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PubMed1997

Differential activation of protein kinase C delta and epsilon gene expression by gonadotropin-releasing hormone in alphaT3-1 cells. Autoregulation by protein kinase C.

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.

Animals↗
PubMed1997

Polarized expression of Ca2+ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca2+]i waves.

The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca2+ pumps and characterizing their role in initiation and propagation of Ca2+ waves. Specific and polarized expression of Ca2+ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca2+ release with ionomycin on Ca2+ release evoked by agonists and Ca2+ uptake induced by antagonists. Despite their polarized expression, Ca2+ uptake by SERCA pumps and Ca2+ efflux by PMCA resulted in uniform reduction in [Ca2+]i. Surprisingly, inhibition of the SERCA pumps, but not Ca2+ release by ionomycin, eliminated the distinct initiation sites and propagated Ca2+ waves, leading to a uniform increase in [Ca2+]i. In addition, inhibition of SERCA pumps reduced the rate of Ca2+ release from internal stores. The implication of these findings to rates of Ca2+ diffusion in the cytosol, compartmentalization of Ca2+ signaling complexes, and mechanism of Ca2+ wave propagation are discussed.

Animals↗
PubMed1998

Related adhesion focal tyrosine kinase and the epidermal growth factor receptor mediate the stimulation of mitogen-activated protein kinase by the G-protein-coupled P2Y2 receptor. Phorbol ester or [Ca2+]i elevation can substitute for receptor activation.

The activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells. Previously, we demonstrated that the activation of G-protein-coupled P2Y2 receptors by extracellular ATP and UTP stimulated MAP (p42 ERK2) kinase by a mechanism that was dependent on the elevation of [Ca2+]i and the activation of related adhesion focal tyrosine kinase (RAFTK) (also called PYK2, CAKbeta, and CADTK) and protein kinase C (PKC). Here, we examine further the signaling cascade between the P2Y2 receptor and MAP kinase. MAP kinase was transiently activated by exposure of PC12 cells to UTP. UTP, ionomycin, and phorbol ester (phorbol 12-myristate 13-acetate) increased MAP kinase activity and also promoted the tyrosine phosphorylation of RAFTK, the epidermal growth factor (EGF) receptor, SHC, and p120(cbl). Down-regulation of PKC and inhibition of the elevation of [Ca2+]i, conditions that block the activation of MAP kinase, also blocked the increases in the tyrosine phosphorylation of RAFTK and the EGF receptor. AG1478, a tyrphostin selective for the EGF receptor, reduced the activation of MAP kinase, the tyrosine phosphorylation of SHC, the association of Grb2 with SHC, and the tyrosine phosphorylation of the EGF receptor and p120(cbl) but did not block the tyrosine phosphorylation of RAFTK. The similar effects of UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA) on these signaling proteins demonstrate that the two signaling molecules from phosphatidylinositol 4,5-bisphosphate hydrolysis ([Ca2+]i, from inositol 1,4,5-trisphosphate production, and diacylglycerol) can individually initiate the activation of MAP kinase in an EGF receptor-dependent manner. These results demonstrate that the P2Y2 receptor-mediated transactivation of the EGF receptor occurs at a point downstream of RAFTK and indicate that the EGF receptor is required for P2Y2 receptor-mediated MAP kinase activation. Although P2Y2 and EGF receptors may both activate a similar multiprotein signaling cascade immediately upstream of MAP kinase, the P2Y2 receptor appears to uniquely utilize [Ca2+]i, PKC, and, subsequently, RAFTK.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k.

Activation of the phosphatidylinositol 3-kinase (PI3K) plays an important role in the mitogenic response of many cell types. Recently, two serine/threonine kinases Akt and p70(S6k) have been identified as physiological targets of PI3K. Observations that expression of activated forms of Akt led to the activation of p70(S6k) implied Akt might mediate mitogenic signaling through activation of p70(S6k). To clarify the relationship between signaling through these two kinases, we have examined their regulation by various mitogenic stimuli. In this study we have focused on the role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts. Depletion of intracellular calcium stores by EGTA pretreatment has no effect on growth factor-induced Akt activation but completely abolishes p70(S6k) stimulation. Increase of intracellular calcium induced by ionomycin or thapsigargin results in a full activation of p70(S6k), whereas little or no activation of Akt is observed. Furthermore, although PI3K in anti-phosphotyrosine immunoprecipitates is only very weakly activated by ionomycin, the calcium-induced stimulation of p70(S6k) is completely inhibited by the specific PI3K inhibitor wortmannin. We conclude Akt and p70(S6k) lie on separate signaling pathways. Activation of signaling to Akt is insufficient for the activation of p70(S6k), which can be achieved independently of Akt. p70(S6k) requires a separate calcium-dependent and wortmannin-sensitive process that is likely to be independent of type IA PI3K family members.

