- and threefold activation of chromatin cleavage, respectively, that was also blocked by forskolin. In VSMC-E1A, inhibition of apoptosis was observed with other activators of cAMP signaling (cholera toxin, isoproterenol, adenosine, 8-Br-cAMP), whereas 6 h incubation with modulators of cGMP signaling (8-Br-cGMP, nitroprusside, atrial natriuretic peptide, L-NAME) did not affect the development of apoptotic machinery. The antiapoptotic effect of forskolin was abolished in 24 h of serum deprivation that was accompanied by normalization of intracellular cAMP content and protein kinase A (PKA) activity. Protection of VSMC-E1A from apoptosis by forskolin was blunted by PKA inhibitors (H-89 and KT5720), whereas transfection of cells with PKA catalytic subunit attenuated apoptosis triggered by serum withdrawal. The protection of VSMC-E1A by forskolin from apoptosis was insensitive to modulators of cytoskeleton assembly (cytochalasin B, colchicine). Neither acute (30 min) nor chronic (24 h) exposure of VSMC to forskolin modified basal and serum-induced phosphorylation of the MAP kinase ERK1/2. Thus, our results show that activation of cAMP signaling delays the development of apoptosis in serum-deprived VSMC at a site upstream of caspase-3 via activation of PKA and independently of cAMP-induced reorganization of the cytoskeleton network and the ERK1/2-terminated MAPK signaling cascade.     

3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein

[³H]-thymidine is commonly used to analyze the accumulation of [³H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [³H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [³H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [³H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [³H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [³H]-thymidine-labeled DNA. They also demonstrate that [³H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na⁺] _i /[K⁺] _i ratio.     

Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3.

Long term elevation of the intracellular Na+/K+ ratio inhibits macromolecule synthesis and proliferation in the majority of cell types studied so far, including vascular smooth muscle cells (VSMC). We report here that inhibition of the Na+,K+ pump in VSMC by ouabain or a 1-h preincubation in K+-depleted medium attenuated apoptosis triggered by serum withdrawal, staurosporine, or okadaic acid. In the absence of ouabain, both DNA degradation and Caspase-3 activation in VSMC undergoing apoptosis were insensitive to modification of the extracellular Na+/K+ ratio as well as to hyperosmotic cell shrinkage. In contrast, protection of VSMC from apoptosis by ouabain was abolished under equimolar substitution of Na+o with K+o, showing that the antiapoptotic action of Na+,K+ pump inhibition was caused by inversion of the intracellular Na+/K+ ratio. Unlike VSMC, the same level of increment of the [Na+]i/[K+]i ratio caused by a 2-h preincubation of Jurkat cells with ouabain did not affect chromatin cleavage and Caspase-3 activity triggered by treatment with Fas ligand, staurosporine, or hyperosmotic shrinkage. Thus, our results show for the first time that similar to cell proliferation, maintenance of a physiologically low intracellular Na+/K+ ratio is required for progression of VSMC apoptosis.     

Hyperosmotic and isosmotic shrinkage differentially affect protein phosphorylation and ion transport

In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ～7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.     

Swelling rather than shrinkage precedes apoptosis in serum-deprived vascular smooth muscle cells

Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10–15 min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na⁺,K⁺-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.     

Arachidonic acid-derived oxidation products initiate apoptosis in vascular smooth muscle cells

The mechanism of arachidonic acid (AA)-induced apoptosis in vascular smooth muscle cells (VSMCs) was studied in the A-10 rat aortic smooth muscle cell line. Treatment of serum-deprived VSMCs with 50 microM AA for 24 h resulted in a loss of cell viability. The apoptotic effect of AA was characterized by annexin V binding, sub-G1 population of cells, cell shrinkage and chromatin condensation. AA-induced VSMC death was attenuated by antioxidants alpha-tocopherol and glutathione, the hydrogen peroxide (H2O2) scavenger catalase and by serum proteins, albumin and gamma globulins. Moreover, the AA peroxidation products, 12(S)-hydroperoxyeicosatetraenoic acid (HPETE), 15(S)-HPETE, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) caused VSMC apoptosis. These data suggest an oxidative mechanism of AA-induced VSMC death. The apoptotic effect of AA was pH-dependent, being inhibited by extracellular alkalinization to pH 8.0. AA inhibited serum-stimulated cell cycle progression in quiescent cells, but not in proliferating cells. In conclusion, AA, through its oxidation products causes VSMC apoptosis. Antioxidants, by inhibiting VSMC apoptosis, may prevent consequent pathological events such as atherosclerotic plaque rupture.     

3-Dimensional microscopy as a method for volume measurement in cells undergoing apoptosis

Different patterns of cell volume perturbations are commonly used for modes of cell death: necrosis (cell swelling) and apoptosis (cell shrinkage). In this study we employed recently developed three dimensional microscopy for the measurement of the volume of attached vascular smooth muscle cells transfected with E1A-adenoviral protein. These cells undergo rapid apoptosis in the absence of growth factors or in the presence of staurosporine. In 30–60 min of serum deprivation the volume of these cells is increased by ～40% that corresponds to the time point of maximal activation of caspase 3 and chromatin cleavage. In 10–15 min swollen cells exhibit morphological collapse indicated by formation of apoptotic bodies. In contrast to serum-deprived cells, staurosporine leads to attenuation of cell volume by 30%. In this case, apoptotic bodies are detected in ～2.5 h after maximal shrinkage. Thus, our results show that cell shrinkage can not be considered as universal hallmark of apoptosis. The role of stimulus-specific cell volume perturbation in the triggering of the cell death machinery should be examined further.     

Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase

Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-α, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.     

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

Temperature-Induced Inactivation of Cytoplasmic Biogel Osmosensing Properties is Associated with Suppression of Regulatory Volume Decrease in A549 Cells

Upstream intermediates of intracellular signaling involved in cell volume regulation remain poorly explored. Recently, we demonstrated that osmolarity-induced volume changes in permeabilized cells were several-fold higher than those observed with intact cells, indicating the osmosensing properties of cytoplasmic gel. To further examine the role of cytoplasmic biogel in cell volume regulation, we compared the action of short-term heat treatment on volume changes in intact and permeabilized A549 cells. Pretreatment of A549 cells at 48 °C suppressed swelling triggered by dissipation of Donnan’s equilibrium as well as by hyposmotic medium. Significantly, heat treatment completely abolished the action of hyposomotic medium on volume changes in permeabilized cells, showing that temperature elevation suppresses osmosensing properties via its effect on biogel rather than on plasma membrane water permeability. Identical heat treatment blocked the regulatory volume decrease (RVD) as well as the increment of Ba²⁺-sensitive K⁺-channel activity seen in control cells exposed to hyposmotic swelling. Unlike swelling, hyperosmotic shrinkage was decreased by twofold in cells subjected to 10-min preincubation at 50 °C. Our results disclose that osmosensing by cytoplasmic gel is a key event in the RVD triggered by hypotonic swelling. The role of biogel and plasma membrane in intracellular signaling triggered by hyperosmotic shrinkage should be further investigated.     

Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress

The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.     

持续性细胞皱缩在人上皮细胞凋亡过程中的必要性

持续性细胞皱缩是凋亡发生的一个主要标志。近期研究发现细胞皱缩在细胞凋亡过程中并不是一个被动的次要事件。在各种细胞中,包括人上皮细胞,凋亡因子（apoptogen）刺激后马上发生全细胞皱缩,又称为凋亡性容积减小（apoptotic　volumede　crease,AVD）,继而发生caspase激活、DNA片段化、细胞破裂死亡。K^＋和Cl^-通道的激活导致KCl外流,诱导AVD发生。抑制AVD发生可以抑制细胞凋亡。AVD与调节性容积增加（regulatory　volume　increase,RVI）异常相伴发生时,人上皮性HeLa细胞发生持续性细胞皱缩。RVI功能受损时,高渗本身就能诱导HeLa细胞持续性细胞皱缩,继而凋亡。即使在正常渗透压、无凋亡因子刺激的情况下,将HeLa细胞置于缺乏Na^＋或Cl。的溶液也会导致细胞持续性皱缩,继而凋亡。因此,AVD诱导和RVI异常所导致的持续性细胞皱缩是人上皮细胞发生凋亡的首要条件。     

Glutamine deprivation-mediated cell shrinkage induces ligand-independent CD95 receptor signaling and apoptosis

Cell shrinkage and loss of cell viability by apoptosis have been examined in cultured CD95(Fas/Apo-1)-expressing leukemia-derived CEM and HL-60 cells subjected to acute deprivation of glutamine, a major compatible osmolyte engaged in cell volume control. Glutamine deprivation-mediated cell shrinkage promoted a ligand-independent activation of the CD95-mediated apoptotic pathway. Cell transfection with plasmids expressing FADD-DN or v-Flip viral proteins pointed to a functional clustering of CD95 receptors at the cell surface with activation of the 'extrinsic pathway' caspase cascade. Accordingly, cell shrinkage did not induce apoptosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with surrogate compatible osmolytes counteracted cell volume decrement and protected the CD95-expressing cells from apoptosis. A glutamine deprivation-dependent cell shrinkage with activation of the CD95-mediated pathway was also observed when asparaginase was added to the medium. Asparagine depletion had no role in this process. The cell-size shrinkage-dependent apoptosis induced by glutamine restriction in CD95-expressing leukemic cells may therefore be of clinical relevance in amidohydrolase enzyme therapies.     

p38 MAPK activation and function following osmotic shock of erythrocytes

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.     

Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth

We have previously hypothesized that the development of severe angioproliferative pulmonary hypertension is associated with not only initial endothelial cell (EC) apoptosis followed by the emergence of apoptosis-resistant proliferating EC but also with proliferation of vascular smooth muscle cells (VSMC). We have demonstrated that EC death results in the selection of an apoptosis-resistant, proliferating, and phenotypically altered EC phenotype. We postulate here that the initial apoptosis of EC induces the release of mediators that cause VSMC proliferation. We cultured EC in an artificial capillary CellMax system designed to simulate the highly efficient functions of the human capillary system. We induced apoptosis of microvascular EC using shear stress and the combined VEGF receptor (VEGFR-1 and -2) inhibitor SU-5416. Flow cytometry for the proliferation marker bromodeoxyuridine showed that serum-free medium conditioned by apoptosed EC induced proliferation of VSMC, whereas serum-free medium conditioned by nonapoptosed EC did not. We also show that medium conditioned by apoptosed EC is characterized by increased concentrations of transforming growth factor (TGF)-beta1 and VEGF compared with medium conditioned by nonapoptosed EC and that TGF-beta1 blockade prevented the proliferation of cultured VSMC. In conclusion, EC death induced by high shear stress and VEGFR blockade leads to the production of factors, in particular TGF-beta1, that activate VSMC proliferation.     

Energy-dependent volume regulation in primary cultured cerebral astrocytes

Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.     

Significance of adenosine metabolism of coronary smooth muscle cells

A detailed understanding of adenosine metabolism of vascular smooth muscle cells (VSMC) is highly desirable to critically evaluate possible autocrine effects of adenosine in this cell species. Therefore, this study quantified intra- and extracellular adenosine flux rates, the transmembrane concentration gradient, and the adenosine surface concentration in porcine VSMC and, for comparison, aortic endothelial cells (PAEC). Cell-covered microcarrier beads packed in a chromatography column were superfused with a HEPES buffer. With the use of specific inhibitors of adenosine kinase (iodotubericidine, 10 microM), adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)-adenine, 5 microM], ecto-5'-nucleotidase (alpha,beta-methylene-adenosine 5'-diphosphate, 50 microM), and adenosine membrane transport (n-nitrobenzylthioinosine, 1 microM), total production rates of 12.3 +/- 2.7 and 7.5 +/- 1.3 pmol x min(-1) x microl cell volume(-1) were obtained for VSMC and PAEC, respectively. Despite prevailing intracellular adenosine production (76 and 70% of total production, respectively), transmembrane concentration gradients under control conditions were directed toward the cytosol as a result of rapid intracellular adenosine rephosphorylation and continuous extracellular hydrolysis from 5'-AMP. Surface concentrations were approximately 18 nM in VSMC and PAEC under control conditions and increased to approximately 60 nM during partial inhibition of adenosine metabolism. Simultaneously, the transmembrane adenosine concentration gradient was reversed. We conclude that adenosine flux rates in VSMC and PAEC are quantitatively similar and that VSMC may influence the interstitial adenosine concentration under basal steady-state conditions.     

Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis

Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.