Cd2+-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca2+ uniporter, K+ cycling, and protonmotive force

The nephrotoxic metal Cd²⁺ causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K⁺ cycle involving a K⁺ uniporter and a K⁺/H⁺ exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd²⁺ on K⁺ cycling in rat kidney cortex mitochondria. Cd²⁺ (EC₅₀ 19 µM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl >> choline chloride. Cd²⁺-induced swelling of energized mitochondria had a similar EC₅₀ value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca²⁺ uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd²⁺ influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (_m) induced by K⁺ influx did not prevent contraction, but addition of the K⁺/H⁺ exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 µM), abolished contraction, suggesting the mitochondrial pH gradient (pH_m) driving contraction. Accordingly, a quinine-sensitive partial dissipation of pH_m was coincident with the swelling-contraction phase. The data indicate that Cd²⁺ enters the matrix through the MCU to activate a K⁺ cycle. Initial K⁺ load via a Cd²⁺-activated K⁺ uniporter in the IMM causes osmotic swelling and breakdown of _m and triggers quinine-sensitive K⁺/H⁺ exchange and contraction. Thus Cd²⁺-induced activation of a K⁺ cycle contributes to the dissipation of the mitochondrial protonmotive force. bongkrekic acid; cyclosporin A; lanthanum; Ru360; ruthenium red     

{micro}-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

µ-calpain and calpain-3 are Ca²⁺-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that µ-calpain becomes proteolytically active in the presence of 2–200 µM Ca²⁺. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca²⁺ concentration levels ([Ca²⁺]_i; 1 µM) than the levels at which µ-calpain activation occurs. We have demonstrated the Ca²⁺-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca²⁺] levels (0, 2.5, 10, and 25 µM). Autolysis of calpain-3 was found to occur across a [Ca²⁺] range similar to that for µ-calpain, and both calpains displayed a seemingly higher Ca²⁺ sensitivity in human than in rat muscle homogenates, with 15% autolysis observed after 1-min exposure to 2.5 µM Ca²⁺ in human muscle and almost none after 1- to 2-min exposure to the same [Ca²⁺]_i level in rat muscle. During muscle activity, [Ca²⁺]_i may transiently peak in the range found to autolyze µ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% O_{2 peak} until fatigue, n = 3) on the amount of autolyzed µ-calpain or calpain-3 in human muscle. No significant autolysis of µ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca²⁺]_i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo. Ca²⁺-dependent proteases; proteolysis     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Complex regulation of store-operated Ca2+ entry pathway by PKC-{varepsilon} in vascular SMCs

The role of PKC in the regulation of store-operated Ca²⁺ entry (SOCE) is rather controversial. Here, we used Ca²⁺-imaging, biochemical, pharmacological, and molecular techniques to test if Ca²⁺-independent PLA₂β (iPLA₂β), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca²⁺ stores and triggered Ca²⁺ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA₂β and Ca²⁺ entry that were absent in SMCs in which iPLA₂β was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca²⁺ entry only in proliferating SMCs, which correlates with the expression of the specific PKC- isoform. Molecular downregulation of PKC- impaired TG- and OAG-induced Ca²⁺ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca²⁺ stores as well as direct PKC--dependent activation of iPLA₂β, resulting in a complex regulation of SOCE in proliferating and confluent SMCs. protein kinase C-; Ca²⁺-independent phospholipase A₂; diacylglycerol; smooth muscle cells     

Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway

Certain angina and coronary artery disease forms do not respond to Ca²⁺ channel blockers, and a role for vasoactive eicosanoids such as PGF₂ in Ca²⁺ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF₂-induced coronary smooth muscle contraction is Ca²⁺ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca²⁺), PGF₂ (10^-5 M) caused transient [Ca²⁺]_i increase followed by maintained [Ca²⁺]_i increase and 34% cell contraction. Ca²⁺ channel blockers verapamil and diltiazem (10^-6 M) abolished maintained PGF₂-induced [Ca²⁺]_i increase but only partially inhibited PGF₂-induced cell contraction to 17%. Verapamil-insensitive PGF₂ contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF₂ caused Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF₂-induced -but not -PKC translocation. PMA (10^-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca²⁺]_i increase and -PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca²⁺ influx, caused 36% cell contraction and [Ca²⁺]_i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause - or -PKC translocation. Thus a significant component of PGF₂-induced contraction of coronary smooth muscle is Ca²⁺ antagonist insensitive, involves Ca²⁺-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle     

Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation

Expression of TNF-, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF- regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF- regulates the diameter of resistance-sized (150-µm diameter) cerebral arteries by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF- increased Ca²⁺ spark and Ca²⁺ wave frequency but reduced global intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells of intact arteries. TNF- elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (–40 mV) cells, TNF- increased the frequency and amplitude of Ca²⁺ spark-induced, large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient K_Ca current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF- induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca²⁺-ATPase blocker that inhibits Ca²⁺ sparks, and iberiotoxin, a K_Ca channel blocker, reduced TNF--induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF- activates NAD(P)H oxidase, resulting in an increase in intracellular H₂O₂ that stimulates Ca²⁺ sparks and transient K_Ca currents, leading to a reduction in global [Ca²⁺]_i, and vasodilation. cerebrovascular circulation; ryanodine-sensitive Ca²⁺ release channel; Ca²⁺-activated K⁺ channel; reactive oxygen species; vasodilation     

Elevated ceramide is downstream of altered calcium homeostasis in low serum-induced apoptosis

Two immortalized cell lines, sup (+)and sup (), derived from mutagenized Syrian hamster embryo cells,were used to study the relationship and temporal order between calciumand ceramide signals during apoptosis. The early preneoplastic cells,termed sup (+), suppress tumorigenicity when hybridized with tumorcells, whereas later-stage sup () cells do not. In reduced serumconditions, sup (+) cells cease proliferating and undergo apoptosis; incontrast, sup () cells continue slow growth and undergo necrosis. Insup (+) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serumtreatment of sup (+) cells but are not found in sup () cells.Inhibiting ER calcium depletion in low serum-treated sup (+) cells bytreating with high levels of calcium prevents both ceramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup ()cells by treating with low serum plus thapsigargin results in elevatedceramide levels and apoptosis. Furthermore, C₆-ceramide treatment induced apoptosis of sup () cells in low serum, a condition that does not normally cause apoptosis. C₆-ceramidetreatment did not induce apoptosis in either sup (+) or sup () cellsin 10% serum but did cause G₂/M arrest. These studies showthat ceramide production is downstream of ER calcium release.

     

Focal adhesion kinase mediates cell survival via NF-kappaB and ERK signaling pathways

Focal adhesion kinase (FAK) is important to cellular functions such as proliferation, migration, and survival of anchorage-dependent cells. We investigated the role of FAK in modulating normal cellular responses, specifically cell survival in response to inflammatory stimuli and serum withdrawal, using FAK-knockout (FAK^–/–) embryonic fibroblasts. FAK^–/– fibroblasts were more vulnerable to TNF--induced apoptosis, as measured by terminal deoxynucleotidyl transferase positivity. FAK^–/– fibroblasts also demonstrated increased procaspase-3 cleavage to p17 subunit, whereas this was undetectable in FAK^+/+ fibroblasts. Insulin receptor substrate-1 expression was completely abolished and NF-B activity was reduced, with a concomitant decrease in abundance of the anti-apoptotic protein Bcl-x_L in FAK^–/– cells. Upon serum withdrawal, FAK^+/+ cells exhibited marked attenuation of basal ERK phosphorylation, while FAK^–/– cells, in contrast, maintained high basal ERK phosphorylation. Moreover, inhibition of ERK phosphorylation potentiated serum withdrawal-induced caspase-3 activity. This was paralleled by increased insulin receptor substrate (IRS)-2 expression in FAK^–/– cells, although both insulin- and IGF-1-mediated phosphorylation of Akt/PKB and GSK-3 were impaired. This suggests that IRS-2 protects against apoptosis upon serum withdrawal via the ERK signaling pathway. The specific role of FAK to protect cells from apoptosis is regulated by activation and phosphorylation of NF-B and interaction between activated growth factor anti-apoptotic signaling pathways involving both phosphatidylinositol 3-kinase/Akt and MAPK/ERK1/2. We demonstrate that FAK is necessary for upregulation of the anti-apoptotic NF-B response, as well as for normal expression of growth factor signaling proteins. Thus we propose a novel role for FAK in protection from cytokine-mediated apoptosis. apoptosis; ERK1/2; insulin; TNF-; IGF-1     