3T3 Cells↗
PubMed1999

Regulation of the human interleukin-5 promoter by Ets transcription factors. Ets1 and Ets2, but not Elf-1, cooperate with GATA3 and HTLV-I Tax1.

Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.

Base Sequence↗
PubMed2002

Calcineurin and NFAT4 induce chondrogenesis.

Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.

Animals↗
PubMed2000

Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets.

We have investigated the restoration of [Ca(2+)](i) in human platelets following the discharge of the intracellular Ca(2+) stores. We found that the plasma membrane Ca(2+)-ATPase is the main mechanism involved in Ca(2+) extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-l-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca(2+)](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca(2+) extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca(2+)](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca(2+) extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca(2+)](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca(2+) extrusion in human platelets.

1-Phosphatidylinositol 4-Kinase↗
PubMed2000

Induction of persistent sodium current by exogenous and endogenous nitric oxide.

Most voltage-gated Na(+) channels inactivate almost completely at depolarized membrane potentials, but in some cells a residual Na(+) current is seen that is resistant to inactivation. This persistent Na(+) current can have a profound impact on the electrical behavior of excitable cells, and the regulation of this property could have important biological consequences. However, the biological signaling mechanisms that regulate the persistence of Na(+) channels are not well understood. This study showed that in nerve terminals and ventricular myocytes nitric oxide (NO) reduced the inactivation of Na(+) current. This effect was independent of cGMP, was blocked by N-ethylmaleimide, and could be elicited in cell-free outside-out patches. Thus, a reactive nitrogen species acts directly on the channel or closely associated protein. Persistent Na(+) current could also be induced by endogenous NO generated enzymatically by NO synthase (NOS). Application of ionomycin to raise the intracellular Ca(2+) concentration in myocytes activated NOS. The NO produced in response to ionomycin was detected with an NO-sensitive fluorescent dye. Persistent Na(+) current was enhanced by the same treatment, and NOS inhibitors abolished both the elevation of NO and the induction of persistent Na(+) current. These experiments show that NO is a potential endogenous regulator of persistent Na(+) current under physiological and pathophysiological conditions.

Animals↗
PubMed2001

Regulation of nuclear factor kappa B transactivation. Implication of phosphatidylinositol 3-kinase and protein kinase C zeta in c-Rel activation by tumor necrosis factor alpha.

Transactivation by c-Rel (nuclear factor kappaB) was dependent on phosphorylation of several serines in the transactivation domain, indicating that it is a phosphorylation-dependent Ser-rich domain. By Ser --> Ala mutational and deletion analysis, we have identified two regions in this domain: 1) a C-terminal region (amino acids 540-588), which is required for basal activity; and 2) the 422-540 region, which responds to external stimuli as tumor necrosis factor (TNF) alpha or phorbol myristate acetate plus ionomycin. Ser from 454 to 473 were shown to be required for TNFalpha-induced activation, whereas Ser between 492 and 519 were required for phorbol myristate acetate plus ionomycin activation. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) zeta were identified as downstream signaling molecules of TNFalpha-activation of c-Rel transactivating activity. Interestingly, dominant negative forms of PI3K inhibited PKCzeta activation and dominant negative PKCzeta inhibited PI3K-mediated activation of c-Rel transactivating activity, indicating a cross-talk between both enzymes. We have identified the critical role of different Ser for PKCzeta- and PI3K-mediated responses. Interestingly, those c-Rel mutants not only did not respond to TNFalpha but also acted as dominant negative forms of nuclear factor kappaB activation.

Amino Acid Sequence↗
PubMed2001

Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP.

The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25(Mn) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family GTPase Cdc42 through still unknown mechanisms. Here, we report that retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomycin-stimulated activities were unaffected by a constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF performance. Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inhibitory effects on Ras-GRF function. These results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded. Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.

Animals↗
PubMed2001

A novel C-terminal kinesin is essential for maintaining functional acidocalcisomes in Trypanosoma brucei.

Kinesins are cytoskeletal motor proteins that play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned, and functionally characterized in Trypanosoma brucei a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 microm for the microtubule dissociation constant (K(d)) with a k(cat) of 0.2 s(-1). Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH(4)Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo, we produced an inducible kinesin-deficient strain by double-stranded RNA interference methodology. Mutant cells were loaded with the fluorescent reagent fura2/acetoxymethylester to measure intracellular free calcium ([Ca(2+)](i)). The resting [Ca(2+)](i) was unchanged in mutant cells; however, alkalinization of acidic vesicles induced by NH(4)Cl or nigericin was not followed by release of Ca(2+). These data and the relative importance of the ionomycin-releasable and the ionomycin-plus-NH(4)Cl-releasable Ca(2+) pools suggest a lower Ca(2+) content in acidocalcisomes and dysfunctional Ca(2+) release.

Ammonium Chloride↗
PubMed2002

Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKCalpha-GFP.

The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha.

Adenosine Triphosphate↗
PubMed2003

Calcium induces cell survival and proliferation through the activation of the MAPK pathway in a human hormone-dependent leukemia cell line, TF-1.

Survival and proliferation of cells of a human myelo-erythroid CD34+ leukemia cell line (TF-1) depend on the presence of granulocyte-macrophage colony-stimulating factor or interleukin-3. Upon hormone withdrawal these cells stop proliferating and undergo apoptotic process. In this report we demonstrate that a controlled increase in [Ca2+]i induces hormone-independent survival and proliferation of TF-1 cells. We found that moderate elevation of [Ca2+]i by the addition of cyclopiasonic-acid protected TF1 cells from apoptosis. Furthermore, a higher, but transient elevation of [Ca2+]i by ionomycin treatment induced cell proliferation. In both cases caspase-3 activity was reduced, and Bcl-2 was up-regulated. Higher elevation of [Ca2+]i by ionomycin induced MEK-dependent biphasic ERK1/2 activation, sufficient to move the cells from G0/G1 to S/M phases. Meanwhile, activation of ERK1/2, phosphorylation of the Elk-1 transcription factor, and, consequently, a substantial elevation of Egr-1 and c-Fos levels and AP-1 DNA binding were observed. Moderate elevation of [Ca2+]i, on the other hand, caused a delayed monophasic activation of ERK1/2 and Elk-1 that was accompanied with only a small increase of Egr-1 and c-Fos levels and AP-1 DNA binding. The specific MEK-1 kinase inhibitor, PD98059, inhibited all the effects of increasing [Ca2+]i, indicating that the MAPK/ERK pathway activation is essential for TF-1 cell survival and proliferation. Based on these results we suggest that the elevation of the [Ca2+]i may influence the cytokine dependence of hemopoietic progenitors and may contribute to pathological hematopoiesis.

Antigens, CD34↗
PubMed2004

A novel human Cl(-) channel family related to Drosophila flightless locus.

Large conductance chloride (maxi-Cl(-)) currents have been recorded in some cells, but there is still little information on the molecular nature of the channel underlying this conductance. We report here that tweety, a gene located in Drosophila flightless, has a structure similar to those of known channels and that human homologues of tweety (hTTYH1-3) are novel maxi-Cl(-) channels. hTTYH3 mRNA was found to be distributed in excitable tissues. The whole cell current of hTTYH3 was large enough to be discriminated from the control but emerged only after treatment with ionomycin. Analysis of pore mutants suggested that positively charged amino acids contributed to anion selectivity. Like a maxi-Cl(-) channel in situ, the hTTYH3 single channel showed 26-picosiemen linear current voltage, complex kinetics, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid sensitivity, subconductance, and the permeability order of I(-) > Br(-) > Cl(-). Similarly, hTTYH2 encoded an ionomycin-induced maxi-Cl(-) channel, but TTYH1 encoded a Ca(2+)-independent and swelling-activated maxi-Cl(-) channel. Therefore, the hTTYH family encoded maxi-Cl(-) channels of mammals. Further studies on the hTTYH family should lead to the elucidation of physiological and pathophysiological roles of novel Cl(-) channel molecules.

Amino Acid Sequence↗
PubMed2004

Proteolytic conversion of STAT3alpha to STAT3gamma in human neutrophils: role of granule-derived serine proteases.

Four isoforms (alpha, beta, gamma, and delta) have been identified for signal transducer and activator of transcription 3 (STAT3). It has been reported that STAT3gamma, which is derived from STAT3alpha by limited proteolysis during granulocytic differentiation, is a major STAT3 isoform expressed in human neutrophils. We confirmed that STAT3gamma was a major STAT3 isoform detected in human neutrophil lysates prepared with the conventional lysis buffer. The enzymes capable of converting STAT3alpha to STAT3gamma in vitro were localized in neutrophil granule fraction and were released into the medium upon ionomycin stimulation. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, CuSO(4), and ONO-5046 (a specific inhibitor of neutrophil elastase), but not by aprotinin, leupeptin, benzamidine, and EDTA. STAT3gamma was effectively generated in vitro from STAT3alpha by limited proteolysis with human neutrophil elastase or proteinase 3 but not cathepsin G. The converting activity in neutrophil lysates was reduced by immunodepletion of elastase but not proteinase 3. Unexpectedly, STAT3gamma was undetected in the lysates of neutrophil-derived cytoplasts, which lack granules, and the cytosol fraction prepared by nitrogen cavitation. The STAT3 isoform detected in these preparations was primarily STAT3alpha. STAT3gamma was also undetected in the lysates of PMSF-pretreated neutrophils and was markedly decreased in the lysates of ionomycin-pretreated neutrophils. These findings indicate that, in contrast to the previous reports, STAT3alpha, but not STAT3gamma, is primarily expressed in human neutrophils, and STAT3gamma is rapidly generated from STAT3alpha by limited proteolysis with granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer.

