Role of mitochondrial Bax, caspases, and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells

It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3–6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.     

Inhibitory effect of ganglioside GD1b on K+ current in hippocampal neurons and its involvement in apoptosis suppression

Gangliosides are endogenous membrane components enriched in neuronal cells. They have been shown to play regulatory roles in many cellular processes. Here, we show for the first time that ganglioside GD1b plays an antiapoptotic role in cultured hippocampal neurons. GD1b inhibited the voltage-dependent outward delayed rectifier current (I(K)) but not the transient outward A-type current in a dose-dependent manner, with an IC50 value of 15.2 microM. This effect appears to be somehow specific, because GD1b, but not GM1, GM2, GM3, GD1a, GD3, or GT1b, was effective in inhibiting I(K). Intracellular application of staurosporine (STS; 0.1 microM) resulted in rapid activation of I(K), which was partially reversed upon addition of the K+ channel blocker tetraethylammonium (TEA; 5 mM) and GD1b (10 microM). Furthermore, GD1b (10 microM) attenuated STS-induced neuronal apoptosis by nearly the same amount as 5 mM TEA. In addition, GD1b suppressed the apoptosis-associated caspase 3 activation that was activated by STS. Collectively, these findings suggest that GD1b plays an antiapoptotic role in cultured hippocampal neurons through its inhibitory effect on the I(K) and caspase activity.     

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells

The sphingomyelin-derived messenger ceramides provoke neuronal apoptosis through caspase-3 activation, while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase-3 activity. However, the mechanisms leading to the opposite regulation of caspase-3 by C2-ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2-ceramide-induced inhibition of mitochondrial potential and C2-ceramide-evoked cytochrome c release. C2-ceramide stimulated Bax expression, but had no effect on Bcl-2, while PACAP abrogated the action of C2-ceramide on Bax and stimulated Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked, respectively, by the JNK inhibitor L-JNKI1 and the MEK inhibitor U0126. L-JNKI1 prevented the alteration of mitochondria induced by C2-ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2-ceramide on mitochondrial potential. Moreover, L-JNKI1 inhibited the stimulatory effect of C2-ceramide on caspase-9 and -3 and prevented C2-ceramide-induced cell death. U0126 blocked PACAP-induced Bcl-2 expression, abrogated the inhibitory effect of PACAP on ceramide-induced caspase-9 activity, and promoted granule cell death. The present study reveals that C2-ceramide and PACAP exert opposite effects on Bax and Bcl-2 through, respectively, JNK- and ERK-dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro- and anti-apoptotic effects of C2-ceramide and PACAP.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.     

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.     

DAP kinase activity is critical for C(2)-ceramide-induced apoptosis in PC12 cells.

Exposure of PC12 cells to C(2)-ceramide results in dose- dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-gamma-induced apoptosis of HeLa cells, in the C(2)-ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 microm C(2)-ceramide, both the total (assayed in the presence of Ca(2+)/calmodulin) and Ca(2+)/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 microm C(2)-ceramide, the Ca(2+)/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C(2)-ceramide concentration. C(8)-ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C(2)-dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C(2)- and C(8)-ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C(2)-, and C(8)-ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis.     

Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.     

Ceramide triggers metacaspase-independent mitochondrial cell death in yeast

The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.     

C(6)-ceramide inhibited Na(+) currents by intracellular Ca(2+) release in rat myoblasts

Ceramides are novel second messengers that may mediate signaling leading to apoptosis and the regulation of cell cycle progression. Moreover, ceramide analogs have been reported to directly modulate K(+) and Ca(2+) channels in different cell types. In this report, the effect of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)) in cultured rat myoblasts was investigated using whole-cell current recording and a fluorescent Ca(2+) imaging experiment. At concentrations of 1-100 microM, ceramide produced a dose-independent and reversible inhibition of I(Na). Ceramide also significantly shifted the steady-state inactivation curve of I(Na) by 16 mV toward the hyperpolarizing potential, but did not alter the steady-state activation properties. C(2)-ceramide caused a similar inhibitory effect on I(Na) amplitude. However, dihydro-C(6)-ceramide, the inactive analog of ceramide, failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent BAPTA, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with xestospongin C or heparin, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, ruthenium red, which is a blocker of the ryanodine-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide on I(Na). Intracellular application of the G-protein agonist GTPgammaS also induced a gradual decrease in I(Na) amplitude, while the G-protein antagonist GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Calcium imaging showed that C(6)-ceramide could give rise to a significant elevation of intracellular calcium. Our data show that increased calcium release through the IP(3)-sensitive Ca(2+) receptor, which probably occurred through the G-protein and phospholipase C pathway, may be responsible for C(6)-ceramide-induced inhibition of the I(Na) of rat myoblasts.     

Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ

This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ.     

Reduction of airway anion secretion via CFTR in sphingomyelin pathway

The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (Isc) measurement revealed that isoproterenol (ISO, 0.1 microM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca2+-activated K+ channel opener)-induced Isc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 microM) or 8-bromo-cAMP (2 mM). C2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.     

Modulation of spontaneous and stimulation-evoked transmitter release from rat sympathetic neurons by the cognition enhancer linopirdine: insights into its mechanisms of action

The mechanisms by which the cognition enhancer linopirdine may affect transmitter release were investigated in cultures of rat superior cervical ganglion neurons. Overflow of previously incorporated [3H]noradrenaline evoked by 10 microM UTP or 0.1 microM bradykinin was enhanced by linopirdine at > or =3 microM, overflow evoked by 25 mM K(-), 100 microM nicotine, or 300 microM ATP was enhanced by linopirdine at > or =10 microM, and overflow due to 40 mM K+ or electrical field stimulation was not altered by linopirdine. Ba2+ (0.3 mM) augmented the same types of stimulation-evoked overflow to a similar extent as linopirdine. K+ (25 mM), nicotine (100 microM), and ATP (300 microM) triggered transmitter release in a partially tetrodotoxin-resistant manner, and the release-enhancing action of linopirdine was lost in the presence of tetrodotoxin (1 microM). Linopirdine (10 microM) raised spontaneous tritium outflow and reduced currents through muscarinic K+ (K(M)) channels with a similar time course. The secretagogue action of linopirdine was concentration- and Ca2(+)-dependent and abolished by tetrodotoxin (1 microM) or Cd2+ (100 microM). Linopirdine (10 microM) added to the partial inhibition of K(M) channels by 1 or 3 mM Ba2(+) but not to the complete inhibition by 10 mM Ba2(+). Likewise, the secretagogue action of 1 and 3 mM, but not that of 10 mM, Ba2+ was enhanced by linopirdine. These results indicate that linopirdine facilitates and triggers transmitter release via blockade of K(M) channels and suggest that these K+ channels are located at neuronal somata rather than at presynaptic sites.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK

Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.     

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.     

Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

Control of ceramide-induced apoptosis by IGF-1: involvement of PI-3 kinase,caspase-3 and catalase

Insulin-like growth factor-1 (IGF-1) inhibited N-acetylsphingosine (C2-ceramide)-induced HL-60 cell apoptosis via relieving oxidative damage. This inhibitory action of IGF-1 was blocked by a phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin and enhanced by overexpression of the p110 catalytic subunit of PI-3 kinase. Either IGF-1 pretreatment or PI-3 kinase overexpression restored ceramide-depleted catalase function, and this restoration was inhibited by wortmannin. A catalase inhibitor 3-amino-1h-1, 2, 4-triazole (ATZ) blocked the inhibitory action of IGF-1 on ceramide-induced apoptosis, whereas exogenous purified catalase enhanced it. Ceramide-activated caspase-3 was inhibited by IGF-1/PI-3 kinase and enhanced by wortmannin, while the addition of a specific caspase-3 inhibitor DMQD-CHO significantly enhanced the restoration by IGF-1 of ceramide-depleted catalase function. Moreover, IGF-1 inhibited C2-ceramide-induced decrease of mitochondrial membrane potential, and increase of cytochrome c release, caspase-3 cleavage and caspase-3 activity as judged by PhiPhiLux cleaving method. In summary, these results suggest that IGF-1/PI-3 kinase inhibited C2-ceramide-induced apoptosis due to relieving oxidative damage, which resulted from the inhibition of catalase by activated caspase-3.     

Redistribution of cytochrome c is not an essential requirement in C2-ceramide induced apoptosis in HL-60 cells

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis. The present study investigates the potential role of bcl-2 on apoptosis in HL-60 cells induced by different agents. HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Staurosporine (STS) promoted DNA fragmentation dose-dependently in the 6 h exposure assay while C2-ceramide was relatively slow in the induction of apoptosis (approximately 40% after 24 h) and required higher concentrations (> 20 microM). Caspases inhibitors, Ac-YVAD-cmk (100 microM) and zVAD-fmk (20 microM) had no effect on DNA fragmentation themselves. However, they blocked C2-ceramide-induced caspase-3 cleavage and apoptosis, but not the release of cytochrome c from the mitochondria. In addition, we found that both Ac-YVAD-cmk and zVAD-fmk failed to protect STS-induced apoptosis in HL-60 cells. Overexpression of bcl-2 inhibited STS and C2-ceramide induced cytochrome c redistribution, caspase-3 activation and apoptosis. These results suggest a protective role of bcl-2 in the regulation of apoptosis and cytochrome c release is unlikely to be involved in the final common pathway in apoptosis.