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gbeta gamma -subunits in Balb/c-3T3 cells

Diacylglycerol (DAG)and ceramide are important second messengers affecting cell growth,differentiation, and apoptosis. Balb/c-3T3 fibroblast cellsexpressing dopamine-D2S (short) receptors (Balb-D2S cells) provide amodel of G protein-mediated cell growth and transformation. In Balb-D2Scells, apomorphine (EC₅₀ = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicatingG_i/G_o proteins. To address which G proteins areinvolved, Balb-D2S clones expressing individual PTX-insensitiveG_i proteins were treated with PTX and tested forapomorphine-induced responses. Neither PTX-insensitive G_i2 nor G_i3 rescued D2S-induced DAG orceramide formation. Both D2S-induced DAG and ceramide signals requiredG-subunits and were blocked by inhibitors of phospholipaseC[1-(6-[([17]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione(U-73122) and partially by D609]. The similar G protein specificity ofD2S-induced calcium mobilization, DAG, and ceramide formation indicatesa common G-dependent phospholipase C-mediated pathway. Both D2agonists and ceramide specifically induced mitogen-activated proteinkinase (ERK1/2), suggesting that ceramide mediates a novel pathway ofD2S-induced ERK1/2 activation, leading to cell growth.

     

cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct

It is generally believed thatcAMP-dependent phosphorylation is the principle mechanism foractivating cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. However, we showed that activating Gproteins in the sweat duct stimulated CFTR Cl conductance(G_Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G_Cl is independent ofprotein kinase A. We activated G proteins and monitored CFTRG_Cl in basolaterally permeabilized sweat duct.Activating G proteins with guanosine5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTRG_Cl in the presence of 5 mM ATP alone withoutcAMP. G protein activation of CFTR G_Cl requiredMg²⁺ and ATP hydrolysis (5'-adenylylimidodiphosphate couldnot substitute for ATP). G protein activation of CFTRG_Cl was 1) sensitive to inhibition bythe kinase inhibitor staurosporine (1 µM), indicating that theactivation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM)and SQ-22536 (100 µM); and 3) independent ofCa²⁺, suggesting that Ca²⁺-dependent proteinkinase C and Ca²⁺/calmodulin-dependent kinase(s) are notinvolved in the activation process. Activating AC with10⁶ M forskolin plus 10⁶ M IBMX (in thepresence of 5 mM ATP) did not activate CFTR, indicating that cAMPcannot accumulate sufficiently to activate CFTR in permeabilized cells.We concluded that heterotrimeric G proteins activate CFTR G_Cl endogenously via a cAMP-independent pathwayin this native absorptive epithelium.