Adult↗
PubMed2006

Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons.

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.

ATP Synthetase Complexes↗
PubMed2000

Effects of phorbol ester treatment on dibutyryl cyclic adenosine-5' monophosphate- and carbachol-stimulated aminopyrine accumulation in isolated rat parietal cells.

BACKGROUND: The functional role of the intracellular diacylglycerol/protein kinase C second-messenger pathway in the regulation of gastric acid secretion and the effects on the involved inositotrisphosphate/Ca2+/calmodulin system are not well understood, and contradictory data have been reported. We therefore evaluated the effects of phorbol ester treatment (tetradecanoylphorbol-12,13-acetate (TPA)) on dibutyryl cyclic adenosine-5' monophosphate (dBcAMP)- and carbachol-stimulated aminopyrine (AP) accumulation in comparison with intracellular alterations of the phospholipase C/inostol phosphate signal transduction pathway in isolated rat gastric parietal cells. METHODS: [14C]AP accumulation was determined as an indirect measure of gastric acid secretion. Inositolphosphate second-messenger activation was investigated with [3H]inositolmonophosphate release in [3H]-myoinositol prelabeled rat gastric parietal cells. RESULTS: TPA at a low concentration of 5 nM caused a small (45%) but significant increase in carbachol (0.1 mM)-stimulated AP accumulation, which was dose-dependently inhibited by higher concentrations of TPA with corresponding shifts in the dose-response curve for carbachol-stimulated AP accumulation. AP uptake stimulated by dBcAMP (0.1 mM) and the synergistic stimulatory effect induced by carbachol together with dBcAMP were inhibited by TPA at all concentrations investigated. In the presence of increasing concentrations of the calcium ionophore ionomycin (10(-8)-10(-5) M) TPA at 5 nM increased AP accumulation (AP ratio was 4.02 with 5 nM TPA versus 1.23 in the absence of TPA; P < 0.05), indicating that phorbol ester stimulates AP uptake in rat parietal cells. Simultaneous investigation of [14C]AP accumulation and [3H]inositol monophosphate release showed that inhibitory effects of TPA on carbachol- and carbachol plus dBcAMP-stimulated cells are mediated by an inhibition of the receptor/G-protein/phospholipase C interaction, leading to a reduction of inositolphosphate release. The costimulation of rat parietal cells with dBcAMP, ionomycin, and TPA (5 nM) did not reproduce the synergistic effects of carbachol together with dBcAMP on AP accumulation, suggesting that carbachol-stimulated AP uptake seems to be additionally mediated by a still unknown pathway independent of intracellular calcium release or protein kinase C activation.

Aminopyrine↗
PubMedDate not supplied

Enhanced production of macrophage inhibitory protein-1alpha in patients with Behçet's disease.

OBJECTIVE: Macrophage inhibitory protein-1alpha (MIP-1alpha), a C-C chemokine, stimulates the activation and migration of leukocytes. We investigated the expression of MIP-1alpha in patients with Behçet's disease (BD) and evaluated the association of the MIP-1alpha levels with disease activity of BD. METHODS: Serum samples were obtained from 67 BD patients and 30 healthy controls. Simultaneously, whole blood cells were isolated from BD patients (n = 25) and healthy controls (n = 11) and cultured in the absence or presence of lipopolysaccharide (LPS), phytohaemagglutinin (PHA), and phorbol 12-myristate 13-acetate (PMA) plus ionomycin. The concentrations of MIP-1alpha, interleukin-8 (IL-8), regulated on activation, normally T cell expressed and secreted (RANTES), and monocyte chemoattractant protein-1 (MCP-1) were measured in the sera and culture supernatants by enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum levels of MIP-1alpha were higher in BD patients than in healthy controls. When whole blood cells were stimulated with LPS or PMA plus ionomycin, but not PHA, BD patients had higher levels of MIP-1alpha in the culture supernatants compared to healthy controls. In sera and culture supernatants of whole blood cells, MIP-1alpha levels correlated well with those of RANTES, MCP-1, and IL-8 in BD patients. Moreover, patients with active disease had significantly higher levels of serum MIP-1alpha levels compared with those with inactive disease. CONCLUSION: MIP-1alpha levels were elevated in patients with BD, and correlated well with IL-8, RANTES, and MCP-1 levels. These results suggest that the increased MIP-1alpha levels in serum of BD patients may lead to activation and migration of leukocytes, playing a role, like other chemokines, in the pathogenesis of BD.

Adult↗