Induced Cell Shrinkage Research Articles

Glucocorticoid-induced plasma membrane depolarization during thymocyte apoptosis: association with cell shrinkage and degradation of the Na(+)/K(+)-adenosine triphosphatase.

Multiple signaling pathways are known to induce apoptosis in thymocytes through mechanisms that include the loss of mitochondrial membrane potential, cell shrinkage, caspase activation, and DNA degradation but little is known about the consequences of apoptosis on the properties of the plasma membrane. We have previously shown that apoptotic signals, including survival factor withdrawal and glucocorticoids, induce plasma membrane depolarization during rat thymocyte apoptosis, but the mechanisms involved in this process are unknown. We report here that inhibition of the Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase) with ouabain similarly depolarized control thymocytes and enhanced glucocorticoid-induced membrane depolarization, suggesting a link between Na(+)/K(+)-ATPase and plasma membrane depolarization of thymocytes. To determine whether repression of Na(+)/K(+)-ATPase levels within cells can account for the loss of plasma membrane potential, we assessed protein levels of the Na(+)/K(+)-ATPase in apoptotic thymocytes. Spontaneously dying thymocytes had decreased levels of both catalytic and regulatory subunits of Na(+)/K(+)-ATPase, and glucocorticoid treatment enhanced the loss of Na(+)/K(+)-ATPase protein. The pan caspase inhibitor (z-VAD) blocked both cellular depolarization and repression of Na(+)/K(+)-ATPase in both spontaneously dying and glucocorticoid-treated thymocytes; however, specific inhibitors of caspase 8, 9, and caspase 3 did not. Interestingly, glucocorticoid treatment simultaneously induced cell shrinkage and depolarization. Furthermore, depolarization and the loss of Na(+)/K(+)-ATPase protein were limited to the shrunken population of cells. The data indicate an important role for Na(+)/K(+)-ATPase in both spontaneous and glucocorticoid-induced apoptosis of rat thymocytes.

Effect of Radiologic Contrast Media on Cell Volume Regulation in Rabbit Proximal Renal Tubules

Rationale and Objectives Most radiographic contrast media are hyperosmotic and able to shrink cells with which they are in contact. The authors studied cell volume control in rabbit proximal renal tubules after incubation with three contrast media: iohexol, ioxaglate, and iodixanol. Materials and Methods Proximal renal tubules were isolated from rabbit kidneys. The tubules were exposed to Ringer solutions containing 5% vol/vol iohexol (final osmolality, 330 mOsm), ioxaglate (323 mOsm), iodixanol (305 mOsm), or mannitol (control solutions with identical osmolalities), and tubule volumes were monitored. After 2 hours of incubation, the tubules were stimulated with a hyposmotic Ringer solution (165 mOsm). Three groups of 10 experiments were performed. Results All solutions induced cell shrinkage (8.3% ± 3.8 [standard error] to 15.4% ± 0.5), which was completely or partly reversible in most experiments (volume increase, 44.8% ± 14.7 to 149.9% ± 107.3) but not those with iohexol and iodixanol. With exposure to the hyposmotic solution, the cells swelled by 11.0% ± 1.8 to 39.7% ± 4.8. In general, the tubules that had been exposed to the most hyperosmotic solution swelled the most. Those exposed to contrast media showed less swelling than the mannitol-exposed controls. In all control experiments, the cells exhibited a gradual shrinkage (43.6% ± 28.5 to 87.0% ± 13). This regulatory response was partly inhibited in tubules exposed to iohexol (39.9% ± 15.8 shrinkage) or iodixanol (8.9% ± 15.8) and completely inhibited in those exposed to ioxaglate. Iohexol and ioxaglate exposure also led to a decrease in water permeability. Conclusion Exposure to hyperosmotic contrast medium tends to induce prolonged cell shrinkage, decrease the water permeability of the cellular plasma membranes, and compromise the ability to regulate cellular volume. These changes seem to reflect both the hyperosmolality of the solutions and their inherent chemical properties.

Prodigiosin induces cell death and morphological changes indicative of apoptosis in gastric cancer cell line HGT-1.

Gastric cancer is one of the most frequent malignancies and its treatment is far from satisfactory. The challenge to oncologists is the characterization of novel chemical entities with greater effectiveness. Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Here we characterize the apoptotic action of prodigiosin in human gastric carcinoma cell line (HGT-1). Cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of actin microfilament architecture. Treatment of these cells with prodigiosin showed a constant decrease in viability by apoptosis. Morphological analysis of prodigiosin-treated cells demonstrated that prodigiosin induces cell shrinkage, chromatin condensation, reorganization of actin microfilament architecture, and detachment of cells from the cell culture substrate. Altogether these results suggest that prodigiosin induces apoptosis in HGT-1 human gastric cancer cells and raises the possibility of its use as a new chemotherapeutic drug.

Docosahexaenoic acid, a peroxisome proliferator-activated receptor-alpha ligand, induces apoptosis in vascular smooth muscle cells by stimulation of p38 mitogen-activated protein kinase.

Omega-3 fatty acids (n-3 FAs) have been shown to exert a blood pressure-lowering effect in hypertension, possibly in part by influencing vascular structure. We previously demonstrated that n-3 FAs induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on the structure of blood vessels. In the present study, we investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured mesenteric VSMCs from Sprague-Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FAs. Morphological changes in apoptosis and DNA fragmentation were examined with phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, we evaluated the expression of phosphorylated p38 mitogen-activated protein kinases, bax, bcl-2, cytochrome c, and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) with Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing, and apoptotic bodies in VSMCs. DHA time-dependently activated p38 mitogen-activated protein kinases, bax, PPAR-alpha, and cytochrome c, with maximal effects obtained after 5 and 30 minutes and 1 and 3 hours, respectively. SB-203580 and SB-202190, selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPAR-alpha but had no effect on the expression of bax or cytochrome c. The present results indicate that DHA induces apoptosis in VSMCs through >/=2 distinct mechanisms: (1) a p38-dependent pathway that regulates PPAR-alpha and (2) a p38-independent pathway via dissipation of mitochondrial membrane potential and cytochrome c release. The death-signaling pathway stimulated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA may play a pathophysiological role in vascular remodeling in cardiovascular disease.

Docosahexaenoic Acid, a Peroxisome Proliferator-Activated Receptor-α Activator, Induces Apoptosis in Vascular Smooth Muscle Cells by Activation of P38 Mitogen-Activated Protein Kinase

9 Omega-3 fatty acids (n-3 FAs) exert a blood pressure-lowering effect in hypertension, possibly by influencing vascular structure. We previously demonstrated that n-3 FAs might induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on structure of blood vessels. This study investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured Mesenteric VSMCs from Sprague Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FA. Morphological changes of apoptosis and DNA fragmentation were examined by phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, expression of phosphorylated p38 mitogen-activated protein kinases (p38 MAPKs), bax, bcl-2, cytochrome C and peroxisome proliferator-activated receptors-α (PPARs-α) was evaluated by Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing and apoptotic bodies in VSMCs. DHA increased apoptosis (%) in a time-dependent manner to 1.5±0.1, 3.6±0.5, 7.1±0.4, 22.5±0.6, 50.8±1.8 and 61.4±0.9 after 0, 1, 3, 6, 17, and 24 h, respectively. DHA time-dependently activated p38 MAPKs, bax, PPARs-α and cytochrome C with maximal effects obtained after 5, 30 min, 1 h and 3 h, respectively to 551±42, 245±55, 310±12 and 407±14.7 % of controls, respectively. SB-203580 (10 -5 M) and SB-202190 (10 -5 M), selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPARs-α, but had no effect on expression of bax or cytochrome C. The present results indicate that DHA induces apoptosis in VSMCs through at least two distinct mechanisms: (i) a p38-dependent pathway that regulates PPAR-α and (ii) a p38-independent pathway via dissipation of mitochondrial transmembrane potential. The death-signaling pathway mediated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA could play a pathophysiological role in vascular remodeling in cardiovascular disease.

Phenotypic effects of overexpression of the MMAC1 gene in prostate epithelial cells.

The prostate cancer cell lines PC3 and LNCaP have been shown to lack expression of the tumour suppressor gene MMAC1/PTEN, in contrast to the immortalized non-tumorigenic epithelial lines PNT1a and PNT2. We have measured the effects of reintroduction of wild type (wt) and mutant MMAC1 genes on to these genetic backgrounds, using gene constructs expressing either wt MMAC1 or various mutants deficient in the dual specificity phosphatase domain of the protein. Over-expression of wild type PTEN protein induced cell shrinkage and rounding, but did not result in increased levels of classical apoptosis. Permanently transfected lines containing the MMAC1 gene could only be obtained from the PNT cells, as PTEN expression resulted in rapid loss of both tumour lines. In contrast, mutation of the phosphatase domain resulted in partial attenuation of the phenotypic effects of MMAC1 after transient transfection, and also allowed the derivation of permanent tumour cell lines containing the mutated MMAC1 gene. The results suggest that re-expression of wt PTEN is incompatible with survival of human prostate cancer cells in vitro, and that the full biological activity of this common tumour suppressor requires functions additional to the established protein and lipid phosphatase activities in epithelial systems. © 2000 Cancer Research Campaign

Protein Kinase C (PKC) Inhibits Fas Receptor-induced Apoptosis through Modulation of the Loss of K+ and Cell Shrinkage: A ROLE FOR PKC UPSTREAM OF CASPASES

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with Gö6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and Gö6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with Gö6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.

Induction of apoptosis in mouse brain capillary endothelial cells by cyclosporin a and tacrolimus

Although cyclosporin A and tacrolimus are used clinically as potent immunosuppressants, there have been reports of neurotoxicity and encephalopathy. A possible mechanism is that these drugs damage the blood-brain barrier (BBB), inducing dysfunction and increased permeability and are then able to enter the brain. We studied the cytotoxicity of cyclosporin A and tacrolimus, focused on apoptosis induction, using an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells (MBEC4). We found that these two drugs induced cell shrinkage, chromatin condensation and DNA fragmentation, which are characteristics of apoptosis. Our data suggest that the induction of apoptosis on the brain capillary endothelial cells may be at least partly involved in the occurrence of immunosuppressant-induced encephalopathy.

Acute modulation of the extent of apoB mRNA editing and the relative rates of syntheses of apoB48 and apoB100 in cultured rat hepatocytes by osmotic and other stress stimuli.

The mRNA for apolipoprotein B (apoB) is edited by the enzyme APOBEC-1, which acts as part of a multiprotein complex or editosome. In cultured rat hepatocytes obtained from fed animals this results in the presence of edited and unedited apoB mRNA in a ratio of approximately 3:2 in the basal state. In this study we show that hyper-osmotic media, which induce cell shrinkage, resulted in an acute increase in the degree of editing of apoB mRNA (hypo-osmotic conditions had no effect). This increase was accompanied by a parallel and highly positively correlated change in the ratio of the rate of synthesis of apoB48 relative to that of apoB100. These changes occurred in the absence of any changes in the overall APOBEC-1 mRNA levels, indicating that the activation of editing occurred at a post-transcriptional level. Levels of total apoB mRNA were also unaffected by hyper-osmotic exposure of the cells indicating that changes in the relative rates of synthesis of apoB48 and apoB100 were due to post/translational events. Exposure of cells to anisomycin at concentrations (50 micrograms/ml) that inhibit protein synthesis or to the transcriptional inhibitor actinomycin D produced changes in the degree of apoB mRNA editing that were similar to those given by hyper-osmotic shock indicating that editing is able to respond acutely to transcriptional or translational inhibition. Anisomycin, at concentrations (50 ng/ml) that activate SAPK/JNK but do not inhibit protein synthesis, gave only a fraction of the effect of hyper-osmotic shock. SB203580, an inhibitor of p38 kinase, did not attenuate the effects of hyper-osmotic conditions on APOBEC-1 editing. These observations suggest that these MAPkinase pathways play a relatively minor part in the transduction of the osmotic stimulus to the editing mechanism. The hyper-osmotically-induced increase in apoB mRNA editing was also insensitive to PD98059 and wortmannin (inhibitors of MEK and PI3 kinase, respectively). These data provide evidence that apoB mRNA editing is capable of acute modulation independently of transcriptional or translational mechanisms and suggest that one or more components of the editosome may undergo post-translational activation.

Na+/K+-ATPase inhibition during cardiac myocyte swelling: involvement of intracellular pH and Ca2+.

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na+/K+-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pHi), intracellular Ca2+ ([Ca2+]i) and Na+/K+-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca2+ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 microM) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca2+]i (200%). A decrease in pHi (from 7.3 +/- 0.09 to 6.4 +/- 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca2+. Inhibition of Na+/H+ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca2+ using CCCP (10 microM) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K+/H+ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca2+ influx via Na+/Ca2+ exchange, which under hyposmotic conditions activates the K+ and Ca2+ mitochondrial exchange systems (K+/H+ and Ca2+/H+).

Sodium-potassium-chloride cotransport.

Obligatory, coupled cotransport of Na(+), K(+), and Cl(-) by cell membranes has been reported in nearly every animal cell type. This review examines the current status of our knowledge about this ion transport mechanism. Two isoforms of the Na(+)-K(+)-Cl(-) cotransporter (NKCC) protein (approximately 120-130 kDa, unglycosylated) are currently known. One isoform (NKCC2) has at least three alternatively spliced variants and is found exclusively in the kidney. The other (NKCC1) is found in nearly all cell types. The NKCC maintains intracellular Cl(-) concentration ([Cl(-)](i)) at levels above the predicted electrochemical equilibrium. The high [Cl(-)](i) is used by epithelial tissues to promote net salt transport and by neural cells to set synaptic potentials; its function in other cells is unknown. There is substantial evidence in some cells that the NKCC functions to offset osmotically induced cell shrinkage by mediating the net influx of osmotically active ions. Whether it serves to maintain cell volume under euvolemic conditons is less clear. The NKCC may play an important role in the cell cycle. Evidence that each cotransport cycle of the NKCC is electrically silent is discussed along with evidence for the electrically neutral stoichiometries of 1 Na(+):1 K(+):2 Cl- (for most cells) and 2 Na(+):1 K(+):3 Cl(-) (in squid axon). Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/dephosphorylation mechanisms is decribed. Interestingly, the presumed protein kinase(s) responsible has not been identified. An unusual form of NKCC regulation is by [Cl(-)](i). [Cl(-)](i) in the physiological range and above strongly inhibits the NKCC. This effect may be mediated by a decrease of protein phosphorylation. Although the NKCC has been studied for approximately 20 years, we are only beginning to frame the broad outlines of the structure, function, and regulation of this ubiquitous ion transport mechanism.

Apoptotic cell death and caspase-3 activation induced by N-methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I.

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.

Cyclothiazide and GYKI 52466 modulate AMPA receptor-mediated apoptosis in cortical neuronal cultures

In neocortical neuronal cultures, (S)-AMPA caused neurotoxicity which was concentration-dependent, receptor-mediated, slow and apoptotic in nature. (S)-AMPA (3–600 μM) failed to produce rapid neuronal swelling, but morphological observations and monitoring of viability at 24–72 h revealed 50% cell death consistent with apoptosis. (S)-AMPA induced cell shrinkage, neurite blebbing and nuclear condensation. Cyclothiazide (50 and 100 μM), which blocks AMPA receptor desensitization potentiated excitotoxicity with 75% of neurones undergoing slow death. The AMPA-selective antagonist GYKI 52466 (10–50 μM), attenuated (S)-AMPA-mediated neurotoxicity. DNA condensation, a hallmark of apoptosis, was found by labelling neurones with the DNA binding dye 4,6-diamidino-2-phenylindole HCl (DAPI). Gel electrophoresis revealed DNA fragmentation, which was increased by cyclothiazide and reduced by GYKI 52466 and cycloheximide. Overstimulation of the AMPA receptor produces a novel form of neuronal death, which is apoptotic, very slow in nature, and which could contribute to various neuropathologies.

Osmotic shrinkage activates nonselective cation (NSC) channels in various cell types.

Osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells (Volk, Frömter & Korbmacher, 1995, Proc. Natl. Acad. Sci. USA 92: 8478-8482). To see whether shrinkage-activated NSC channels are an ubiquitous phenomenon, we tested the effect of hypertonic extracellular solution on whole-cell currents of HT29 human colon carcinoma cells, BSC-1 renal epithelial cells, A10 vascular smooth muscle cells, and Neuro-2a neuroblastoma cells. Addition of 100 mm sucrose to an isotonic NaCl bath solution induced cell shrinkage of HT29 cells as evidenced by a decrease in cell diameter from 18 +/- 1 microm to 12 +/- 1 microm (n = 13). Upon cell shrinkage whole-cell currents of HT29 cells increased within 8 +/- 1 min by about 30-fold (n = 13). Cell shrinkage and current activation were reversible upon return to isotonic solution. Replacement of bath Na+ by K+ or Li+ had almost no effect on the stimulated inward current. In contrast, replacement by N-methyl-d-glucamine (NMDG) completely abolished it and shifted the reversal potential from -4.5 +/- 0.7 mV to -57 +/- 4.1 mV (n = 10). Thus, the stimulated conductance is nonselective for alkali cations but highly selective for cations over anions with a cation-to-anion permeability ratio of about 13. Flufenamic acid (100 microm) inhibited the stimulated current by 84 +/- 4.7% (n = 8). During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 12 and more individual channels with a single-channel conductance of 17.6 +/- 0.9 pS (n = 4) could be resolved. In analogous experiments similar shrinkage-activated NSC channels were also observed in BSC-1 renal epithelial cells, A10 vascular smooth muscle cells, and Neuro-2a neuroblastoma cells. These findings indicate that shrinkage-activated NSC channels are an ubiquitous phenomenon and may play a role in volume regulation.

Stereo-specific cytotoxic effects of gossypol enantiomers and gossypolone in tumour cell lines

The naturally occurring compound, gossypol, has been previously used as a male oral contraceptive, for the treatment of benign gynaecological conditions and cancer patients. Long-term daily dosing with gossypol is associated with minimal side effects and no myelosuppression. Since gossypol exhibits atropisomerism due to the restricted rotation about the 2,2′ carbon bond, we have isolated the l- and d-isomers by Schiff's base formation using a chiral amine and regenerated the enantiomers by acid hydrolysis. The enantiomers and the proposed oxidative metabolite, gossypolone, were characterized by HPLC, 1H-NMR and optical rotation. The cytotoxicity was assessed in cell cultures derived from melanoma, lung, breast, cervix, and leukaemia using the MTT viability assay. The cytotoxicity of gossypolone was similar to racemic gossypol in five out of the six cell lines studied. The l-enantiomer of gossypol induced a dose-dependent cell kill in all cell lines with a mean IC 50 of 20 μM and was significantly more potent than racemic gossypol, the d-enantiomer of gossypol and gossypolone. In addition, when the leukaemia line was exposed to l-gossypol (0.5–10 μM) over a 4-day period, a schedule-dependent decrease in cell viability was observed. l-Gossypol was also compared with respective drugs used to treat patients with melanoma, lung cancer and leukaemia. The data indicate that l-gossypol was significantly more active than cisplatin, melphalan and dacarbazine in the two melanoma lines, cisplatin and daunorubicin in the lung line and hydroxyurea and busulphan in the leukaemia line. Preliminary studies using one melanoma line showed that the l-isomer induced cell shrinkage, membrane blebbing and DNA fragmentation, characteristics suggestive of apoptotic cell death.

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells

The mechanisms, by which the P2 receptor agonists adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca 2+] i) and in intracellular pH (pH i), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca 2+] i evoked by ATP or UTP is abolished after depletion of intracellular Ca 2+ stores with thapsigargin in Ca 2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca 2+] i is primarily due to release from intracellular, Ins(1,4,5)P 3-sensitive Ca 2+ stores. ATP also activates a capacitative Ca 2+-entry pathway. ATP as well as UTP evokes a biphasic change in pH i, consisting of an initial acidification followed by alkalinization. Suramin and 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pH i, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5′-( N-ethyl- N-isopropyl)amiloride (EIPA)-sensitive Na +/H + exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca 2+ sensitive K + and Cl − efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K + concentration, or by adding the K +-channel blocker, charybdotoxin, does not affect the increase in [Ca 2+] i, but abolishes the activation of the Na +/H + exchanger, indicating that activation of the Na +/H + exchanger is secondary to the Ca 2+-induced cell shrinkage.

Persistent activation of a swelling-activated cation current in ventricular myocytes from dogs with tachycardia-induced congestive heart failure.

The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) channels was tested. Digital video microscopy and amphotericin-perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing-induced CHF. In normal myocytes, osmotic swelling in 0.9x to 0.6x isosmotic solution (296 mOsm/L) was required to elicit an inwardly rectifying swelling-activated cation current (I(Cir,swell)) that reversed near -60 mV and was inhibited by 10 micromol/L Gd3+, a mechanosensitive channel blocker. Block of I(Cir,swell) by Gd3+ simultaneously reduced the volume of normal cells in hyposmotic solutions by up to approximately 10%, but Gd3+ had no effect on volume in isosmotic solution. In contrast, I(Cir,swell) was persistently activated under isosmotic conditions in CHF myocytes, and Gd3+ decreased cell volume by approximately 8%. Osmotic shrinkage in 1.1x to 1.5x isosmotic solution inhibited both I(Cir,swell) and Gd3+-induced cell shrinkage in CHF cells, whereas osmotic swelling only slightly increased I(Cir,swell). The K0.5 and Hill coefficient for Gd3+ block of I(Cir,swell) and Gd3+-induced cell shrinkage were estimated as approximately 2.0 micromol/L and approximately 1.9, respectively, for both normal and CHF cells. In both groups, the effects of Gd3+ on current and volume were blocked by replacing bath Na+ and K+ and were linearly related with varying Gd3+ concentration and the degree of cell swelling. CHF thus altered the set point for and caused persistent activation of I(Cir,swell). This current may contribute to dysrhythmias, hypertrophy, and altered contractile function in CHF and may be a novel target for therapy.

Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells.

1. The possible mechanisms of the antiproliferative and apoptotic effects of curcumin (diferuloylmethane), a polyphenol in the spice turmeric, on vascular smooth muscle cells were studied in rat aortic smooth muscle cell line (A7r5). 2. The proliferative response was determined from the uptake of [3H]-thymidine. Curcumin (10(-6)-10(-4) M) inhibited serum-stimulated [3H]-thymidine incorporation of both A7r5 cells and rabbit cultured vascular smooth muscle cells in a concentration-dependent manner. Cell viability, as determined by the trypan blue dye exclusion method, was unaffected by curcumin at the concentration range 10(-6) to 10(-5) M in A7r5 cells. However, the number of viable cells after 10(-4) M curcumin treatment was less than the basal value (2 x 10(5) cells). 3. To analyse the various stages of the cell cycle, [3H]-thymidine incorporation into DNA was determined every 3 h. After stimulation with foetal calf serum, quiescent A7r5 cells started DNA synthesis in 9 to 12 h (G1/S phase), then reached a maximum at 15 to 18 h (S phase). Curcumin (10(-6)-10(-4) M) added during either the G1/S phase or S phase significantly inhibited [3H]-thymidine incorporation. 4. Following curcumin (10(-6)-10(-4) M) treatment, cell cycle analysis utilizing flow cytometry of propidium iodide stained cells revealed a G0/G1 arrest and a reduction in the percentage of cells in S phase. Curcumin at 10(-4) M also induced cell apoptosis. It is suggested that curcumin arrested cell proliferation and induced cell apoptosis, and hence reduced the [3H]-thymidine incorporation. 5. The apoptotic effect of 10(-4) M curcumin was also demonstrated by haematoxylin-eosin staining, TdT-mediated dUTP nick end labelling (TUNEL), and DNA laddering. Curcumin (10(-4) M) induced cell shrinkage, chromatin condensation, and DNA fragmentation. 6. The membranous protein tyrosine kinase activity stimulated by serum in A7r5 cells was significantly reduced by curcumin at the concentration range 10(-5) to 10(-4) M. On the other hand, the cytosolic protein kinase C activity stimulated by phorbol ester was reduced by 10(-4) M curcumin, but unaffected by lower concentrations (10(-6)-10(-5) M). 7. The levels of c-myc, p53 and bcl-2 mRNA were analysed using a reverse transcription-polymerase chain reaction (RT-PCR) technique. The level of c-myc mRNA was significantly reduced by curcumin (10(-5)-10(-4) M) treatment. And, the level of bcl-2 mRNA was significantly reduced by 10(-4) M curcumin. However, the alteration of the p53 mRNA level by curcumin (10(-5)-10(-4) M) treatment did not achieve significance. The effects of curcumin on the levels of c-myc and bcl-2 mRNA were then confirmed by Northern blotting. 8. Our results demonstrate that curcumin inhibited cell proliferation, arrested the cell cycle progression and induced cell apoptosis in vascular smooth muscle cells. Curcumin may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post-angioplasty restenosis. Our results suggest that the antiproliferative effect of curcumin may partly be mediated through inhibition of protein tyrosine kinase activity and c-myc mRNA expression. And, the apoptotic effect may partly be mediated through inhibition of protein tyrosine kinase activity, protein kinase C activity, c-myc mRNA expression and bcl-2 mRNA expression.

Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl − which might be consistent with a H 2PO 4 −/Cl − exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K + and Cl − channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na + and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl − channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Swelling-activated Gd3+-sensitive cation current and cell volume regulation in rabbit ventricular myocytes.

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6-0.9x normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (ICir,swell) that was reversibly blocked by Gd3+, but ICir,swell was not detected in isotonic or hypertonic media. This current was not related to IK1 because it was insensitive to Ba2+. The PK/PNa ratio for ICir,swell was 5.9 +/- 0.3, implying that inward current is largely Na+ under physiological conditions. Increasing bath K+ increased gCir,swell but decreased rectification. Gd3+ block was fitted with a K0.5 of 1.7 +/- 0.3 microM and Hill coefficient, n, of 1.7 +/- 0.4. Exposure to Gd3+ also reduced hypotonic swelling by up to approximately 30%, and block of current preceded the volume change by approximately 1 min. Gd3+-induced cell shrinkage was proportional to ICir,swell when ICir,swell was varied by graded swelling or Gd3+ concentration and was voltage dependent, reflecting the voltage dependence of ICir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that ICir,swell was sufficient to explain the majority of the volume change at -80 mV. In addition, swelling activated an outwardly rectifying Cl- current, ICl,swell. This current was absent after Cl- replacement, reversed at ECl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of ICl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, ICir,swell is expected to cause diastolic depolarization. Activation of ICir, swell also is likely to affect contraction and other physiological processes in myocytes.