     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Although the importance of estradiol-17 (E₂) in many physiological processes has been reported, to date no researchers have investigated the effects of E₂ on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E₂ on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E₂ (10^–9 M) significantly increased [³H]thymidine incorporation at >4 h and that E₂ (>10^–12 M) induced an increase of [³H]thymidine incorporation after 8-h incubation. Moreover, E₂ (>10^–12 M) also increased 5'-bromo-2'-deoxyuridine (BrdU) incorporation and cell number. Indeed, E₂ stimulated estrogen receptor (ER)- and - protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases in [³H]thymidine incorporation. In addition, estradiol-6-O-carboxymethyl oxime-BSA (E₂-BSA; 10^–9 M) increased [³H]thymidine incorporation at >1 h, and E₂-BSA (>10^–12 M) increased [³H]thymidine incorporation after 1-h incubation. E₂-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H]thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E₂ increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10^–5 M PD-98059 (MEK inhibitor). Moreover, E₂-induced increase of [³H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17 stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes. cyclin-dependent kinase; mitogen-activated protein kinase     

PKCdelta mediates anti-proliferative, pro-apoptic effects of testosterone on coronary smooth muscle

Sex hormone status has emerged as an important modulator of coronary physiology and cardiovascular disease risk in both males and females. Our previous studies have demonstrated that testosterone increases protein kinase C (PKC) expression and activity in coronary smooth muscle (CSMC). Because PKC has been implicated in regulation of proliferation and apoptosis in other cell types, we sought to determine if testosterone modulates CSMC proliferation and/or apoptosis through PKC. Porcine CSMC cultures (passages 2–6) from castrated males were treated with testosterone for 24 h. Testosterone (20 and 100 nM) decreased [³H]thymidine incorporation in proliferating CSMC to 59 ± 5.3 and 33.1 ± 4.5% of control. Flow cytometric analysis demonstrated that testosterone induced G₁ arrest in CSMC with a concomitant reduction in the S phase cells. Testosterone reduced protein levels of cyclins D₁ and E and phosphorylation of retinoblastoma protein while elevating levels of p21^cip1 and p27^kip1. There were no significant differences in the levels of cyclins D₃, CDK2, CDK4, or CDK6. Testosterone significantly reduced kinase activity of CDK2 and -6, but not CDK4, -7, or -1. PKC small interfering RNA (siRNA) prevented testosterone-mediated G₁ arrest, p21^cip1 upregulation, and cyclin D₁ and E downregulation. Furthermore, testosterone increased CSMC apoptosis in a dose-dependent manner, which was blocked by either PKC siRNA or caspase 3 inhibition. These findings demonstrate that the anti-proliferative, pro-apoptotic effects of testosterone on CSMCs are substantially mediated by PKC. androgens; coronary; smooth muscle; cell cycle     

Mechanisms of interleukin-1beta-induced Ca2+ signals in mouse cortical astrocytes: roles of store- and receptor-operated Ca2+ entry

Many neurodegenerative disorders are accompanied by chronic glial activation, which is characterized by the abundant production of proinflammatory cytokines, such as IL-1. IL-1 disrupts Ca²⁺ homeostasis and stimulates astrocyte reactivity. The mechanisms by which IL-1 induces Ca²⁺ dysregulation are not completely defined. Here, we examined how acute and chronic (24–48 h) treatment with IL-1 affect Ca²⁺ homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) was measured with fura-2 using digital imaging. An acute application of 10 ng/ml IL-1 induced Ca²⁺ mobilization from intracellular stores and activated store-operated Ca²⁺ entry (SOCE) and receptor-operated Ca²⁺ entry (ROCE) in both freshly dissociated and cultured actrocytes. Treatment of cultured astrocytes with IL-1 for 24 and 48 h elevated resting [Ca²⁺]_cyt, decreased Ca²⁺ store content [associated with sarco(endo)plasmic reticulum Ca²⁺-ATPase 2b downregulation], and augmented ROCE. Based on evidence that receptor-operated, but not store-operated Ca²⁺ channels are Ba²⁺ permeable, Ba²⁺ entry was used to distinguish receptor-operated Ca²⁺ channels from store-operated Ca²⁺ channels. ROCE was activated by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba²⁺, OAG-induced elevations of cytosolic Ba²⁺ (fura-2 340-to-380-nm ratio) were significantly larger in astrocytes treated with IL-1. These changes in IL-1-treated astrocytes correlate with augmented expression of transient receptor potential cation channel (TRPC)6 protein, which likely mediates ROCE. Knockdown of the TRPC6 gene markedly reduced ROCE. The data suggest that IL-1-induced dysregulation of Ca²⁺ homeostasis is the result of enhanced ROCE and TRPC6 expression. The disruption of Ca²⁺ homeostasis appears to be an upstream component in the cascade of IL-1-activated pathways leading to neurodegeneration. transient receptor potential cation channel proteins     

Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca²⁺-sensitive K⁺ (mtK_Ca) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK_Ca channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K⁺ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (_m). To test this hypothesis, we measured effects of NS-1619, a putative mtK_Ca channel opener, and valinomycin, a K⁺ ionophore, on mitochondrial respiration, _m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O₂·min^–1·mg protein^–1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O₂·min^–1·mg protein^–1, respectively, with the FADH₂-linked substrate succinate (+ rotenone); these effects were abolished by the mtK_Ca channel blocker paxilline. _m was not decreased by 10–30 µM NS-1619 with either substrate, but H₂O₂ release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H₂O₂ release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained _m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K⁺ influx during states 2 and 4 increases mitochondrial respiration while maintaining _m; this allows singlet electron uptake by O₂ and ROS generation. mitochondrial bioenergetics; heart mitochondria     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca²⁺ accumulation resulting from functional coupling of Na⁺/Ca²⁺ exchange (NCE) with stimulated Na⁺/H⁺ exchange (NHE1) and 2) 17-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na⁺ and therefore Ca²⁺ accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH_i), Na⁺ (Na), and calcium concentration ([Ca²⁺]_i) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH_i, Na and [Ca²⁺]_i during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH_i fell from 6.93 ± 0.03 to 5.98 ± 0.04 vs. 6.96 ± 0.04 to 6.68 ± 0.07; Na rose from 25 ± 6 to 109 ± 14 meq/kg dry wt vs. 25 ± 1 to 76 ± 3; [Ca²⁺]_i changed from 365 ± 69 to 1,248 ± 180 nM vs. 293 ± 66 to 202 ± 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca²⁺]_i is greater than can be accounted for by the thermodynamic effect of reduced Na accumulation on NCE. myocardial ischemia; Na⁺/H⁺ exchange; Na⁺/Ca²⁺ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation     

Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra

Freshly dispersedinterstitial cells from the rabbit urethra were studied by using theperforated-patch technique. When cells were voltage clamped at 60 mVand exposed to 10 µM norepinephrine (NE) at 80-s intervals, eitherlarge single inward currents or a series of oscillatory inward currentsof diminishing amplitude were evoked. These currents were blocked byeither phentolamine (1 µM) or prazosin (1 µM), suggesting that theeffects of NE were mediated via ₁-adrenoceptors.NE-evoked currents were depressed by the blockers ofCa²⁺-activated Cl currents, niflumic acid (10 µM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversalpotential of the above currents changed in a predictable manner whenthe Cl equilibrium potential was altered, againsuggesting that they were due to activation of a Clconductance. NE-evoked currents were decreased by 10 µM cyclopiazonic acid, suggesting that they were dependent on store-releasedCa²⁺. Inhibition of NE-evoked currents by the phospholipaseC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate(100 µM) suggested that NE releases Ca²⁺ via an inositol1,4,5-trisphosphate (IP₃)-dependent mechanism. Theseresults support the idea that stimulation of₁-adrenoceptors releases Ca²⁺ from anIP₃-sensitive store, which in turn activatesCa²⁺-activated Cl current in freshlydispersed interstitial cells of the rabbit urethra. This elevates slowwave frequency in these cells and may underlie the mechanismresponsible for increased urethral tone during nerve stimulation.

     

Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes

The effect of -adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented I_NCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl^– current (CFTR-Cl^– current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl^– bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 µM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion